title: A rapid method for semiquantitative analysis of the human V beta-repertoire using TaqManR PCR.
abstract: Analysis of the V beta-repertoire of antigen-reactive T cell populations can be approached using either flow-cytometry or PCR-based techniques. While the former method requires a complete set of V beta-specific monoclonal antibodies (mAbs) and large cell numbers for analysis, the latter is both time-consuming and labour-intensive. To circumvent the drawbacks of both these methods we have employed the recently developed technique of TaqManR PCR to analyse the V beta-usage of human T cell populations. TaqManR PCR is based on the 5'-->3' nuclease activity of Taq polymerase. During PCR amplification an internal oligonucleotide probe, that is labelled with a fluorescent reporter and a quencher dye, is cleaved by Taq polymerase. After cleavage, quenching of the reporter dye is lost and reporter fluorescence can be detected with a fluorescence plate reader. Using one C beta-specific fluorogenic probe and a panel of V beta-specific primers, we show that fluorescence-detected amplification of TCR beta cDNA is V beta-specific and linear within a 2-3-log range of template concentration. The sensitivity of TaqManR PCR is comparable to conventional detection of PCR-products by agarose gel staining, while processing time is reduced. Furthermore, superantigen-induced skewing of the V beta-repertoire of human T cells is readily detected with this method. Thus TaqManR PCR is a reliable and fast method for semiquantitative analysis of the V beta-repertoire of human T cell populations.
authors.affiliation: Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Lang
authors.forename: K
authors.initials: K
authors.lastname: Pfeffer
authors.forename: H
authors.initials: H
authors.lastname: Wagner
authors.forename: K
authors.initials: K
authors.lastname: Heeg
mesh_terms: D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D008274:Magnesium / Q000502:physiology; D005810:Multigene Family / Q000276:immunology*; D015345:Oligonucleotide Probes / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis* / Q000235:genetics*; D012016:Reference Values; D012680:Sensitivity and Specificity; D016176:T-Lymphocyte Subsets / Q000737:chemistry / Q000378:metabolism*; D019914:Taq Polymerase
chemical_list: D015345:Oligonucleotide Probes; D016693:Receptors, Antigen, T-Cell, alpha-beta; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium