Comparative study between direct mild acid extraction and immunobead purification technique for isolation of HLA class I-associated peptides.
Identification of tumour-specific peptide(s) hidden within the groove of human leucocyte antigens is a crucial prerequisite for peptide vaccine therapy. Conventionally, the peptide(s) are isolated by mild acid extraction (MA) technique followed by sequential ultra-filtrations. Here we describe a new approach for peptide isolation using the immunobead purification (IB-P) technique in conjunction with reverse-phase high-performance liquid chromatography. The obtained data were validated by SDS-PAGE followed by the silver staining technique. The results can be summarised as follows: (1) Comparison of class I-associated peptides isolated from a bladder cell line before and after the correction of class I antigens by gene transfection followed by IB-P technique showed the presence of peptides only from the class I-corrected cells. The data were confirmed using the silver staining technique as a way of detecting individual peptide bands. (2) Whilst peptides could be isolated by both techniques, the MA method led to the isolation of peptides from both class I-negative and class I-positive Fen cell lysates. (3) The IB-P approach could be used for isolation of class I-associated peptides from a normal kidney tissue. The data showed the high efficiency of the IB-P approach for isolation of class I-associated peptides. Unlike the MA technique, where the presence of non- class I-associated peptides was a problem, the IB-P approach isolated only peptides associated with the class I antigens. In addition, the data showed the feasibility of extracting peptides from tissue fragments by the IB-P method. The approach presented here may assist the future development of peptide vaccine therapy in urological cancers.
Torabi-Pour
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Uro/Oncology Research Unit, Department of Medical Oncology, St. Bartholomew's and the Royal London School of Medicine, London, UK. n.torabi-pour@mds.qmw.ac.uk
Nouri
A M E
AM
Saffie
R
R
Oliver
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RT
D017951:Antigen Presentation; D014408:Biomarkers, Tumor / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015395:Histocompatibility Antigens Class I / Q000302:isolation & purification*; D006801:Humans; D007668:Kidney / Q000166:cytology; D012016:Reference Values; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014462:Ultrafiltration / Q000379:methods*; D001749:Urinary Bladder Neoplasms / Q000276:immunology* / Q000473:pathology
D014408:Biomarkers, Tumor; D015395:Histocompatibility Antigens Class I