Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP,8 probes and a DPa probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of PPwl, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defimed and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies.Polymorphism in the HLA-DP subregiont has until recently only been detectable by primed lymphocyte typing (PLT) (1). Two other methods are now available to identify these polymorphic differences, one by Southern blotting with a series of DP DNA probes and restriction enzymes and the other using monoclonal antibodies in an ELISA. In this paper, we describe studies using these two methods to determine both the amount of DP polymorphism they can define and whether the alleles seen correspond to those identified using PLT. One of the problems with using full-length cDNA clones as probes in the HLA system is that there is cross-hybridization between the sequences at the DP, DQ, and DR loci. We therefore chose to use two short DP/3 probes ( Fig. 1) to analyze DP restriction fragment length polymorphisms (RFLP) as well as a DPa probe.Three monoclonal antibodies were used in the study. Two were directed against polymorphic determinants of the DP region and the third was a monomorphic antibody. The cells were grown in hydrogen carbonate-buffered RPMI 1640 medium (Flow Laboratories) supplemented with 10% fetal calf serum/penicillin (100 units/ml)/streptomycin (100 Ag/ml). MATERIALS AND METHODSThe antibodies used in this study are listed in Table 1 (2-4). DP11.1, one of the two polymorphic DP antibodies used, was produced by immunizing a C3H mouse (2) with C3H-derived L cells transfected with cosmid LC11, which contains DPJA and DPIB genes (5).The antibody has been shown by immunoblotting to be directed against the DPa chain and reacts by ELISA only with DPw2 and DPw4 cells. The other polymorphic antibody, ILR1, was produced as a result of immunization with ascites tumor cells from a Burkitt lymphoma patient (3). The monomorphic antibody, B7/21, was produced in response to immunization with the pre-B-cell leukemic cell line Nalm-6 (4).Screening of the Cell Lines by ELISA. The cell lines were screened using an ELISA galactosidase-antigalactosidase complex technique (6) with poly(L-lysine)-embedded gluteraldehyde-fixed cells as antigen.Extraction of DNA, Southern Blotting, and Hybridization. High molecular weight genomic DNA was extracted from either peripheral blood or from Epstein-Barr virus-transformed B lymphoblastoid cell lines essentia...

Mouse L cells transfected with human HLA-DP (DPw4) a and f3 genes were used to make monoclonal antibodies in C3H mice. A polymorphic antibody, DPl1. Alloantisera, mainly produced by fetal maternal stimulation, were the original basis for the definition of the HLA-DR and -DQ region products and their polymorphism. It is clear that most of the serologically detected variation is in the DR and DQ 83 chains. The DP specificities were identified by cellular typing techniques and there is, so far, no corresponding serological identification. One monoclonal antibody, IRL1, has been identified, which appears to react preferentially to DPw2 and DPw3 (4). Monoclonal antibodies that are monomorphic have made a major contribution to the biochemical analysis of the HLA-D region products (5, 6), but useful polymorphic antibodies have been harder to obtain (7). A wider range of polymorphic HLA-D monoclonal antibodies, particularly for the definition of HLA-DP types, would be extremely useful both for serological typing and for biochemical and functional studies.The production by transfection of mouse L cells expressing a single human HLA-D region product, in this case HLA-DP (8), provides the basis for a novel approach to making anti-HLA class II monoclonal antibodies. If an HLA-DPexpressing L-cell transfectant is used to immunize C3H mice, the strain from which L cells were derived, then the only product the mice should react to, apart from L-cell-specific determinants, is HLA-DP. This principle is an extension of that used to make human-specific antisera (9) and monoclonal antibodies (10) by immunizing mice with human-mouse somatic cell hybrids containing a single human chromosome. In this paper, we describe the recognition of polymorphic specificities on L-cell DP transfectants and the use of these transfectants to make a polymorphic monoclonal anti-DP antibody with, surprisingly, specificity for the a chain of DPw4 and to an apparently lesser extent, DPw2.

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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.