The following video tutorial described how to run a sandwich ELISA, also known as enzyme-linked immunosorbent assay, using a general PeproTech protocol. 021-64288051/2 021-64869486 021-64869486(传真) 13611693188美国PeproTech (派普泰克) 公司是世界上领先的重组细胞因子和蛋白生产商，同时也生产多种细胞因子和蛋白的ELISA法检测试剂盒，详细信息请查看www.peprotech.com，或与PeproTech中国代表处 (电话：0512-6832 5993，Email：china@peprotechasia.com) 联系。 为让中国用户能够更好的熟悉和掌握PeproTech的ELISA试剂盒及其技术，PeproTech专门制作了\"ELISA操作指南视频\"，供各位老师和同学观摩！ 视频网址为：http://v.youku.com/v_show/id_XMzQ3Mzk3NTg4.html或http://www.tudou.com/programs/view/tzU8m93o0tI/，欢迎大家的批评和指正！ 该视频为英文讲解，为便于大家理解，请看下面的英文原文及翻译：The following video tutorial described how to run a sandwich ELISA, also known as enzyme-linked immunosorbent assay, using a general PeproTech protocol.接下来的实验指导讲述PeproTech夹心法ELISA，即酶联免疫吸附试验的通用步骤The capture, standard, and detection are supplied as stable lyophilized products, and should bestored at -20℃ until ready for use.捕获抗体、标准品和检测抗体均为冻干粉，使用前应保存于-20℃Reconstituted Capture, Standard, and Detection components are only guaranteed to be stable forup to 2 weeks when stored at 4 ℃.捕获抗体、标准品和检测抗体重悬后，在4℃时最长保存2周。If you have reconstituted the EDK, and plan on using it for a duration greater than two weeks, aliquotand store at -20℃ for up to 6 months.如果您已重悬了EDK的各组份，并准备在2周之后使用，请将重悬的组分分装并冻存于-20℃，最长可保存6个月。In contrast to the other three EDK components, the Avidin-HRP vial comes ready to use与EDK的其它三个组分不同，Avidin-HRP为即用型In order to avoid harmful repeated freeze/thaw cycles, or long-term storage at 4 ℃ which mean advisory functionality. Aliquot the Avidin-HRP into ten 6uL vials upon receipt and stored at -20 ℃ are stable for up 2 years from the date of receipt.目的是避免反复冻融或长期4℃保存对该组分的功能损害。收到Avidin-HRP后，立即将其分装为6ul/管，共10管，冻存于-20℃，最长可保存2年Phase 1:第1阶段：Coating the plate with capture antibody捕获抗体包板Centrifuge the vial briefly to bring the capture antibody to the bottom将捕获抗体稍作离心，使抗体集中于管底Reconstitute the capture body in sterile water to the concentration specified on the datasheet. And allowthe vial to be reconstituted for minimum 10 minutes.用无菌水将捕获抗体重悬至说明书上要求的浓度，静置10分钟以使抗体完全溶解Centrifuge the reconstituted vial for 3 minutes at maximum speed重悬的抗体以最高速度离心3分钟Dilute the capture antibody in 1×PBS to the concentration specified on the datasheet.用1xPBS稀释捕获抗体至说明书上要求的浓度Gently mix or vortex the vial, try to ensure the air bubbles don’t mix into the solution,轻轻颠倒或振荡混匀，一定不要产生气泡Immediately add 100ul of the capture antibody solution into the ELISA plate wells.立即在每个ELISA板孔中加入100uL捕获抗体Press firmly to seal the plate, take care not to let the reagent splash on the film将封板膜用力压盖在ELISA板上，注意不要使抗体滴溅在封板膜上Incubated the plate overnight at 25 ℃, alternatively the incubation for this phase can be done at 37℃ for 2-4 hours25 ℃孵育过夜，或者37℃孵育2-4个小时To wash the plate, discard the liquid and blot on a clean paper towel,洗板时需将液体倒掉，并在干净的吸水纸上拍干Add 300ul of the wash buffer to every well and then aspirate the plate每孔加入300ul 洗液，然后再将液体吸除Repeat the step three additional times, totaling four washes in all.该步骤再重复3次，总共洗涤4次After the last wash, invert the plate to move liquid, and blot on a clean paper towel.最后一次洗涤后，将板倒置以去除液体，并在干净的吸水纸上拍干There are several other methods to wash an ELISA plate.ELISA板还有其它几种洗涤方法。Whatever you choose, be consistent with your washing technique throughout the whole process.无论使用哪种方法，请在整个ELISA实验过程中保持一致Phase 2: Blocking non-specific binding第二阶段：封闭非特异性结合Bovine serum albumin is used as the blocking reagent to block any unbound open sites within the plastic wells牛血清白蛋白作为封闭试剂，可封闭塑料孔内任何未结合蛋白的位点Add 300ul of the blocking buffer to each well.每孔中加入300ul封闭液Seal the plate and incubate for at least 1 hour at 25 ℃.封板，并在25 ℃孵育至少1小时Phase 3:第3阶段：Specific binding of antigen抗原的特异性结合Priority to the end of the previous incubation period在上一孵育过程结束前Centrifuge the standard vial briefly将标准品稍作离心And reconstitute to the specified concentration in sterile water.并用无菌水重悬至要求的浓度Allow the vial to reconstitute for minimum 10 minutes.静置至少10分钟，以使标准品完全溶解When the incubation period is over, remove the plate and wash four times重悬结束后，取出板并洗涤4次Centrifuge the reconstituted standard vial for 3 minutes at maximum speed重悬的标准品以最高速度离心3分钟Dilute to the specified concentration, and gently mix or vortex the solution.稀释到所需浓度，轻轻颠倒或振荡混匀The standard curve is the area on the plate dedicate to fixed concentrations of our protein standard标准曲线在ELISA板上的区域专用于检测已知浓度的蛋白标准品If using a multi-channel pipette, firstly add 100ul of the diluent to all standard curve wells except for the first row如果使用多道移液器，除第一列外的所有标准曲线孔中均加入100ul稀释液Next, add 200ul of standard solution to the first row of standard curve wells.然后，在标准曲线孔的第一列中加入200ul标准品溶液Dilute 1:2 down the plate to a minimum of six concentrations依次倍比稀释，标准品至少有6个浓度Each standard curve well should contain the final volume of 100ul of the solvent.每个标准曲线孔中液体的终体积应为100ulUsing the minimum six concentrations in the triplicate, plus a minimum six wells of blanks containing only diluent.标准品至少有6个浓度，每个浓度为3个复孔，再设6个空白孔，其中仅含稀释液These blanks are essential and later determining the efficacy and ability of the measurements of the samples.空白孔是必须的，它们将用来评价试剂盒检测样本的效能Next, add your specific samples of interest in triplicate to the remaining wells然后，在剩余孔中加入待测样本，每个样本设3个复孔Depending on the concentration of the analyte, dilution may be necessary for optimal results样本中若待检抗原的浓度过高，要得到最佳结果可能需对样本进行稀释Seal the plate and incubate for two hours at 25 ℃.封板，25 ℃孵育2小时Phase 4:第4阶段：Sandwich formation via the addition of biotinylated detection antibody.加入生物素标记的检测抗体以形成夹心Priority to the end of the incubation, centrifuge briefly and reconstitute the detection antibody for ten minutesin sterile water.孵育结束前，稍离心检测抗体，并用无菌水重悬，静置10分钟When the incubation is over, remove the plate and wash four times.孵育结束后，取出板，洗涤4次Centrifuge for 3 minutes at maximum speed最高速度离心3分钟And dilute the detection antibody in diluent to the specified concentration.用稀释液将检测抗体稀释至要求的浓度Gently mix the solution and immediately add 100ul per well轻轻混匀，立即在每孔中加入100ul检测抗体液Seal the plate and incubate for 2 hours at 25 ℃封板，25 ℃孵育2小时Phase 5:第5阶段：Addition of the enzyme-linked Avidin-HRP to the sandwich夹心上加酶联亲合素(Avidin-HRP)While the plate is incubating, remove one 6ul aliquot of Avidin-HRP from the freezer and let it thaw.板在孵育过时，从冰箱中取出一管Avindin-HRP (6ul)，使其融化When the incubation is over, wash the plate 4 times板孵育结束后，洗涤4次Dilute the Avidin-HRP 1:2000 in diluent用稀释液1:2000稀释Avidin-HRPGently mix the solution and immediately add 100ul per well.轻轻混匀，立即向每孔中加入100ulSeal the plate and incubate for 30 minutes at 25 ℃.封板，25 ℃孵育30分钟To reduce the background interference, ensure the incubation is for 30 minutes only.为降低背景干扰，请确保孵育时间正好为30分钟Phase 6: Conversion of the colorless substrate into a colored solution.阶段6：底物显色Promptly washed the plate after incubation and add 100ul of ABTS substrate solution to each well.孵育结束后，立即洗板。每孔加入100ul ABTS底物溶液Use an Avidin-HRP in conjunction with ABTS only.Avidin-HRP仅可与ABTS配合使用Using it in conjunction with TMB or other substrate solutions, mainly to a dramatic rise in background development如与TMB或其它底物溶液配合使用，会显著提高背景显色Incubate at room temperature for color development室温孵育显色Monitor the plate at 5-minute intervals for up to 60 minutes.每隔5分钟读板一次，最长可监测60分钟Using a ELISA plate reader set at appropriate wavelength correction for your selected plate在酶标仪上设置合适的校正波长OD readings will vary depending on the specific EDK and its reading time provided on the product datasheet.EDK不同，以及说明书上所要求的读板时间不同均可能导致OD值的差异