Description
During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus or the available sulfur (e.g. Fab’ fraction or thiolated antibodies). Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge.
Copper-free click chemistry is a fairly new chemistry that has been commercialized during the past few years. More and more click chemistry-based reagents are becoming available commercially which makes the formulation development much easier for scientists. The great advantage of this chemistry is biocompatibility since no cytotoxic copper catalyst is required. By far, click chemistry is the most efficient and easiest conjugation chemistry available for coupling of antibodies and other reactive ligands to the surface of the liposomes. The conjugation chemistry is based on the reaction of the dibenzocyclooctyne (DBCO) reagent with an azide linker to form a stable triazole. DBCO moiety can be on the antibody and azide moiety can be on liposomes and vice versa. This conjugation protocol is based on the reaction of the dibenzocyclooctyne (DBCO) group of the liposomes with an azide linker on the antibody, peptide or proteins.
There are many commercialized reagents that can be used for azide modification of proteins, peptides and antibodies. To see the list of commercialized reagents for azide modification see here.
For other reactive (PEGylated and non-PEGyalated products) Immunosome® products suitable for other types conjugation methods see here.
Formulation Information
Immunosome®-DBCO (PEGylated)
Lipid Composition | Concentration (mg/ml) | Concentration (mM) | Molar Ratio Percentage |
---|---|---|---|
Total | 15.95 mg/ml | 21.58 mM | 100 |
Hydrogenated Soy PC | 9.58 | 12.22 | 57 |
Cholesterol | 3.19 | 8.25 | 38 |
DSPE-PEG(2000) | 2.5 | 0.89 | 4 |
DSPE-PEG(2000)-DBCO | 0.68 | 0.22 | 1 |
Buffer and Liposome Size | Specification |
---|---|
Buffer | Phosphate Buffered Saline |
pH | 7.4 |
Liposome Size | 100 nm |
Conjugation Protocol
Materials and Equipment
You need the following materials and equipment in order to use the kit.
- Laboratory vortex mixer is recommended to have.
- Laboratory magnetic stirrer is needed for dialysis.
- Float-A-Lyzer® with a proper MWCO that easily allows the cleanup of your liposome conjugated ligand from free and non-conjugated protein/peptide/ligand. You need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton, the pore size on the dialysis membrane gets close to 100 nm and therefore your liposomes can be dialyzed out. You cannot use dialysis cassettes blindly. Please understand the technique before using either spin column or dialysis cassette. If you do not use the correct MWCO, you can lose your entire prep. For this protocol, we recommend MWCO of 300,000 dalton.
Preparation Method
- The total lipid concentration in Immunosome®-DBCO is 21.58 mM. 1% mol of the lipid in liposomes contains DBCO group and only half of them are exposed to the outside of the liposomes, which is equal to 0.11 mM of reactive conjugable lipid. For 2 ml volume liposomes, this is equal to 2.20×10-7 mol, and for 5 ml volume liposomes, this is equal to 5.50×10-7 mol of DBCO. Add 2.5 mol equivalents of DBCO-lipids in liposomes to 1 mol equivalent of Azide containing protein. Incubate the mixture of liposome and antibody at room temperature for 4 h followed by overnight incubation at 4 °C in a refrigerator.
- Remove the non-conjugated protein, peptide or antibody from the immunoliposomes by dialysis. We prefer dialysis to size exclusion columns. Dialysis is a much slower process but there will be minimum loss of immunoliposomes after the prep is cleaned from non-conjugated protein/peptide/ligand. Spin columns are much faster; however, you can easily lose over 50% of the liposomes on the spin column. We recommend using Float-A-Lyzer® dialysis cassette from Spectrum Labs. You will need to choose a cassette with proper MWCO depending on the MW of your protein, peptide, antibody or antibody fragment. NOTE: If you decide to use a dialysis cassette, you will need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton, the pore size on the dialysis membrane gets close to 100 nm and therefore, your liposomes can be dialyzed out. You cannot use dialysis cassettes and spin columns blindly. They come in various sizes and you need to choose the correct size wisely. Dialyze the immunoliposome solution in 1 liter of PBS at pH 7.4 for 8 hours. Change the dialysis buffer with a fresh 1 liter of PBS and let is dialyze for another 8 hours. After this step, your cleaned up immunoliposome is ready to be used.
Liposome Particle Calculator
Immunosomes are unilamellar liposomes and sized to 100 nm. The molar concentration of liposome is 21.58 mM. By having liposome diameter (nm) and lipid concentration (µM), you can calculate the total number of the lipids in one liposome and the number of the liposomes in one milliliter of the liposome solution. To use the calculator click here.
Technical Notes
- Before starting the conjugation process please make sure to avoid buffers that contain azides, which can react with DBCO.
- DBCO group is known to be hydrophobic and it buries itself in the lipid bilayer of the liposomes. Direct conjugation of a ligand to the liposomes containing DBCO has produced immunoliposomes with yield of above 60% which is quite acceptable and much higher than many other conjugation chemistries. Post-insertion of DBCO lipid conjugated ligands into the liposomes increases the yield to above 80%. For more information see reference 11.
- Reactions of DBCO and azides are more efficient at high concentrations and temperatures (i.e., up to 37 °C). In order to avoid denaturation of proteins, peptides and antibodies it is recommended to incubate molecules with liposomes at room temperature followed by refrigeration (see step 1).
- Typical reaction times are less than 12 h, however, incubating for longer can improve efficiency.
- Spin columns can be used for the immunoliposome separation, and they are very fast method for purification. However, a large quantity of the samples is lost on the column. Dialysis is a slower process with minimal sample loss and therefore, we recommend dialysis over spin columns.
- If you are using a ligand or peptide that is hydrophobic then it is recommended to solubilize it in DMSO or DMF and then add the buffer to it. It is recommended not to use more than 5% volume of DMSO or DMF in the solution. DMF and DMSO are both compatible with liposomes and they are also miscible in water. Other organic solvent such as ethanol and chloroform are not compatible with liposomes and will cause the liposomes to lyse. If you end up using DMSO or DMF then after the conjugation reaction is done, you need to remove DMSO and DMF from the liposomes. In order to do that you need to use a dialysis cassette that is made from REGENERATED CELLULOSE MEMBRANE. NOTE: Not all membranes are compatible with DMF and DMSO. We recommend using a Slide-A-Lyzer™ MINI Dialysis Device with MWCO of 2K made from regenerated cellulose membrane manufactured by ThermoFisher. After DMSO or DMF is removed, you can use Float-A-Lyzer® dialysis device for the final step of cleaning up the prep.
- Liposomes should be kept at 4°C and NEVER be frozen.
Database
Direct link to the database page for easy navigation: Immunoliposomes Conjugation Database
Appearance
Immunosome®-DBCO is a white translucent liquid made of nano size unilamellar liposomes. Usually due to the small size of liposomes no settling will occur in the bottom of the vial. The liposomes are packaged in an amber vial.
Ordering/Shipping Information
- All liposome based formulations are shipped on blue ice at 4°C in insulated packages using overnight shipping or international express shipping.
- Liposomes should NEVER be frozen. Ice crystals that form in the lipid membrane can rupture the membrane, change the size of the liposomes and cause the encapsulated drug to leak out. Liposomes in liquid form should always be kept in the refrigerator.
- Clients who order from outside of the United States of America are responsible for their government import taxes and customs paperwork. Encapsula NanoSciences is NOT responsible for importation fees to countries outside of the United States of America.
- We strongly encourage the clients in Japan, Korea, Taiwan and China to order via a distributor. Tough customs clearance regulations in these countries will cause delay in custom clearance of these perishable formulations if ordered directly through us. Distributors can easily clear the packages from customs. To see the list of the distributors click here.
- Clients ordering from universities and research institutes in Australia should keep in mind that the liposome formulations are made from synthetic material and the formulations do not require a “permit to import quarantine material”. Liposomes are NOT biological products.
- If you would like your institute’s FedEx or DHL account to be charged for shipping, then please provide the account number at the time of ordering.
- Encapsula NanoSciences has no control over delays due to inclement weather or customs clearance delays. You will receive a FedEx or DHL tracking number once your order is confirmed. Contact FedEx or DHL in advance and make sure that the paperwork for customs is done on time. All subsequent shipping inquiries should be directed to Federal Express or DHL.
Storage and Shelf Life
Storage
Immunosome® products should always be stored at in the dark at 4°C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. If the suspension is frozen, the encapsulated drug can be released from the liposomes thus limiting its effectiveness. In addition, the size of the liposomes will also change upon freezing and thawing.
Shelf Life
Immunosome®-DBCO is made on daily basis. The batch that is shipped is manufactured on the same day. It is advised to use the products within 4 months of the manufacturing date.
References and background reading
1. Simon M, Zangemeister-Wittke U, Plückthun A. Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries. Bioconjugate chemistry. 2012 Jan 20;23(2):279-86.
2. Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, Lo A, Codelli JA, Bertozzi CR. Copper-free click chemistry for dynamic in vivo imaging. Proceedings of the National Academy of Sciences. 2007 Oct 23;104(43):16793-7.
3. Marqués-Gallego P, de Kroon AI. Ligation strategies for targeting liposomal nanocarriers. BioMed research international. 2014 Jul 14;2014.
4. Debets MF, van Berkel SS, Schoffelen S, Rutjes FP, van Hest JC, van Delft FL. Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. Chemical communications. 2010;46(1):97-9.
5. Agard NJ, Baskin JM, Prescher JA, Lo A, Bertozzi CR. A comparative study of bioorthogonal reactions with azides. ACS chemical biology. 2006 Oct 20;1(10):644-8.
6. Ma Y, Zhang H, Gruzdys V, Sun XL. Azide-reactive liposome for chemoselective and biocompatible liposomal surface functionalization and glyco-liposomal microarray fabrication. Langmuir. 2011 Oct 7;27(21):13097-103.
7. Xu J, Filion TM, Prifti F, Song J. Cytocompatible Poly (ethylene glycol)‐co‐polycarbonate Hydrogels Cross‐Linked by Copper‐Free, Strain‐Promoted Click Chemistry. Chemistry–An Asian Journal. 2011 Oct 4;6(10):2730-7.
8. Sletten EM, Bertozzi CR. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angewandte Chemie International Edition. 2009 Sep 7;48(38):6974-98.
9. Campbell‐Verduyn LS, Mirfeizi L, Schoonen AK, Dierckx RA, Elsinga PH, Feringa BL. Strain‐Promoted Copper‐Free “Click” Chemistry for 18F Radiolabeling of Bombesin. Angewandte Chemie International Edition. 2011 Nov 18;50(47):11117-20.
10. Jewett JC, Sletten EM, Bertozzi CR. Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones. Journal of the American Chemical Society. 2010 Feb 26;132(11):3688-90.
11. Wang L, Jiang R, Liu Y, Cheng M, Wu Q, Sun XL. Recombinant and chemo-/bio-orthogonal synthesis of liposomal thrombomodulin and its antithrombotic activity. Journal of bioscience and bioengineering. 2017 Oct 1;124(4):445-51.
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首先要什么有什么的,你得好好考虑一下。
2、看生产地址
根本没有生产地址,我们知道做实验做产品需要很多的仪器、试剂、耗材,没有人相信一间简单的屋子可以生产各种样的试剂盒。
3、看产品包装
没有任何的生产地址、联系方式等信息,这种产品有问题了连个投诉的地方都没有。
4、看公司网站
有些打着国外原装旗号,整个公司网站为英文页面,实际注册IP地址在中国。如果写着国外的地址,让你国外的朋友实地去看一下!
5、做交叉验证
拿对方提供的几个种类的试剂盒,把里面的关键组份相互替换做做实验,如果交叉严重,只能说明是一种原料生产的试剂盒贴了不同的标签。
6、看价格
价格低得离谱,却打着进口大公司原料分装,核算成本,这种低得离谱的价格是连原料都买不起的。
现在国内最差也是用3代试剂,有些地方会用4代试剂。
4代试剂(检查抗原+抗体)——窗口期为4周。因为抗原于3-4周达到复制的峰值,此时通过4代试剂检查,如果感染了HIV,抗原/抗体至少有一个为阳性,如果都是阴就排除了。
3代试剂(只查抗体)——窗口期为6周。
以上为理论分析+临床经验的结果,可以说是99.9%的准确度。
但是目前FDA、CDC和试剂生产商统一达成的共识,也就是针对普通人,最保守的窗口期是3个月。无论什么试剂,3个月都100%排除。
ELISA的基础是抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性,酶标记的抗原或抗体既保留其免疫学活性,又保留酶的活性。在测定时,受检标本(测定其中的抗体或抗原)与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开。再加入酶标记的抗原或抗体,也通过反应而结合在固相载体上。此时固相上的酶量与标本中受检物质的量呈一定的比例。加入酶反应的底物后,底物被酶催化成为有色产物,产物的量与标本中受检物质的量直接相关,故可根据呈色的深浅进行定性或定量分析。由于酶的催化效率很高,间接地放大了免疫反应的结果,使测定方法达到很高的敏感度。
1.取出试剂盒室温平衡30min,取出血样放至室温。
2.配标准品:取150uL标准品加入150uL标准品稀释液稀释,依次稀释5次。
3.加样:分别于各反应孔中加入标准品50uL,样品40UL,标准品做复孔,样品做3孔。
4.分别于样品孔中加入10UL抗体。
5.标准品和样品孔中分别加入50uL链酶亲和素-HRP,盖上封板膜,轻轻震荡混匀,37℃温育60min。
6.配洗涤液:将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。
7.洗涤:小心揭开封板膜,弃去液体,甩干,每孔加200uL洗涤液,静置30s后弃去,如此重复5次,拍干。
8.显色:每孔先加入显色剂A 50uL,再加显色剂B 50uL,轻轻震荡混匀,37℃避光显色6min。
9. 终止:每孔加入终止液50uL,终止反应(此时蓝色立即转为黄色)。
10.测定:以空白孔调零,450nm波长依序测量各孔的吸光度。测定应在加终止液10min之内进行。
11.保存结果,收拾桌面。
12.分析处理数据
注意事项
1. 取出板条前恢复到室温后再打开外包装袋,实验中不用的板条立即放回包装中,密闭封口,其余不用试剂应盖好。
2. 实验操作中请使用一次性的吸头,避免交叉污染。
3.实验板孔加入试剂的顺序应一致,以保证所有反应孔的孵育时间一致。
4.洗涤过程中反应孔中残留的洗涤液应在滤纸上充分拍干,勿将滤纸直接放入反应孔中吸水。
5.试剂盒内试剂请在保质期内使用,不同批号试剂不要混用。
6.1000pg/ml以上的结果为非线性的,根据此标准曲线无法得到精确的结果。大于1000pg/ml 的样品应以标准稀释缓冲液稀释后重做。在结果分析时,结合考虑相应的稀释度。
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