A PCR-SSP method to specifically select HLA-A0201 individuals for immunotherapeutic studies

• Published in: Tissue antigens 2000-06, Vol.55 (6), p.532-547
• Main Authors: Gatz, S.A., Pohla, H., Schendel, D.J.
• Format: Article
• Language: English
• Subjects: Alleles; Cell Line; DNA Primers - genetics; DNA-typing; Flow Cytometry; Genetic Testing; Genotype; histocompatibility antigen HLA; Histocompatibility Testing - methods; HLA-A02 subtyping; HLA-A0201; HLA-A0201 subtyping; HLA-A02011; HLA-A2 Antigen - analysis; HLA-A2 Antigen - genetics; Humans; immunotherapy; Immunotherapy - methods; PCR-SSP; Polymerase Chain Reaction - methods
• ISSN: 0001-2815; 1399-0039
• DOI: 10.1034/j.1399-0039.2000.550604.x

: 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 subtype‐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*0209 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 ( Note).