Thorsen L I, Gaudernack G, Brosstad F, Pedersen T M, Solum N O
Thromb Haemost. 1987 Apr 7;57(2):212-6.
A method is described for the identification of antigens by monoclonal antibodies. This is applicable whenever precipitating antibodies to the same antigens from a different species are available. The method is based upon: Separation and immunoprecipitation of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody. Coprecipitation of the monoclonal antibody with its antigen. Subsequent passive transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose membrane. Visualization of the blotted antibody using an enzyme-linked secondary antibody and a chromogenic substrate. Identification of the corresponding antigen by comparisons to the immunoprecipitate pattern of the original immunoplate. To test this method we have analyzed the detection of the antigens recognized by six previously described monoclonal antibodies against platelet membrane proteins and von Willebrand factor. Specific immunoblots were obtained in each case using small amounts of monoclonal antibodies. Thus, the technique provides an alternative when epitopes are denatured by SDS, and avoids the use of radioactively labelled monoclonal antibodies.
描述了一种用单克隆抗体鉴定抗原的方法。只要能从不同物种中获得针对相同抗原的沉淀抗体，该方法就适用。该方法基于：在非变性去污剂Triton X - 100和单克隆抗体存在的情况下，用多特异性抗血清在交叉免疫电泳中分离和免疫沉淀细胞蛋白。单克隆抗体与其抗原的共沉淀。随后将抗体 - 抗原复合物中的单克隆抗体被动转移到硝酸纤维素膜上。使用酶联二抗和显色底物对印迹抗体进行可视化。通过与原始免疫板的免疫沉淀模式进行比较来鉴定相应抗原。为了测试该方法，我们分析了六种先前描述的针对血小板膜蛋白和血管性血友病因子的单克隆抗体所识别的抗原的检测情况。在每种情况下，使用少量单克隆抗体都获得了特异性免疫印迹。因此，当表位被SDS变性时，该技术提供了一种替代方法，并且避免了使用放射性标记的单克隆抗体。
