Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR-CD3 signalling

Immunol Lett. 2010 May 4;130(1-2):43-50. doi: 10.1016/j.imlet.2009.12.011. Epub 2010 Jan 6.

Abstract

The T cell antigen receptor (TCR-CD3) complex contains 12 different cytoplasmic tyrosines, each of which is part of an immunoreceptor tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-tyrosines of the TCR-CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR-CD3 subunit zeta. Expressing TCR-CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of zeta (phospho-zetaY1) and antisera raised against the first and the second phospho-tyrosine of CD3epsilon (phospho-epsilonY1 and phospho-epsilonY2).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Phospho-Specific / immunology*
  • Cell Line
  • Drosophila
  • Enzyme-Linked Immunosorbent Assay
  • Gene Expression Regulation
  • Genetic Vectors
  • Immunoblotting
  • Mice
  • Phosphorylation
  • Receptor-CD3 Complex, Antigen, T-Cell / immunology*
  • Signal Transduction*
  • T-Lymphocytes / immunology*

Substances

  • Antibodies, Phospho-Specific
  • Receptor-CD3 Complex, Antigen, T-Cell