A New Assay of Serum Complement Activity, C42-Tmax

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A new assay of serum complement activity was developed for complement analysis of serum with a low total complement activity level (CHSO). The principle of this method is to detect amounts of C3 convertase in the classical complement pathway (C42) generated by the test serum. Hemolysis of sensitized sheep erythrocytes (EA) was measured after incubation with diluted test serum for various incubation periods, followed by another 60 min incubation with guinea-pig serum diluted in EDTA buffer. Formation and decay of C42 on EA was estimated from the C42-Tmax curve, obtained by plotting the hemolytic ratio on the ordinate against the incubation period on the abscissa. For pooled normal human serum (NHS) diluted 1: 600 in gelatin veronal buffer, the C42-Tmax. curve showed a Tmax (time of maximal hemolysis) at 4-5 min and Ymax (hemolytic ratio at Tmax) of 60-90% lysis. The decrease in hemolytic ratio after Tmax is probably due to the decay of active C2 from EA. C42-Tmax obtained with various sera showed that Ymax was decreased in low CH50 serum after classical complement pathway activation, depending on the degree of complement activation. Furthermore, low CH50 serum after alternative complement pathway activation as well as C7-or C9-deficient serum showed C42-Tmax curves similar to that by NHS. Thus, C42-Tmax is a simple and rapid assay for serum complement activity as a combination activity of C1, C4, and C2.