Measurement of modulation of mouse complement levels in vivo, utilizing a microtiter hemolytic assay

The complement system, with its protein components, plays a fundamental role in host defense as the major immunological mechanism of innate immunity. Effects of immunotoxic agents on serum complement have not been studied due to the lack of an assay capable of measuring the in vivo effects of compounds in the mouse, the species of choice for immunotoxicological testing. A microtiter hemolytic assay was developed and utilized to measure modulation of serum complement in vivo in B6C3F1 mice. In order to validate this functional assay. Cobra Venom Factor (CVF) was used to produce decomplementation. CVF administered intravenously produced a dose-dependent suppression of complement activity when evaluated 24 hours after a single injection. Administration of 25 or more anticomplementary units (ACU/kg) completely abolished functional hemolytic activity. The duration of the effect was dose dependent with 100 ACU/kg suppressing the response for 48 hours. A decomplemented state could be maintained for up to 6 days, with a second injection. A third injection failed to prolong the decomplementation. Elevated complement levels were obtained following a single injection with Pyran Copolymer. Pyran, a macrophage activator, produce a dose dependent increase in serum complement levels 8 days after administration. The microtiter hemolytic complement assay represents a useful tool capable of evaluating the in vivo effects of agents on serum complement in the mouse.