Immunochemical Studies of the Rh System

Abstract

Summary A method of antibody isolation based upon absorption with intact erythrocytes, lysis of washed sensitized cells with digitonin, and acid elution of antibodies from hemoglobin-free stroma, has been described. Antibody activity of eluates was correlated with the firmness of the erythrocytic antigen-antibody bond during absorption and with the amount of γ-globulin found in the eluate. The γ-globulin in eluates was characterized by column chromatography, double gel diffusion, immunoelectrophoresis and analytical ultracentrifugation, and was measured by specific precipitin method. Ficin treatment of red cells was found to increase the capacity of Rh antigenic sites to bind some Rh antibodies. On gradient DEAE chromatographic fractionation of sera, poorly binding Rh antibodies in one serum were recovered at a slightly higher buffer concentration than the more common firmly binding antibodies.