Kilchherr E, Schumaker V N, Phillips M L, Curtiss L K
J Immunol. 1986 Jul 1;137(1):255-62.
Two monoclonal antibodies directed against C1q, and their (Fab)2 and Fab fragments, were used to study the mechanism of C1 activation. Monoclonal antibody 2A10, an IgG2a, was digested by pepsin to yield fully immunoreactive (Fab')2. Monoclonal antibody 1H11, an IgG1, was digested by papain to yield fully immunoreactive, bivalent (Fab)2. Previously 1H11 had been shown to bind to the C1q "heads," whereas 2A10 bound to stalks. Activation of C1 was followed by the cleavage of 125I-C1s in the presence of C1 inhibitor (C1-Inh) at 37 degrees C. Spontaneous activation was minimal at inhibitor concentrations above 0.4 micron (1.3 X physiologic inhibitor concentration); all results were corrected for the spontaneous activation background. Heat-aggregated IgG activated completely in this system and was taken as 100% activation. Monoclonal antibody 2A10 caused precipitation of C1 and slow activation; neither the (Fab')2 nor the Fab' derived from 2A10-caused activation. Probably, aggregates of intact 2A10 and C1 were serving as immune complexes to activate other molecules of C1. In contrast, both 1H11 and its (Fab)2 activated completely and stoichiometrically; that is, maximal activation was achieved at a ratio of one C1q head to one antibody combining site. The monovalent Fab derived from 1H11 bound well to C1q, but no activation of C1 was observed. Thus, bivalent binding of this head-binding monoclonal is required for C1 activation, but not the presence of the antibody Fc portion. Neither 1H11 nor its (Fab)2 fragments caused C1 precipitation; however, the 1H11 did form complexes composed of two C1q cross-linked by multiple 1H11, which were visualized by electron microscopy. The presence of these dimeric complexes correlated well with activation. A model for C1 activation is proposed in which two C1q subcomponents are held together by multiple (Fab)2 bridging C1q heads. The model is roughly analogous to touching opposing pairs of fingers and thumb tips, the two hands representing the two C1q, forming a cage. C1-Inh, which probably binds to C1r through the open end of the C1 cone, is too long asymmetric to be included within the cage. Thus, according to this model, the dimers of C1 are released from the inhibitory action of C1-Inh, and activation proceeds spontaneously and rapidly at 37 degrees C.
使用两种针对C1q的单克隆抗体及其（Fab）2和Fab片段来研究C1激活的机制。IgG2a型单克隆抗体2A10经胃蛋白酶消化产生完全具有免疫反应性的（Fab'）2。IgG1型单克隆抗体1H11经木瓜蛋白酶消化产生完全具有免疫反应性的二价（Fab）2。先前已证明1H11与C1q的“头部”结合，而2A10与柄部结合。在37℃下，在存在C1抑制剂（C1-Inh）的情况下，通过125I-C1s的裂解来跟踪C1的激活。当抑制剂浓度高于0.4微米（1.3倍生理抑制剂浓度）时，自发激活最小；所有结果均针对自发激活背景进行了校正。热聚集的IgG在该系统中完全激活，并被视为100%激活。单克隆抗体2A10导致C1沉淀和缓慢激活；源自2A10的（Fab'）2和Fab'均未引起激活。完整的2A10和C1的聚集体可能作为免疫复合物来激活其他C1分子。相比之下，1H11及其（Fab）2均完全且按化学计量激活；也就是说，在一个C1q头部与一个抗体结合位点的比例下实现最大激活。源自1H11的单价Fab与C1q结合良好，但未观察到C1的激活。因此，这种与头部结合的单克隆抗体的二价结合是C1激活所必需的，但不是抗体Fc部分的存在。1H11及其（Fab）2片段均未引起C1沉淀；然而，1H11确实形成了由多个1H11交联的两个C1q组成的复合物，通过电子显微镜可以看到。这些二聚体复合物的存在与激活密切相关。提出了一种C1激活模型，其中两个C1q亚组分通过多个（Fab）2桥接C1q头部而结合在一起。该模型大致类似于触摸相对的手指和拇指尖对，两只手代表两个C1q，形成一个笼子。C1-Inh可能通过C1锥体的开口端与C1r结合，其太长且不对称，无法包含在笼子内。因此，根据该模型，C1的二聚体从C1-Inh的抑制作用中释放出来，并且在37℃下自发且快速地进行激活。
