Biesecker G, Wagner J L, Hugli T E
Department of Pathology, Hahnemann University, Philadelphia, PA 19102.
J Immunol. 1989 Aug 15;143(4):1228-32.
The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.
通过比较补体激活血清中C5a的水平与补体C6缺失血清中产生的C5a水平，研究了末端补体成分对补体C5a片段生成和释放的影响。为了研究C5a的释放，开发了一种改良的C5a检测方法，该方法在进行C5a检测之前，利用抗C5b单克隆抗体从样品中去除C5、C5b和C5b - C5a复合物。开发这种改良检测方法是因为标准方法（包括旨在解离C5a和C5b的酸沉淀步骤）无法区分游离C5a和与C5b结合的C5a。因此，标准方法无法监测末端成分对C5a/C5b解离的影响。使用改良的C5a检测方法和标准方法，分别检测了补体激活的全人血清、C6缺失血清以及含有抑制水平抗C6 Fab的血清中的C5a水平。血清用酵母聚糖激活替代补体途径或用抗体包被的绵羊红细胞激活经典途径进行补体激活。激活后C6缺失血清中游离C5a的水平与激活的全血清中C5a的水平相当，表明从C5b释放C5a不需要C6。此外，使用标准酸沉淀方法在酵母聚糖激活血清中检测到的C5a量大于使用改良免疫吸附技术检测时的C5a水平，证实酸处理增强了C5a解离并促进了C5a回收。由于其他末端成分C7、C8和C9仅在C6结合后才与C5b结合，这些结果表明末端成分都不是C5a释放所必需的。虽然末端成分可能影响C5a释放的速率，但血清中释放的C5a量完全独立于末端成分。
