Newman S L, Mikus L K
J Exp Med. 1985 Jun 1;161(6):1414-31. doi: 10.1084/jem.161.6.1414.
Monoclonal antibodies were used to determine the number and molecular form of C3 bound to particulate activators of the complement (C) system by human serum. Sheep erythrocytes (E) coated with IgM (EIgM) and IgG (EIgG) were used to study activation of the classical pathway (CP). Yeast (Y), rabbit erythrocytes (ER), and five species of bacteria (Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae type 3, Streptococcus pyogenes, and Hemophilus influenzae type b) were used to study activation of the alternative pathway (AP). The deposition of C3b onto EIgM and EIgG incubated in C7-deficient human serum was dependent on the serum concentration. At all serum concentrations tested, there was complete conversion of C3b to iC3b. Kinetic analysis of C3b deposition and conversion to iC3b indicated that these events occurred almost simultaneously; the reaction was completed by 15 min. The deposition of C3 onto the AP activators ER and Y was also dependent on serum concentration, and ER, but not Y, required the presence of Mg-EGTA and thus the activation of only the AP. C3b deposition and conversion to iC3b on Y was complete in 15 min, with 82% of bound C3 converted to iC3b. For ER, maximum C3 deposition required 30 min in both the presence and absence of Mg-EGTA. However, after 1 h of incubation, 74% of bound C2 was iC3b in the absence of Mg-EGTA, compared with only 52% in the presence of Mg-EGTA. Thus, even on AP activators, a large portion of C3b may be converted to iC3b, and this conversion is probably controlled by elements on the particle's surface. Studies with the five species of bacteria yielded similar results. Approximately 3-5 X 10(4) molecules of C3 were bound per microorganism, with opsonization being completed in 30 min. Remarkably, only 16-28% of bound C3 was in the form of iC3b, even after 2 h of incubation. The presence or absence of Mg-EGTA, or the addition of purified CR1 to the reaction mixture, did not significantly effect the ratio of C3b to iC3b. Finally, SDS-PAGE and autoradiography of particle-bound 125I-C3 fragments confirmed that there was no conversion of iC3b to C3d,g or C3d. The data obtained about the opsonization of bacteria suggest that the predominant form of C3 that is encountered by inflammatory phagocytes may be C3b.
单克隆抗体用于确定人血清中与补体（C）系统的颗粒激活剂结合的C3的数量和分子形式。用包被有IgM（EIgM）和IgG（EIgG）的绵羊红细胞（E）来研究经典途径（CP）的激活。用酵母（Y）、兔红细胞（ER）以及五种细菌（大肠杆菌、金黄色葡萄球菌、3型肺炎链球菌、化脓性链球菌和b型流感嗜血杆菌）来研究替代途径（AP）的激活。在C7缺陷的人血清中孵育时，C3b在EIgM和EIgG上的沉积取决于血清浓度。在所有测试的血清浓度下，C3b完全转化为iC3b。对C3b沉积和转化为iC3b的动力学分析表明，这些事件几乎同时发生；反应在15分钟内完成。C3在AP激活剂ER和Y上的沉积也取决于血清浓度，并且ER（而非Y）需要Mg-EGTA的存在，因此仅激活AP。C3b在Y上的沉积和转化为iC3b在15分钟内完成，82%的结合C3转化为iC3b。对于ER，在有和没有Mg-EGTA的情况下，最大C3沉积都需要30分钟。然而，孵育1小时后，在没有Mg-EGTA的情况下，74%的结合C3是iC3b，而在有Mg-EGTA的情况下仅为52%。因此，即使在AP激活剂上，很大一部分C3b也可能转化为iC3b，并且这种转化可能受颗粒表面成分的控制。对这五种细菌的研究得出了类似的结果。每个微生物结合约3 - 5×10⁴个C3分子，调理作用在30分钟内完成。值得注意的是，即使孵育2小时后，仅16 - 28%的结合C3是iC3b的形式。Mg-EGTA的存在与否，或向反应混合物中添加纯化的CR1，均未显著影响C3b与iC3b的比例。最后，对颗粒结合的¹²⁵I-C3片段进行SDS-PAGE和放射自显影证实，iC3b没有转化为C3d,g或C3d。关于细菌调理作用获得的数据表明，炎性吞噬细胞遇到的C3的主要形式可能是C3b。
