Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A.
Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A.
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DOI：
10.4049/jimmunol.115.6.1617
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发表时间：
1975-12
影响因子：
4.4
通讯作者：
S. Kessler
中科院分区：
文献类型：
--
作者：
S. Kessler
金黄色葡萄球菌的 Cowan I 菌株已被用作与细胞裂解物中放射性标记抗原复合的抗体的吸附剂。该应用是一种优于其他免疫沉淀分离抗原方法的替代方法。它利用了某些葡萄球菌菌株细胞壁上蛋白 A 分子对 IgG 分子的高吸附能力，以及细菌有利的沉降特性。免疫复合物与吸附剂的相互作用最初是使用牛血清白蛋白和高过量的兔抗牛血清白蛋白抗体（IgG）的模型系统来定义的。在这些条件下，免疫复合物的吸收速度非常快，在几秒钟内发生，而游离 IgG 的最大结合则慢得多。此外，一旦结合，复合抗原就不能被大量的正常 IgG 或额外的游离抗体从吸附剂上取代。使用多种溶剂系统，例如与尿素和高温相结合的去污剂SDS以及具有强溶致盐化性质的中性盐，可以将抗原几乎完全从惰性吸附剂中洗脱出来，用于分析或制备目的。通过与传统双抗体沉淀法分离小鼠淋巴细胞 IgM 的直接比较，测试了蛋白 A-抗体吸附技术的功效。细菌吸附剂不仅在抗原分离速度方面具有明显的优势，而且通过 SDS 中的聚丙烯酰胺凝胶电泳进行的分析也显示出始终较高的抗原回收率、较低的背景放射性水平，并且不存在可能与传统免疫沉淀物非特异性结合并使分析复杂化的其他细胞成分。
The Cowan I strain of the bacterium Staphylococcus aureus has been used as an adsorbent for antibodies complexed with radiolabeled antigens from cell lysates. This application is advanced as a superior alternative to other methods of immune precipitation for the isolation of antigens. It exploits the high adsorption capacity for IgG molecules by protein A molecules on the cell walls of certain strains of staphylococci, along with the advantageous sedimentation properties of the bacteria. The interaction of immune complexes with the adsorbent was defined initially using a model system of bovine serum albumin with a high excess of rabbit anti-bovine serum albumin antibodies (IgG). The uptake of immune complexes under these conditions was extremely rapid, occurring within seconds, whereas maximum binding of free IgG was much slower. In addition, once bound the complexed antigen could not be displaced from the adsorbent either by large amounts of normal IgG or by extra free antibody. Antigen could be eluted almost completely from the inert adsorbent for analytic or preparative purposes with a variety of solvent systems, such as the detergent SDS in combination with urea and high temperature, and neutral salts with strong lyotropic salting in properties. The efficacy of the protein A-antibody adsorption technique was tested in direct comparisons with a conventional double antibody precipitation method for the isolation of mouse lymphocyte IgM. The bacterial adsorbent not only had a distinct advantage in speed of antigen isolation, but analyses by polyacrylamide gel electrophoresis in SDS also revealed consistently higher antigen recoveries, lower levels of background radioactivity, and an absence of other cell components which may nonspecifically bind to and complicate analyses using conventional immune precipitates.