A New Assay of Serum Complement Activity, C42-T_max

Abstract

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-T_max 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-T_max. curve showed a T_max (time of maximal hemolysis) at 4-5 min and Y_max (hemolytic ratio at T_max) of 60-90% lysis. The decrease in hemolytic ratio after T_max is probably due to the decay of active C2 from EA. C42-T_max obtained with various sera showed that Y_max 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-T_max curves similar to that by NHS. Thus, C42-T_max is a simple and rapid assay for serum complement activity as a combination activity of C1, C4, and C2.