Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules
ABSTRACT Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets.
Immunological Reviews 04/2006; 47(1):3 - 61. DOI:10.1111/j.1600-065X.1979.tb00288.x · 12.91 Impact Factor
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ABSTRACT: The experiments reported here show that W6/32 antigenic activity is retained in papain-solubilized HLA antigens and that the W6/32 antibody provides a useful standard reagent to detect and assay HLA-A,B,C antigens. The W6/32 antibody was purified and used to construct an immunoaffinity column. Soluble HLA-A,B,C antigens from papain or detergent-treated membranes could be highly purified in a single step with this column and serologic analysis showed that HLA-A,B and C antigens were bound to the column. Thus, the W6/32 antigenic determinant is present on gene products of all three loci. The amount of HLA-A,B,C antigens on the surface of human B cell lines and peripheral blood lymphocytes was measured with W6/32 antibody. B cell lines expressed, on average, 9 times as much cell surface HLA-A,B,C antigens as peripheral lymphocytes, although there was appreciable variation within each group. The B cell line Bri 8, for example, expressed 1.5 × 106 W6/32 antigenic sites, and by inference, HLA-A,B,C molecules per cell. Equal amounts of β2m and HLA-A,B,C chain were found on both cell types. The isolated HLA-A chain from intact 125I-HLA-A2 antigens weakly bound to W6/32 antibody in contrast to 125I-β2-microglobulin (β2m) isolated from the same preparation of HLA-A2 antigens that showed no demonstrable binding. when an excess of cold β2m was added to the isolated 125I-HLA-A2 chain, the binding to W6/32 antibody was considerably enhanced. These results suggest that the W6/32 antigenic determinant involves only amino acids of the HLA-A,B,C chain and is a product of their three dimensional configuration. Stable maintenance of this configuration appears to be dependent on the association of the HLA-A,B,C chain with β2m.The Journal of Immunology 08/1979; 123(1):342-9. · 5.36 Impact Factor
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ABSTRACT: Antibody-secreting hybrid cells have been derived from a fusion between mouse myeloma cells and spleen cells from a mouse immunized with membrane from human tonsil lymphocyte preparations. Hybrids secreting antibodies to cell surface antigens were detected by assaying culture supernatants for antibody binding to human tonsil cells. Six different antibodies (called W6/1, /28, /32, /34, /45 and /46 were analyzed. These were either against antigens of wide tissue distribution (W6/32, /34, and /46) or mainly on erythrocytes (W6/1 and W6/28). One of the anti-erythrocyte antibodies (W6/1) detected a polymorphic antigen, since blood group A1 and A2 erythrocytes were labeled while B and O were not. Antibodies W6/34, /45 and /46 were all against antigens which were mapped to the short arm of chromosome 11 by segregation analysis of mouse-human hybrids. Immunoprecipitation studies suggest that W6/45 antigen may be a protein of 16,000 dalton, apparent molecular weight, while W6/34 and /46 antigens could not be detected by this technique. Antibody W6/32 is against a determinant common to most, if not all, of the 43,000 dalton molecular weight chains of HLA-A, B and C antigens. This was established by somatic cell genetic techniques and by immunoprecipitation analysis. Tonsil leucocytes bound 370,000 W6/32 antibody molecules per cell at saturation. The hybrid myelomas W6/32 and W6/34 have been cloned, and both secrete an IgG2 antibody. W6/32 cells were grown in mice, and the serum of the tumor-bearing animals contained greater than 10 mg/ml of monoclonal antibody. The experiments established the usefulness of the bybrid myeloma technique in preparing monospecific antibodies against human cell surface antigens. In particular, this study highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymorphisms.Cell 06/1978; 14(1):9-20. DOI:10.1016/0092-8674(78)90296-9 · 33.12 Impact Factor