Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1995-10-23
1999-03-16
Jones, W. Gary
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 243, 435 6, C07H 2102, C07H 2104, C12Q 168
Patent
active
058832386
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a process and reagents for DNA typing of HLA-B alleles.
The technical problem underlying the present invention is to provide a DNA typing method using specific primer and probe sets enabling the discrimination of HLA-B alleles, especially those which are difficult to discriminate by serological means.
The Human Leukocyte Antigen (HLA) system comprises a series of linked genes on the short arm of chromosome 6. Three classes of genes are defined: class I antigens (HLA A, B, C) composed of an .alpha. chain non covalently associated with .beta.2 microglobulin, encoded on chromosome 15; class II antigens (DP, DQ, DR) composed of an .alpha. and a .beta. chain; class III products which correspond to components of the complement system. Class I and class II antigens are polymorphic transmembrane glycoproteins and share a common immunologic role in antigen presentation. HLA class I restricted presentation of foreign antigens leads to cytotoxic T cell receptors in mature T lymphocytes. In addition, class I and class II antigens play a crucial role in transplantation immunology and in the susceptibility to autoimmune diseases.
Extensive polymorphisms exist at most loci. In view of the biological and medical importance of these antigens a highly sensitive and rapid technique for HLA typing is required. Different protocols have been used until now: serologic, cellular and DNA based restriction fragment polymorphism (RFLP) and recently also sequence specific oligonucleotide (SSO) hybridization methods. DNA typing by oligonucleotide hybridization provides the best direct definition of HLA polymorphisms next to complete sequence analysis. However, sequence analysis is expensive and time consuming and hence not the method of choice for routine applications.
Polymorphisms are of fundamental significance for the function of HLA antigens and will be mostly localized in exons coding for the functionally important extracellular domains.
For class I genes most of the polymorphisms are localized in the aminoterminal .alpha.1 and .alpha.2 domains. The .alpha.3 domain is a highly conserved immunoglobin-like domain. A total of 40 HLA A, 64 HLA B, and 24 HLA C alleles have been identified (Zemmour and Parham, Tissue Antigens, 40: 221-228,1992). Diversity between different alleles occurs in specific regions of the .alpha.1 and .alpha.2 domain. A patchwork pattern with short stretches of homology between different alleles occurs. Genetic mechanisms such as homologous recombination and exon shuffling have lead to locus specific allelic diversity (Parham et al. PNAS (USA) 85:4005-4009, 1988).
Different typing methods have been developed to discriminate between the different alleles of the very polymorphic class I and class II loci. An overview of these different typing methods is given below:
Serology: In a microtoxicity test antisera to different HLA class I or class II antigens are incubated with lysed purified lymphocytes. Lysed cells will be stained with eosin or other dyes while not-lysed cells will remain unstained. This method is used for class I A, B, C alleles and class II DR and DQ alleles. DP alleles cannot be typed due to too low level of expression and a limited availability of antisera. The reaction for class II alleles is performed on purified B lymphocytes. A limited determination of supertypic groups of alleles is possible without further subtyping. Three alleles of the HLA C locus remain serologically undefined. Since epitopes on the HLA molecule are detected, discrimination between the .alpha. and the .beta. chains is impossible and the .alpha..beta. hetrodimer is identified. Allosera against class II molecules are often anti-class I contaminated and need to be absorbed before use. Crossreactions between alleles on the same or on a different locus occur, which makes analysis of the result difficult. Even if monoclonal antibodies are used the problem of crossreactions is not solved. Although this is a very rapid method (3 hrs for complete typing), incomplete and erroneous results are the main pr
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Andrien Marc
De Canck Ilse
Dupont Etienne
Rossau Rudi
Jones W. Gary
N.V. Innogenetics S.A.
Whisenant Ethan
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