title: Isolation of human leukocyte antigen (HLA)-associated peptide(s) in the absence of HLA-restricted specific cytolytic T lymphocytes.
abstract: METHODS
  In this study, immunobead purification, dot-blot, immunocytochemical staining, and SDS-PAGE techniques in combination with high-performance liquid chromatography were used to isolate human leukocyte antigen (HLA) class I antigens and associated peptides from a bladder tumour cell line (Fen) before and after gene transfection.
  
  
  RESULTS
  The results showed that: (1) Transfection of the class I negative Fen cell line with normal beta-microglobulin (beta(2)-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the beta(2)-m gene transfected cells using Sepharose CNBr-W6/32 beads. (3) Dissociation of class I antigens from beads and analysis by the SDS-PAGE showed the presence of both free heavy and light chains of class I antigens. (4) More than 22 class I-associated peptides with a molecular weight of 700-3,000 daltons could be isolated from W6/32-loaded beads but only from lysate of HLA-positive Fen cell line. The data also showed that 1 x 10(6) of positive Fen cells contained about 200 microg total protein of which about 0.10 microg was class I and about 2 ng was class I-associated peptides.
  
  
  CONCLUSIONS
  These findings demonstrated that the gene transfection approach could be used to restore missing class I antigens on an otherwise class I negative bladder tumour cell line. The results also showed the feasibility of using above techniques for isolation of HLA-associated peptides. These approaches may provide a realistic possibility for identification of putative tumour-specific peptide(s) from tumour specimens with the long-term aim to use such peptide(s) for immunotherapy in cancer patients.
authors.affiliation: Uro/Oncology Research Unit, Department of Medical Oncology and Urology, The Royal London Trust, UK. n.torabi-pour@mds.qmw.ac.uk
authors.forename: N
authors.initials: N
authors.lastname: Torabi-Pour
authors.forename: A M E
authors.initials: AM
authors.lastname: Nouri
authors.forename: R
authors.initials: R
authors.lastname: Saffie
authors.forename: W J W
authors.initials: WJ
authors.lastname: Morrow
authors.forename: R T D
authors.initials: RT
authors.lastname: Oliver
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015395:Histocompatibility Antigens Class I / Q000302:isolation & purification*; D006801:Humans; D010455:Peptides / Q000302:isolation & purification*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000276:immunology* / Q000473:pathology
chemical_list: D015395:Histocompatibility Antigens Class I; D010455:Peptides