Thompson Nancy E, Foley Katherine M, Stalder Elizabeth S, Burgess Richard R
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Methods Enzymol. 2009;463:475-94. doi: 10.1016/S0076-6879(09)63028-7.
Immunoaffinity chromatography is a powerful tool for purification of proteins and protein complexes. The availability of monoclonal antibodies (mAbs) has revolutionized the field of immunoaffinity chromatography by providing a continuous supply of highly uniform antibody. Before the availability of mAbs, the recovery of the target protein from immobilized polyclonal antibodies usually required very harsh, often denaturing conditions. Although harsh conditions are often still used to disrupt the antigen-antibody interaction when using a mAb, various methods have been developed to exploit the uniformity of the antigen-antibody reaction in order to identify agents or conditions that gently disrupt this interaction and thus result in higher recovery of active protein from immunoaffinity chromatography. We discuss here the use of a specific type of monoclonal antibody that we have designated "polyol-responsive monoclonal antibodies" (PR-mAbs). These are naturally occurring mAbs that have high affinity for the antigen under binding conditions, but have low affinity in the presence of a combination of low molecular weight hydroxylated compounds (polyols) and nonchaotropic salts. Therefore, these PR-mAbs can be used for gentle immunoaffinity chromatography. PR-mAbs can be easily identified and adapted to a powerful protein purification method for a target protein.
免疫亲和色谱法是用于纯化蛋白质和蛋白质复合物的强大工具。单克隆抗体（mAb）的出现彻底改变了免疫亲和色谱领域，因为它能持续提供高度均一的抗体。在单克隆抗体出现之前，从固定化多克隆抗体中回收目标蛋白通常需要非常苛刻的条件，往往是变性条件。尽管在使用单克隆抗体时通常仍会使用苛刻条件来破坏抗原 - 抗体相互作用，但已开发出各种方法来利用抗原 - 抗体反应的均一性，以识别能温和破坏这种相互作用从而提高免疫亲和色谱法中活性蛋白回收率的试剂或条件。我们在此讨论一种特定类型的单克隆抗体，我们将其命名为“多元醇反应性单克隆抗体”（PR - mAb）。这些是天然存在的单克隆抗体，在结合条件下对抗原具有高亲和力，但在存在低分子量羟基化化合物（多元醇）和非离液盐的组合时亲和力较低。因此，这些PR - mAb可用于温和的免疫亲和色谱法。PR - mAb可以很容易地被识别并适用于一种针对目标蛋白的强大蛋白质纯化方法。
