Isaacs I J, Steiner J P, Roche P A, Pizzo S V, Strickland D K
Biochemistry Laboratory, American Red Cross Biomedical Research and Development, Rockville, Maryland 20855.
J Biol Chem. 1988 May 15;263(14):6709-14.
Studies were performed to determine if monoclonal antibody 7H11D6 binds to the region of alpha 2-macroglobulin (alpha 2M) that interacts with cell-surface receptors. F(ab')2 fragments prepared from this antibody delayed the in vivo clearance of alpha 2M-trypsin complexes from the murine circulation and blocked the in vitro binding of 125I-labeled alpha 2M-trypsin to rat kidney fibroblasts. Chemical modification studies revealed that the epitope region for 7H11D6 is sensitive to modification of the inhibitor with cis-dichlorodiammineplatinum (II). To determine if the epitope for 7H11D6 is comprised of residues involved in the alpha 2M receptor determinant, anti-idiotypic antibodies against 7H11D6 were prepared. A competitive enzyme-linked immunosorbent assay demonstrated that the anti-idiotypic IgG competed with alpha 2M-trypsin complexes for binding to 7H11D6. The anti-idiotypic IgG inhibited the binding of 125I-labeled alpha 2M-trypsin complexes to normal rat kidney fibroblasts, and a Kd of 194 pM for the binding of the anti-idiotype to these cells was derived from a fit of the data to a model involving a single class of sites. Binding of the anti-idiotypic IgG to the alpha 2M receptor provides unequivocal evidence that 7H11D6 is binding to residues within the receptor recognition site on alpha 2M and not merely to residues sufficiently close to that region to cause steric hindrance.
开展了多项研究以确定单克隆抗体7H11D6是否与α2-巨球蛋白（α2M）与细胞表面受体相互作用的区域结合。从该抗体制备的F(ab')2片段延缓了α2M-胰蛋白酶复合物在小鼠循环中的体内清除，并阻断了125I标记的α2M-胰蛋白酶与大鼠肾成纤维细胞的体外结合。化学修饰研究表明，7H11D6的表位区域对顺二氯二氨铂（II）对抑制剂的修饰敏感。为了确定7H11D6的表位是否由参与α2M受体决定簇的残基组成，制备了针对7H11D6的抗独特型抗体。竞争性酶联免疫吸附试验表明，抗独特型IgG与α2M-胰蛋白酶复合物竞争结合7H11D6。抗独特型IgG抑制了125I标记的α2M-胰蛋白酶复合物与正常大鼠肾成纤维细胞的结合，并且通过将数据拟合到涉及单一类位点的模型中得出抗独特型与这些细胞结合的解离常数为194 pM。抗独特型IgG与α2M受体的结合提供了明确的证据，表明7H11D6与α2M上受体识别位点内的残基结合，而不仅仅是与足够靠近该区域以引起空间位阻的残基结合。
