Affinity purification of antibodies using antigens immobilized on solid supports.
Affinity purification of antibodies using antigens immobilized on solid supports.
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使用固定在固体支持物上的抗原对抗体进行亲和纯化。
DOI：
10.1042/bst0160134
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发表时间：
1988
影响因子：
3.9
通讯作者：
Smith,DE
中科院分区：
文献类型：
--
作者：
Fisher,PA;Smith,DE
Olmsted（1981,1）首次报道了一种亲和纯化抗体的方法，该方法将抗体与从SDS/聚丙烯酰胺凝胶转移后固定在重氮纸印迹上的相应抗原结合。我们采用了她的方法，利用硝化纤维素作为固体载体，并用它来代替重氮化纸，并利用与小牛碱性磷酸酶偶联的二抗的比色检测，探索了直接可视化反应性一抗体的潜力。这些研究的结果之前已经发表过（Smith & Fisher, 1984）。我们还研究了溴化氰（CNBr）活化的Sepharose作为固体载体取代硝化纤维素进行大规模免疫亲和纯化的适用性。用SDS/聚丙烯酰胺凝胶电泳（PAGE）固定在硝化纤维素上的多肽对抗体进行亲和纯化的基本方法已经详细描述，这里只简要回顾一下。SDS/PAGE基本上根据Laemmli（1970）进行，并进行了少量修改（Fisher et al., 1982）。电泳后，蛋白质通过印迹法从凝胶被动转移到硝化纤维素上。我们发现，被动转移程序，虽然相对耗时，结果始终较高质量的印迹分析和制备目的。电泳转移可以在没有严重不良后果的情况下使用。当使用梯度聚丙烯酰胺凝胶时，被动转移在多肽大小范围内是均匀的（Fisher等，1982）；在连续浓缩凝胶中，传递效率随着质量的减小而提高。在最坏的情况下，大于200 kDa的蛋白质需要48-72小时才能转移约50%。然而，并非所有蛋白质的行为都相同。一些大的蛋白质似乎更有效地转移，可能与它们在特定的转移缓冲液中的溶解度有关。除了我们认为所得免疫印迹的技术质量优越外，出于以下原因，被动转移也是亲和纯化的首选方法。我们用于印迹转移的缓冲液包括75 mM-Tris碱基和570 mm -甘氨酸，不含SDS和甲醇。我们假设在一开始，凝胶中的蛋白质与SDS完全络合。因此，递送到硝化纤维素的第一配体分子处于变性构象中。随着转移的进行，SDS被从凝胶中冲洗出来，理论上蛋白质有机会再生，至少部分再生。在过程后期转移的配体可能在硝化纤维素上具有更多的天然构象，从而允许在固体载体上广泛代表抗原决定因子。抗体针对许多不同的多肽抗原已亲和纯化在我们的实验室使用
Olmsted (198 1) first reported a method for affinity purification of antibodies by binding them to their respective antigens that had been immobilized on diazotized paper blots following transfer from SDS/polyacrylamide gels. We have adapted her method to take advantage of nitrocellulose as a solid support and used it to replace diazotized paper, and have explored the potential for direct visualization of reactive primary antibodies using colorimetric detection of a secondary antibody coupled to calf alkaline phosphatase. The results of these studies have previously been published (Smith & Fisher, 1984). We have also investigated the suitability of cyanogen bromide (CNBr)-activated Sepharose as a solid support to replace nitrocellulose for large-scale immunoaffinity purification. The basic methodology for affinity purification of antibodies using polypeptides immobilized on nitrocellulose following SDS/polyacrylamide gel electrophoresis (PAGE) has been described in detail and will only be reviewed briefly. SDS/PAGE is performed essentially according to Laemmli (1970) with minor modifications (Fisher et al., 1982). After electrophoresis, proteins are blot-transferred from the gel to nitrocellulose passively. We find that passive transfer procedures, while relatively time consuming, result in consistently higher quality blots for analytical as well as preparative purposes. Electrophoretic transfer may nevertheless be used without serious adverse consequences. When gradient polyacrylamide gels are used, passive transfer is uniform throughout the range of polypeptide size (Fisher et al., 1982); on continuous concentration gels, transfer efficiency improves with decreasing mass. At worst, proteins larger than 200 kDa require 48-72 h for about 50% transfer. However, not all proteins behave identically. Some large proteins seem to transfer more efficiently, perhaps relating to their solubility in the particular transfer buffer used. In addition to what we feel is the superior technical quality of the resulting immunoblots, passive transfer is also preferred for affinity purification purposes for the following reason. The buffer that we use for blot-transfer includes 75 mM-Tris base and 570 mM-glycine and contains neither SDS nor methanol. We presume that at the outset, the proteins in the gel are fully complexed with SDS. The first ligand molecules delivered to the nitrocellulose are therefore in a denatured conformation. As transfer proceeds, the SDS is washed out of the gel and proteins theoretically have the opportunity to renature, at least partially. Ligands transferred late in the process may assume more native conformations on the nitrocellulose, thus allowing a broad representation of antigenic determinants on the solid support. Antibodies against a number of different polypeptide antigens have been affinity purified in our laboratory using