Partial Purification of ABO Blood Group Antigens: Sodium Deoxycholate Fractionation of Human Erythrocyte Membranes

Abstract

A method has been developed for the fractionation of human erythrocyte membranes, using sodium deoxycholate, EDTA, differential ultracentrifugation, and DEAE-cellulose column chromatography. Serologic studies demonstrate the presence of ABO blood group antigens in a 50,000 g supernatant. This fraction is further purified on DEAE-cellulose, and in type AB ghost preparations results in the separation of A and B antigens. Phytohemagglutinin binding activity is also in the same supernatant and after DEAE chromatography is located in the fraction that exhibited A activity. Rh and MN activity is associated with a larger membrane fragment which is pelleted at 30,000 g. The S, s, and I antigens are present in a soluble form, and remain in the supernatant following centrifugation at 100,000 g.

Dissociation of MNSs Antigens Using Sodium Deoxycholate Fraction of Erythrocyte Membranes

“…Sodium deoxycholate (E. Merck) fractionation of ghosts followed by differential ultracentrifugation resulted in a pellet at 30,000 gx 1 h (P-30) and a supernatant (S-30); ultracentrifugation of the S-30 at 100,000 g x 2 h resulted in a pellet, (P-100) and a supernatant (S-100) [15]. The pellets (P-30 and P-100) were resuspended using a Dounce homogenizer, and all fractions were dialyzed exhaustively against isotonic phosphate buffer pH 7.4.…”

“…In addition to the MN serologic activity of these glycoproteins, most preparations, irrespective of the fractionation system, have S and s blood group activity [9,12,13] and influenza hemagglutination inhibition activity, thought to indicate the presence of virus receptor substance. Recently, we have de monstrated the separation of the MN antigens and the Ss blood group antigens into two distinct fractions [15].…”

“…We have recently developed a procedure for fractionation of blood group systems using partial solubilization with sodium deoxycholate and differ ential ultracentrifugation [15], The ABO blood group antigens remained soluble after centrifugation at 30,000 g and were pelleted at 100,000 g, whereas the antigens of the Rh system were pelleted in a 30,000-g membrane fraction. In contrast, the antigens of the MNSs blood group system were detected in either the 30,000 g pellet or remained in a soluble form following 100.000 g centrifugation.…”