酶联免疫吸附试验(Enzyme-linked immunosorbent assay,简称ELISA)是一种基于抗原抗体之间专一键结特性的分子实验。结合在固体承载物(通常为塑胶孔盘)上的抗原或抗体仍然保持蛋白质的完整天然结构,利用键结机制,再配合酶催化的呈色反应,不但可以检测抗原或抗体的存在,而且可以利用颜色深浅来进行定量。根据键结机制的不同,ELISA可设计出各种不同类型的检测方式,小编在这里着重给大家介绍两种常用的方法:间接法(indirect)和三明治法(sandwich)。
间接法(Indirect ELISA)
- 把蛋白质样品固着于塑胶孔盘上 (这里我们是希望有想要的抗原哟),完成后洗去多余样品;
- 加入一次抗体,如果抗原存在并且天然结构正确的话,就会和一次抗体进行专一性键结;
- 洗去多余的一次抗体,加入酶连接的二次抗体,和一次抗体键结;
- 洗去多余没有键结的二次抗体,加入催化反应的底物进行显色,孔板检测仪(plate reader)会测量塑胶盘中的吸光值(Optical Density, 简称 OD),对比标准曲线,我们就可以测量样品中抗原的含量啦。
间接法跟直接法比较的的好处就是敏感度加大,因为单一的一次抗体有很多二次抗体结合。而且实验成本比三明治法较低,因为只有一种一次抗体。
三明治法(Sandwich ELISA)
- 把具有专一性的抗体(也叫吸附抗体)固着于塑胶孔盘上,完成后洗掉多余的抗体;
- 加入待测蛋白样品,样品中如果有我们期待的抗原的话,就会和塑胶孔盘上的吸附抗体进行专一性键结;
- 洗去多余的待测样品,加入另一种对抗原专一的一次抗体(检测抗体),和待测抗原进行键结;
- 洗去多余没有键结的一次抗体,加入酶连接的二次抗体,和一次抗体键结;
- 洗去多余没有键结的二次抗体,加入催化反应的底物进行显色,孔板检测仪(plate reader)会测量塑胶盘中的吸光值(Optical Density, 简称 OD),对比标准曲线,我们就可以测量样品中抗原的含量啦。三明治法用两种抗体对样品中的抗原进行专一性辨认,所以专一性是很高的。但这对抗原的要求也就更高,必须是多价抗原,这样才能获得两种以上的专一性抗体,来分别进行“夹心”。
很多做实验的小伙伴可能分不清楚ELISA和蛋白质印迹法(Western Blot, 简称WB)的区别。其实主要区别在于ELISA检测的是天然蛋白质,需要保持完整的三级结构。这让它在探究蛋白质相互作用时起了至关重用的作用。而通常WB都是检测失活的蛋白,使用的抗体可以辨别失活抗原一级结构中的抗原决定基(epitope)。与此同时,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,简称SDS-PAGE)是WB的第一个步骤,它可以根据蛋白质的分子量将大小不同的蛋白分开。这一步ELISA是不包含的,虽然省时,但敏感度会下降。另外,ELISA通常是在96/384孔板上进行操作,相对于WB显示单一结果的特点,可以大大提高实验的生产量。正是因为很大的生产量,它在自动化实验和检测有着很大的潜力。
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