IgG was eluted from RBCs of patients with WAIHA and healthy blood donors and purified by affinity chromatography on protein G. The in vitro binding of purified anti-RBC IgG to homologous RBCs when coincubated with different reagents was assessed by means of an indirect Coombs assay. Purified anti-RBC IgG from eluates of patients and of healthy subjects was tested at a concentration of 1 ng/10⁷ RBCs. It was coincubated with normal plasma at a mol/mol ratio of purified anti-RBC IgG to plasma IgM of 1:350. Purified plasma IgG and purified plasma IgM were used at a concentration of 1 g/L. In those experiments in which IgG and IgM were mixed, a ratio of 1:1 (wt/wt) was used. Serum albumin was used at a concentration of 50 g/L.