The purification of murine histocompatibility antigens (H-2b) from RBL-5 tumor cells using detergents.

A detergent-solubilized form of H-2b (dH-2b) has been purified 1500-fold from RBL-5 tumor cells. The purification was accomplished by deoxycholate solubilization of purified plasma membranes, gel filtration, Lens culinaris lectin affinity chromatography, and affinity chromatography on a sheep anti-dH-2b immunoadsorbent. Both alloantigen and beta 2-microglobulin were monitored by radioimmunoassay during purification. The final product was judged to be greater than 90% pure by the following criteria: 1) sodium dodecyl sulfate-polyacrylamide electrophoresis which showed the expected 2-component structure of histocompatibility antigens, i.e. a heavy chain and beta 2-microglobulin; 2) amino acid composition which was comparable to the known compositions of other H-2 and HLA molecules; 3) NH2-terminal sequencing which gave a unique sequence for the heavy chain, and the reported sequence for beta 2-microglobulin; and 4) immunoprecipitation of the bulk of the preparation by appropriate alloantisera.