Development of a hemolytic assay for mouse C2 and determination of its genetic control

A previously described one-step hemolytic assay for human C2 with C2-deficient human serum (C2D) was adapted to provide an accurate, reproducible, and sensitive assay for mouse C2. Hemolytic activity was maximized by using rabbit IgG anti-sheep cell antibody as amboceptor instead of hemolysin. Other factors examined for their effects on the assay for mouse C2 and found to be similar to the human assay included the dilution of C2D, cation concentration, ionic strength of the buffer, and time and temperature of incubation. Oxidation of mouse serum with iodine did not enhance hemolysis. Depletion of murine C4, Factor B, or C3 by prior treatment of plasma with specific antisera did not alter C2 functional activity. Mouse blood allowed to clot for several hours at 4°C, for 60 min at 37°C, or mouse plasma (5 mM ethylenediaminetetraacetic acid) maintained for several hours at 4°C or 37°C demonstrated minimal depletion of C2 activity. Mouse serum or plasma stored at -76°C for up to 12 months also showed only minimal loss of C2 activity. In all strains tested, males had greater C2 activity than females. Young (<6 weeks age) and old (> 20 weeks) mice had lower activity than 6- to 12-week-old mice. There was a several-fold range of hemolytic activity among standard strains on a B10 background, with animals of the k haplotype having relatively high levels, d and s haplotypes intermediate values, and u and f haplotypes low concentrations. Further analysis of these differences in functional activity employing H-2-congenics and critical recombinants demonstrated that C2 hemolytic activity is controlled at least in part by the S region of the H-2 complex.