A PCR-SSP method to specifically select HLA-A*0201 individuals for immunotherapeutic studies.
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.
Gatz
S A
SA
Institute of Molecular Immunology, GSF National Research Center for the Environment and Health, Munich, Germany.
Pohla
H
H
Schendel
D J
DJ
D000483:Alleles; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D005820:Genetic Testing; D005838:Genotype; D015789:HLA-A2 Antigen / Q000032:analysis / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; D015789:HLA-A2 Antigen