Background/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 β-microglobulin (β2-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the β2-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 × 106 of positive Fen cells contained about 200 µg total protein of which about 0.10 µg 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.

Keywords:
HLA, Peptide, HPLC, Bladder tumour, Gene transfection
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© 2002 S. Karger AG, Basel
2002
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