An enzyme-linked immunoabsorbent assay for the quantitation of the terminal complement complex from cell membranes or in activated human sera.
A sensitive and simple enzyme-linked immunoabsorbent assay (ELISA) has been developed to measure the terminal complement complex (TCC) in solution. Commercially available antibodies to the native complement (C) components C5 and C9 were used in a double antibody sandwich technique sensitive enough to detect 0.3 microgram/ml of purified TCC. The TCC was not detected in normal human serum (NHS) nor was it generated when sera from patients with a genetic deficiency of functional C5, C7, C8 beta or C9 were activated with cobra venom factor (CVF). If the C8 beta deficient serum was reconstituted with the C8 beta chain and incubated with CVF, TCC were formed and detected by the assay. In in vitro experiments, the TCC was detected in NHS activated by either the classical or alternative pathway even when there was no measurable consumption of C5, C8 or C9. In addition, adaptation of a detergent extraction procedure permitted the quantitation by the assay, of TCC which were generated on sensitized sheep erythrocyte membranes. Experiments to test sample handling conditions showed no generation of TCC in NHS after four freeze/thaw cycles and spontaneous formation only if NHS had been incubated at 37 degrees C for 48 h. The TCC in zymosan-activated NHS were stable at 37 degrees C for 1 week. Patients with C activation associated diseases such as SLE and rheumatoid arthritis had increased levels of TCC that correlated with positive clinical tests for inflammation, even though C levels were normal when measured by routine techniques. These results suggest that this ELISA will provide a valuable tool for studying the role of C in the pathogenesis of C-mediated diseases and in examining the mechanism of tissue injury in in vitro experimental systems.
D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000276:immunology*; D003167:Complement Activation; D003182:Complement C5 / Q000276:immunology; D003186:Complement C9 / Q000276:immunology; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D012756:Sheep
D000906:Antibodies; D003182:Complement C5; D003186:Complement C9; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins