【共享】胶体金经典文献 IVD 有时间多发点，慢慢会多的！ng protein binding in nitrocellulose membranes.doc (197.5k)Assay Development Immunogold conjugation for IVD applicationsImmunogold conjugates created by linking proteins to gold colloid (above) are used as detector reagents in a wide array of IVD test systems.To create viable immunogold complexes for use in diagnostic applications, manufacturers must be capable of overcoming a variety of technical challenges. Nikki Robinson Immunogold conjugation is a technique whereby any protein, including antibodies and antigens, can be coupled to gold colloid to produce an immunogold complex. Although a variety of applications for such immunogold conjugates are still emerging, the current primary applications are in rapid-test devices used for the diagnosis and monitoring of disease.1Over the past decade, the growth of membrane-based rapid-test technologies—including both lateral-flow and flow-through formats—has created a vast market for gold-linked immunological reagents. Conjugates created using this technique are employed as detector reagents in test systems for allergies, infectious diseases, environmental contaminants, drugs, cardiac markers, fertility, and veterinary applications.2Like any other conjugation, a successful outcome is only achieved through the use of quality reagents (protein and colloid), sufficient manufacturer experience, and thorough knowledge of the final assay for which the conjugate is intended—together with end-user education in the handling of the immunogold complex. Proteins for ConjugationFor IVD applications, the types of proteins that are most commonly used to create immunogold conjugates are antibodies and antigens. One factor that can affect antibody conjugations is whether monoclonal or polyclonal antibodies are used.Although most antibody conjugates for diagnostics are created using immunoglobulin G (IgG) antibodies, IgM, IgE, and IgA can be conjugated successfully to colloidal gold. Further consideration should also be given to the subclass of antibody that is used for conjugation. Among the IgG subclasses of mouse antibodies, for example, IgG1 has the best success rate, while IgG3 has proven to be technically challenging. Preparing conjugates using antigens can also be technically challenging. Conjugators typically encounter no difficulties in creating bonds to link the antigen to the colloid. But because of the ways that antigens bind to gold colloid, the success rate for creating a conjugate in which the gold label does not mask the working reactive epitopes of the protein can be as little as 50%. For this reason, it is important for conjugate developers to give consideration to the ways in which proteins bind to gold.3Binding MechanismsResearchers generally accept the theory that mechanisms related to the residues of three particular amino acids play an important role in binding proteins to gold particles. Each of these amino acids—lysine, tryptophan, and cysteine—operates by a different mechanism to bring about conjugation.4·Lysine is highly positively charged and therefore naturally attracted to a negatively charged gold particle.·Tryptophan works through hydrophobic interactions.·Cysteine creates dative bonds through the formation of sulphur bridges with the gold surface, such that the antibody and gold particles share electrons. Of all the forces controlling the attachment of antibodies to gold particles, this is the strongest, most permanent, and most difficult to break. The success of the conjugation process depends to a large extent upon the location of these amino acid residues in the protein that is being conjugated. In some cases, poorly located amino acids can bind to gold molecules in such a way that the gold actually interferes with the binding capacity of the protein. Termed steric hindrance, this type of interference can occur when the amino acids are located in the antigen-binding (Fab) region of antibodies, or among the working reactive epitopes of antigens (that is, epitopes recognized by specific antibodies). Such interference is almost impossible to overcome without compromising the integrity of the protein molecule being conjugated.In order for an immunoassay to achieve optimal sensitivity, it is therefore important that the three amino acids involved in conjugation be located appropriately. For antibodies, they should be located in the Fc region. For antigens, they should be physically isolated from the working reactive epitopes.Optimizing Conjugation In order to develop and optimize a single gold conjugate, it is not unusual for researchers to produce small-scale batches of as many as 60 different conjugates. Each of these conjugates differs from the others in one key way, so that each can be tested to determine which conjugate is likely to give the best response in the final assay for which it is intended. Such testing should encompass all the parameters that moderate the sensitivity, cross-reactivity, and potential stability of the conjugate. Characteristics to be tested include, but are not limited to, antibody vehicle buffer, preservative type, salt ratio, surfactant content, gold colloid size, blocking agent type, total protein concentration, final conjugate buffer, and conjugate concentration. To determine the effects of different blocking agents, for instance, tests for the following should also be considered.·Type of blocking agent (e.g., casein, bovine serum albumin, ovalbumin, human albumin, PEG, nonspecific IgG). ·Purification grade of the blocking agent. ·Concentration of the blocking agent. ·Compatibility of the blocking agent with the completed assay.Figure 1. Diagrammatic representation of a half dipstick used for screening conjugates.Developers should conduct experiments to determine the optimal values for all such factors prior to scaling up for prototype production. Ultimately, however, the only way to determine which conjugate should be scaled up for pilot production is to develop a mini-assay that mimics the final test conditions as closely as possible. For membrane-based assays, the easiest way to do this is to use a half-dipstick format that requires only a capture antibody and a purified antigen (see Figure 1). By using such a simplified test format, many small-scale conjugates can be assessed to determine which is most suitable without encountering common problems such as those related to drying techniques or sample buffer preparation.In order to carry out all of this testing, conjugators usually require 3 mg of antibody or 5 mg of antigen. Using these small samples, the conjugator can then optimize the manufacturing parameters for the conjugate and provide a preliminary sample for assessment. Once a quality antibody or antigen has been conjugated, and its manufacturing parameters have been set, it is usually straightforward for the manufacturer to faithfully reproduce and scale up the chosen conjugate.Quality of Materials for Conjugation Figure 2. The important aspects of antibody pairs include steric separation of epitopes, adequate titer of stocks, high affinity, high specificity, high avidity, and purity.The suitability of a particular antibody for use in creating an immunogold conjugate depends largely on the technical specifications of the assay in question. For lateral-flow assays, the best types of antibodies to use are generally monoclonal pairs. In this case, one monoclonal antibody is labeled with the gold colloid as the detector reagent, and the other is immobilized on the nitrocellulose membrane as the capture reagent. Each antibody should be specific to a different reactive epitope on the antigen surface, and these epitopes in turn should be physically isolated from one another (see Figure 2). Polyclonal antibodies can also be used, but they should be at least protein-A purified. In most cases, affinity purification provides the most sensitive and specific conjugate. This depends of course on the level of acceptable cross-reactivity in the assay specifications.Affinity PurificationAmmonium sulphate precipitation, or DEAE purified serum, contains a mass of protein that will compete for binding sites on the surface of the gold colloid. Those proteins with the highest levels of the three amino acid residues that control protein binding to the colloid will conjugate most readily to the naked, negatively charged colloid. However, these may not be the proteins required to drive the assay. When this occurs, the detector reagent of the assay can have a decreased degree of sensitivity. Similarly, if the antibody preparation is protein-A or protein-G purified, then the IgG fraction may contain high levels of interfering IgG, and not just the specific IgG required. All of the IgG will attempt to bind to the gold colloid, and a low level of sensitivity may result. Affinity purification—with careful elution of the highest-affinity antibodies from the column—should yield the most-specific antibodies for antibody preparation and therefore produce the most-specific gold conjugate. These antibodies may require further absorption in order to modify cross-reactivity characteristics. Although affinity purification can be costly in terms of time, money, and serum, it produces a quality conjugate which should be considered a key raw material for any assay. Antigen ConjugationSuccessful creation of antigen conjugates depends on two factors: size and the situation of the three amino acid residues that control the binding of the antigen to the colloid. Most antigen conjugates are requested for use in rapid tests, such as serological sandwich or competition assays. For such assays, a 40-nm colloid is often chosen. Until recently, it was considered that the smallest particle that could be conjugated to a 40-nm colloid had a molecular cutoff weight of 30 kDa. Under some conditions, however, advances in conjugation techniques can cut this limit in half to 15 kDa. The primary restriction for conjugates using such small particles is that the protein to be conjugated must contain sufficient amounts of lysine, tryptophan, and cysteine residues. Antigens frequently do not contain enough cysteine, so the strength of the bond between the antigen and the colloid is relatively weak and easy to break, particularly in the presence of high-affinity antibodies in the sample.For antigens with a molecular weight of less than 30 kDa, other techniques may be applied, such as conjugation to smaller gold particles. This too may result in a loss of sensitivity due to the reduced visibility of smaller gold colloids at the capture site. For antigens containing little or none of the three binding residues, an efficient solution is to preconjugate the antigen to a carrier molecule, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). However, this technique is not as simple as it sounds and is not something that the amateur protein chemist should attempt. The type of linker used, the length of the linker used, the molar ratio of the hapten to the BSA, and the type of carrier molecule used are only some of the factors that must be carefully considered in order to carry out a reproducible conjugation that exposes the working reactive epitopes of the antigen—and therefore maximizes the potential sensitivity of the protein-carrier-gold conjugate in the assay.Size MattersSelection of the size of the colloid depends upon the application for which the conjugate is intended. The following sections describe relevant issues for two of the most common applications for gold conjugates: microscopy and rapid-test devices.Microscopy. Any gold conjugate that is 1 to 40 nm may be used for electron microscopy and light microscopy. However, it may not be possible to view 1- or 2-nm colloids even under a transmission electron microscope that provides 250,000 magnification. To make it possible for end-users to visualize the gold-labeled antigenic sites on such small particles, the gold colloid can be enhanced with silver. Silver enhancement involves the precipitation of silver salts on the gold surface, which allows the gold particle to grow in size until it can be seen under the microscope. This process is most effective and easily controlled when all of the gold particles are the same size and shape, with a low coefficient of variation. Using small particles, such as 1- and 2-nm gold particles, facilitates access to reactive epitopes that larger gold particles would not be able to label due to steric hindrance. Therefore, the gold conjugate will bind easily and is grown by silver enhancement once the antigen has been located. For electron microscopy without silver enhancement, several different reactive epitopes may be labeled on the same section of the conjugate. In this case, colloids with distinct size separations are used to label antibodies specific to each of the different reactive epitopes.Rapid tests (lateral-flow and flow-through devices). The most common size of gold colloid used for this application is 40 nm.4 A 40-nm colloid offers maximum visibility with the least steric hindrance in the case of IgG conjugations (see Figure 3). An IgG antibody with a molecular weight of 160 kDa is approximately 8 nm long. Gold particles between 40 and 100 nm can also be conjugated successfully to IgG antibodies. Such larger particles are certainly more visible than the 40 nm particles, but there are fewer particles in a 1-ml solution at an optical density of 520 nm, so fewer of them can be packed onto the capture line. Overall, this usually expresses itself as a loss of signal at lower levels of analyte (between 1 and 10 ng/ml), when compared with the same antibody conjugated to a 40- or 60-nm colloid.Figure 3. The relationship between particle size, visibility, and steric hindrance. Note that as the particle size increases, signal visibility increases, but steric hindrance causes an overall decrease in visual sensitivity. A 60-nm colloid is not routinely employed but can be useful for rapid-test assays. Its color is slightly different compared with a 40-nm colloid. Good quality 40-nm colloid should be cherry red, while 60-nm colloid is deep pink.4 For some applications in which the sample type may cause background staining of the membrane, this subtle color difference may make a signal easier to read. For example, samples containing bilirubin will leave a brown background stain, and it is easier to visualize pink on brown than red on brown. If the molecule to be conjugated has a molecular weight less than 160 kDa, then a 20-nm colloid may be more appropriate. Colloids of 20 nm are commonly used for streptavidin, protein A, and antigen conjugations in which the protein that is being presented for conjugation has a molecular weight of less than 60 kDa. Sensitivity Advances in rapid-test technologies may increase the sensitivity limits of gold conjugates. At present, the sensitivity limit in most assays is 1 ng/ml, with high-quality antibodies reaching as low as 10 pg/ml. However, the development of silver enhancement techniques may increase sensitivity still further, perhaps from 10 to 100 times.4Conclusion Conjugation of proteins to colloidal gold is a process that should not be undertaken lightly. While it is relatively straightforward to make an immunogold conjugate, it is difficult to make a quality gold conjugate, and many inexperienced manufacturers encounter problems in batch reproducibility and scale-up. References1. S Brunelle, \"Electroimmunoassay Technology for Food-Borne Pathogen Detection,\" IVD Technology 7, no. 5 (2001): 55–66.2. S Wallace, \"Bioterrorism Tests in Demand,\" IVD Technology 7, no. 6 (2001): 14.3. J Chandler, N Robinson, and K Whiting, \"Handling False Signals in Gold-Based Rapid Tests,\" IVD Technology 7, no. 2 (2001): 34–45.4. J Chandler, T Gurmin, and N Robinson, \"\"The Place of Gold in Rapid Tests,\" IVD Technology 6, no. 2 (2000): 37–49.Nikki Robinson, BSc, is custom conjugation manager at British BioCell International (Cardiff, Wales, UK). She can be reached via nikkirobinson@bbigold.com.Troubleshooting protein binding in nitrocellulose membranes Part 2: Common problems Kevin D. Jones Manufacturers can ensure good protein binding results if they perform carefully designed and controlled experiments during product development. The first installment of this article (IVD Technology, March/April 1999) covered the basic principles behind protein binding.1 This second installment discusses common problems and the most common solutions. For clarity, the problems have been broken down into six generic groups (see Table I). Because IVD technology is wide and varied, there are no universal answers. No amount of discussion can replace experimental results. The purpose of this article is, therefore, to suggest starting points for experiments. ProblemPossible CausesNonspecific signalNonspecific protein bindingNonspecific conjugate bindingWeak or diffuse capture lineInsufficient protein appliedCapture reagent spreading after applicationCapture reagent being washed off the membraneLateral wicking rate too fastCapture reagent binding only weakly to the membraneAffinity constant between capture reagent and target analyte too lowUneven capture line wettingUneven membrane dryingUneven hydrophobicityUneven pore structureCapture line too thickCapture reagent spreading too far after applicationCapture reagent being washed away when sample is appliedToo much protein addedApplication aperture too largeInsufficient membrane drying after applicationCapture line too thinCapture reagent binding too rapidly after applicationInsufficient proteinApplication aperture too narrowUneven line intensityMembrane hydrophobicity variationPressure variation in application systemPoorly mixed protein solutionsProtein precipitationFlow problemsSuboptimal storage conditionsUneven membrane pore structureTable I. Common defects associated with nitrocellulose membrane–based test development and the most likely causes.The decrease in nitrocellulose surface area available for protein immobilization can be seen as the membrane pore size increases from 0.45 m through 3.0 m to 8.0 m . Increased pore size leads to a faster wicking rate, but also means that the protein being applied travels a greater distance before it comes into contact with the nitrocellulose membrane wall. The capture line will thus be wider on an 8- m membrane than on a 0.45- m membrane, because of both the increased lateral wicking rate and the increased distance traveled by the protein before immobilization. Nonspecific Signal Nonspecific signal in nitrocellulose-based assays is a significant problem for the diagnostic industry. It can appear either generally over the entire membrane surface or, more seriously, at the capture line. Causes. Nitrocellulose will bind proteins, which is the mechanism behind the capture line. The reasons for this binding were discussed in Part 1. Nonspecific protein binding. Nonspecific protein binding can be caused by an unwanted interaction between the sample or conjugate and the capture line. Such interactions at the capture line can be the result of any of the common causes of protein attachment, including charge attraction, hydrophobicity, disulfide bridging, or a genuine nonspecific immunogenic binding. Conjugate binding. Since conjugate particles are covered by a protein layer, the causes of nonspecific conjugate binding are similar to the causes of nonspecific protein binding. Additional problems can occur. The conjugate particles themselves are often permanently charged. In the case of gold conjugate, the conjugate is very susceptible to interaction with sulfhydryl moieties on the surfaces of proteins and membranes. The causes of and remedies for the interaction of conjugates are discussed in literature available from conjugate manufacturers.2–4 Solutions. Nonspecific attachment to the membrane can normally be reduced by blocking with a protein (e.g., bovine serum albumin [BSA]), surfactant (Tween 20, Triton X-100, or sodium dodecyl sulfate [SDS]). The effect of charge can be overcome by either changing the pH of the test or increasing the ionic strength of the system. Disulfide bridging is rarely seen in diagnostic products, although nonspecific immunogenic interactions do regularly occur. Standard immunologic blocking techniques, such as use of a similar protein as a blocking reagent, can solve this problem. For example, if a nonspecific interaction occurs between a specific mouse IgG and a human sample, blocking the sample with mouse serum would remove the false-positive signal.5–7 Weak or Diffuse Capture-Line Intensity If the level of capture reagent per unit area of membrane surface is too low, the capture line may be weak or diffuse. The problem may be simply mechanical; for instance, too-rapid movement of the membrane through the application striping system. However, the cause may be more complex. Causes. It is often impossible to determine the cause of this problem by merely examining the test strip. The only solution is to work through the potential causes and find by experiment where the major problem lies. Concentration of the capture reagent in the application solution is too low. The application solution diffuses away from the point of application. The capture reagent spreads with the application solution to an extent defined by the partition coefficient between the solid phase and the solute. If attachment to the solid phase is preferred, the capture reagent will spread less. If solubilization in the solute is preferential, then the capture reagent will spread further (see Figure 1). Figure 1. A weak capture line indicates that the amount of protein bound to the membrane is too low. Figure 2. A diffuse capture line can result when the capture reagent is washed away by the passage of analyte proteins and surfactant solutions. Capture reagent is washed off the membrane by the sample. If the strength of attachment between the capture reagent and the membrane is too low, or the surfactant level in the running buffer is too high, the capture reagent may be physically removed from the membrane surface by the sample (see Figure 2). The lateral wicking rate of the membrane is too high, causing spread of the capture reagent after application. If the membrane used has a very high lateral wicking rate, the applied protein will diffuse very rapidly from the point of application. As in all physical interactions, there is a time-related function—the faster the protein is moving in a lateral direction, the wider the capture line will be. The protein solution will also penetrate vertically into the membrane, and most of the protein that penetrates will be wasted, as only the material trapped in the top 10 m of the nitrocellulose membrane will be visible. This combination of lateral and vertical wicking can cause a weak capture line. The apparent concentration of the analyte in solution is a function of the lateral wicking rate of the test (apparent concentration 1/wicking rate).2 This equation means that as the wicking rate increases, the apparent concentration of the analyte drops dramatically.8 A reduction in the lateral wicking rate (by use of a smaller-pore nitrocellulose or a viscosity modifier) results in a stronger test line. However, it may cause an increase in nonspecific signal, a change in the test sensitivity cut-off, or a significant increase in test time. The capture reagent binds only weakly to the membrane. The binding of proteins to membranes can be influenced by optimization of the application buffer. However, some capture reagents are difficult to attach to membranes, either because of their size or their surface properties. In these cases where the binding is very weak, there are only a limited number of solutions. The most common is cross-linking the required reagent to a carrier protein. The affinity constant between the capture reagent and the target analyte is too low to support efficient capture. The use of low-affinity antibodies is, fortunately, normally avoidable. Careful screening of antibodies during development is an absolute necessity. In cases where the use of a low-affinity antibody is the only option, then the use of a small-pore membrane coupled with a low lateral wicking rate will maximize the interaction between the sample and the analyte. Solutions. Whatever the cause of weak or diffuse capture-line intensity, the solution lies in achieving a higher concentration of capture reagent at the desired point. Use a smaller-pore membrane. The use of a smaller-pore membrane will increase line sharpness and intensity for two reasons. First, the smaller the pore, the greater the surface area of the material, and the greater the surface area, the higher the concentration of capture reagent that attaches to it. Second, the smaller the pore, the slower the wicking rate. Use a different membrane. Different membranes have different binding characteristics for different capture materials. Selecting a membrane that has better binding characteristics for the particular capture reagent chosen improves the capture line appearance. Use a higher protein concentration. Increasing the concentration of the capture reagent in the application buffer may cause a higher concentration of the capture reagent to attach to the membrane. This would allow a higher level of analyte to stick in the capture zone and hence improve signal intensity. Change the application buffer. Modification of the application buffer adjusts the point of equilibrium between the amount of capture reagent attached to the membrane and the amount remaining in solution. Optimization of the buffer following the principles outlined in Part 1 of this article gives the maximum adsorption of the capture reagent to the membrane.1 Reduce the lateral wicking rate. Slowing the lateral wicking rate by the introduction of a viscosity modifier or by the use of a smaller-pore membrane effectively increases the concentration of the analyte in solution and hence improves the appearance of the capture line. Cross-link the capture reagents. If the molecular weight of the applied protein is low (or if there are unfavorable surface properties), the use of a cross-linking agent can significantly enhance the level of protein binding observed. The cross-linking itself can either precede or follow application of the sample to the membrane. The most common method is to cross-link the capture reagent to a carrier protein that will not cause any nonspecific interactions prior to application. The proteins typically used for this purpose are BSA or keyhole limpet haemocyanin (KLH). It is possible to use a cross-linking agent after capture reagent application (e.g., a glutaraldehyde wash step). However, the developer should ensure that all active groups introduced to the membrane are efficiently blocked before performing the test (e.g., in the case of glutaraldehyde, an ammonium sulfate wash blocks any free aldehyde groups). More specific examples of cross-linking chemistries can be found in the literature or in suppliers\' catalogs.10–12 Uneven Capture-Line Wetting The uneven rewetting of the capture line can have very serious consequences for the developers of rapid diagnostic assays. If the membrane rewets unevenly, the capture line will be seen to be striped. In the worst case, it is possible for the capture line to be more hydrophobic than the surrounding membrane (normally due to the removal of membrane surfactant in washes). In this case it is possible for \"submarining\" to occur—this is when the sample runs through the membrane until it reaches the capture line, the higher hydrophobicity of the capture line effectively stopping lateral flow. The sample may then run along the plastic support of the membrane (which is more hydrophilic), and then reenter the membrane above the capture line where the membrane is more hydrophilic. This can result in a capture line that has no or very little sample penetration, with obvious problems for test sensitivity and selectivity. Causes. Capture-line wetting may be uneven because of a defect in the membrane itself or because of a flaw in the test-strip manufacturing process. Membrane drying is uneven. The rewetting of a nitrocellulose membrane is usually dependent upon the degree of drying the membrane has undergone. If the relative rates of drying vary across a membrane due to variability of the drying conditions to which the membrane was exposed during manufacture, the rate at which the membrane rehydrates will vary across the membrane. Blocking MethodEfficiencyProsConsDip blocking of entire membraneHighVery even rewettingHigh batch-to-batch consistencyGood storage propertiesRequires expensive coating equipmentMembrane must be blocked after protein application but before attachment to the sample padsMay redissolve capture reagentsInclusion of a blocking agent may reduce capture reagent antigenicity or shelf lifeInclusion of blocking agent in a sample pad or conjugate padModerateCheap and easy to performNo redissolving of capture reagentsSeparation of capture reagent and blocking agent reduces chance of an unfavorable interactionNot as efficient as blocking the membrane itselfInclusion of a surfactant in the capture reagent application bufferCapture line: HighMembrane: LowCheap and easy to performNot as efficient as blocking the entire membraneInclusion of a blocking agent may reduce capture reagent antigenicity or shelf lifeInclusion of a blocking agent may cause the capture reagent to spread significantly on applicationTable II. The efficiency of different blocking techniques. The application of an aqueous sample can also affect the distribution by washing any water-soluble residues away from the point of application. Any variation in the character or concentration of these residues will affect the rewetting rate of the membrane. Hydrophobicity of the membrane is uneven. Perhaps the most significant factor for uneven capture-line wetting is hydrophobicity variation in the membrane. The rate of membrane rehydration is strongly influenced by the presence of hydrophobic or hydrophilic residues on the membrane surface. These residues can be introduced by membrane posttreatments (e.g., the introduction of a rewetting agent), hydrophilic materials added during manufacture, or additives in the striping buffer. The distribution of these hydrophilic materials is a factor in the evenness of the initial application and any subsequent migration of the hydrophilic materials through the membrane during storage. Membrane pore structure is inconsistent. The pore structure of nitrocellulose membranes is a function of the parameters in the casting machine during the casting process. Uneven airflow within the casting machine may cause a variation within the membrane. As nitrocellulose-casting machines normally use laminar airflow, uneven airflow is typically seen as a variation across the width of the machine. This can be seen as a variation in performance between adjacent rolls cut from the larger master roll. The developer cannot solve this problem without performing 100% quality control checks on incoming materials. The best compromise is to ensure that across-machine variation is evaluated adequately during the development process. Solutions. As mentioned above, if a defect in the membrane is the cause of uneven rewetting, the only recourse is more-rigorous inspection of raw materials. If the problem is a result of the test manufacturing technique, however, there are several possible solutions. Change the application buffer. Introduction of a mild surfactant to the striping buffer ensures that the capture line is in an evenly hydrophilic environment. This encourages even rewetting of the capture line. The choice of surfactant and its concentration is critical; effective results can be obtained using a low-concentration (~0.1%) SDS or sodium dodecylbenzolylsulfonate solution. Perform a membrane-blocking step. The most common way to ensure even wetting of the capture line is to use a blocking technique. Blocking the membrane with a material that promotes rewetting of the membrane ensures rapid and even membrane rewetting. The effect of these blocking agents has been evaluated in product support literature.13,14 The developer should investigate a range of blocking agents to find the most efficient for any particular test. The method used for membrane blocking is also a significant factor in the success or failure of the blocking step. There are three points where a blocking agent can be applied (see Table II). The choice of method for inclusion of the blocking agent depends on the efficiency of the blocking step coupled with the cost of achieving the solution. As a compromise, therefore, the second technique (inclusion of the blocking agent in the conjugate pad or in a sample application pad) is often chosen. While it is less efficient than blocking the entire membrane, the procedure is operationally simple. Change the membrane. A membrane with a surfactant posttreatment is more likely to show uneven capture line rewetting than a membrane without one. Application of the capture reagent solution may wash the surfactant away from the membrane surface. The removal of the additional rewetting agent can significantly affect the rewetting properties of the capture line. When no additional rewetting agent is added to the membrane surface, the line rewetting is likely to be consistent. However, the overall speed of rewetting may be slow. Capture Line Too Thick If the capture line is too thick, test results may be difficult to interpret (see Figure 3). The capture line may well show significant intensity variation across its width, probably with an intense front and back edge. For an inexperienced user the potential variation across the width may be confusing. Figure 3. Problems with protein binding are typically visible in the capture line of an assay\'s test result, as in these examples.Causes. At first glance the cause of too thick a capture line may seem obvious: too thick a line is being applied. In reality, this is only one of many possible causes. The capture line is spreading too far after its application. If the protein being applied favors remaining in solution rather than attaching to the solid phase, it is possible that the protein molecules will move with the solvent front of the application buffer. In such a case the capture line may be extremely wide or have sharp edges with a relatively diffuse middle portion. The latter effect (colloquially known as a \"coffee ring effect\") is caused by migration of the protein with the solvent and increasing concentration of the protein as the solvent evaporates. The capture reagent is washed away when the sample is applied. If the physical attachment of the protein to the membrane is too weak, or if a surfactant is present in the system, the capture line itself can be washed away as the sample wicks up the membrane. The displaced protein can then reattach to the nitrocellulose above the capture line. Too much protein is applied. If we assume that the protein saturates the available nitrocellulose, the effect of excess protein levels will be the spread of the protein capture line. The application aperture is set too wide. The protein will normally attach to the membrane at the point of application. If the settings are such that the sample is applied over a wide area, then the capture line will be wide. The membrane is dried insufficiently after capture line application. If the membrane is dried insufficiently, the capture reagent will not be efficiently immobilized.1 The application of the test sample may wash the capture reagent away from the point of application. The capture line may therefore become significantly wider due to the spreading of the capture reagent. Solutions. The possible means of achieving a line of the desired thickness involve varying many of the parameters already discussed. Changes in the membrane, the buffer, the blocking agent, or the manufacturing conditions may be appropriate. Change the application buffer. Optimization of the application buffer, both to minimize protein stability in solution and to maximize the viscosity of the application buffer, will produce the sharpest capture line on the membrane. The slower the protein solution flows, the greater the chance that the protein will bind close to the point of application (see Figure 4).Figure 4. Varied results from capture lines of 1mg/ml mouse IgG applied using different buffers: 10 mmol phosphate, pH 7.2; 10 mmol phosphate + 3% methanol, pH 7.2; 10 mmol phosphate + 150 mmol NaCl + 3% methanol, pH 7.2; 50 mmol phosphate + 150 mmol NaCl + 1% BSA, pH 7.2; 50 mmol phosphate + 150 mmol NaCl, pH 7.2; 50 mmol phosphate + 150 mmol NaCl, pH 6.0. All samples were detected by a 40 nmol gold-conjugated goat antimouse IgG antibody.Use a smaller-pore membrane. Smaller-pore membranes have a greater surface area of nitrocellulose per unit area of membrane, and the wicking away from the point of application is therefore slower than in larger-pore membranes. The combination of these effects means that the protein line is relatively narrow. Change the membrane. Selecting a membrane with higher binding capacity will improve line sharpness. Change the membrane blocking conditions. The blocking agents used are normally chosen because they interfere with protein binding. If the blocking materials are present in high quantities, the capture line binding may experience some interference effects. Lowering the concentrations of the blocking agents and investigating alternatives may achieve a better result. Use less protein. Reducing the protein content of the system will reduce the area of nitrocellulose occupied by the capture reagent. The protein binds to the first available unoccupied surface of nitrocellulose. Set application aperture more narrowly. Applying the protein solution from a narrower aperture will initially apply the protein to a smaller area of membrane, encouraging the formation of a thinner line. Increase drying of the membrane. The strength of protein binding to nitrocellulose has long been linked to drying. Drying the membrane more vigorously after the protein reagent has been applied may well therefore reduce the chance of the capture protein being washed away when the sample is applied. Capture Line Too Thin A capture line that is too thin may give a false-negative test result. This case may well be true for cases where there is significant proportion of eye disease in the target market, this would be especially prevalent where the target market is the elderly of many third world areas. A line that is very thin would be very difficult to read accurately. Perhaps the optimal line width would be in the region of 0.8 to 1 mm wide. If the capture line is of the order of 0.2-mm wide then reading even a strong positive result can be difficult. Causes. The causes of too thin a capture line are mainly the opposites of those mentioned above. Protein binds too rapidly after application. Binding of the protein to the membrane immediately after application results in a capture line that is very narrow, although the application-buffer front will be significantly wider than the capture line itself. Protein in the application system is insufficient. If the protein shows good binding properties to the membrane surface, a low concentration of protein in the capture reagent gives narrow lines following application. All the protein will bind in a very small area before it has had a chance to diffuse away from the application point. The application aperture setting is too narrow. With optimal protein binding, the protein binds immediately to the area to which it is applied and does not spread. If the capture reagent is applied over a very narrow area, the protein is unlikely to move away from the application point before binding. Solutions. The solutions to the problem of a too-thin capture line are essentially the opposite of those to a too-thick line. Add bulking protein. Introduction into the application buffer of a nonspecific protein, with membrane-binding qualities similar to those of the capture reagent, will provide competition for the protein-binding sites on the membrane and hence encourage formation of a wider line. Use more protein. Increasing the protein content in the application buffer will cause a wider line. The excess protein will move farther to find empty binding sites on the nitrocellulose, leading to saturation of a larger area of membrane. Use a larger-pore membrane. A larger-pore membrane accelerates lateral diffusion of the capture reagent and also has a lower available surface area of nitrocellulose than a smaller-pore membrane. The line will therefore be wider. Change the protein buffer. If the buffer is adjusted to increase protein solubility, the protein will remain in solution longer and travel farther before binding to the membrane. The capture line width therefore increases with increasing protein solubility. Change the membrane. A membrane that shows a lower binding affinity for the chosen protein will allow the formation of a wider protein line for a given level of protein applied. Use a wider application aperture. Spraying a wider line of the protein solution onto the membrane will cause the protein to bind over a wider area. Uneven Line Intensity A test with uneven line intensity can be a quality-control nightmare. This problem also drives up cost, since many batches will be rejected. Unless 100% of the devices undergo QC testing, the problems may not be detected before the product reaches the customers. The least damaging effect may be wastage due to rejection of batches where the majority of the product works acceptably. A more significant problem will be lack of selectivity in test kits sold, which can lead to loss of market confidence or the removal of regulatory approval. Causes. While a line of uneven intensity may not look much different from a line that is too thick or too thin, the possible causes of this problem are quite distinct from those already discussed. Membrane hydrophobicity varies. Hydrophobicity in the nitrocellulose membrane, either before or after capture line application, can result in the formation of a variable capture line (see Figure 5). Depending upon the reason for the hydrophobicity, the problem can manifest itself as a striped or uneven capture line or as a significant variation in line widths between tests. Common causes of membrane hydrophobicity include poor storage of unblocked material, solvent interaction, or the washing away of the blocking agent during capture line application. Pressure varies in application system. A pressure (or flow-rate) variation in the application system can often cause uneven line width (sometimes called \"blobbing\") or line intensity. This effect is caused by variation in the quantity of protein applied. Blobbing or differences in line intensity reflect the protein saturation limit for the membrane used. The result is a test with very poor within-batch reproducibility. Protein solution is poorly mixed. Variations in the concentration of the protein solution applied result in problems similar to those caused by pressure variation. The line width and intensity vary along the length of the applied line. Protein precipitates. Precipitation of the protein in the application system results in variation of the protein concentration (see effects mentioned previously) or clogging of the membrane by particulates (see next section). The particles can also cause blocking of small-bore print heads, such as those found in many piezoelectric printing systems. Flow in the strip is interrupted. Flow problems in nitrocellulose membranes can be due to two major causes: clogging of pores by particulates and poor contact between components in the lateral flow strip. Clogging of pores results in the membrane appearing striped, particularly at the capture line. Since the flow along a membrane strip is largely linear, blocked pores result in areas of the capture line where the sample does not penetrate and therefore no color develops. The formation of particulates (often by protein precipitation) should therefore be eliminated if possible. The flow in a lateral flow assay is dependent upon continuous capillary contact among the various components of the test system. If contact among the test components is poor or nonexistent, the amount of sample passing through the affected area of the lateral flow device will be significantly reduced when compared with an area that has good contact. This problem can result in a capture line that appears striped.Figure 5. Water present during the application of posttreatments can make sections of the membrane hydrophobic, resulting in striations or intensity variations in the capture line.Storage conditions are suboptimal. The major effect of improper storage conditions is the loss of hydrophilicity from the membrane. This loss can result from any of several defects in the manufacturing process. Volatile rewetting agents may migrate (if added during manufacture), residual moisture may be lost from the surface (if no rewetting agent is added), or solvent, adhesive, or plasticizers may interact with the membrane. The exact cause can be hard to identify. The effect is often seen as a reduction in the lateral flow rate of the membrane or the appearance of hydrophobic spots on the surface. Membrane pore structure is uneven. As mentioned previously, a variation of the pore size across the membrane will result in flow rate and surface area variations across the width of the membrane. Both of these factors can result in a significant variation in the line intensity seen at the capture line. Solutions. The solution to this problem may be chemical, mechanical, or operational, depending on the cause. If the underlying cause is a variation in the membrane pore structure across the roll of membrane, there is no solution that can be practiced by the end user. The membrane will have to be replaced with a batch that has a more consistent pore structure. Treat the membrane. Pretreating a nitrocellulose membrane with carefully selected rewetting agents can solve problems caused by hydrophobicity and membrane storage.13 The rewetting agents added by the blocking step will ensure an even capture line. Treatment of the membrane will result in more consistent performance throughout the roll, which will significantly improve the test consistency and performance after long-term storage. Optimize the membrane storage. For nitrocellulose membranes that have no added rewetting agent, storing the membranes in controlled conditions, both before and after capture line striping, results in more consistent line intensity in the finished product. Suggested storage conditions for nitrocellulose membranes are 40 to 60% relative humidity and between 20° and 25°C. Under these conditions, nitrocellulose membranes are stable for several years. Equilibration of the membrane in a humid atmosphere before striping will result in more even and consistent line application. Optimize the application system. For an even capture line across a membrane, the application system should have good pressure or volume control and allow mixing of the solution being applied to ensure consistency. Minimize protein precipitation. If the buffer conditions used for protein application are too aggressive, the protein may precipitate. This event will result in the formation of particulates and reduce the total amount of protein in solution. While it is preferential to make the protein unstable in solution to ensure rapid and complete binding to the membrane, if the protein is too unstable the system becomes unusable. The buffer should be made more favorable for protein solubility (e.g., increase salt content slightly, adjust pH, or reduce the level of coprecipitating agent). Ensure contact among system components. The level of contact among the various components is difficult to measure. However, the pressure in the system at the overlap between pads is critical. If the pressure is too high, the materials can be crushed, potentially blocking pores. If the pressure at the junction is too low, contact among the various pads may be poor. The amount of pressure required should be determined by experimentation, as differences in pad thickness and the relative compressibility of the materials will both be relevant in optimizing the system. Different tests therefore require reoptimization if any of the materials are changed. Conclusion While many problems can occur with protein application to a nitrocellulose membrane, it is possible to produce a highly consistent line that has very high intensity with no nonspecific interactions. The basic techniques for optimization of protein binding are straightforward. Good results can be obtained if a carefully designed and controlled series of experiments is performed. One common mistake is use of the same conditions for a range of different proteins and tests. Most membrane-based rapid immunochromatographic assays on the market are unique, and the conditions required to give the optimal results are equally unique. A willingness to investigate the optimization of protein binding for each assay developed is key to obtaining the best results. References 1. KD Jones, \"Troubleshooting Protein Binding in Nitrocellulose Membranes, Part 1: Principles,\" IVD Technology 5, no. 2 (1999): 32 41. 2. The Latex Course, 1994 (Fisher, IN: Bangs Laboratories, 1994). 3. Immunogold Reagents, catalogue 2 (Cardiff, UK: BBInternational, 1996). 4. Gold Conjugates, Diagnostic Products Guide (Durham, UK: Jamare Biotest International, 1998). 5. E Harlow and D Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor, NY: CSHL Press, 1988). 6. TG Wreghitt and P Morgan-Capner (eds.), ELISA in the Clinical Microbiological Laboratory (London, UK: PHLS, 1990). 7. JM Polak and S Van Noorden, Immunochemistry: Modern Methods and Applications (Bristol, UK: Wright, 1987). 8. Short Guide for Developing Immunochromatographic Test Strips (Bedford, MA: Millipore Corp., 1996). 9. KD Jones and AK Hopkins, \"Protein Binding in Nitrocellulose Membranes 0.2 to 12 m: A Comparison of Commercially Available Membranes for a Novel Flow-Through Immunoassay,\" poster no. 21 (presented at the 1998 Annual Meeting of the American Association for Clinical Chemistry, Chicago, August 2–6, 1998). 10. G Hermanson, Bioconjugate Techniques (San Diego: Academic Press, 1996). 11. Products Catalogue, 1999 (Rockland, IL: Pierce Chemical Company, 1998). 12. RP Haugland, Handbook of Fluorescent Probes and Research Chemicals (Eugene, OR: Molecular Probes Inc., 1996). 13. KD Jones and AK Hopkins, \"Evaluation of the Efficiency of a Range of Membrane Blocking Agents for Nitrocellulose Membrane Based In Vitro Diagnostic Disease,\" poster no. 3 (presented at the 1998 Annual Meeting of the American Association for Clinical Chemistry, Chicago, August 2—6, 1998). 14. KD Jones, Technical Application Notes, nos. 1–3 (Maidstone, UK: Whatman International Ltd., 1997–1998). Kevin D. Jones, PhD, is a research scientist for diagnostics at Whatman International Ltd. (Maidstone, Kent, UK).呵呵！我公司还可以提供胶体金＆酶免诊断试剂的研发！提供蛋白质组学相关技术服务！希望大家多多支持！这么好的文献竟然没人顶一下，实在是看不过去，我定！谢谢！继续努力！Originally Published May 2000In-line manufacturing for rapid-flow diagnostic devicesAdvanced manufacturing technologies are helping IVD manufacturers achieve quantitative testing in lateral-flow formats. Thomas C. TisoneBecause market demand for immunoassays in rapid, lateral-flow formats is continuing to increase, IVD manufacturers are gradually making the transition from batch manufacturing to automated processing systems capable of producing greater volumes of such tests. At the same time, IVD companies are also seeking to satisfy a growing demand for the development of immunoassays capable of providing quantitative test results. Because achievement of quantitative test results often depends on the repeatability of the individual processes used in manufacturing a test, greater control over such processes is essential. Together, these trends are placing new demands on manufacturing technologies. This article discusses new technologies and equipment for achieving higher throughput, exercising greater control over individual steps in the manufacturing process, and achieving the level of repeatability necessary for the creation of quantitative tests in lateral-flow formats. Traditional Processing A typical lateral-flow immunoassay device employs a number of different material layers and associated reagent locations (see Figure 1). The components of such a device and their typical dimensions include a plastic backing (60–90 mm wide x 300 mm long), a membrane (25 mm wide), sample and absorbent pads (15–20 mm wide), and a conjugate pad (8–12 mm wide). The cut test strip is in the range of 3–8 mm wide x 60–90 mm long. Processing of these components is typically accomplished as described below. Figure 1. Schematic of a lateral-flow test device showing laminate structure and reagent placements.Membrane. The membrane substrate is usually made of nitrocellulose or a similar material. A positive-displacement dispensing system is normally used to apply test and control reagents to this substrate in the form of a line. An impregnation process can then be used to block the membrane, which is then dried in a batch or conveyor oven (see Figure 2). During test development, membrane blocking can also be done dynamically by impregnating the sample pad with blocking reagents. Figure 2. Typical equipment and process flow for batch and in-line processing of lateral-flow test strips.Conjugate Pad. This component is typically a polyester or glass fiber material that is treated with a conjugate reagent. A typical process for treating a conjugate pad is to use impregnation followed by drying. In use, the liquid sample added to the test must redissolve the conjugate so that it will flow into the membrane. Sample Pad. The sample pad is treated with chemicals such as buffers or salts, which, when redissolved, optimize the chemistry of the sample for reaction with the conjugate, test, and control reagents. Wicking Pad. This layer is used in tests where blood plasma must be separated from whole blood. An impregnation process is usually used to treat this pad with reagents intended to condition the sample and promote cell separation. Absorbent Pad. This material acts as a reservoir for collecting fluids that have flowed through the device. It is not normally treated. Plastic Backing and Adhesive. This material serves as a structural member for the above layers. Whether the above components begin in sheet, strip, or web format, all are usually treated with the applicable reagents and processes before they are laminated together. However, some types of processing enable manufacturers to apply reagents after the test components have been partly laminated. Whichever process is used, reagent application is a critical step in achieving high-throughput, controlled test manufacturing. Materials. Because the materials described above can exhibit large variations in liquid-holding capacity, they can represent a major source of problems for achieving controlled processes. On the typical scale of such lateral-flow tests, material thickness and density can vary as much as 30 to 50%. Such problems are most evident with the wicking, sample, and conjugate pads, which have traditionally been treated using impregnation processes. If not performed correctly, drying of impregnated materials can lead to nonuniformities among the test strips produced by the manufacturing process. Density and thickness variations in test membranes are typically controlled much more readily than are variations in other components. When applying test and control reagents onto the test membrane, variations in membrane tolerances can generally be eliminated by using dispensing technologies that take advantage of very rapid protein-binding kinetics. For conjugate pads, a significant reduction in processing variations has been achieved by replacing the impregnation process with a dispensing process.1 To make use of such an approach, it has been necessary to develop specially formulated high-density conjugate solutions. Dimensional Tolerances. The functioning of a lateral-flow test is based on the unidirectional flow of the fluid sample through the different pad structures to the test membrane, and ultimately to collection in the absorbent pad. As the sample flows, it must redissolve dried reagents in each of the pads before entering the membrane. A typical flow response in the membrane is nonlinear and decreases rapidly with flow distance (see Figure 3). Figure 3. Flow rate of liquid in a nitrocellulose membrane as a function of distance.The effective concentrations and reaction rates of reagents at the test and control lines are dependent upon the flow of the sample in the test membrane. If reagent loading in the pads varies as a result of the materials issues described above, it can become a source of variation in the reagent concentration, and hence of variation in the reaction rate at the test and control lines. Similarly, the signal levels generated by the test and control lines are especially sensitive to their positioning within the device. For these reasons, the reagent loading of the various pads, the geometry of the lamination assembly, and the placement of test and control lines are critical factors in achieving controlled and quantitative test results. High Throughput Lateral-flow tests have historically been manufactured in relatively low volumes using a batch-processing approach, with a typical manufacturing line producing somewhere between 2 million and 10 million devices per year. The substrates used for batch processing can be in either a sheet or strip format, and are usually about 300 mm in length. In order to effectively handle the large number of materials involved in such manufacturing, batch processing is somewhat labor intensive. To increase production volumes using this approach, the manufacturer must dramatically increase the amount of floor space dedicated to equipment and operators. For manufacturers seeking to increase production, the next logical step is to move toward in-line approaches that can reduce labor requirements and speed up processing. One of the most effective approaches to in-line processing is the use of webs and web-handling systems (see Figure 4). The objective is to design processes that can be carried out in parallel, taking advantage of the relatively rapid speed of web processing (in the range of 10–100 mm/sec). Such an approach can produce 5-mm-wide test strips at rates in the range of 2–20 parts/sec, which is equivalent to 12 million to 120 million parts per shift per year. Figure 4. Layout of a reel-to-reel reagent-processing system incorporating units for dispensing, impregnation, and drying.However, scaling up to such higher production volumes can also require changes in both the reagent application and lamination processes. Reagent Processing. The batch approach to reagent application often includes impregnation dwell and drying times that are in the range of minutes to hours. Using web-based systems can dramatically reduce the time required for such reagent application. When in-line impregnation tanks and drying tunnels are used to process webs of about 100 mm width, for example, sample and conjugate pads can be produced at speeds in the range of 5–10 mm/sec. When such 100-mm webs are later slit to typical widths of 10 mm, this type of processing can achieve effective throughput in the same range as for dispensed processes. However, because impregnation commonly results in the application of 10–100 times more liquid than does dispensed processing, impregnation remains a much slower method. With so much more liquid to be evaporated, reagent drying is the most critical step in impregnation processing. The time required for this step can be reduced by limiting the liquid volume absorbed into the substrate and by maximizing the drying temperature (see Figure 5). Figure 5. Material temperature as a function of time during the drying process for impregnated substrates.Drying is accomplished in two stages: evaporation and desorption. During the evaporation stage, the temperature of the substrate remains lower than the ambient air temperature because of the cooling associated with evaporation. During the transition to desorption, the temperature of the substrate begins to increase, reaching air temperature when the drying process is complete. Because high temperatures can damage protein-based reagents, the general practice for batch processing is to maintain drying temperatures at less than 40°C over a period of many hours. This practice minimizes the problems of temperature gradients and water-loading variations that generally exist in batch-drying processes. To keep pace with web speeds in the range of 10 mm/sec, however, an accelerated drying process is required. At such processing speeds, a drying tunnel of 72 in. can provide an effective drying time of three minutes, but complete reagent drying within that time requires higher temperatures than are usually permitted. By using an in-line approach, each portion of the web is exposed to the same drying conditions, making it easier for the manufacturer to dry the substrate according to specified parameters. Because exposure to high drying temperatures can thus be controlled and limited to a very short time, manufacturers can use temperatures in excess of 50°C without causing damage to proteins. To minimize the footprint required for such equipment, the necessary drying path can be constructed in the form of a vertical drying tower (see Figure 4). In contrast to impregnation, dispensing processes for applying test and control reagents can be quite rapid. Replacing impregnation processes with the dispensing of conjugates can greatly reduce the volume of liquid required, hence allowing very rapid application and drying times.1 Because both application and drying times can be quite fast, dispensing processes for test, control, and conjugate reagents can easily be scaled to web speeds in the range of 10–100 mm/sec. Typical drying times are in the range of one to two minutes. Lamination. In the batch mode, lamination is basically a manual process using tooling and fixtures. One of the strengths of the lateral-flow format is the simplicity of its mechanical assembly, which involves the lamination of a membrane strip to a single adhesive layer on a plastic backing. An experienced operator can create one or two such laminated master strips per minute, equivalent to a throughput of 1–2 parts/sec, or 6 million to 12 million parts per shift per year. In reality, such production rates may not be achievable because of the potential for operator fatigue. In-line approaches using combinations of pretreated web or strip stock can provide lamination speeds in the range of 50–100 mm/sec. This is equivalent to 5–10 parts/sec, or 30 million to 60 million parts per shift per year. Figure 6 illustrates an in-line lamination machine using web materials with reagents already applied to produce a laminated master strip cut to 300–500 mm. The laminate is cut to a master strip to avoid subjecting it to bending stresses that would cause damage. Figure 6. Layout of a lamination system with combined web and strip feed. System output is a fully laminated master strip.Economics of Manufacture. For production rates in excess of 2 million units per year, the use of in-line processing methods can increase output per employee by a factor of 10 or more. Such processes can reduce materials and reagent use by 10% or more, while also improving manufacturing yields and product quality. However, the transition to in-line processing also requires the manufacturer to underwrite the costs of equipment, which increases product costs by the amounts associated with capital amortization. Controlled Processes Manufacturers seeking to establish very tightly controlled manufacturing processes often face significant challenges caused by the substrates and reagents in use or by the processing technologies themselves. As in the case of increased throughput, increased control can require manufacturers to pay special attention to their reagent application and lamination processes. Reagent Processing. When batch processing is employed, reagent application through impregnation is subject to several major problems. First, the substrates can become distorted during drying. Second, depending on the orientation of the material in the drying oven, reagents can become segregated or redistributed as a result of surface tension and gravity effects during the drying of strip materials. And finally, changes in the chemistry of the reagent bath over time can lead to different absorption rates for the various chemistries in the bath, resulting in nonuniform coating of the substrates. The last of these problems can be solved through in-line processing using a small-volume reagent bath that is continuously replenished with fresh solution (see Figure 7). Another in-line solution is to use saturation dispensing, which adds a constant volume of reagent per unit length. The saturation dispensing method can result in microscopic gradients in reagent concentration caused by chromatographic effects as the solution flows transversely in the web. However, such gradients may not be significant for lateral-flow tests, since the direction of sample flow is normal to the web direction. Figure 7. Close-up of the impregnation station for the reagent-processing system shown in Figure 4.Lamination. The essential requirement of the lamination process is for the materials to be capable of sustaining process- and use-related stresses while maintaining their specified functionality. To exercise increased control over lamination, the primary challenges are maintaining relative dimensional tolerances and constant material tensions so that every test will function within a specified coefficient of variance (CV). Regardless of whether batch or in-line processing is employed, the lamination process is subject to three major variables: material tolerances, material mechanics, and material stresses. When implementing tightly controlled processes, the dimensions and straightness of the substrate materials must be consistent with the tolerances established as part of the device design. Moreover, such tolerances must be maintained after reagent treatments. The applying and drying of reagents can significantly alter the dimensions of materials and distort their shapes. Compared with full impregnation, small-volume dispensing has less of an effect on the dimensions and shapes of substrates. Because in-line processing exerts a constant tension on the substrate throughout the reagent application and drying steps, it can help to maintain the shape and dimensional uniformity of the substrate along the web length. Using wide webs followed by slitting to smaller widths can also help to control the dimensional stability and shape of materials during lamination processes. The mechanical properties of the substrates used for a test must be capable of meeting the design tolerances established for the device. Moreover, such materials must be chosen not only for their ability to function in the test device, but also for their ability to be processed. Many materials that function well from the point of view of a test\'s chemistry cannot be processed according to the required dimensional tolerances. In many instances, such as in the case of an unbacked nitrocellulose membrane, the problem is that the edges of the material are not mechanically well defined. One method of dealing with an unbacked membrane is to first laminate it to the plastic backing, and then to carry out the reagent dispensing and additional lamination steps. In this way, tolerances of pad placement relative to the test and control line positions can be better controlled using the plastic backing as a mechanical reference. Other considerations include the ability of materials to withstand the bending and shear stresses associated with the lamination and cutting processes. Again, many materials that function well chemically cannot withstand manufacturing processes and maintain test functionality. Fine fiberglass mat materials used for conjugate or sample pads are examples of materials that fail under bending stresses. Before selecting a material to be used in product development, the manufacturer should test it for manufacturability using the processes that will be employed in actual production. The third consideration in lamination is that of material stresses, particularly stresses on the membrane as a result of web tension generated by mechanical handling of the materials. Such stresses can alter the effective porosity of a material and hence the flow rate of sample through that material. When compared with batch processing of short strips of materials, in-line lamination using web formats can provide more-effective control over material stresses. Web-handling equipment is generally designed to laminate materials under a condition of constant tension, which can be adjusted as necessary to avoid over-stressing the substrates (see Figure 6). Quantitation The development of lateral-flow tests from simple yes/no and threshold formats to true quantitative tests is an active area of R&D. To be useful, most test analytes require some level of quantitative readout. One of the key factors necessary for achieving quantitative test formats is the development of controlled and quantitative manufacturing processes. Reagent Processing. When developing quantitative tests, manufacturers must go beyond merely controlling their reagent processing to achieving uniform and quantitated distribution of reagents on the various test substrates. Such precision cannot be accomplished using impregnation processing because that method is subject to variations caused by differences in the liquid-holding capacities of the materials or by reagent redistribution during drying. The best approach to achieving truly quantitative reagent application seems to be through dispensed processing using a positive-displacement system. Three such platforms include a drop-on-demand ink jet (BioJet), an aerosol dispensing system (AirJet), and a contact or near-contact syringe needle.2–6 Each of these systems can be programmed to deliver a specified volume per unit length, and can deliver a dispensed line with a CV of less than 5% (see Figure 8). The BioJet and syringe needle can dispense narrow lines, with profiles determined respectively by the drop size or syringe orifice size. The AirJet delivers droplets in the picoliter range with an angular distribution; line width is controlled by varying the height above the substrate, the aerosol pressure, and the flow rate of the liquid being dispensed. Typical line widths for an AirJet are in the range of 0.5 to 10 mm, but jets with wider angles can produce line widths up to 25 mm. Figure 8. Dispensed lines of conjugate on a fiberglass sheet: lines dispensed using an ink jet and lines dispensed using an aerosol system. In both examples, the liquid volume dispensed is 2 l/cm of gold conjugate concentrated to an optical density of 100.By using dispensed processing, manufacturers can achieve quantitative delivery of reagents. Such precision enables the company to reduce the liquid volume applied to test substrates, thereby speeding up drying processes and reducing the risk of material changes associated with drying. Thus applied, the reagent should appear on the substrate as a uniform band, with penetration through the pad so that it offers a uniform front to the flow of the test sample. In addition, the dried reagent should be readily redissolved by the test sample. Lamination. For quantitative tests, the major issue relating to lamination is the precision placement of parts relative to a reference such as a plastic backing edge (see Figure 9). When batch processing is used, placement precision is limited by the mechanical stability of the substrate edges. Placement of materials with good edge definition can be controlled to within tolerances of ±1 mm. However, the placement of dispensed reagents on such batch-processed materials can also vary, thereby increasing the potential for placement error relative to actual flow position on the membrane. When the placement errors from batch processing and reagent dispensing are combined, the effective placement error between the entry point of a sample into the membrane and the test and control lines can easily be as much as ±2 mm—a measure far too great to be acceptable for a quantitative device. Figure 9. Schematic of a lateral-flow device showing the position of a dispensed conjugate line in the conjugate pad relative to the membrane and sample pads.The placement tolerances of both reagents and lamination can be better controlled by employing an in-line processing system that makes use of adjustable mechanical guides and sensor-based tracking systems. Figure 11 shows an in-line dispensing module in which the dispensing heads are located on a servo-driven slide that operates in conjunction with a sensor system for detecting the edge of the substrate. When processing materials with well-defined edges, systems such as this can control the accuracy of reagent placement to within ±0.25 mm. This type of dispensing system can work well even with unbacked nitrocellulose membranes. Tracking systems similar to that used on the illustrated system are also used to maintain edge alignment during the rewinding of processed roll-stock and for in-line lamination. Figure 11. Schematic of QC inspection for a master strip, showing defects and related inspection marking.QC Inspection Quality control (QC) is one of the most neglected areas in the manufacturing of lateral-flow test devices. Such neglect is due, in part, to the lateral-flow format itself, which makes it difficult for manufacturers to inspect, detect, and remove bad components during processing. Even when the manufacturer recognizes that a processing step has exceeded its assigned parameters, the resulting faulty portions or components often cannot be physically removed until the master strip or webs have been cut into individual parts. For manufacturers, the difficulty of carrying out QC operations implies several related challenges, including how to inspect for process defects, how to track identified defects through the manufacturing process, and how to remove faulty components or bad sections of processed materials. Although such operations can be carried out manually—by cutting the master strips and then sorting out the good parts from the bad—such an approach adds processing steps and requires additional labor and equipment. An alternative strategy for tracking bad parts is illustrated in Figure 12, which shows an uncut master strip that has been inspected for defects in reagent application and lamination processes. The presence of a defect is indicated using a master mark on a specified portion of the laminate, which can then be automatically read during the cutting process. As the part is passed through the cutter, faulty portions of the laminate are automatically detected and removed from the process. Methods of inspection and marking are discussed below. Figure 12. An in-line system for reagent dispensing on sheet or strip substrates, combined with an automated QC inspection sensor and marking system.Reagent Processing. When impregnation processing is used, the problems of inspecting and marking defects can be difficult to overcome. With impregnated substrates, defects tend to be related to gradients in the composition of the reagent rather than to the simple absence or misplacement of the reagent. For this reason, there is often no clear-cut correlation between a defect and an optical readout. Such complexities in carrying out QC on impregnated substrates are among the many reasons that the industry trend is toward the increased use of dispensing processes. When dispensed processing is used, inspection and marking can be done either manually or with machine control. Inspection can be performed by means of an optical readout such as a dye or by an optical sensor that detects the presence or absence of reagents using the contrast between wet and dry areas as an indicator. When the sensor detects an area in which reagent is absent or has been misapplied, software in the inspection system triggers an ink jet to mark the exact position of the defect (see Figure 10). It is also possible to use combinations of sensors to measure the width of dispensed reagent lines, thereby providing an indirect measurement of the reagent volume dispensed. Figure 10. Close-up of the dispensing station for the reagent-processing system shown in Figure 4. The station includes ink-jet and aerosol dispensers and automated edge tracking.The next step up in QC measurement is to use an in-line vision inspection system that measures the width of the reagent line, its position, and its intensity profile.7 Vision inspection systems offer a more-quantitative approach to QC inspection. Depending on the visual resolution required and the speed of the manufacturing process, such systems may use more than one camera. Both optical and vision inspection systems can be adapted for use with either batch (x-y motion) or in-line web systems. Lamination. QC for laminated substrates can also be accomplished using sensor arrays, vision inspection systems, and automated marking methods. In this application, such systems inspect the placement of the various materials during lamination. Vision inspection systems generally offer better resolution and sensitivity. Defects should be marked in a standard position on the membrane. This practice ensures that defect marks can be detected during later processing steps, so that faulty parts can be rejected. Hybrid Systems At the high end of the equipment spectrum are hybrid systems that combine dispensing and lamination into a single process, working at speeds up to 100 mm/sec (see Figure 13). Such systems enable manufacturers to exercise greater control over the relative positioning of both substrates and reagents. Figure 13. Layout of an in-line hybrid processing system. Such systems laminate adhesive and membrane to strips of bare plastic backing, and then dispense test and control reagents.The use of hybrid systems represents a new paradigm for the manufacture of lateral-flow tests because it allows for the dispensing of different reagents on different substrates while maintaining high relative positional accuracy (in the range of ±0.1 mm). Thus the placement of the conjugate line can be very accurate relative to the test and control lines and independent of the limitations of material dimensional tolerances. The same is true for the placement of dispensed reagents on sample and wicking pads. Conclusion IVD manufacturers are currently using both batch and in-line processing for the manufacture of lateral-flow tests (see Table I). As demand for such tests continues to grow, companies will increasingly be seeking to employ processing technologies that enable them to produce greater volumes of tests. At the same time, product developers are looking for ways to better control their processes, so that the lateral-flow format can be used for quantitative tests. Batch Processing In-Line Processing Strip format for material and lamination: 150–500-mm strip length Roll format for material andlaminate: 50–100-m roll length Process strips in groups Process rolls in continuous process Process time is noncritical Process time is critical and limitedby web speed and equipment size Poor process symmetry Excellent process symmetry Low-to-medium volume: fromR&D to 6 million tests per year Medium-to-high volume: from ~4 million to 1 billion tests per year High labor content Low labor content Table I. Comparison of the key characteristics of batch and in-line manufacturing strategies for lateral-flow test devices. Regardless of the processing method used to manufacture such tests, manufacturers should ensure that they select reagents, substrates, and other materials that are compatible with those processes. Correct selection of materials and components is the essential basis for developing controlled manufacturing processes. Advancing technologies are offering manufacturers significant alternatives for improving their manufacturing methods. Both batch and in-line processing can be improved by replacing impregnation with quantitative dispensing of reagents. Reagent dispensing and lamination can be performed using continuous in-line processes that now offer considerable advantages in throughput, quality, and cost. And new hybrid systems are showing potential for additional improvements in the development of more-quantitative process technologies. Together, these major trends in the manufacture of lateral-flow tests suggest a continually evolving technology that IVD manufacturers should be keeping abreast of. Staying informed about the latest processing technologies can help manufacturers gain production efficiencies that can ultimately improve their bottom line. References 1. J Colanduoni, unpublished research, Arista Biologicals Inc., 115 Research Drive, Bethlehem, PA 18015. 2. TC Tisone, Dispensing Systems for Miniaturized Diagnostics, IVD Technology 4, no. 3 (1998): 40–46. 3. TC Tisone, Precision-metered solenoid valve dispenser, U.S. Pat. 5,743,960, April 28, 1998. 4. TC Tisone, Precision-metered aerosol dispensing apparatus, U.S. Pat. 5,738,728, April 14, 1998. 5. TC Tisone, Method of dispensing a liquid reagent, U.S. Pat. 5,741,554, April 21, 1998. 6. TC Tisone, Dispensing apparatus having improved dynamic range, U.S. Pat. 5,916,524, June 29, 1999. 7. TC Tisone et al., Image Analysis for Rapid-Flow Diagnostics, IVD Technology 5, no. 5 (1999): 52–58.--------------------------------------------------------------------------------Thomas C. Tisone is vice president for R&D and engineering at BioDot Inc. (Irvine, CA)Manufacturing high-quality gold sol Understanding key engineering aspects of the production of colloidal gold can optimize the quality and stability of gold labeling components.Basab Chaudhuri and Syamal Raychaudhuri As early as the first decade of the twentieth century, colloidal gold sols containing particles smaller than 10 nm were being produced by chemical methods.1 However, these inorganic suspensions were not applied to protein labeling until 1971, when Faulk and Taylor invented the immunogold staining procedure.2 Since that time, the labeling of targeting molecules, especially proteins, with gold nanoparticles has revolutionized the visualization of cellular and tissue components by electron microscopy. The silver enhancement method has extended the range of application of gold labeling to include light microscopy. The electron-dense and visually dense nature of gold labels also facilitates detection in such techniques as blotting, flow cytometry, and hybridization assays. Double- and triple-labeling systems involving immunogold methods have been used successfully to detect more than one antigen at the same time.3 A recent article nicely explained the place of gold in the development of rapid diagnostic tests.4 Table I reproduces from that article a useful comparison of the characteristics of labels commonly employed in rapid tests. A significant topic not mentioned in the piece, and deserving discussion, is the role of various process parameters in determining the quality of the gold suspension. The first step toward manufacturing a consistent gold-protein conjugate is to make a gold sol having particles of proper size and dimension. Basically, colloidal gold sols consist of small granules of this transition metal in a stable, uniform dispersion. Most preparations of colloidal gold are made up of particles varying in diameter from about 5 to around 150 nm. For the development of diagnostic assays that make use of gold conjugates, typical particle sizes in the gold sol range between 20 and 40 nm. Since these are very small particles, the surface area of the gold in the sol is remarkably high. This means that production of colloidal gold sol involves the creation of a large surface area having a very high surface energy. Any colloidal suspension with high surface energy can lose its stability if proper operating conditions are not maintained during its production. This article discusses some process engineering aspects of gold sol manufacture that have considerable influence over the quality and stability of the suspension. It highlights the physical, rather than chemical, factors that play important roles in gold sol production. But first, a look at process chemistry is in order. Basic Chemistry A variety of chemical methods can be employed to produce monodisperse colloidal gold suspensions. However, three procedures have become the most common for making particles that fall into predictable size ranges. In all three processes, tetrachloroauric acid (HAuCl4) in a 1% aqueous solution is reduced by an agent in order to produce spheroidal gold particles. The greater the power and concentration of the reducing agent, generally, the smaller the resultant gold particles in the suspension (see Table II).Figure 1. Vortex formation in a magnetically stirred system.To create large-particle colloidal gold dispersions, an aqueous solution of tetrachloroauric acid is treated with trisodium citrate in aqueous solution. This results in particles sized 15–150 nm, the final range depending on the concentration of the citrate used in the reduction process. Medium-sized gold particles with diameters between 6 and 15 nm and an average size of 12 nm are formed by reducing the tetrachloroauric acid solution with an aqueous sodium ascorbate solution. The smallest particles, measuring less than 5 nm in diameter, are produced by reduction with either white or yellow phosphorus in diethyl ether.3,5 The following discussion focuses mainly on the process-development aspects of making colloidal gold with 20- to 40-nm particles. Gold sols with particles in that range are the most suitable candidates for use in developing rapid diagnostic tests. It can be seen from the foregoing that the starting raw materials for production of these gold sols are an aqueous solution of tetrachloroauric acid and an aqueous solution of trisodium citrate, which are brought together to create a chemical reaction. As described below, such chemical reactions are broadly classified as either homogeneous or heterogeneous. Homogeneous Reaction. In a homogeneous reaction, all the reactants are miscible and form a homogeneous solution. The products that are formed from the reaction are also soluble; therefore, there is no phase separation at any time during the course of reaction. The rate of such a reaction depends on the concentration of the reactants and on the operating temperature. The temperature influences the rate constant of the reaction. Usually, a 10° rise in the operating temperature enhances the rate of reaction by a factor of two. This effect of operating temperature on the rate of reaction is exploited in order to increase reaction speed or achieve greater production throughput with a single reactor. For irreversible reactions—reactions that produce almost complete conversion of reactants to products—the upper temperature limit is established by the highest operating temperature of the material from which the reactor is constructed. The effect of temperature on reversible reactions is more complicated, because both the reaction rate and the equilibrium conversion are functions of temperature and both need to be taken into consideration. For a reversible endothermic reaction, the rate of reaction and the equilibrium conversion increase with an increase in temperature; therefore, the highest possible temperature is suitable for large-scale production. For a reversible exothermic reaction, however, the equilibrium conversion decreases while the rate increases with an increase in temperature. These are opposing effects, and the reaction is thus conducted at varying temperatures so as to reach the optimum rate of reaction and achieve the best conversion of reactants into products. The transport factors—heat transfer, mass transfer, and so on—do not play major roles in homogeneous reaction kinetics. Heterogeneous Reaction. In a heterogeneous reaction, more than one phase is present. The reaction might be gas-liquid, liquid-liquid, gas-solid, liquid-solid, gas-liquid-solid, or liquid-liquid-solid, or display some other progression of phases. Any solid involved could be a catalyst or a reactant. The relatively simple rules for controlling homogeneous reactions are not applicable to the control of heterogeneous reactions. In addition to concentrations and temperature, the physical shift of reactant from one phase to another assumes great importance in heterogeneous reactions. The chemical reaction between an aqueous solution of tetrachloroauric acid and an aqueous solution of trisodium citrate is interesting in that the reaction begins as a homogeneous one, but then, within a minute, the reaction mixture becomes heterogeneous. This phase transition from homogeneous to heterogeneous occurs very rapidly, making the sol-manufacturing process difficult to monitor or control effectively. Moreover, the reaction is completed so quickly that operators do not have much time to take any corrective action necessary to ensure reproducible product. Producing Gold Colloids Before the addition of the reducing agent, 100% gold ions exist in solution. Immediately after the reducing agent is added, gold atoms start to form in the solution, and their concentration rises rapidly until the solution reaches supersaturation. Aggregation subsequently occurs, in a process called nucleation. Central icosahedral gold cores of 11 atoms are formed at nucleation sites. The formation of nucleation sites, in response to the supersaturation of gold atoms in solution, occurs very quickly. Once it is achieved, the remaining dissolved gold atoms continue to bind to the nucleation sites under an energy-reducing gradient until all atoms are removed from solution. The number of nuclei formed initially determines how many particles finally grow in solution. At a fixed concentration of tetrachloroauric acid in solution, as the concentration of the reducing agent is increased the number of nuclei that form grows larger. The more nuclei, the smaller the gold particles produced. Finding the optimal concentration of the citrate in solution is therefore an important, even crucial, task. If manufacturing conditions are optimized, all nucleation sites will be formed instantaneously and simultaneously, resulting in formation of final gold particles of exactly the same size (monodisperse gold). This is indeed difficult to achieve. Most manufacturing methods fail to accommodate this ideal and generate irreproducible gold (gold inconsistent from batch to batch) that gives unstable gold conjugates in most situations. Gold colloids are composed of an internal core of pure gold that is surrounded by a surface layer of adsorbed AuCl–2 ions. These negatively charged ions confer a negative charge to the colloidal gold and thus, through electrostatic repulsion, prevent particle aggregation. All colloidal gold suspensions are sensitive to electrolytes. Electrolytes compress the ionic double layer and thereby reduce electrostatic repulsion. This destabilizing effect results in particle aggregation, which is accompanied by a color change and eventual sedimentation of the gold. The detrimental effect of chloride, bromide, and iodide electrolytes on the stability of the gold colloid is greatest for chlorides and least with iodides. All gold colloids display a single absorption peak in the visible range between 510 and 550 nm. With increasing particle size, the absorption maximum shifts to a longer wavelength, while the width of the absorption spectra relates to the size range. The smallest gold colloids (2–5 nm) are yellow-orange, midrange particles (10–20 nm) are wine red, and larger particles (30–64 nm) are blue-green. Smaller gold particles are basically spherical, while particles in the range of 30–80 nm show more shape eccentricity related to the ratio of major to minor axes. Researchers have observed several factors that affect the quality and stability of the gold colloid. An important consideration leading to the preparation of stable gold colloids is employment of thoroughly cleaned glass apparatus, 0.2- m-filtered solutions, and, ideally, triple-glass-distilled water.5 The use of nanopure water is recommended. These precautions suggest the adverse effect that even trace contaminants have on the preparation of colloidal gold. Although the use of siliconized glassware is often recommended, good results have consistently been obtained without any special glassware. The effect of the order of reagent addition—that is, adding citrate solution to the tetrachloroauric acid solution or vice versa—on the quality of the gold colloid formed has been noted by researchers.6 However, no clear indication of how addition order might relate to methods of manufacturing colloidal gold suspensions reproducibly has been given. Researchers have not explicated the role of mixing in the formation of the suspension, nor have they mentioned the negative impact of the use of a stir bar (for laboratory-scale preparation) in a magnetically agitated system on the quality and stability of the gold sol. It must be kept in mind that, in a large-scale operation, it is not only the chemistry of the process that is important, but also its perhaps seemingly insignificant physical parameters. Small changes in process conditions can so adversely affect the quality of the product that its utility to end-users will be minimal. Batch or Continuous Process? Generally, manufacturing processes can be run either batchwise or in continuous mode. Certain considerations dictate the best choice. The scale of operation is an important factor. Small-scale production calls for batch operation. The reactor used in such a case is called a batch reactor. Reactants are added to the batch reactor at a suitable temperature, the reaction proceeds, and then, at the end of the batch time, the reactor\'s contents are removed. The reaction products are subsequently recovered by means of a separation process. Unconverted reactants can be reused in certain cases. Whenever the unconverted reactants are discarded, they must be disposed of in accordance with pertinent environmental and safety rules. In a batch reactor, the concentrations of reactants and products change continuously with time. Thus, the reaction can be tracked by noting carefully the fall in reactant concentration or the rise in product concentration as a function of time. The continuous mode of production is used for high-volume manufacture. In a continuous process, the reactants are fed into the reactor steadily and the products form and come out continuously. The flow pattern of fluid in a continuous reactor will take one of three forms. A mixed flow pattern is characterized by the reactor contents being completely mixed and the exit concentration being equal to the reactor concentration. In a plug-flow reactor, the concentrations of reactants and products change progressively as the materials pass through the reactor. There is no mixing of fluid in the longitudinal direction, although the radial mixing is complete. A flow pattern between the mixed- and plug-flow configurations is possible. In such a case, the fluid in the reactor is partially rather than completely mixed. Sound experimental techniques are available for determining the specific flow pattern in an unknown vessel, and modes of describing the vessel characteristics have been established. Without proper characterization of fluid flow within a reactor, it is practically impossible to predict the behavior of the reaction taking place there. Typical production volumes for colloidal gold are in the range of 1 to 100 L. Batch processing is appropriate for such production. Manufacturers producing gold suspension in the range of 10 to 100 L (production volume depends on the order size and the availability of suitable reactor) sell it to customers for subsequent conjugation with proteins. Typically, for a 100-L batch, the power consumption for stirring the liquid contents will be substantially large. The reactor needs an adequate piping arrangement for pumping reactants, washing solutions, etc., into the reactor. After every batch of production, the reactor must be cleaned thoroughly with a sodium bicarbonate and detergent solution, distilled water, and then with some volatile organic solvent, such as acetone. A further wash with nanopure water is recommended. Many diagnostic companies produce gold suspension in the range of 2 to 5 L for their in-house use (captive consumption). For such small-volume production, power consumed in stirring the reactor contents can be expected to be minimal, and it is easy to clean the reactor thoroughly after every batch by dismantling the entire assembly. Complicated piping arrangements that are necessary for large-batch production or in continuous processes do not figure in small-batch processes, making cleaning simple. The important parts of the batch reactor used for making colloidal gold are the reaction vessel, the agitation or stirring system, and a constant-temperature bath that keeps the reactor contents at a uniform and suitable operating temperature throughout the reaction. The reaction assembly is easy to set up; the only precaution that needs to be taken is that the assembly components must be cleaned scrupulously. It is best that all glassware be autoclaved before each use. Factors Affecting the Quality of the Final Gold Suspension A variety of physical parameters affect the quality of the final gold suspension that is produced by the reaction of aqueous tetrachloroauric acid with an aqueous solution of trisodium citrate. Key factors worthy of consideration are: The concentration of reactants. Mixing of the reactants. The order in which reactants are added. Operating temperature. Liquid head in the reactor. The reactor\'s material of construction. Concentration of Reactants. The rate of any homogeneous chemical reaction depends on the concentration of the reactants and the operating temperature. A low reactant concentration will result in low rates. A high concentration is therefore desirable for realizing high rates. Too high a concentration might yield other problems, however, particularly for competing reactions. In such cases, the desired product might not form in sufficient quantity and the reaction might produce a large amount of byproducts. Only one reaction is involved when tetrachloroauric acid and trisodium citrate are combined, but inadequate reactant concentration would result in gold particles of undesirable size and a broad distribution of particle sizes. The procedure developed by Frens is most commonly used to produce 40-nm gold particles.7 In accordance with this procedure, to 50 ml of tetrachloroauric acid in 0.01% solution (weight to volume) that is at a boil, 0.5 ml of a 1% solution of trisodium citrate is added. The solution initially has a gray color which changes to lavender and then, with continued boiling, after 1 to 3 minutes develops a red hue. The resulting particle size is 41 nm. Once the colloid is formed, neither prolonged heating nor further addition of the citrate solution will produce any change in particle diameter. The use of proportionally larger reaction volumes is accompanied by an increase of some 20% in the final particle size. A 20% difference in the amount of tetrachloroauric acid or trisodium citrate used has been observed not to affect the particle size substantially. Rates of initial nuclei formation are practically uniform throughout this range of concentrations. Mixing of Reactants. This is perhaps the most crucial physical process parameter. The reactants must be well mixed in order for nucleation to occur. Adequate mixing results in uniform concentration and temperature in every part of the reactor. If concentration varies from location to location, the rates of reaction in different places will be different, too. All chemical reactions are accompanied by either heat generation or heat absorption, which gives rise to either an increase or a decrease in temperature from the desired value. Different temperatures within the reactor cause heat- and mass-transfer gradients. With an intrinsically slow reaction, how the reactants are mixed is not going to be a cause of substantial distortion in the product. But in the gold-making process the reaction occurs in seconds, and the reactants must be brought to uniform concentration before it happens. A challenge of rapid mixing is therefore encountered here; special arrangement must be made for agitation of the liquid contents of the reactor. In small-scale processes a small stir bar is used to agitate the liquid in the reactor. The reactor is placed on a magnetic stirrer, and, in order to promote fast mixing, the stirrer is rotated at high speed. Careful observation reveals that under such a condition a vortex is formed in the reactor (see Figure 1). The formation of this vortex is counterproductive to proper mixing because liquid revolves in a narrow zone and the circulation current does not spread throughout the reactor. The larger the diameter of the reactor, the more pronounced will be such a deleterious effect. In a small reactor having a volume of, say, 500 ml, the quality of the colloidal gold produced is not too bad even when a vortex has formed. But under otherwise uniform conditions, when the scale of production is increased to 4 L, the effect of bad mixing is evident. The gold produced will have poor characteristics; particles become larger and display greater eccentricities.A magnetically stirred system is not recommended for making gold even in small-scale processes, simply because the mixing characteristics in the liquid undergo batch-to-batch variation and it is hard to achieve reproducible process conditions. Use of a mechanically agitated reactor is to be preferred. In such a reactor an agitator fitted to a motor imparts motion to the liquid and effects mixing. The reactor should be equipped with baffles in order to avoid any formation of a vortex. Baffles are basically rods attached to the wall of the vessel that promote turbulence in the reactor and stifle the tendency toward vortex creation. Achievement of reproducible mixing in the reactor depends on several important factors, including the diameter of the reactor, the diameter of the stirrer, the thickness of the baffles, and the speed of rotation or agitation of the reactor. In addition to these, the properties of the liquid contents are important. The higher the viscosity of the liquid being stirred, the more power must be applied for good mixing. Liquid viscosity is a strong function of temperature; if the process allows an increase in operating temperature, then making such an adjustment can reduce both viscosity and power consumption. For a given liquid at a given temperature, if the reactor diameter, stirrer diameter, baffle size, and speed of agitation are constant, the mixing characteristics produced in the reactor also will be reasonably constant. An irreproducible equipment setup is more likely than a constant configuration to result in an irreproducible product. Therefore, the reactor needs to be designed correctly and operated uniformly. Simple experiments, commonly called tracer studies, can be devised to find the mixing characteristics of a particular vessel. In these studies, a tracer, usually a known amount of dye, is injected into a known volume of water in the reactor. The concentration of the dye in water can be calculated readily when the tracer is dispersed uniformly. The time required to achieve this uniform dispersion is determined by noting the change in concentration of the dye as a function of time at different speeds of agitation and with different stirrer designs. The combination that takes the least time to reach uniform dispersion is chosen for the process. In making gold suspensions, it is first necessary to bring the two reactants to uniform concentration as soon as possible. Once this is achieved, the speed of agitation must be reduced; otherwise, the gold particles will collide with each other and form larger particles. Rapid mixing of the reactants thus is recommended, followed by slow agitation designed to impart flow to the liquid, but not much turbulence. Order of Reactant Addition. The order in which reactants are combined in the reactor is another crucial factor that determines the quality of the gold sol. Is it better to add the aqueous solution of trisodium citrate to the tetrachloroauric acid, or vice versa? There is not much apparent difference between these two modes of addition. But in reality the difference is substantial. A thought experiment may help to bring out the difference. Consider a homogeneous reaction that develops as follows: A + B = C and A + C = D. Now suppose a beaker containing only substance A to which substance B is added quantum by quantum. A will react with B to produce C, and since C will find itself in a large excess of A, D will be the predominant product to form in the beaker. On the other hand, suppose A is added quantum by quantum to a beaker containing a large excess of B. The concentration of C will rise gradually. With the continued addition of A, the concentration of C will pass through a maximum. At some point a competition will develop between B and C to react with A. This stage of competitive reactions presents a choice: If the desired product is D, then the former contacting pattern--adding B to the beaker--should be chosen. If, however, the desired product is C, then the second contacting pattern is better; substance A is added quantum by quantum, the concentration of C reaches the maximum, and then C is separated from the product mix. This is one way to engineer the chemistry so as to obtain most efficiently the product desired. The gold-making process does not involve the multiple reactions in this illustration. Still, the order of reactant addition is important. If a small quantity of trisodium citrate is introduced to the tetrachloroauric acid, it takes a long time for it to uniformly disperse in the large volume. The reactor might contain pockets of notably high or low concentration. Such a circumstance promotes unequal rates of reaction, a condition that in turn leads to unequal rates of nucleation and, hence, bad gold as the finished product. On the other hand, if the tetrachloroauric acid is quickly added to the citrate solution, the chances of pockets of varying concentration forming are rather remote. Operating Temperature. All standard textbooks prescribe adding trisodium citrate to a boiling tetrachloroauric acid solution, and continuing the boiling for about 15 minutes thereafter. The reaction usually is complete within 5 minutes, and there is no subsequent change in the particle size of the gold. How is the boiling achieved, and is it at all necessary? In small laboratories the reactor is placed on a combination magnetic stirrer and hot plate and the liquid is heated by means of the hot plate. This form of heating has been observed to be detrimental for colloidal gold. The reason is that tiny bubbles form at the bottom of the reactor, and the moment they disengage from the hot surface they briefly leave dry spots behind. Any colloidal gold particles at these dry spots become dehydrated and lose their desirable characteristics. The liquid in the reactor does not have to be boiled in order for good gold to be produced. As long as the temperature in the liquid phase is controlled at around 95°C, the gold-making reaction proceeds smoothly. Control of the temperature is what is important. Operating the reactor at a constant temperature requires the selection of a proper heating system. In order to maintain uniform temperature and avoid hot spots, hot liquid should be circulated around the reactor. The liquid could be high-molecular-weight oil. For small-scale production a thermostat employing hot oil may be used. The reactor should be placed inside the thermostat. The oil temperature can be controlled by means of a temperature controller. Liquid Head in the Reactor. The liquid head is a consideration important only in large-scale operation. Suppose a batch reactor is employed to make 100 L of gold. In such a situation the depth of liquid in the reactor is substantial. The reaction involved in making gold suspension takes place at atmospheric pressure, which is the pressure at the top of the liquid in the reactor. But at the bottom of the reactor, the pressure is the sum of the atmospheric pressure and the pressure due to the liquid head. Pressure at the bottom of the reactor is therefore higher than that of the atmosphere. Consequently, the boiling point of the liquid at the bottom of the reactor is higher than it would be at atmospheric pressure. Exposure to this high-boiling-point temperature could destroy the gold suspension. With rapid mixing, the temperature of the liquid should be the same throughout the reactor. But after nucleation, when slower agitation is necessary, an unusually high temperature gradient can develop. In addition, dry spots might form on the inner surface of the reactor. The gold suspension becomes dehydrated where it comes into contact with these dry spots. When the process is complete, suspended particulates appearing at the surface of the liquid will suggest that the quality of the gold produced is not likely to be good. Careful prior consideration of the liquid head in the reactor is therefore essential to any attempt to make gold suspension in large batches. Material of Construction. Finally, the possible effect of the materials from which the reactor and the agitation assembly are made on the quality and stability of the gold sol must be carefully considered. In small-scale batch production, small Teflon-coated stir bars are employed for agitation. The use of Teflon is supposed to ensure a clean system. However, the shear between the stir bar and the reactor generated during rotation and arising at the point of contact between them exposes the inner metallic region of the stir bar, imperfections that close observation will reveal. The colloidal gold interacts with the exposed metal at these tiny flaws, and particles form as a result. It must be kept in mind that any contaminant can destroy the gold sol. Therefore, an all-glass assembly is recommended. If steel reactors are used, they should be provided with a glass lining. The lining should be tested from time to time to ensure that no crack has developed. The material of construction of the agitator requires similar consideration. Agitators generally should be made of Teflon, which reacts adversely with virtually no chemical. Conclusion The manufacture of gold suspensions involves combining simple chemistry with some difficult process engineering. Chemists and biotechnologists need to understand the engineering considerations discussed in this article in order to make gold sols of dependable quality. An understanding of the effects of different process parameters on the quality of the gold suspension is a great help in troubleshooting as well. The problems discussed above are actual challenges encountered by the authors. It is hoped that small manufacturers heeding the lessons learned and reported here will see improvement in the quality of the gold suspensions they produce.Basab Chaudhuri, PhD, is a research associate with InBios International Inc. (Seattle) and a reader in the department of chemical engineering at the University of Calcutta (India). Syamal Raychaudhuri, PhD, is chief technology officer at InBios. --------------------------------------------------------------------------------References1. R Zsigmondy, Zur Erkenntnis der Kolloide (Jena, Germany, 1905). 2. WP Faulk and GM Taylor, \"An Immunocolloid Method for the Electron Microscope,\" Immunochemistry 8 (1971): 1081 1983. 3. GT Hermanson, Bioconjugate Techniques (San Diego: Academic Press, 1996). 4. J Chandler, T Gurmin, and N Robinson, \"The Place of Gold in Rapid Tests,\" IVD Technology 6, no. 2 (2000): 37 49. 5. MA Hayat, ed., Colloidal Gold: Principles, Methods, and Applications, vol. 1 (San Diego: Academic Press, 1989). 6. DA Handley, \"Methods for Synthesis of Colloidal Gold,\" in Colloidal Gold: Principles, Methods, and Applications, vol. 1, ed. MA Hayat (San Diego: Academic Press, 1989), 22. 7. G Frens, \"Controlled Nucleation for the Regulation of Particle Size in Monodisperse Gold Solutions,\" Nature Physical Science 20 (1973): 241.Copyright 2001 IVD Technology还没仔细看,不过楼主这么努力,值得表扬啊.另,有没有中文版.....有时间翻译一下，不过水平有限怕影响各位！确实好东西啊！coesar：谢谢你的支持，我相信能够做的更好看来我做的还不够好！Processing Technologies Dealing with humidityStefan Dick and Jean Thomas WoynickiExposure to moisture can reduce the shelf life and performance of IVD products, but manufacturers can minimize such risks by selecting an appropriate desiccant. Tubes and desiccant stoppers provide ideal moisture and product protection for diagnostic test strips. During the early phases of development, IVD manufacturers should consider the variety of protective packaging options available in order to ensure product integrity and maintain product quality. For IVD manufacturers, maintaining quality and reliability in their products is of utmost importance. To ensure that they are producing high-quality products, companies implement stringent quality assurance systems that comply with international quality systems standards for key steps such as raw-material sourcing, production, and software validation. Another equally important task is to ensure that the end-user has the full benefit of this high quality long after the product has been manufactured. Companies maintain this quality during shipping, storage, and use of the product by choosing the proper packaging materials and methods.Packaging provides solutions for IVD manufacturers in four functional areas: protection (e.g., from moisture, light, and for mechanical stability), dispensing, safety (e.g., child resistance, tamper evidence), and information. Although this article focuses primarily on desiccants as components of a protective packaging system, references to the other functions of packaging are made when different packaging methods are compared.Moisture DamageMoisture can harm IVD products in a number of ways, mostly by reducing their shelf life and quality. For example, experiments have shown the negative influence of moisture on the reliability of gold-based rapid tests.1 In addition to damaging conjugates and rendering capture antibodies inactive by hydrolyzing antibodies, excess moisture during storage may lead to the crystallization of sugar and subsequent poor conjugate release.Humidity may also influence the aging and migration of adhesives that are used in the construction of lateral-flow assays. It has been demonstrated that the aging of such adhesive components has a significant impact on shelf life.2 Membrane components can suffer from moisture-related aging, membrane immobilization effects, and the interference of water through hydrogen bond protein–membrane binding.3Finally, without moisture control, nonsterile IVD systems can be ruined by microbial activity.Desiccant TypesThree types of desiccants are currently used for moisture protection in IVDs: silica gel, molecular sieves, and desiccant clay. Each of these desiccants exhibits different properties under various temperature and humidity conditions (see Table I). Figure 1. Absorption capacity of desiccants as a function of relative humidity (temperature = 25°C; constant humidity conditions; maximum airflow). (Click to enlarge) Silica Gel. Among these desiccants, silica gel is the most widely used for IVD applications. This desiccant is a synthetic amorphous modification of silicon dioxide (SiO2) and is characterized by a large number of mesopores that allow for effective binding of water molecules. When tested at a constant temperature, silica gel\'s absorption capacity for water is relatively small at low humidity levels, but rises with increasing humidity (see Figure 1). Since silica gel reacts slowly in low humidity and quickly in high humidity, it slowly reduces low levels of humidity in a closed environment (e.g., bottle, pouch, tube), but rapidly loses this capacity if exposed to high levels of humidity (e.g., during dispensing).Molecular Sieves. Generally 25–50% more expensive than the equivalent amount of silica gel, molecular sieves are synthetic crystalline zeolites with a general formula of Myx/y[AlxSi1–xO2]. The 4A-zeolite in which the metal ion in this formula is sodium is the most widely used molecular sieve for IVDs. These 4A molecular sieves contain a network of interconnecting micropores with a uniform diameter of 4 (0.4 nm) and can capture and effectively bind water molecules even at very low humidity levels. In fact, the absorption capacity of molecular sieves is almost independent of relative humidity at constant temperature. Since molecular sieves react rapidly at both high and low humidity levels, they quickly reduce the humidity in closed environments to a very low level, but can be difficult to handle in uncontrolled manufacturing environments (see Figure 2). Figure 2. Development of relative humidity in a closed container after the addition of desiccant (200-ml vial; 1 g of desiccant; and starting conditions of 25°C, 20% relative humidity). The time axis is on a square-root scale. (Click to enlarge) Desiccant Clay. In contrast, desiccant clay is a cheaper option and generally 5–15% less expensive than the same amount of silica gel. This desiccant is derived from naturally occurring bentonite clay, and its main component is the layered mineral Ca-montmorillonite, Ca0.16 [Al1.68Mg0.32(OH)2(Si4O10)]. With water molecules binding predominantly to the cation interlayers of the fine clay crystals, the absorption capacity of clay increases with rising humidity and is higher than the absorption capacity of silica gel when conditions are below 30% relative humidity. Since clay reacts relatively slowly at low as well as high humidity levels, it slowly reduces the humidity in closed containers, but it is easy to handle. In addition, desiccant clay granules have up to 30% greater density than either silica gel or molecular sieve beads, thereby occupying less space.Desiccant ProductsTo meet the requirements of different industries, desiccants are prepared using particular materials in a variety of sizes and shapes. Manufacturers should be careful to select the proper desiccant type and product, according to their required performance, insertion process, packaging preferences, and cost considerations. In the IVD industry, desiccant products are available as packets, canisters, tablets, cap inserts, desiccant stoppers, and desiccant polymer blends (see Figure 3). A description of each of these desiccant products follows.Canisters. Desiccant canisters are considered the gold standard among the desiccants used in the pharmaceutical industry. In the United States, the most popular canister sizes are 13.9 x 17.8 mm, 13.9 x 25.7 mm, and 19.4 x 15.8 mm. Also available are canisters as thin as 3.6 mm, and as large as 62.8 mm in diameter. In addition to being able to contain between 0.25 g and 27 g of desiccant, canisters are specifically designed for high-speed product insertion and have less potential for contamination and machine jamming than desiccant packets. Figure 3. Desiccant products for IVD packaging (clockwise: tube and desiccant stoppers, desiccant canisters, desiccant packets, and desiccant tablets). Packets. Desiccant packets are available as either individual packets or long strips that are cut into separate packets upon being inserted into a package. While these packets have fill weights that contain between 0.25 g and 10 g of desiccant, various facestock materials are used to form the actual packets, with GDTII (polyester and polyolefin nonwoven) and Tyvek (spunbonded polyolefin) being the most commonly used in the United States. Each facestock material has different effects on the appearance, seal strength, absorption rate, and dusting properties of the desiccant packet. IVD manufacturers should especially consider dusting properties since it has been reported that desiccant dust contamination can affect the reliability of test strips.Tablets. Desiccant tablets can be made into virtually any size and shape in order to fit the packaging needs of IVD devices. Inserting these preformed desiccants is relatively easy and can be automated to accommodate high-speed production. Some IVD manufacturers have found that using desiccant tablets instead of packets allows them to speed up their packaging processes tremendously.4,5Cap Inserts. Desiccant cap inserts are used in conjunction with screw-on caps for rigid containers. Since desiccant-filled inserts fit directly into the cap, the desiccant does not take up any extra space in the container itself, and no additional equipment is needed to insert the desiccant during the packaging process. A desiccant cap insert is always designed to fit exactly into a special packaging configuration, so it cannot be considered an off-the-shelf item.Tube and Stopper Systems. The tube and desiccant stopper system is the most advanced moisture-protection packaging solution for IVD test strips. Similar to the cap insert, the desiccant is contained in the stopper, does not consume any extra space in the tube, and requires no additional desiccant feeding system. The desiccation and protection functions of the stopper can also be combined with such safety features as child resistance and tamper evidence.Desiccant-Filled Polymers. Another moisture-protection option is to incorporate a desiccant into the polymer material that is used in tubes for strip storage and housings for test kits. These tubes and devices consist of an outer layer of moisture-impermeable material and an inner layer of desiccant-filled polymer material. Absorption capacity and rate can be adjusted by choosing certain plastic materials, desiccant types, desiccant amounts, and additives. Extreme care has to be exercised when choosing such a system to ensure that the additives used by some suppliers in their desiccant-entrained plastics do not react with the test strips. Contamination can occur because of direct contact between strips and tubes, and through the air that is contained in the tube if an additive exhibits considerable vapor pressure under shelf life conditions. Other possible side effects include interaction with adhesives and membranes, and protein attachment.3Quality ParametersDesiccant manufacturers usually specify the absorption capacity and residual moisture of their products. Absorption capacity is measured by exposing the desiccant to specified, static conditions and observing its weight increase, which is given as a percentage of the desiccant weight. Residual moisture is measured by heating the desiccant to its reactivation temperature and observing the weight loss, which is also given as a percentage of the desiccant weight.Dessicants 25°C, 10% relativehumidity (hours) 30°C, 60% relative humidity (hours) Clay 48.0 3.5 Silica gel 35.0 1.0 Molecular sieve 12.0 1.0 Table I: Reaction rates of desiccants under high and low-humidity conditions. The table indicates the time needed to absorb 5% of water by weight under constant humidity and temperature conditions. In most cases, these two parameters are redundant. They measure the same quality parameter of the desiccant, because absorption capacity decreases the same amount as residual moisture increases. When desiccant users set up incoming-quality procedures or want to compare quality data from different suppliers, they should not depend solely on the data provided by the supplier. They should also confirm the suppliers\' quality procedures. For example, residual moisture data, especially for molecular sieve products, is extremely sensitive to changes in testing temperatures and procedures.Choosing the Right SolutionA manufacturer\'s choice of desiccant must ultimately be based on the performance characteristics required to protect the IVD product. Key measures of desiccant performance include absorption capacity, absorption rate, and the relative humidity level it is capable of maintaining within the packaging. Other important factors to consider are weight, size, and shape restrictions of the package; dust requirements; process restrictions; and cost.Absorption Capacity. The required absorption capacity for moisture (Mtotal) is calculated by taking the sum of the initial moisture in the package (Minitial) and the amount of water flowing into the package during shelf life (Minflow). Minitial is the sum of headspace air humidity (Mheadspace) and available residual moisture in the packaged good (Mrm). Mtotal = Minitial + Minflow = (Mheadspace + Mrm) + MinflowMheadspace is the product of headspace volume and absolute air humidity in the environment during the packaging operation. This contribution to total moisture is usually negligible, because headspace is minimized during packaging design or when packaging is done under controlled low-humidity conditions.Mrm is the amount of water that can be desorbed from the packaged good by the desiccant during shelf life. This amount depends on the overall moisture content of the good after manufacturing, the desorption energy for water from the packaged good, and the desorption kinetics. The product\'s residual moisture is usually determined at 110°C and gives an upper limit for this amount.Minflow can be calculated from the water ingress data of the package (in mg/day) under shelf life conditions for the desired number of days. This factor very much depends on the type of packaging (e.g., flexible, rigid) and the packaging materials that are used. For example, various flexible packaging materials exhibit different water-vapor transmission rates (WVTRs; see Table II).Water ingress into flexible packaging can only be roughly estimated from such data. The reason is that even though the seal area is the most likely spot for moisture ingress, its contribution is not included in the pure-material data. Another source of uncertainty is that WVTRs are sometimes reported for conditions that are unlike shelf life conditions and therefore have to be recalculated for specific requirements.On the other hand, rigid packaging solutions, such as tube and stopper systems, have an advantage in that moisture ingress, rather than WVTR, is specified by the supplier and does not require extra testing.Relative Humidity. In order to calculate the minimum amount of desiccant that is needed based on the total amount of moisture to be absorbed (Mtotal), the maximum allowable relative humidity in the packaging during shelf life must also be known. The amount of desiccant needed is then the quotient of Mtotal and the absorption capacity of the desiccant at the maximum allowable relative humidity.For example, if Mtotal is 0.1 g and the maximum allowable relative humidity is 20%, the absorption capacity of desiccant clay is 13%, so the minimum amount of clay needed is 0.78 g (0.1 / 0.13). Under these same conditions, 0.87 g of silica gel or 0.55 g of molecular sieve would be needed. It must be kept in mind that the data used for these calculations are valid only for pure desiccants.The effect of residual moisture, which is usually specified by the desiccant supplier, also needs to be taken into account. In most cases, it is sufficient to round up to the nearest standard desiccant product size. For the above example, 1 g of clay, 1 g of silica gel, or 0.75 g of molecular sieve would suffice.In addition, stability tests usually establish the maximum moisture level that should not be exceeded during shelf life. It is also useful to check whether there is a minimum moisture level to be maintained during shelf life in order to avoid problems as a result of moisture loss in the product (e.g., altered membrane and adhesive aging). For example, molecular sieves are able to reduce the humidity level in a closed environment to virtually zero, while silica gel and clay leave a small amount of residual moisture in the air and consequently in the product (see Figure 2). Thus, knowledge of minimum and maximum humidity levels that should be maintained can further enhance the correct choice of desiccant.Absorption Rate. Another parameter to consider is absorption rate. Molecular sieves reduce the relative humidity in a closed container from 20% to virtually 0% within 5 minutes; desiccant clay absorbs the moisture within 20 minutes; and silica gel needs about 60 minutes (see Figure 2). In most cases, however, these time differences are irrelevant because the products packaged are not so moisture sensitive that they would suffer from the 15-minute difference between molecular sieve and clay.Under real-life conditions, the products being packaged contain some water, or residual moisture, which has to be picked up by the desiccant and therefore increases the reaction time. In most cases, the kinetics for desorption of water from the product are slower than the absorption kinetics of the desiccant, such that the desorption reaction is the rate-limiting step for overall dehumidification. This factor eliminates the differences between the desiccant reaction rates and leads to reaction times of hours and days instead of minutes. Process RestrictionsAs discussed above, at elevated humidity levels, different desiccants exhibit different reaction rates, a factor that influences the moisture uptake of the desiccant product during exposure on the packaging line. Moisture uptake during this exposure time reduces the desiccant\'s absorption capacity. Consequently, this loss of capacity has to be considered when the required amount of desiccant is calculated, or the desiccant must be dispensed under moisture-controlled conditions. For example, small molecular sieve packets are extremely difficult to handle and require humidity control during dispensing.In addition, the choice of desiccant product determines the loss of capacity during exposure. Generally, desiccant canisters, inserts, and stoppers react more slowly than packets or tablets. Humidity IndicatorsAs a value-added feature, a humidity indicator can be added to packaged IVD test strips. This indicator allows the consumer to read the humidity level inside the packaging and gives an indication of whether the test strips can still be used or should be discarded. Custom indicators can be designed to change color at specific critical humidity levels of a product or packaging combination. The indicator may also contain additional consumer information and instructions, and thus add informational value to the packaging solution.ConclusionMany options exist for protective packaging of moisture-sensitive IVD products. It is important to consider the desiccant as part of the whole packaging solution. The choice of desiccant should be taken into account in the early stages of packaging development. Such early planning enables the IVD manufacturer and the desiccant supplier to find an optimum packaging solution in terms of performance, appearance, market acceptance, and overall cost. A complete cost comparison includes cost of packaging materials as well as investments and process costs that are associated with packaging speed, potential downtime, and overall efficiency. --------------------------------------------------------------------------------References1. J Chandler, N Robinson, and K Whiting, \"Handling False Signals in Gold-Based Rapid Tests,\" IVD Technology 7, no. 2 (2001): 34–45. 2. K Jones, and A Hopkins, \"Effects of Adhesive Migration in Lateral-Flow Assays,\" IVD Technology 6, no. 5 (2000): 57–63.3. K Jones, \"Troubleshooting Protein Binding in Nitrocellulose Membranes, Part 1: Principles,\" IVD Technology 5, no. 2 (1999): 32–41.4. \"Desiccant Tablet Boosts Throughput 3000%,\" Pharmaceutical & Medical Packaging News 7, no. 5 (1999): 38–41.5. \"Desiccant Tablet Helps Streamline Production,\" Pharmaceutical & Medical Packaging News 9, no. 3 (2001): 94. Stefan Dick, PhD, is product group manager, and Jean Thomas Woynicki is the key account manager for the U.S. pharmaceutical and diagnostics industries at Süd-Chemie Performance Packaging (Albuquerque, NM). The authors can be reached at sdick@sud-chemieinc.com and jwoynicki@sud-chemieinc.com, respectively.Copyright 2002 IVD Technology谢谢！希望大家能够喜欢！一定要把这个版面的内容丰富一下Effects of adhesive migration in lateral-flow assaysSelection of an appropriate laminating adhesive can help manufacturers improve processing and increase the shelf-life of membrane-based diagnostics. Kevin D. Jones, Anne K. Hopkins The materials used for lateral-flow diagnostic tests have two common characteristics: they are porous and relatively weak. To compensate for their lack of physical strength, such materials have traditionally been mounted onto support cards, usually by means of an adhesive (see Figure 1). In the past, product developers have used almost anything sticky to mount their materials, including thermal setting adhesives. Today, advances in adhesive technology have made available a wide range of adhesive products that are appropriate for use in lateral-flow test applications. By selecting an adhesive that functions compatibly with the porous materials used in lateral-flow test strips, product developers can help to ensure the proper operation of such tests throughout their planned shelf life. For the vast majority of lateral-flow applications, most product developers now specify pressure-sensitive adhesives (PSAs), which present relatively few complications for test design, processing, or use. When an appropriate adhesive formulation is used, PSAs have proven to be a reliable means of bonding together the component materials of lateral-flow tests.Figure 1. Diagram of a typical lateral-flow assay showing the location of adhesive layer used to bond porous membrane materials to a supporting substrate.In a typical lateral-flow application, bonding of the component materials is accomplished when the adhesive penetrates into the porous materials, thereby linking them together. This process of adhesive migration under normal conditions is known as cold flow. Because no heat is applied during the process of laminating a PSA to other components of a lateral-flow test, some degree of cold flow is essential for the formation of a bond between the materials. However, an adhesive that exhibits either inadequate or excessive cold flow can present challenges for the developers of such diagnostic products. To ensure proper manufacturing and performance of their lateral-flow tests, product developers should therefore study what type of adhesive to use and select one that offers the best compromise between bond strength and the degree of adhesive migration. When product developers study, test, and allow for the influences of adhesive migration, the effects resulting from this phenomenon are usually minimal. Adhesive Relative Hardness Propensity for Cold Flow RHA High Low RHB Moderate Moderate RHC Low High Table I. Hardness and cold-flow properties of the adhesives used for the study described in this article. The Scope of Migration Effects If left unexamined, however, excessive adhesive migration can affect many of the components of a lateral-flow assay. If the level of cold flow is too low, the result can be a low initial bond strength that may result in inadequate bonding of the component materials. To obtain a higher bond strength, the product developer must therefore specify an adhesive that offers a higher degree of cold flow. On the other hand, a level of cold flow that is too high can result in blocked pores, hydrophobic patches, and material rewetting problems that may interfere with the performance of the test. These symptoms can result from the migration of the adhesive into the porous materials of the test after they have been bonded together, especially during long periods of shelf storage. In many cases, problems associated with cold flow can be resolved through the use of direct-cast membranes. For instance, such membranes can eliminate bottom-up adhesive migration during storage because the supporting plastic sheet prevents adhesives from entering the pores of the membrane. Nevertheless, there are cases when the differences in processing or performance characteristics between supported and unsupported membranes are so great that developers must use unsupported membranes. Grade Composition Thickness ( m) Nominal pore ( m) Lateral wicking (sec) GFD Glass fiber 675 6 70 F075-14 PVA/Glass fiber 380 20 40 3MM Cellulose 335 4 640 PuraBind AS Nitrocellulose 125 3 50 PuraBind AF Nitrocellulose 125 5 37 PuraBind AR Nitrocellulose 125 8 27 Table II. Characteristics of the porous sustrates used for the study described in this article. Even when direct-cast membranes are used, however, excessive adhesive migration can interfere with the proper functioning of other test components—including sample wicks, conjugate pads, and absorbents. If a sample wick is used to control sample volume within a test, for instance, adhesive migration into the wick can affect the total volume of sample available. Similarly, when a test requires a filtration step (as in the case of tests requiring blood separation), migrating adhesive can block a membrane filter, making less sample available for the test. Adhesive migration can also occur in tests where the manufacturer has specified a plastic film to be used as a protective layer on top of the test membrane—a practice that is becoming increasingly common. Any top-down adhesive migration occurring in tests with these protective laminates will have a more significant effect than in unprotected tests. Excessive adhesive migration can interfere with the development of the test line that must be viewed through the laminate, resulting in a visible reduction in the intensity of the line. Manufacturers that are developing membrane-based quantitative tests can also encounter challenges in dealing with cold flow. If migrating adhesive blocks part of the porous structure it can reduce the bed volume of the material, thus affecting the calibration of the test. The current generation of quantitative instruments read tests by optical means (either reflectance or transmittance). In either case, obstruction of part of the membrane can change the amount of sample and conjugate flowing through the capture zone, and thus alter the sensitivity of the test. Migrating adhesive that blocks a part of the membrane structure can also change the amount of material available to absorb or reflect light, thereby affecting test readings. Similar effects can occur in magnetic particle-based systems, where test readings are based on the total amount of conjugate bound in the capture zone. If the available area of the capture zone is blocked with migrating adhesive, the test reading will decrease accordingly. Moreover, such readings can deteriorate further with continued adhesive migration during storage, so that over time the same concentration of sample will result in different readings. The presence of hydrophobic spots on test membranes can have a significant effect on test operation regardless of the method used for quantification. Patches in the membrane that do not wet out will lead to variations in results, especially if the hydrophobic patch is within the region of the capture zone that is being measured. Adhesive Migration Studies Pressure-sensitive adhesives are categorized according to their relative degree of hardness. An adhesive with high hardness possesses a low level of cold flow and low initial bond strength. An adhesive with low hardness possesses a high level of cold flow and higher initial bond strength. To study the effects of different adhesive formulations on lateral-flow materials, three adhesives were produced with differing levels of hardness (see Table I). A 23- m layer of each adhesive under investigation was applied to a plastic support card, onto which typical diagnostic materials were then laminated. These materials included nitrocellulose membranes in a range of pore sizes, cellulose membranes, and glass fiber products (see Table II). The diagnostic materials were laminated at a constant pressure (20 kPa) and placed in sealed foil pouches. Sets of the prepared pouches were then stored in a temperature-controlled room (at 20°C) or placed in an incubator (at 37°C). Samples were removed at regular intervals and tested for the range of properties under investigation. A model lateral-flow immunoassay was used to assess the effects of the various adhesive formulations. Lateral wicking rate was assessed by measuring the time required for deionized water to migrate a known distance (7.5 cm for glass fiber and cellulose membranes; 2 cm for nitrocellulose membranes). The performance of the nitrocellulose membranes was also tested for protein application, capture-line intensity, and hydrophobic patches by using a model lateral-flow assay (monoclonal anti- hCG capture and conjugate with samples of urine spiked with hCG). All tests were repeated 10 times on two separate pieces of the material. The laminated materials were also analyzed using a scanning electron microscope (SEM). For this examination, cross sections of the laminated materials were cut to permit measurement of the thickness of the adhesive layer. Study Results When compared with the results of wicking tests conducted on unlaminated materials, the results of this study indicated that the lateral wicking rate increased over time for all the materials that were laminated onto an adhesive card (see Table III). The amount of the increase depended upon the hardness of the adhesive used in laminating the materials. Following is a summary of the test results for wicking time, inspection for hydrophobic spots, capture-line intensity, and depth of adhesive migration.Figure 2. The effect of adhesive type on lateral wicking of PuraBind membranes. Figure 3. The number of hydrophobic patches seen on PuraBind membranes after three months storage at 37°C.Wicking Time. The time required for a sample to wick a predetermined distance was measured for all samples after storage for three months at 37°C (see Figure 2). In this study, the most serious effects on wicking time were those involving nitrocellulose membranes. When an 8- m nitrocellulose membrane was laminated using the adhesive with the lowest hardness rating (RHC), the time required for the sample to wick 2 cm increased by as much as 45%. Use of the intermediate adhesive (RH resulted in an increase of 14%, while use of the hardest adhesive (RHA) resulted in an increase of only 6%. Similar effects were observed for all the other laminated membranes evaluated in this study. In all cases, use of the adhesive with the lowest hardness rating (RHC) resulted in significantly longer wicking times. Inspected after three months at 37°C, the laminated cellulose membranes (3MM) showed very little change in wicking time. There was no significant observed increase in wicking time for the samples that used either the hardest (RHA) or intermediate (RH adhesives. The samples that used RHC adhesive showed only a 5% increase in lateral wicking time. The laminated glass fiber membranes exhibited an intermediate level of performance. In the samples laminated using RHC, the observed increases in wicking time were significant (GFD membranes incurred a 20% increase; F075-174 membranes a 27% increase). Hydrophobic Spots. When a lateral-flow test is performed, users occasionally observe small spots that do not wet out. This occurrence commonly results from imperfections in the way that the liquid runs through the membrane, which can be caused by either pore occlusion or an increase in the hydrophobic character of the membrane.Figure 4. SEM showing migration of RHA adhesive into nitrocellulose membrane (PuraBind AF) after one month at 37°C. Figure 5. SEM showing migration of RHB adhesive into nitrocellulose membrane (PuraBind AF) after one month at 37°C. Figure 6. SEM showing migration of RHC adhesive into nitrocellulose membrane (PuraBind AF) after one month at 37°C.The occurrence of hydrophobic spots can typically be prevented by blocking the membrane after the capture line has been applied. In this study, however, the laminated membranes were left unblocked in order to permit comparisons to be made. For evaluation purposes, 20 5 x 25-mm test strips were cut for each membrane. The test was then performed, and the hydrophobic spots were counted (see Figure 3 and Table IV). The fewest number of hydrophobic spots appeared on the control sample and on those membranes laminated using the hardest adhesive (RHA). The membranes laminated with RHB adhesive showed a slightly greater number of spots. The greatest number of spots appeared on the membranes laminated using the adhesive with the lowest hardness rating (RHC). The hydrophobic character of those laminated membranes also continued to change during storage. Capture-Line Intensity. In this study, performance of the model assay as measured by the intensity of the capture line was also found to be directly related to the type of adhesive used to laminate the test components. Laminates formed using the hardest adhesive (RHA) exhibited no capture-line problems. However, laminates formed using the adhesive with the lowest hardness rating (RHC) exhibited capture lines that appeared uneven and patchy. These membranes also showed areas of inconsistent rewetting and in some cases white spots that had not rewetted when the sample was applied. Depth of Migration. After the prepared samples had been stored for one month at 37°C, SEM analyses were performed to evaluate the degree of adhesive migration (see Figures 4–6 and Table V). In all cases the original coating of the adhesive was 23 m thick. The SEM examinations showed that the depth of the adhesive layer increased for all of the samples during the month of storage. Although it was initially hypothesized that the degree of adhesive migration would be determined by the matrix through which the adhesive was moving, the SEM examinations revealed that the extent of adhesive migration was largely independent of the substrate or its characteristics. Instead, the degree of adhesive migration turned out to depend mostly upon the nature of the adhesive under investigation. The hardest adhesive (RHA) showed the least migration, with the average depth of the adhesive layer increasing from 23 to 25 m. With decreasing hardness, the depth of adhesive migration increased: for materials laminated with RHB the layer increased to approximately 30 m; for materials laminated with RHC the layer increased to approximately 45 m. Study Implications The results of this study indicate that porous materials can be affected by cold flow when product developers specify the use of an adhesive with a relative hardness that is too low. As the relative hardness of the adhesive increases, the degree of migration is reduced. Similarly, the results suggest that adhesives with the highest levels of migration also cause the largest number of related effects. For instance, the adhesive with the lowest hardness level (RHC) not only had the highest level of migration, but also caused the greatest increase in wick time and produced the greatest number of hydrophobic spots. Membrane Adhesive Spots per 20 Strips (no.) AS None 3 AS RHA 4 AS RHB 6 AS RHC 25 AF None 2 AF RHA 4 AF RHB 8 AF RHC 21 AR None 4 AR RHA 3 AR RHB 7 AR RHC 19 Table IV. Number of hydrophobic spots, by membrane and adhesive type. The most significant real-time effects of adhesive migration are due to the increase in lateral wicking rate, the development of hydrophobic spots, and the reduction of the bed volume of the membrane. In line with the third of these causes, all of the effects observed were greatest when the membrane material was thinnest (e.g., nitrocellulose membranes were affected more than either the cellulose or the glass fiber grades that were investigated). Although the membrane and conjugate-release pads are the most critical components for the performance of a lateral-flow test—and the ones studied for this article—it should be noted that adhesive migration occurs in all directions.1,2 Thus, adhesives may also migrate sideways to the cut edges of the laminate, where they can then come into contact with and potentially adhere to other materials. When assays are stored at elevated temperatures, the effects associated with selection of an inappropriate adhesive can be seen relatively rapidly (approximately one month). Under real-time conditions, however, such effects take longer to develop. And some effects are more readily observed than others. For laminates produced with RHC adhesives, wicking and hydrophobic effects can often be observed only after approximately five months (at 20°C), and it can take even longer for laminates produced with RHB adhesives. Importantly, the effects observed in this study all appear within the period commonly accepted as a reasonable shelf life for a lateral-flow test. Most lateral-flow tests have a shelf life in excess of 12 months. But if it is going to cause any problems at all, excessive adhesive migration will likely begin to show its effects within that time. Such migration will also have a significant impact upon semiquantitative or quantitative assays. In qualitative tests the bed volume of the membrane and change in wicking rate may have limited significance. But in quantitative assays any change in bed volume will also change the amount of sample passing through the capture zone, and this factor can affect the calibration of the test. Any change in wicking rate will also have a direct impact on the sensitivity of such tests. Obviously, these are issues that will need to be addressed by those who are developing the new generation of quantitative lateral-flow assays. Conclusion Product developers have many options to consider when they are designing a lateral-flow assay.3 They must select not only the conjugates and reagents that ultimately provide the test\'s diagnostic information, but also the substrates and other components that enable the chemistry to work. Whenever necessary, developers should undertake design testing to ensure that all components of their test systems are compatible with one another and are optimized for factors such as manufacturability, stability, and reliability. Material Adhesive Thickness at 20°C Change (%) Thickness at 37°C Change (%) GFD RHA 23 7.5 23 7.5 GFD RHB 25 12.5 25 12.5 GFD RHC 31 27.5 40 50 F075-14 RHA 24 10 26 15 F075-14 RHB 29 22.5 32 30 F075-14 RHC 36 40 46 65 3MM RHA 25 12.5 26 15 3MM RHB 28 20 28 20 3MM RHC 36 40 41 52.5 AS RHA 25 12.5 24 10 AS RHB 28 20 28 20 AS RHC 35 37.5 42 55 AF RHA 24 10 25 12.5 AF RHB 28 20 29 22.5 AF RHC 40 50 47 68.5 AR RHA 26 15 27 17.5 AR RHB 29 22.5 31 27.5 AR RHC 42 30 50 75 Table V. SEM analysis of changes in thickness of the adhesive layer after storage at 20° and at 37°C By investigating the relative hardness of adhesives chosen for use in diagnostic assays, researchers should be able to predict the ways in which such adhesives will interact with attached substrates. With such information in hand, product developers can then select an adhesive that will provide the bond strength required by their test without incurring excessive migration or its related effects. Selecting an appropriate adhesive can significantly increase the shelf life of finished diagnostic tests. Accelerated aging tests can provide useful information about the potential compatibility and functioning of test components over extended periods. To firmly establish the shelf life of a particular lateral-flow test, however, researchers should perform adequate shelf-life testing. As suggested by this study, such testing should include examination of the test device for effects related to adhesive migration. References 1. Kevin D. Jones, \"Troubleshooting Protein Binding in Nitrocellulose Membranes, Part 1: Principles,\" IVD Technology 5, no. 2 (1999): 32–41. 2. Kevin D. Jones, \"Troubleshooting Protein Binding in Nitrocellulose Membranes, Part 2: Common Problems,\" IVD Technology 5, no. 3 (1999): 26–35. 3. Alan Weiss, \"Concurrent Engineering for Lateral-Flow Diagnostics,\" IVD Technology 5, no. 7 (1999): 48–57. Kevin D. Jones is manager for diagnostic technology and Anne K. Hopkins is a senior technologist at Whatman International (Maidstone, Kent, UK). This article originated in a poster presentation at the American Association for Clinical Chemistry Oak Ridge Conference (Boston, MA, May 2000), which was cosponsored by IVD Technology.太好了，终于找到了！！楼主要继续努力啊好文章 支持...强烈支持..顺便请教一下 lzsgxl ,表面活性剂和 buffer 的选择 不同,为什么会对最后的结果有 影响.表面活性对胶体金结合物的洗脱、稳定性、特异性有一定的关系， buffer 的选择对结果的影响我目前还没有很好的结论，但我常用PB系统，还不错表面活性剂我常用T－20再接再利！ASSAY DEVELOPMENTHandling false signals in gold-based rapid tests John Chandler, Nicola Robinson, and Karen WhitingA guide to the systematic approach needed to overcome false signals and optimize test performance. In recent years the IVD industry has increased enormously its work to develop membrane-based lateral-flow tests. Such tests have found applications in both clinical and nonclinical fields. A review of the wide range of applications for these devices has been reported in an earlier article.1 While the concept of membrane-based lateral-flow immunoassay tests is quite simple, how well such tests perform depends on a number of critical parameters. Earlier articles in IVD Technology have discussed the importance of the quality of the gold conjugate and the way that capture proteins bind to membranes.1– 3 This article explores the possible causes of poor test performance that result in the generation of false-positive and false-negative signals, and provides a systematic approach to overcoming such problems and optimizing test performance. While this article specifically describes such problems with specific reference to gold-based lateral-flow tests, very similar problems are also found in latex-based tests. In order to avoid repetition, it is assumed that the reader is already familiar with the mechanics of a lateral-flow test and with the basic properties of the lateral-flow test components—specifically the antibodies, gold conjugate, membrane, conjugate release pad, and sample and absorbing pads—as well as the standard chemical components that are used to treat the various papers and membranes used in the test. For the reader unfamiliar with these concepts, they have been described in detail elsewhere.1 Symptoms of False-Positive and False-Negative ResultsA false-positive result is the appearance of a colored line on the test strip (red for gold conjugates) in the absence of analyte in the sample. The line may appear early in the test or after a significant time delay. A false-negative result is the absence of a visible line at the capture antibody (for a sandwich assay) in the presence of a detectable positive sample. Either type of false signal may occur with a wide variety of samples, or may arise only with certain types or sources of sample. The problem may appear in every test strip from a single batch of devices, or it may only occur in a random number of strips within a sample. The latter situation demonstrates the importance of performing large trials of test strips at each stage of development before moving on to the next stage toward full manufacture. Even at the level of full manufacture, it may be found that occasional false-positive results are obtained from standard negative samples and vice versa. Because of the enormous cost of batch failure, it is therefore mandatory to test thoroughly for, and understand the causes of, false-positive and false-negative results well before reaching large-volume production. False-positive and false-negative signals may have many different causes. Such signals may be caused by either the sample or by the poor or inadequate design and manufacture of the test itself. The potential for generating false signals should be eliminated during the course of a test\'s development through a thorough, iterative trial of large numbers of test devices at each stage of development. The cause of a false-positive or false-negative result can sometimes be determined from the visual appearance of the signal, or of the flow characteristics leading to the signal, as the test is performed. However, the reason a false signal has arisen will not always be evident. Only experience, a systematic and scientific approach, and a detailed troubleshooting schedule can eliminate such signals and prevent them from happening. Before adjusting any particular parameter, the test developer should be thoroughly familiar with what makes a test work reliably and what can cause it to go wrong. It is not necessary to adopt an empirical approach if a full understanding of the mechanics of a lateral-flow test has been achieved. A detailed description of the workings of a membrane-based lateral-flow test and a detailed troubleshooting manual for false-positive and false-negative results is outside the scope of this short article.4 Instead, this article summarizes typical symptoms and describes a methodological approach for determining the cause of the problem. Suggested causes and cures are also provided. Basic Causes of False PositivesMost causes of false-positive signals arise for the very same reasons that proteins bind specifically to gold particles during a routine conjugation procedure. During such a conjugation, proteins are adsorbed onto the surface of the gold by three major forces (see Figure 1). Figure 1. Binding forces between an anitbody and a gold particle. Charge Attraction. Gold particles are negatively charged because of a layer of negative ions adsorbed onto their surfaces from the reducer (often citrate) used to convert the gold salt into gold colloid. This negative charge will attract positively charged proteins and bring them close enough to the surface for the binding forces to take effect. Proteins that are more acidic than their isoionic point will be positively charged and are thus likely to be strongly attracted to the gold surface. Regions of the protein particularly rich in lysine or arginine will be strongly positively charged at pH values lower than the pH of lysine (pH 10.4) and arginine (pH 12.5). Hydrophobic Binding. Once the proteins are close enough, (that is, closer than approximately 1 nm), any hydrophobic areas of the protein will be more likely to make contact with and bind to the hydrophobic gold surface. Any protein rich in apolar amino acids (e.g., tryptophan, valine, leucine, isoleucine, or phenylalanine) will thus be strongly bound to the gold surface. Dative Bonding. Dative bonding provides the strongest binding of all. Proteins with a high content of sulphur-containing amino acid groups (due to cysteine and methionine residues) will bind strongly to the surface of gold. This is because of the attraction between gold atoms (having conductive electrons) and sulphur atoms (having valence electrons). While these three types of binding forces work in favor of the production of stable gold conjugates, they also work against the performance of the assay system by creating false-positive signals as described below. Before adopting a systematic approach to diagnosing and curing false-positive signals, it is important to be aware of most, if not all, of the possible reasons for such signals. Figure 2 schematically illustrates the major areas of concern, while the descriptions below summarize the most common causes. However, these sources are not at all exhaustive. Generally, all possible causes of false signals will be connected with either the gold conjugate, the capture antibody, the membrane, the added chemicals, or the sample. By understanding these potential causes, a speedy diagnosis can be made from the symptoms that arise during the performance of the test. Figure 2. Main sources of false positives. Problems Arising from the Gold Conjugate. Areas of the gold particles may become partly uncovered by proteins for any of several reasons. Antibodies may be removed while drying the gold conjugate or during the test procedure, or the conjugate may have been poorly coated in the first place. For any of these reasons, naked gold regions will be attracted to any positively charged protein or nitrocellulose or nylon membrane. This will be especially true as the gold passes through the capture line, since distances are very small and the likelihood of physical contact is great. It is therefore very important to coat the gold particles effectively and thoroughly so that the proteins do not detach during storage, handling, or the performance of the test. Apart from the attraction of gold to the capture line, the conjugated antibody itself may be attracted to the capture line under certain operating conditions. This may again be due to charge or hydrophobic interactions, and may depend on the acidic or ionic environment during the procedure. Excess gold conjugate will create several problems. Primarily it will increase the possibility of false-positive signals at the capture line simply because of the large quantity of gold conjugate passing the line. It will also greatly increase the likelihood of backflow of gold after the test period has elapsed. A good-quality gold conjugate should not need to be used in excess. Gold conjugate might also be clustered, for a variety of reasons, usually through poor manufacturing. Large-enough clusters may block the membrane at any point where there is a restriction. If clustering is caused by the gold being hydrophobic, then the gold particles may also stick to the capture antibody during the flow of the conjugate. The gold-labeled antibody may react nonspecifically and immunologically with the capture antibody regardless of whether analyte is present. While such reactions between a pair of antibodies may not always be detected in ELISA techniques, the method of forcing labeled antibody to flow very close to capture antibody in a nitrocellulose membrane increases this possibility. In addition, especially where polyclonal antibodies are used, there may be some nonspecific reactivity with other analytes in the sample, depending on the purity of the antibodies. False-Positive Signals Associated with the Capture Antibody. A variety of factors cause some capture antibodies to behave in a nonspecific sticky manner. Such behavior may be due to hydrophobicity, nonspecific immune reactions, additives within the antibody, high positive charge, or a high concentration of sulphur-containing amino acids in the capture protein which will attract the gold. The same forces that cause antibodies to adsorb and bind onto the surfaces of gold particles during conjugation can actually work here with the capture antibody to the detriment of the lateral-flow test performance. Difficulties Associated with the Solid-Phase Materials. Nitrocellulose membranes are extremely fragile and are easily damaged by contact. It is therefore very important to avoid any mechanical contact with the membrane surface during the procedure for striping the capture antibody. If the membrane is compressed at the capture line while the capture antibody is being applied, there is an increased risk of nonspecific trapping of gold conjugate at that point during the flow of the sample. Two things can cause residual gold to stick at the capture line—too slow a flow of the gold along the membrane, or too slow a release from the conjugate pad. These events can happen if the membrane has too small a pore size, if there is not sufficient surfactant in the strip, or if there is poor contact between the membrane and the gold conjugate pad or the absorbing pad. Also, the membrane may be hydrophobic, thus hindering smooth flow of the gold conjugate. In addition, some samples can be very viscous (serum, for instance), which also slows down the flow. Gold can also stick at the capture line when there is an insufficient amount of sample or of surfactant in the system to wash all the gold along the strip. If the test takes too long to read (usually anything longer than 15 minutes), there is a possibility that excess gold conjugate will start flowing back down the strip from the absorbing pad onto the membrane as the latter dries out. Gold returning from the absorbing pad is very likely to dry out at the capture line because during drying the capture antibody becomes very hydrophobic. Risks Associated with Added Chemicals. Some lateral-flow test manufacturers perform blocking of membranes as a matter of course. With certain membranes, and when applying certain types of sample, blocking procedures convert the membrane from a chromatographic separator (bibulous) to a nonchromatographic strip (nonbibulous). This conversion is designed to improve the flow of the sample and the gold conjugate along the membrane strip. However, blocking a membrane by immersion in a protein or surfactant solution is also likely to wash out any additives that the manufacturer incorporated to prevent the membrane from drying completely and becoming hydrophobic. Such blocking may thus cause the membrane to behave in a more hydrophobic manner when dry and can even cause more-general background staining or false-positive signals at the capture line. Alternatively, blocking with excess protein or surfactant can produce high viscosity during sample flow and may reduce the sample clearance rate. Blocking with the wrong kind of reagents can also change the characteristics of the capture antibody, making it more sticky through charge, hydrophobicity, or increased sulfur-hydrogen (SH) attachments. Blocking should only be used for demonstrably good reasons, such as when the flow of sample or gold needs improving, and then only with minimum reagent concentrations. Some preservatives, whether in the capture antibody, labeled antibody, or sample, can produce false positives. Thimerosal, which contains both sulphur and mercury, and lysine, which is always very positively charged at pH 10.4, are particularly troublesome. Problems Specific to the Sample. Many samples contain components which may bind nonspecifically to the gold conjugate or to the capture line, and in doing so may produce nonspecific results. For example, samples may contain bacteria that may be partly broken down into cellular fragments and that may be extremely hydrophobic. These hydrophobic cellular fragments may also produce cross-linking between the capture line and the gold conjugate. Other samples may contain high levels of sulphur or SH groups, or be very positively charged. In addition, some samples may contain large enough molecular or cellular components to block the membrane and disturb the flow of gold along the strip. Samples may vary greatly in their acidity. For example, urine samples may be initially presented at pH 4–7 and may, in the absence of preservative, gradually become more acidic as bacterial contamination increases. Acidic samples will produce a positive charge on the capture antibody during sample flow and, in turn, this will cause the nonspecific attraction of negatively charged gold conjugate. If samples contain large quantities of acidic or positively charged proteins, they may bind nonspecifically to the gold conjugate before ever reaching the capture line. This can either mask the conjugate, thus reducing the specific signal, or enlarge the conjugate complex to such an extent that clustering occurs on the membrane and at the capture antibody. Diagnosis and Remedies for False PositivesWhen faced with either occasional or recurrent false-positive signals, a systematic approach to diagnosis of the problem should be adopted in order to apply the most appropriate remedy (see Figure 3). The most obvious causes should be looked at first. These are problems with reactions between the gold conjugate and the capture antibody; specifically charge attraction, hydrophobicity, and gold-sulphur bonding. Next, nonspecific cross-reactivity between antibodies, specific sample characteristics, and membrane flow characteristics should be observed. The appropriate use of controls will quickly provide a guide to the source of the problem.Following is a description of a typical systematic method of diagnosis, together with reference to the possible causes listed previously. As with any method of systematic diagnosis, only one parameter should be changed at a time when performing controls. In this way, a process of elimination can take place. A suggested sequence of questions follows. 1. Is the problem due to charge? Varying the acid levels in the test system (from pH 5 to 11) will demonstrate whether positive charge is occurring somewhere and attracting gold to the capture line. 2. Is the problem due to hydrophobicity? This may occur in the solid phase, the capture line, or the gold conjugate. Varying surfactant concentrations in the system will give clues to whether hydrophobicity is the cause. 3. Is the problem gold-SH attraction? This is most likely to occur from cysteine and arginine groups within the capture line or the sample. Closer inspection of these two regions as described in the following section will reveal the difficulty. Systematic Approach to Problem Solving for False PositivesFor most of these possible scenarios, systematic testing can be performed using only a dipstick (or \"half stick\"). The fully dried test strip is not necessary. To conduct such tests, the nitrocellulose membrane strip is placed directly into a microwell containing the gold conjugate, the sample, and the chemicals that would normally be dried into the fully assembled device. In this way, several tests may be made quickly and easily without the need for full assembly and drying. However, if the problem arises from the drying procedure, then the full assembly must be performed before testing. The questions to be asked from the systematic approach can be grouped into the following five categories associated with the test assembly and components. Is the Problem Related to the Gold Conjugate? This can be easily determined by using an alternative gold conjugate—for example BSA-gold at the same concentration and at the same acidity level as the original conjugate. If the problem persists, it is most likely to be caused by a charge effect. If the false signal disappears with an alternative gold conjugate, then it was most likely caused by a poorly made original conjugate or a problem with a conjugated antibody. Other controls to use here would be similar-species conjugates and alternative-species conjugates of monoclonal and polyclonal antibodies. The test developer should always have a range of alternative gold conjugates available for such testing. An example of detecting problems stemming from the gold conjugate follows. A test was developed for an infectious disease using clinical samples (serum). False-positive results were observed for all negative samples. The signals disappeared when using a BSA-gold conjugate. With alternative nonspecific conjugates, the signals also disappeared. When changing to nonclinical control samples, however, such as PBS at pH 7.2, the problem persisted with the specific gold conjugate but not with the others. Changing the acidity of the buffer to pH 10 reduced the occurrences of false positives, but did not cause them to disappear. It was thus concluded that the problem was independent of the sample or capture antibody and was due instead to the high sensitivity of the gold conjugate specific to the capture antibody. The gold conjugate was suspected of having naked gold areas which bound to the capture line. Using a freshly and carefully made gold conjugate eliminated the false-positive signals.Is the Problem in the Capture Antibody? Suitable controls to use in these situations would be alternative capture antibodies from similar or alternative species. Before using such alternatives, however, striping a capture protein of BSA alone will demonstrate whether the problem lies elsewhere. It may also be possible that it is not the antibody itself that is causing the false signal, but any preservative that may be in the antibody. In this case, dialysis against a suitable buffer (e.g., 10 mmol PO4) can improve matters. If the problem disappears with alternative antibodies, then clearly the specific capture antibody is the cause of the false signals and must either be replaced or cleaned up. For example, in practice, polyclonal antibodies from rabbits are sometimes particularly troublesome because of their hydrophobicity and should be treated with extra care. A pregnancy test for detecting hCG in urine was found to produce false-positive signals for all samples, regardless of whether they were positive or negative. Altering the acidity levels did not reduce the signal, neither did changing the gold conjugate. The false-positive signals persisted even with a PBS buffer control sample. It was suspected that the problem lay in the capture antibody. Striping the capture line with BSA instead eliminated all signals. Dialysis of the original capture antibody against a 10 mmol PO4 buffer eventually produced signals faithful to the sample. It was concluded that the capture antibody had been suspended in a buffer containing SH components such as thimerosal, and that these need to be removed or avoided. Is the Problem Related to the Membrane? A significant part of any test development process is the correct choice of membrane. Some membranes are more suited to certain types of assay or samples than others. The developer should have a full repertoire of membranes from all manufacturers and in a range of pore sizes for immediate comparison. For example, random false-positive signals have been observed from test strips cut from batches of membrane where macroscopic hydrophobicity has occurred during drying. The choice of membrane for a lateral-flow test should consider not only the desired flow rate and protein-binding characteristics, but also the homogeneity of the membrane during the preparation processes. Suitable controls to use here would be membranes of different pore sizes, different sources, and different areas of the same membrane batch. For a serological test designed to detect antibodies to H. pylori, initial test development up to experimental prototype stage produced no false-positive or false-negative results. Full field trials determined that the test was ready to move on to the production prototype stage. When scaling up of the test assembly process to several thousand devices was performed, however, many random false-positive results were recorded in each batch. Adjustment of acidity levels, use of an alternative gold conjugate, and double-checking the capture antibody did not immediately reveal the problem. Going back to small-scale experimental prototype tests showed that the problem was in the nitrocellulose membrane since the false-positive results occurred together with loss of the control-line intensity. For the experimental prototype studies, a small batch of membrane had been used. This batch was not matched by a similar one for large-scale manufacture. It was discovered that the large batch of membrane had macroscopic hydrophobic regions (i.e., areas that could not properly wet), which caused localized nonspecific binding of gold to the capture line). Is the Problem a Result of Added Chemicals? Both the gold conjugate and the solid-phase materials (membrane, conjugate pad, or sample pad) may be pretreated with various chemicals during the assembly of a lateral-flow test device. Such additives may include salts, surfactants, proteins, sugars, and polymers. Some additives can give rise to false positives as just described. It is a simple matter to adjust the concentration of each of these chemicals one at a time to determine which may be the source of difficulty. Typical concentration ranges used in lateral-flow test manufacture may be as follows: surfactants, 0.1–1%; sugar, 0.1–5%; protein, 0.01–1%; polymers, 0.01–1%; salts, 10–100 mmol. Concentrations lower than or greater than these values may well give rise to problems of the nature described here. In a test development process designed for the study of animal proteins in serum, some false-positive results occurred. During the experimental prototype stage, the test was performed using a dipstick in a microwell by adding a preparative buffer to the serum sample and gold conjugate before applying them to the device. Alternative gold conjugates were used without effect. The acidity level of the buffer was altered but did not affect the results. Changing the capture antibody failed to eliminate the occasional false-positive result. It was then observed that the appearance of the false positives with negative serum samples occurred only when the buffer had been added to the serum sample for several minutes before applying it to the dipstick. The buffer contained 5% of a strong surfactant. Lowering the surfactant concentration to 1% allowed the serum and gold conjugate to mix with the buffer for several hours without any false-positive results being observed. This demonstrated that the excess surfactant had acted aggressively on the gold conjugate, removing antibodies from the surface and creating naked gold regions which interacted with the capture antibody. Is the Problem in the Sample? Because of variations in factors such as acidity, sample contamination, or cystein content, samples can cause unpredictable false-positive signals as previously described. To determine if the sample is at fault, a variety of samples from similar and alternative sources may be tested. Apart from the biological or organic content of the sample, the problem may also lie in the vehicle buffer. If this is the case, alternative buffers should be tested. The simplest adjustment to make is to the acidity of the sample, which will quickly determine if charge attraction is the problem with the sample. With some samples a filtration step may be required, either separate from the test procedure or built into the test assembly itself. A lateral-flow test for urine-based hormones was developed to production prototype with 97% specificity and 98% sensitivity using a wide range of urine samples. During the latter stages of field trials, however, it was discovered that several false-positive results were occurring from stored samples. Measurement of the acidity of the samples showed an increase in acidity, but control-buffer samples of equivalent acidity levels ruled out acidity as the cause. The problem disappeared when the urine samples were microfiltered. A subsequent microscopic check revealed that bacterial contamination of the samples was producing the false positives. It was concluded that the increased bacteria produced a hydrophobic contamination of the sample that caused nonspecific interaction between the gold conjugate and the capture antibody. Fresh samples did not produce the same effect. Possible Causes of False Negatives Creating a flow chart for diagnosing the causes of false-negative signals is much more involved than doing so for false positives, and is beyond the immediate scope of this article. This is because reseachers are often working in the dark with no visible signal to begin with. The approach to solving the problem will also depend on the appearance or nonappearance of the control line, and the sample and gold flow along the membrane strip. Table I giv es a summary of most of the possible causes of false-negative signals.Test Region Cause Reasons Damaged conjugate Antibody lost and competing Poorly made conjugate; insufficient sugar with conjugate Hydrophobic collapse of antibody onto gold Poorly made conjugate; insufficient sugar with conjugate Hydrolysis of antibody Poor drying procedure; poor (moist storage Capture antibody inactive Missing capture antibody Lifted off membrane; surfactant in antibody Hydrophobic collapse of antibody Very hydrophobic antibody; unblocked membrane Excess salt in antibody resists gold conjugate Hydrophic salt barrier Hydrolized capture antibody Poor (moist) storage conditions Impure antibody Excess nonspecific proteins mask specific antibody Poor conjugate release Hydrophobicity of conjugate pad Wrong conjugate pad material; insufficient sugar in conjugate Hydrophobicity in membrane Insufficient surfactant; wrong or dried membrane Crystallization of sugar Excess moisture during storage Poor contact between conjugate pad and membrane Insufficient physical pressure; insufficient surfactant; insufficient sample colume Poor membrane performance Sample too viscous Membrane pore size too small; sample needs diluting Flow rate too fast Membrane pore size too large Gold blocks at base of membrane Membrane hydropholic; insufficient surfactant Table I. Some typical causes of false-negative results. As with the identification of false positives, there are certain symptoms that can help diagnose the cause of false negatives. Examining the most likely causes progressively will enable an analysis and remedy for the problem. A detailed guide to such a systematic approach has been compiled.4 The description here is primarily for occasional false-negative results among the majority of true positives. If all test strips produce false negatives with positive clinical samples, then a much more fundamental design problem has occurred. Figure 4. Main sources of false negatives. The most common sources of false-negative results are shown schematically in Figure 4. The problems most frequently occur in the capture antibody or gold conjugate, but may also be caused by poor flow characteristics of the membrane (too fast or too slow), poor release of the gold, insufficient salt in the system (antibodies will not work without a minimum salt concentration), or incorrect acid levels in the test system. In addition, the sample may need to be treated to allow access to the antigens, or it may need to be diluted to avoid the hook effect, and certain antigens may be masked by variations in sample composition. Figure 5. Instability of capture antibody. The capture antibody sometimes becomes unstable and loses its specific activity during storage, either through hydrolysis (from moisture and inadequate drying), or through hydrophobicity and collapse onto the membrane (see Figure 5). The latter effect may be a characteristic of the antibody itself and may be overcome by substituting the antibody or treating the membrane with surfactant after striping the capture protein. Potential instability of either the capture antibody or the gold conjugate highlights the need to assemble the test in a controlled dry-room environment. False-negative results are frequently caused by the failure of the sample to release the gold conjugate, or even to move along the membrane strip. A number of possible causes can result in such a symptom. There could be insufficient sample to make the test run, insufficient sugar in the gold conjugate or sample pad, poor contact between the gold-conjugate pad and the membrane, insufficient surfactant in the system, or the wrong type of conjugate pad material. These problems would be evident from the absence of the control line, which should always be positive. The most likely cause of gold not moving onto the membrane is crystallization of sugar in the gold conjugate following exposure to excess moisture. Two other common causes of false negatives are failure of the capture antibody or gold conjugate to react immunologically, and the removal of the capture antibody from the membrane by the sample (see Figure 5).2,3 False-negative results are also frequently due to gold conjugate being damaged following drying and rehydration. The presence of a control line will indicate whether the gold has been adequately released by the sample, but it does not necessarily provide a check on the performance of the capture antibody or on the immunological activity of either antibody. ConclusionLateral-flow tests are generally developed in stages, leading up to full manufacturing stage by passing through experimental prototype design, into preproduction prototypes, and then into full production with field trials, quality assurance, and validation at each stage. At any stage of this process, unexpected problems can arise and could set the whole development process back by several weeks or months. It is therefore very cost-effective to have not only a thorough understanding of the mechanism by which the test performs optimally, but also of all the possible sources of error that may occur. Ideally this understanding comes with long experience, but not every developer starts with this prior knowledge. By having a full awareness of the exact mechanism of the test performance and a knowledge of the sources of potential problems, a diagnosis of false-positive and false-negative signals can be quickly achieved. This will result in a considerable savings of time, materials, and money. It is essential to approach problem solving in a systematic manner and not by a quick-fix method that may allow the same fault to unexpectedly arise later. By having a good basic understanding of what each component does, what affects its stability, and how all components interact together, the developer may use a flow chart intelligently to eliminate problems. A random and empirical approach will only provide a short-term solution and can prove to be very costly. Lateral-flow tests are simple in concept and in basic design. While general rules apply, it should not be assumed that steps taken in the optimization of one test will necessarily apply to another. There are a wide variety of samples and analytes to be examined, and not all antibodies behave in the same way. Membranes may also vary from manufacturer to manufacturer, and reproducibility between batches from the same source should always be checked. In the end, the best results are obtained by using the best materials together with the best procedures. High-quality lateral-flow test development requires thorough knowledge and awareness of all the parameters involved, and of what factors affect component stability. The needs of sensitivity and specificity are matched by an equal demand for reliability and reproducibility. Only with a full understanding of the way in which each component works, the hydrodynamics involved, and how to diagnose problems using a systematic approach, will these high standards be maintained. --------------------------------------------------------------------------------REFERENCES 1. J Chandler, T Gurmin, and N Robinson,\"The Place of Gold in Rapid Tests,\" IVD Technology 6, no. 2 (2000): 37–49. 2. K Jones, \"Troubleshooting Protein Binding in Nitrocellulose Membranes, Part 1: Principles,\" IVD Technology 5, no. 2 (1999): 32–41. 3. K Jones,\"Troubleshooting Protein Binding in Nitrocellulose Membranes, Part 2: Common Problems,\" IVD Technology 5, no. 3 (1999): 26–35. 4. A Weiss, \"Concurrent Engineering for Lateral-Flow Diagnostics,\" IVD Technology 5, no. 7 (1999): 48–57. 5. Troubleshooting Guide for False Positives and False Negatives in Lateral-Flow Rapid Tests (Cardiff, UK: British Biocell International, 2000). 6. A Short Guide: Developing Immunochromatographic Test Strips (Bedford, MA: Millipore Corp., 1996).虽然现在没有多少人支持这个帖子，但是我还会努力去丰富里边的内容，相信朋友们能够用的上!谢谢.我目前也用的是PB 还有tris .能给个联系方式吗?有时间向你请教.再支持一下.文章确实不错.基本上所有的步骤都有了.非常好，谢谢！QQ523861910谢谢大家的支持！慢慢的把一些细节的文章都贴上来www.devicelink.com/ivdt 中看原文就是了.lzsgxl 能不能发点往上没有的东东?哈哈！不反对大家去看原文，希望能够加强交流！是否需要翻译,还有很多朋友看不懂. 如果需要可以考虑由你们来组织一个义务翻译团队，将一篇文章分拆,各人翻一部分,合作翻译. 我工作比较多,不能组织和校正,但我可以加入翻译一部分.这样最好不过工作量太大，并且有很多与原文不符，怕影响读者，特别是那些没有做过胶体金的人，翻译出来的差异会更大，不过可以试试有那位愿意参加IVD文章翻译的请于我联系！个人认为不需要翻译，谁能把robust翻译顺达雅？呵呵 ,这个工程 够复杂.tom.近来可好.现在比较忙.有时间和你联系.最近怎么不上msn了.你提供的网站是不错，可是怎么能看到楼主发的原文啊？！没法注册啊？看来我还是在这里看好了！省得麻烦了！要是有翻译好的请楼主发一下谢谢了！看得好累哦！上一篇:Re:【求助】请高手帮帮忙，翻译一下下面的句子下一篇:Re:[资料]几本实验动物电子书的下载地址您的位置：医学教育网 医学资料