Bergh K, Iversen O J
Department of Microbiology, University of Trondheim, Norway.
Scand J Immunol. 1989 Mar;29(3):333-41. doi: 10.1111/j.1365-3083.1989.tb01132.x.
Eight murine monoclonal antibodies (MoAb) raised against the major zymosan-induced chemotactic factor in rabbit serum were found to neutralize the chemotactic activity induced by lipopolysaccharides (LPS) and antigen-antibody complexes. A 15 kDa antigen was identified in plasma incubated with LPS by immunoblot analysis with MoAb. This is similar to the molecular weight of the major zymosan-induced chemotactic factor. Both the generation of this 15 kDa antigen and chemotactic activity were abrogated in a heat-inactivated plasma. A cross-reaction to human C5a was demonstrated for three MoAb (5H8B9, 4B1C11, and 2A5E3) in an indirect enzyme-linked immunosorbent assay (ELISA) of partially purified C5a and by the isolation of zymosan-induced chemotactic activity by affinity chromatography. MoAb 5H8B9 and 4B1C11 were able to neutralize the chemotactic activity in human zymosan-activated serum. MoAb 2A5E3 was able to bind 125I-labelled human C5a des Arg. We conclude that these MoAb are directed against rabbit C5a. MoAb 5B2C5 and 2B1A2, which are directed to different antigenic binding sites on C5a, may be applied in a sandwich ELISA for the detection and quantification of C5a des Arg in rabbit serum or plasma. The sandwich ELISA can be performed directly on serum or plasma samples without having to precipitate native C5. Complement activation is demonstrated by measuring the increased generation of C5a des Arg in rabbit plasma or serum activated with LPS, zymosan, antigen-antibody complexes, or cobra venom factor.
针对兔血清中主要的酵母聚糖诱导趋化因子产生的8种鼠单克隆抗体（MoAb）被发现可中和脂多糖（LPS）和抗原 - 抗体复合物诱导的趋化活性。通过用MoAb进行免疫印迹分析，在与LPS孵育的血浆中鉴定出一种15 kDa的抗原。这与主要的酵母聚糖诱导趋化因子的分子量相似。在热灭活血浆中，这种15 kDa抗原的产生和趋化活性均被消除。在部分纯化的C5a的间接酶联免疫吸附测定（ELISA）中以及通过亲和层析分离酵母聚糖诱导的趋化活性，证实了三种MoAb（5H8B9、4B1C11和2A5E3）与人C5a有交叉反应。MoAb 5H8B9和4B1C11能够中和人酵母聚糖激活血清中的趋化活性。MoAb 2A5E3能够结合125I标记的人C5a去精氨酸。我们得出结论，这些MoAb针对的是兔C5a。针对C5a上不同抗原结合位点的MoAb 5B2C5和2B1A2可用于夹心ELISA，以检测和定量兔血清或血浆中的C5a去精氨酸。夹心ELISA可直接在血清或血浆样本上进行，无需沉淀天然C5。通过测量用LPS、酵母聚糖、抗原 - 抗体复合物或眼镜蛇毒因子激活的兔血浆或血清中C5a去精氨酸生成的增加来证明补体激活。
