A PCR-SSP method to specifically select HLA-A*0201 individuals for immunotherapeutic studies

作者：Gatz SA; Pohla H; Schendel DJ^*

来源：Tissue Antigens, 2000, 55(6): 532-547.

DOI：10.1034/j.1399-0039.2000.550604.x

• DNA-typing
• HLA-A*0201
• HLA-A*02011
• HLA-A*02 subtyping
• HLA-A*0201 subtyping
• immunotherapy
• PCR-SSP

HLA-A*0201 is an important restriction element for peptide presentation to T cells in disease and cancer. Mutation studies and analyses using cytotoxic T lymphocytes have shown the functional relevance of sub type-specific differences in HLA-A2 molecules for peptide binding and T-cell receptor recognition. Therefore, many immunotherapeutic studies need to accurately select HLA-A*0201-positive individuals. We designed an easy, robust approach based on the polymerase chain reaction using sequence-specific primers (PCR-SSP) to specifically distinguish A*0201-positive individuals from other HLA-A2 subtypes described to date. The first step includes reactions that give information whether the sample donor is HLA-A2 and, if so, whether the individual is homozygous or heterozygous for HLA-A2. Further, it is determined whether the sample has an HLA-A*020S or an HLA=A*0201 sequence at the corresponding position in exon 4. Samples that may contain an HLA-A*0201 allele according to the results of this first step are subtyped in a second step nested PCR. Here the strategy is focussed on the discrimination of HLA-A*0201 from the other subtypes by considering divergent nucleotide positions in two ways. One SSP combination amplifies the HLA-A*0201 sequence while a corresponding SSP combination specifically amplifies the subtype or group of subtypes differing from HLA-A*0201 at this position. Thus, at relevant polymorphic nucleotide positions the HLA-A*0201 sequence is both directly and indirectly confirmed. This strategy strongly enhances the reliability of the subtyping and allows better verification of HLA-A*0201-positive patient selection for clinical studies.

• 出版日期2000-6