Gaffar S A, Chen F M, Liu C, Epstein A L
University of Southern California-School of Medicine, Department of Pathology, Los Angeles 90033.
J Immunoassay. 1991;12(1):1-14. doi: 10.1080/01971529108055053.
Direct and indirect radioimmunoassay (RIA) procedures to determine the amount of binding of a mouse monoclonal antibody (MoAb) reactive with an intracellular antigen present in human cells are described. In these RIAs, mouse IgG2a MoAb, designated as Tumor Necrosis Treatment (TNT-1) antibody, paraformaldehyde/acetone fixed cells, and Sephadex beads were used to standardize the assay conditions. In the direct RIA, 83% of the 125I-labeled TNT-1 MoAb was bound to the target cells within 30 min after the addition of reagents. The amount of binding of the MoAb was directly proportional to the amount of antigen present in the assay. When the direct RIA was carried out using different types of target cells, 125I-labeled TNT-1 MoAb showed greater than 70% binding. In the indirect RIA, the amount of binding of secondary 125I-labeled goat anti-mouse IgG antibody to the target cells was linear. These results suggest that the indirect RIA can be used to estimate the immunoreactivity of the unlabeled TNT-1 MoAb present in crude protein preparations. Based on the results of RIAs the following two conclusions are drawn: 1) the direct RIA can be used to estimate rapidly the amount of immunoreactive TNT-1 MoAb present in 125I-labeled antibody preparations and 2) the indirect RIA which estimates the amount of immunoreactivity of unlabeled TNT-1 MoAb can be used to monitor the purification and study the characteristics of the MoAb present in crude protein preparations. These methods enable the quantitative measurement of MoAbs reactive against intracellular antigens using standard RIA procedures.
本文描述了直接和间接放射免疫测定（RIA）程序，用于测定与人类细胞中存在的细胞内抗原发生反应的小鼠单克隆抗体（MoAb）的结合量。在这些RIA中，使用了指定为肿瘤坏死治疗（TNT-1）抗体的小鼠IgG2a MoAb、多聚甲醛/丙酮固定细胞和葡聚糖凝胶珠来标准化测定条件。在直接RIA中，添加试剂后30分钟内，125I标记的TNT-1 MoAb中有83%与靶细胞结合。MoAb的结合量与测定中存在的抗原量成正比。当使用不同类型的靶细胞进行直接RIA时，125I标记的TNT-1 MoAb显示出大于70%的结合率。在间接RIA中，二级125I标记的山羊抗小鼠IgG抗体与靶细胞的结合量呈线性关系。这些结果表明，间接RIA可用于估计粗蛋白制剂中存在的未标记TNT-1 MoAb的免疫反应性。基于RIA的结果得出以下两个结论：1）直接RIA可用于快速估计125I标记抗体制剂中存在的免疫反应性TNT-1 MoAb的量；2）估计未标记TNT-1 MoAb免疫反应性的间接RIA可用于监测粗蛋白制剂中MoAb的纯化并研究其特性。这些方法能够使用标准RIA程序对针对细胞内抗原的MoAb进行定量测量。
