Yan B S, Lee K M, Liu S H, Syu W J
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
Hybridoma. 1996 Dec;15(6):429-33. doi: 10.1089/hyb.1996.15.429.
Six monoclonal antibodies (MAbs) were raised in mice against the 26-kDa glutathione S-transferase (GST) of the parasite Schistosoma japonicum. These MAbs were originally selected for their specific binding to the recombinant GST (r-GST) generated in E. coli by an enzyme-linked immunosorbent assay. A further study demonstrated that all these MAbs bound to plate-coated GST affinity-purified from the parasite Schistosoma japonicum. However, in Western blotting analysis only a single monoclonal antibody (MAb Y3D7) yielded positive binding. The binding of MAb Y3D7 on Western blotting was further characterized; specific binding was found on other GST fusion proteins and on the authentic 26-kDa GST but not the 28-kDa GST in the total soluble worm proteins from Schistosoma japonicum. Using protein-A-mediated immunoprecipitation, MAbs Y3D7 and Y5D5 precipitated r-GST while in parallel experiments the remaining MAbs did not generate r-GST precipitation. In an alternative co-precipitation experiment, r-GST was first bound to glutathione (GSH) Sepharose beads and subsequently tested for interaction with the MAbs. In this manner, all MAbs except MAb Y5D5 were co-precipitated with the complexes. Thus, these select MAbs readily reacted with GST although their binding characteristics were different. Because GST has been widely used in the generation of fusion proteins for various purposes and is a potential vaccine candidate in controlling schistosomiasis, these MAbs should prove valuable for their application to molecular biology and parasitology.
针对日本血吸虫的26 kDa谷胱甘肽S -转移酶（GST），在小鼠体内制备了六种单克隆抗体（MAb）。最初通过酶联免疫吸附测定法，根据这些MAb与大肠杆菌中产生的重组GST（r - GST）的特异性结合来进行筛选。进一步研究表明，所有这些MAb都能与从日本血吸虫中亲和纯化并包被在平板上的GST结合。然而，在蛋白质印迹分析中，只有一种单克隆抗体（MAb Y3D7）产生阳性结合。对MAb Y3D7在蛋白质印迹中的结合进行了进一步表征；发现它能与其他GST融合蛋白以及真实的26 kDa GST特异性结合，但不能与日本血吸虫总可溶性虫体蛋白中的28 kDa GST结合。使用蛋白A介导的免疫沉淀法，MAb Y3D7和Y5D5能沉淀r - GST，而在平行实验中，其余MAb不能产生r - GST沉淀。在另一种共沉淀实验中，r - GST首先与谷胱甘肽（GSH）琼脂糖珠结合，随后测试其与MAb的相互作用。通过这种方式，除MAb Y5D5外的所有MAb都能与复合物共沉淀。因此，尽管这些选择的MAb结合特性不同，但它们都能与GST轻松反应。由于GST已被广泛用于多种目的的融合蛋白制备，并且是控制血吸虫病的潜在疫苗候选物，这些MAb在分子生物学和寄生虫学中的应用应该具有重要价值。
