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浅谈ELISA的那些事儿

发布: 2016-01-16 872阅读 222评论

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）

1

  1. 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；
  2. 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；
  3. 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；
  4. 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）

2

  1. 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；
  2. 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；
  3. 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；
  4. 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；
  5. 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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