Reactivity and epitope mapping of single-chain T cell receptors with monoclonal antibodies

Thomas C. Brodnicki, Philmore O. Holman, David M. Kranz

Research output: Contribution to journalArticlepeer-review


To examine further the structure of the T cell receptor (TCR) and the specificity of mAbs generated against the native protein, the TCR was expressed in Escherichia coli as a single chain in which the variable regions of the α and β chains are joined by a 25 amino acid linker. Five single-chain TCR that have different α and/or β variable CV) regions were examined with the anti-Vβ8 region mAbs KJ16 and F23.1 and the anti-Vαs mAbs KT50, KT65 and B21.14. Each of the mAbs reacted with one or more of the single-chain receptors. Western blot analysis demonstrated that, the intrachain disulfide bonds were required for proper epitope conformation and recognition of the TCR by the antibodies. KT50, KT65 and B21.14 antibodies distinguished between two related Vα regions that differed at only six residues. A model of the V regions of the TCR based on immunoglobulin (Ig) structure suggests that three of these six variant residues are in the putative CDR1 of the receptor and possibly accessible to antibody. To test this possibility, site-directed mutagenesis of the unreactive Vα region demonstrated that the combination of all three residues restored binding by the anti-Vα8 antibodies. In addition, these three complimentarity determining regions (CDR) residues are likely to be in close proximity to the putative CDR3 which also influenced binding of the antibodies. The epitopes recognized by the Vα-specific antibodies are thus predicted to reside closer to the putative binding site than the epitopes previously determined to be recognized by the anti-Vβ8 antibodies, KJ16 and F23.1. Finally, the specificities of KT50 and KT65 as determined with the E. coli expression system suggests an explanation for previous observations about the differences in. the T cell populations that are recognized by these antibodies.

Original languageEnglish (US)
Pages (from-to)253-263
Number of pages11
JournalMolecular Immunology
Issue number3
StatePublished - Feb 1996


  • B21.14
  • Epitopes
  • F23.1
  • KJ16
  • KT50
  • KT65
  • Single-chain TCR
  • mAbs

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology


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