FUNCTIONAL ASSAY OF THE ALTERNATIVE COMPLEMENT PATHWAY OF RAT SERUM

Two functional assays of the alternative pathway of complement activation in rat serum were developed. In the first assay, conditions were established for titration of alternative pathway activity by use of the 50% hemolytic end-point of rabbit red blood cells (RaRBC) in serum treated with ethyleneglycol-bis-(beta-aminoethyl ether)-N, N1-tetraacetic acid (EGTA). Under the conditions of the assay the 50% hemolytic end-point could be determined from a single value in the region of partial lysis of RaRBC by use of the Von Krogh equation. The second assay of alternative pathway activity was based on the opsonization of heat-killed radiolabeled pneumococci of serotype 25 (Pn 25). Opsonization of Pn 25 was shown to proceed entirely via the alternative pathway in rat serum. Both the Pn 25 opsonization test and the RaRBC lysis test were more sensitive than measurement of hemolytic C3-C9 for detecting activation of the alternative pathway. There was excellent correlation between the results obtained with the RaRBC lysis test and those obtained with the opsonization test. Because of its technical simplicity, the RaRBC lysis test appeared to be the single most useful test of alternative pathway activity in rat serum. © 1979.