The products of the human major histocompatibility complex (HLA Class I and II) have historically been detected using serological or cellular assays. With the availability of DNA sequence information for alleles of the HLA system, and with the development of molecular biological techniques it has become possible to tissue type for allelic differences in the HLA genes themselves. We describe here a polymerase chain reaction (PCR) system, based on the principle of the amplification refractory mutation system (ARMS), for low-resolution DNA typing of the HLA-B gene. The technique involves a one-step PCR from genomic DNA using sequence-specific primers in particular combinations that determine the specificity of each reaction. A low-resolution primer panel has been designed, based on published HLA-B gene nucleotide sequences, consisting of 34 sequence-specific primers (SSP) in 24 PCR reactions which cover all known HLA-B alleles, to give allele-specific or group-specific amplification of DNA fragments of defined size (344-784bp). Advantages of the system are that it can be performed in under 4 hours including DNA extraction, results are easy to interpret and it does not require viable cells.
A polymorphic monoclonal antibody (TAL16.1), raised against a mouse L-cell transfectant expressing the human DRB5*0101 gene from the HLA-DR15(2) Dw2 DR51 haplotype was shown to have a complex pattern of reactivity to DRB gene products. The antibody bound to a transfectant expressing the DRB5*0101 allele against which it was produced but not to a transfectant expressing the DRB1*1501 allele. These alleles of the DRB1 and DRB5 genes are usually coexpressed on DR15(2) Dw2 DR51 cells. A comparison of the HLA-DRB amino acid sequences of reactive and non-reactive cells identified an aspartic acid residue at position 70, conserved in all antibody-positive cells and absent in antibody-negative cells, which was postulated as being responsible for conferring the specificity of the antibody. The aspartic acid residue at position 70 is present in DRB5*0101 and DRB5*0102 alleles but absent in DRB5*0201 and DRB5*0202 alleles, allowing the antibody to distinguish between these splits of the DR51 serological specificity. TAL16.1 also binds to the product of the DRB1*0103 allele and discriminates between cells with a DR103 specificity and the other DR1 subtypes, DRB1*0101 and DRB1*0102. In this report the value of transfectants as immunogens for use in the production of monoclonal antibodies of predetermined specificity and as tools for the fine mapping of antibody specificity is discussed.
A monoclonal antibody TAL13.1 was raised against mouse L cells transfected with the human HLA-DRB1*0701 gene. This antibody was found to be polymorphic recognizing a determinant expressed by the DR7, DRB1*0701 and DRB1*0702 gene products. Four polymorphic sites unique to this specificity have been identified within the DR beta 1 domain. These are residues 11-14, 25, 30 and 71-74, one or a combination of which is postulated as being responsible for conferring the specificity of the antibody. In Western blot analysis TAL13.1 was found to react with the DR alpha beta dimer, but not with the free alpha or beta chains. However, in flow cytometry it failed to bind a DR alpha/DQ beta mixed pair transfectant confirming that it recognizes an epitope on the DR beta not the DR alpha chain. Although TAL13.1, a low affinity antibody is negative or only weakly positive on resting peripheral blood lymphocytes (PBLs), we have demonstrated that by interleukin-4 (IL-4) stimulation we can up-regulate the levels of antigen already present and gain a level of binding comparable to that found on B lymphoid cell lines (B-LCLs) where it has been found to be a valuable reagent in their characterization.
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