Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line

The major histocompatibility complex (MHC) class I molecules present proces sed self and nan-self peptides to T lymphocytes. Given that the class I: pe ptide complex plays a critical role in cell-mediated immunity, it is import ant to identify the nature of class I-associated peptides unique to maligna nt cells as a prelude to the development of vaccines. The aim of this study was to combine immune-bead purification (using anti-class T antibody W6/32 ) technique, sequential ultra-filtration and high performance liquid chroma tography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by beta (2)-microglobulin (beta (2)-m) gene tran sfaction. The results were as follows: (a) class I antigens could be separa ted from tumour cell lysate but only from the class I positive Fen cells; C b) treatment of CNBr-W6/32 beads pre exposed to class I positive Fen lysate and eluted with dissociation agent(mild acid) resulted in the release of m ore than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS-PAGE and silver staining analysis, (c) purified and el uted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumou r cell line. The extension of these approaches to isolate peptides from tis sue tumour biopsies may help the future of vaccine therapy in cancer patien ts. Copyright (C) 2001 John Wiley & Sons, Ltd.