Thompson N E, Hager D A, Burgess R R
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706.
Biochemistry. 1992 Aug 4;31(30):7003-8. doi: 10.1021/bi00145a019.
A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.
一种改良的酶联免疫吸附测定法（ELISA）被用于筛选与大肠杆菌RNA聚合酶反应的单克隆抗体（MAb），以检测其在低分子量多羟基化化合物（多元醇）和非离液盐存在的情况下释放RNA聚合酶的能力。这种测定法，称为ELISA洗脱测定法，在总共筛选的218种抗原特异性单克隆抗体中鉴定出19种推定的“多元醇反应性”单克隆抗体。其中一种单克隆抗体，命名为NT73，被详细研究了其对多元醇和盐的各种组合释放抗原的能力。使用与琼脂糖偶联的NT73，通过单一免疫亲和色谱步骤可以快速制备高活性RNA聚合酶，取代了我们传统纯化程序中的两个冗长的色谱步骤。由于NT73与RNA聚合酶的β'亚基反应，从免疫亲和柱中回收了核心聚合酶和全酶的混合物。全酶（E σ70）可以通过随后在Mono Q柱上的色谱法与核心聚合酶分离。这表明通过ELISA洗脱测定法可以轻松鉴定和表征多元醇反应性单克隆抗体。使用多元醇反应性单克隆抗体提供了一种使免疫亲和色谱法适用于纯化不稳定蛋白质的方法。
