Purified HLA antigens to probe human alloantibody specificity

HLA class I antigens were purified in sufficient quantities to probe the antigen specificity of lymphocyte and platelet antibodies. Purification of HLA was achieved by affinity chromatography using the monoclonal W6/32 antibody that recognizes a nonpolymorphic determinant of HLA-A,B,C molecules. Pooled platelets from a large number of donors were used as source of antigen. A highly purified HLA preparation was obtained that produced a dose-dependent inhibition of the binding of the W6/32 antibody to lymphocytes as measured by flow cytometry. The binding of monoclonal antibodies to T-lymphocyte antigens or to HLA class II antigens was not affected. The purified HLA also inhibited the binding of lymphocyte alloantibodies from renal transplant patients, providing a direct and definitive way to probe HLA specificity. HLA also inhibited the binding of the same antibodies to platelets but it did not interfere with the binding of alloantibodies to the platelet-specific PLA1 antigen. This preparation, therefore, can conclusively probe the HLA specificity of both alloantibodies for clinical investigation purposes and monoclonal antibodies for screening purposes, and has the potential of becoming a reagent for routine use in clinical and research laboratories.