Protein A binding assay for the identification of HLA antigens on peripheral blood lymphocytes by monoclonal antibodies: application to HLA B27.
Splenocytes from mice immunized with purified, papain-solubilized HLA B27 antigen and/or human lymphocytes bearing the B27 specificity were fused with myeloma cell lines NSI or Sp2. The screening strategy employed a protein A binding assay in which various target cells were used. First, the hybrid cell supernatants were screened against B lymphocyte cell lines of known HLA specificities and the Daudi cell line, which does not express HLA-A, B, or C antigens. Second, a panel of PBLs were used as target cells. It was necessary to refine the protein A binding assay by preabsorbing the radiolabeled protein A with PBLs and by precoating the test wells with ovalbumin. Clones selected by these criteria were further tested by indirect immunoprecipitation and by inhibition of binding or microcytotoxicty to target call lines with purified HLA antigens or beta 2m. Forty-four clones were selected which showed varying degrees of specificity for allo- and nonallo-specific determinants and one clone was selected which was specific for beta 2m. Clone 27M1 which was previously shown to be specific or HLA-B27 as judged by conventional microcytotoxicity testing (Grumet et al., Lancet No. 8239, II:174, 1981) was compared with other clones using the above parameters for evaluation. Antibody from clone 27M1 showed preferential binding to B27 positive cell lines and PBLs, lesser binding to B7 positive target cells, and no binding to B40 positive target cells. Purified B27 antigen (papain) from two sources including the B27 target cell line, was able to inhibit the binding of antibodies from 27M1 to target cells. The extension of the protein A binding assay to PBLs has made it possible to more accurately quantitate the binding or inhibition of binding of antibodies to panels of PBLs.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D011232:Chemical Precipitation; D000939:Epitopes; D006680:HLA Antigens / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D015796:HLA-B27 Antigen; D006801:Humans; D006825:Hybridomas; D008214:Lymphocytes / Q000276:immunology*; D051379:Mice; D010206:Papain / Q000494:pharmacology; D013205:Staphylococcal Protein A / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006680:HLA Antigens; D015796:HLA-B27 Antigen; D013205:Staphylococcal Protein A; D001613:beta 2-Microglobulin; D010206:Papain