Novel strategy for identification of candidate cytotoxic T-cell epitopes from human preproinsulin

L. Chang, L. Kjer-Nielsen, S. Flynn, A. G. Brooks, S. I. Mannering, M. C. Honeyman, L. C. Harrison, J. McCluskey, A. W. Purcell

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17 Citations (Scopus)

Abstract

We describe a strategy for identifying ligands of human leukocyte antigen (HLA) class I molecules based on a peptide library-mediated in vitro assembly of recombinant class I molecules. We established a microscale class I assembly assay and used a capture ELISA to quantify the assembled HLA-peptide complexes. The identity of the bound ligands was then deduced by mass spectrometry. In this method, HLA complexes assembled in vitro in the presence of components of a mixture of peptides were immunoprecipitated and the bound peptide(s) identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This process of epitope extraction is robust and can be used with complex mixtures containing in excess of 300 candidate ligands. A library of overlapping peptides representing all potential octamers, nonamers and decamers from human preproinsulin was synthesized using unique library chemistry. Peptides from the library were used to initiate assembly of recombinant HLA-B8, HLA-B15 and HLA-A2, facilitating the identification of candidate T-cell epitopes from preproinsulin.

Original languageEnglish
Pages (from-to)408-417
Number of pages10
JournalTissue Antigens
Volume62
Issue number5
DOIs
Publication statusPublished - 1 Nov 2003
Externally publishedYes

Keywords

  • HLA-binding studies
  • Mass spectrometry
  • Preproinsulin
  • T-cell epitope

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