The alternative pathway hemolytic assay

Washed rabbit erythrocytes at 1×10⁸ c/ml (Biotrading) were incubated with 5% pooled normal human serum in HBS plus 10 mM MgCl₂ and 10 mM EGTA for 60 min at 37°C while shaken at 600 rpm. Different concentrations SCIN were preincubated with purified humAb 6D4 (10 µg/ml), with the control human anti-DNP IgG1 (10 µg/ml, Genmab, Utrecht), or with HBS-buffer plus 10 mM MgCl₂ and 10 mM EGTA for 10 min at room temperature before mixing with serum. Erythrocytes were pelleted and the absorbance of supernatants at 450 nm was measured. Data are expressed relative to the mean value measured for erythrocytes incubated with serum only, which was set to 1.²¹