The major histocompatibility complex (MHC) includes the HLS gene complex which is located on the short arm of human chromosome 6, as depicted in FIG. 4. This region encodes cell-surface proteins which regulate the cell-cell interactions of the immune response. The various HLA class I loci encode the HLA antigens, 44,000 dalton polypeptides which associate with 2 microglobulin at the cell surface. These class I molecules are involved in the recognition of target cells by cytotoxic T lymphocytes. HLA class II loci encode cell surface proteins of 29,000 and 34,000 daltons. These class II molecules are also involved in the regulation of the immune response.
The HLA-A and HLA-B loci of the HLA Class I genes exhibit an extremely high degree of polymorphism. The 1984 International Histocompatibility Testing Workshop designated 19 alleles of HLA-A (HLA-A1, A2, etc.) and 36 alleles of HLA-B. See, Human Immunology, 11, 117 (1984). Since this high degree of polymorphism is thought to relate to the function of the HLA-A and HLA-B molecules, much effort has gone into determining its molecular basis. With the recent cloning of certain HLA class I genes this effort has extended to the DNA level.
Currently, serological HLA typing is routinely done in connection with many medical procedures, e.g., organ transplantation. Rejection of organ grafts is believed to be diminished if the HLA alleles of donor and recipient are identical. The numerous alleles of HLA genes in the population also make HLA typing useful for paternity testing.
During pregnancy women can develop antibodies to fetal HLA antigens of paternal origin. Sera obtained from these women are qualitative as these sera contain a mixture of antibodies often directed against several HLA alleles. In addition, a considerable effort must be directed towards the ongoing identification of new sera and the determination of their specificity. Monoclonal antibodies to class I antigens have been described. However, only a few uniquely define the alleles according to locus. Furthermore, the genetic complexity of the HLA class I gene family as indicated by hybridization techniques and genomic cloning is much greater than the HLA-A, -B and -C loci which can be serologically defined. For example, Southern blot analysis using a cross-reactive class I cDNA probe suggests that there are 15 to 20 class I genes in the human genome. This complexity has restricted the correlation of polymorphic restriction endonuclease fragments with HLA class I alleles.
One approace to developing locus specific DNA probes from cross-reactive HLA class I genomic clones is disclosed by F. C. Grumet et al., in Mol. Biol. Med., 1, 501 (1983), for the HLA-B locus. The DNA sequences of the B7 gene and a class I pseudogene were compared to locate non-homologous segments. One such segment, including approximately 180 nucleotides comprising the last (7th) intron of the B7 gene was isolated and subcloned to yield a DNA probe reported to be specific for the HLA-B locus.
Therefore a need exists for improved methods to determine the HLA class I allele or alleles expressed by a given individual, e.g. to facilitate tissue matching. One such method can be based on the ability to assign the restriction fragments obtained from the HLA gene complex to individual HLA alleles. A further need exists for methods to map the restriction sites present in individual HLA alleles, particularly those which are polymorphic.
USPTO Backgrounds
USPTO Backgrounds