Rapid purification of anti-I and anti-i cold antibodies by affinity chromatography

A method is described for the rapid purification of serologically active high titer anti-I and anti-i cold antibodies from the sera of patients with chronic cold agglutinin disease (CCAD). The purification procedure is based on thermal affinity chromatography, using desialated orosomucoid (alpha 1-acid glycoprotein)-Sepharose 4B conjugated beads. The nature of the interaction between the cold agglutinins (CA) and the desialated orosomucoid is unknown. Inhibition studies, however, revealed that the cold hemagglutinating activities of all the anti-i sera were inhibited by desialated orosomucoid while only 1 out of 4 of the anti-I sera was similarly affected. Anti-I or anti-i antibodies were separated from whole sera in 7 out of 7 samples with a recovery in most cases of 100% of the cold hemagglutinating activity. The resultant products were purified monoclonal IgM fractions which could react with anti-kappa and anti-mu but not with anti-lambda sera. The homogeneity, purity and specificity of all preparations were confirmed by immunodiffusion analysis against purified I and i blood group antigens isolated from human erythrocyte membranes, zonal and right-angle electrophoresis and hemagglutination or hemagglutination inhibition studies.