A rapid method for semiquantitative analysis of the human V beta-repertoire using TaqManR PCR

J Immunol Methods. 1997 Apr 25;203(2):181-92. doi: 10.1016/s0022-1759(97)00028-8.

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.

MeSH terms

  • Cells, Cultured
  • DNA-Directed DNA Polymerase
  • Humans
  • Magnesium / physiology
  • Multigene Family / immunology*
  • Oligonucleotide Probes / standards
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • Receptors, Antigen, T-Cell, alpha-beta / analysis*
  • Receptors, Antigen, T-Cell, alpha-beta / genetics*
  • Reference Values
  • Sensitivity and Specificity
  • T-Lymphocyte Subsets / chemistry
  • T-Lymphocyte Subsets / metabolism*
  • Taq Polymerase

Substances

  • Oligonucleotide Probes
  • Receptors, Antigen, T-Cell, alpha-beta
  • Taq Polymerase
  • DNA-Directed DNA Polymerase
  • Magnesium