Hessing M, Kanters D, Heijnen H F, Hackeng T M, Sixma J J, Bouma B N
Department of Haematology, University Hospital Utrecht, The Netherlands.
Eur J Immunol. 1991 Sep;21(9):2077-85. doi: 10.1002/eji.1830210916.
Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP. By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1:1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked beta chain of C4BP were identified that inhibited the binding of protein S to C4BP. In a binding assay using 125I-labeled C4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated.
人C4b结合蛋白（C4BP）是一种多聚体调节性补体成分，可与维生素K依赖性蛋白S和补体C4b相互作用。利用杂交瘤技术，制备了一组针对完整人C4BP及其160 kDa胰凝乳蛋白酶消化的中央核心片段的单克隆抗体（mAb），以研究C4BP的结构-功能关系。通过蛋白质印迹分析和竞争实验，鉴定出四组不同的mAb，并将其定位在C4BP分子上。通过旋转投影，获得了C4BP-抗体复合物的蜘蛛状图像，免疫电子显微镜提供了一些关于抗体与C4BP相互作用化学计量的信息。某些抗体仅以1:1的比例与C4BP分子相互作用。其他抗体则形成了两个或更多抗体与C4BP分子上同源位点结合的复合物。使用酶联免疫吸附夹心测定法测量蛋白S与C4BP之间的复合物形成，鉴定出针对C4BP中央核心和二硫键连接的β链的mAb，它们可抑制蛋白S与C4BP的结合。在使用125I标记的C4BP和固相C4b的结合测定中，证明了一组抗C4BP mAb对C4BP与C4b结合的抑制作用。
