Isolation of serologically active HLA-A, B, C and DR antigens as monitored by a fixed cell radioimmunoassay

Procedures for isolation of antigenically active preparations of human HLA antigens were monitored using a sensitive and quantitative radioimmunoassay for HLA-A, B, C or HLA-DR antigens. These assays involve binding of radiolabeled monoclonal antibodies to appropriate target cells and inhibition of this binding by solubilized antigen preparations. Fixation of the target cells with glutaraldehyde allows quantitation of inhibitors in high concentrations of nonionic detergents. Using this assay, it was possible to examine quantitatively the relative merits of several alternative procedures for isolating both class I and class II antigens. For example, chromatography of cell lysates on columns of Ricinus communis agglutinin gave high recoveries of DR antigens purified from the majority of cell proteins. When Lens culinaris hemagglutinin was used for separation of cell lysates approximately 90% of the A, B, C antigens but only 32% of the DR antigens bound the column and were eluted by alpha-methyl-mannoside. Immunoadsorbent column were then used to recover antigenically active molecules suitable for structural or functional studies from these enriched fractions.