Biomedical subjects
Publications and source records attributed to P Parham.
A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kohler and Milstein [1]. This antibody recognizes a new specificity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.
In our search for monoclonal antibodies defining specific HLA-A,B antigens we have found a significant number of antibodies with polymorphic reactions that are not explicable with our current picture of HLA-A,B interrelationships. Analysis of one antibody (MB40.1) in some detail shows that it recognizes subsets of both A and B antigens and that it is able to split highly cross-reactive pairs of A or B antigens. These results address an outstanding difference in the serological interpretation of H-2, where these types of public reactions have been well established, from HLA, where they have been poorly characterized. Comparison of these types of serological reactions with amino acid sequence data and the apparent evolutionary stability of these broad cross-reacting determinants suggests a simple model for the molecular nature based upon limited amino acid variability and convergent evolution.
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A computer search was carried out for homologies between HLA-A and HLA-B antigen sequences and the sequences of constant and variable regions of immunoglobulins and of all other sequenced proteins. Searches were made both with relatively short peptide sequences from the HLA antigens and with those longer peptide sequences which were available in 1978. Significant homology of HLA antigen sequences to immunoglobulin constant region sequences was found in two cases: (1) a short decapeptide sequence which includes the fourth cysteine residue of HLA-B7 and (2) an 89-amino-acid residue (Ac-2) C-terminal fragment of the papainsolubilized HLA-B7 molecule. The difficulty of establishing statistically significant sequence homology with relatively short peptide sequences is emphasized by computer-based comparisons of the decapeptide sequence with randomly generated peptide sequences. It is concluded that statistically significant homology with short sequences can be assured only when extraordinarily high degrees of homology are present and additional constraints are included in the matches, for example, matches at relatively rare amino acid residues such as Cys, His and Trp. The homology of the 89-amino-acid residue sequence to constant region sequences of immunoglobulins is as great as or greater than that of beta 2-microglobulin. These findings and the unique domain structure involving a disulphide loop of comparable size strongly favour a common evolutionary origin for this region of HLA-A and -B, beta 2-microglobulin and immunoglobulin constant regions.
Two monoclonal antibodies recognizing HLA-DRw (human la) antigens were produced. The DA2 antibody binds a monomorphic determinant, common to all specificities and Genox3.53 antibody binds to a cross-reacting site on the HLA-DRw1,2 and 6 specificities. Both antibodies are IgG1 and show complement dependent cytotoxicity only in the presence of rabbit anti-mouse IgG serum. Specificity of both antibodies for the HLA-DRw molecule was shown by inhibition of antibody binding by preincubation of antibody with detergent solubilized Ia from JY (HLA-DRw4,6) cells and by preincubation of target cells with F(ab')2 fragments of a rabbit anti-la serum. DA2 antibody reacted with all cells of human B cell origin tested and with peripheral blood lymphocytes of several primate species tested. Genox3.53 antibody bound only to human cells expressing HLA-DRw1,2 or 6 antigens, giving a negative reaction with all primates tested. Genox3.53 antibody detected a split in the HLA-DRw6 specificity, showing reduced binding to the Daudi cell (HLA-DRw6) in comparison with binding to several other cell lines typed as HLA-DRw6, under saturating conditions. This low reactivity with Daudi was confirmed by absorption experiments. The ratio of DA2 binding to Genox3.53 binding to homozygous and heterozygous cell lines under saturation conditions was compared. Results suggested that, on some cell lines, DA2 might be reacting with a second population of human Ia antigens in addition to the HLA-DRw antigens. When a mixture of saturating concentrations of DA2 and Genox3.53 antibodies was tested for binding to cells under saturating conditions, the number of counts bound suggested the antibodies could bind simultaneously. Direct binding experiments showed that when each antibody was iodinated, its binding was not inhibited by preincubation with the other antibody, confirming that the DA2 and Genox3.53 determinants are distinct on the Ia molecule.
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Serologically and biochemically pure preparations of detergent and papain-solubilized HLA-A2 and HLA-B7 antigens were isolated by high pH elution from a series of immunoaffinity columns constructed from monoclonal antibodies with specificity for HLA-A2 and HLA-B7 antigens. These preparations retained the immunological activity and quaternary structure of the native molecule and should provide suitable reagents for insertion into liposomes and probing the role of major histocompatibility antigens in lymphocyte interactions.
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A monoclonal anti-beta 2-microglobulin (BBM.1 antibody) was produced by cell fusion between the mouse myeloma, P3-X63-Ag8, and spleen cells from a BALB/c mouse immunized with Molt 4, a human T cell line. BBM.1 antibody was fully inhibited by soluble beta 2-microglobulin and purified HLA-A, B antigens and reacted with human-mouse somatic cell hybrids only if they had chromosome 15 and expressed human beta 2-microglobulin. It was cytotoxic in complement-dependent lysis and of the IgG class. BBM.1 and a monoclonal anti-HLA-A, B, C glycoprotein antibody, W6/32 (Barnstable, C. J. et al., Cell 1978. 14:9.), were used to quantitate relative amounts of beta 2-microglobulin and HLA-A, B, C glycoproteins on different human cell types. Thymocytes and the Molt 4 cell line showed a considerable excess of beta 2-microglobulin over HLA-A, B, C glycoproteins, as measured by W6/32 reactivity. B cell lines, peripheral blood lymphocytes, fibroblasts, a HeLa cell derivative, and HSB2, another T cell line, had equal amounts. Immunological cross-reactions between HLA-A, B, C antigens and beta 2-microglobulin and their homologues in other species were detected with the BBM.1 and W6/32 antibodies. The W6/32 antigenic determinant appears to be more highly conserved than that recognized by the BBM.1 antibody.
The three-dimensional organization of HLA antigens has been investigated by spectroscopic and immunochemical techniques. Measurement of the circular dichroism shows that in papain-solubilized HLA the heavy chain as well as the previously studied light chain (beta 2-microglobulin) consists predominantly of beta-pleated sheet structures. When heavy chain is separated from the light chain under denaturing conditions and is allowed to renature, about 50% of the beta structure is lost, concomitantly with most of the alloantigenic activity. Analysis of the two acid-cleaved fragments of HLA-B7 heavy chain shows that beta structure is preferentially lost from the COOH-terminal region of the heavy chain. Exposure to denaturants per se does not inevitably result in irreversible loss of antigenic activity. However, recovery of antigenic properties does seem to depend on reassociation of the two chains. The results reported here provide further evidence for (i) the similarity of HLA antigens and immunoglobulins at the three-dimensional level and (ii) two distinct and physiologically important conformations of the HLA heavy chain, depending upon whether it is associated with the light chain.
The complete amino acid sequence for papain-solubilized HLA-B7 heavy chain is compared with the partial sequences of HLA-A2 and H-2Kb heavy chains. Although these molecules are highly conserved (i.e., 80% homology in comparing HLA-B7 with HLA-A2; 72% and 74% homology in comparing H-2Kb with HLA-A2 and HLA-B7, respectively), two stretches of greater variability are observed. These clusters of variability are discussed in terms of their possible involvement in the alloantigenic determinant(s) characteristic of these highly polymorphic membrane antigens.
A mouse monoclonal antibody, called MRC OX 3, which detects a polymorphic Ia determinant in the rat and cross reacts with an Ia determinant coded for by the I-A subregion in the mouse, detects a polymorphic determinant on human B cell lines. MRC OX 3 antibody binds to human B cell lines which express HLA-DRw specificities DRw 1, DRw2 and DRw6 but does not bind to those cells which only express other HLA-DRw specificities. No significant binding of MRC OX 3 antibody to either normal or mitogen stimulated human peripheral blood leukocytes, some of which are typed as HLA-DRw1, DRw2 or DRw6, could be detected. The binding of MRC OX 3 antibody to a human B cell line could be completely inhibited by pre-absorbing the antibody with purified rat Ia antigen.
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Explore the source record for details and available documents.