A study of optimal reaction conditions for an assay of the human alternative complement pathway

The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50% lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH₅₀ titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37° C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH₅₀ titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37°C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH₅₀ ± SD for 45 normal adults was 25.3 ± 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH₅₀ by 50% at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH₅₀ titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH₅₀ depletion at low doses of zymosan was at least twice the per cent depletion of CH₅₀ or antigenic P, B, and C3. A striking dichotomy between nearly complete APH₅₀ depletion and normal or near normal CH₅₀ and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH₅₀ than of conventional complement determinations for detecting complement consumption by alternative pathway activators.