Isolation and purification of the Rh(D) blood group receptor component from human erythrocyte membrane

The Rh(D) antigen of human red blood cell membranes has been isolated as a homogeneous immunologically intact component. The method of preparation was as follows: Red cell membranes were prepared free of hemoglobin and components were solubilized using ethylenediaminetetraacetic acid (EDTA) followed by sodium chloride. Diaflo® ultrafilter membranes were used to separate solubilized membrane components into different molecular weight classes. The Rh(D) activity was demonstrated in the molecular weight class between 10 000 and 20 000. This fraction, which had six components by disc gel electrophoresis, was further purified by isoelectric focusing on a pH gradient of 3 to 10, followed by a pH gradient of 5 to 8. The Rh(D) antigen was eluted as a single component, migrated as a discrete band on disc gel electrophoresis, and inhibited the agglutination of anti-Rh(D) antibody. When the Rh(D) antigen was injected into guinea pigs, a high titer of anti-Rh(D) antibody was obtained.