Hugo F, Krämer S, Bhakdi S
J Immunol Methods. 1987 May 20;99(2):243-51. doi: 10.1016/0022-1759(87)90134-7.
Activation of the complement system to completion results either in the generation of a pore-forming, cytolytic C5b-9(m) complex, or of a cytolytically inactive, fluid-phase SC5b-9 complex. In this paper, we describe a sensitive and reliable, sandwich ELISA for C5b-9(m) and SC5b-9, which is based on the use of a monoclonal antibody to a neoantigen of C5b-9 in combination with affinity-purified, polyclonal rabbit antibodies. The ELISA has been calibrated with purified C5b-9(m) and SC5b-9, and can detect 3 ng/ml C5b-9(m) and 20 ng/ml SC5b-9. We show that maximal conversion of C5-C9 in pooled human serum by insulin or zymosan activation generates 220 +/- 40 micrograms/ml SC5b-9. 65 of 100 normal human EDTA plasma samples analyzed in this study contained 100-600 ng/ml SC5b-9, corresponding to 0.04-0.24% of maximal conversion. Levels of circulating SC5b-9 in other donors were below the limit of detection. Incubation of serum at 37 degrees C always led to spontaneous generation of SC5b-9; concentrations ranged from 490-4725 ng/ml after 60 min, 37 degrees C, with a mean of 1848 +/- 1031 (SD) ng/ml amongst 25 donors studied. The terminal complement complex present in EDTA plasma was partially purified by PEG precipitation, DEAE-ion exchange chromatography and sucrose density gradient centrifugation, and was found to contain C8, C9 and S-protein as demonstrable by SDS-PAGE immunoblotting. Thus, the material most probably represented genuine SC5b-9. No significant age- or sex-dependent variations in SC5b-9 levels were noted. The present data call for a critical re-appraisal of several previously published methods for the determination of SC5b-9 levels in human plasma and serum.
补体系统激活至完成会产生一种形成孔道的溶细胞性C5b - 9(m)复合物，或者一种无溶细胞活性的液相SC5b - 9复合物。在本文中，我们描述了一种针对C5b - 9(m)和SC5b - 9的灵敏且可靠的夹心酶联免疫吸附测定法（ELISA），该方法基于使用针对C5b - 9新抗原的单克隆抗体与亲和纯化的多克隆兔抗体相结合。该ELISA已用纯化的C5b - 9(m)和SC5b - 9进行校准，能够检测到3 ng/ml的C5b - 9(m)和20 ng/ml的SC5b - 9。我们发现，胰岛素或酵母聚糖激活混合人血清中的C5 - C9，其最大转化率可产生220±40微克/毫升的SC5b - 9。在本研究中分析的100份正常人乙二胺四乙酸（EDTA）血浆样本中，有65份含有100 - 600 ng/ml的SC5b - 9，相当于最大转化率的0.04 - 0.24%。其他供体的循环SC5b - 9水平低于检测限。血清在37℃孵育总会导致SC5b - 9的自发产生；在37℃孵育60分钟后，浓度范围为490 - 4725 ng/ml，在25名研究供体中，平均浓度为1848±1031（标准差）ng/ml。通过聚乙二醇（PEG）沉淀、二乙氨基乙基（DEAE）离子交换色谱和蔗糖密度梯度离心对EDTA血浆中存在的末端补体复合物进行了部分纯化，并且通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）免疫印迹法发现其含有C8、C9和S蛋白。因此，该物质很可能代表真正的SC5b - 9。未观察到SC5b - 9水平存在显著的年龄或性别依赖性差异。目前的数据要求对先前发表的几种用于测定人血浆和血清中SC5b - 9水平的方法进行批判性重新评估。
