Hooker, A.D. and Green, N.H. and James, D.C. and Strange, P.G. and Baines, A.J. and Bull, A.T. and Jenkins, N. (1997) Epitope determination for antibodies raised against recombinant human interferon-gamma. In: Carrondo, M.J.T. and Griffiths, B. and Moreira, J.L.P., eds. Animal Cell Technology - From Vaccines to Genetic Medicine. Kluwer Academic Publ pp. 277-282. ISBN 0-7923-4321-2.
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Oligosaccharides, like peptides, make excellent antigens due to their hydrophilicity and complexity and can also mask potential antigenic sites on the peptide backbone. Human interferon-gamma (IFN-gamma) has been purified by immunoaffinity chromatography from the supernatant of batch cultures of recombinant Chinese hamster ovary cells to determine its glycosylation profile. To validate this purification procedure and the quantification of IFN-gamma by enzyme-linked immunosorbent assay (ELISA) with mouse monoclonal antibody 20B8, the effects of glycosylation upon antibody recognition were investigated. Preliminary experiments with glycosylated and deglycosylated IFN-gamma demonstrated that there was no difference in antibody binding by ELISA. To confirm this result, IFN-gamma was digested with trypsin and the resulting peptides separated by reverse-phase highperformance liquid chromatography, the isolated peptides being identified by matrix-assisted laser desorption/ionisation mass spectrometry. Glycopeptides containing the Asn(25) and Asn(97) glycosylation sites were not recognised by anti-IFN-gamma 20B8 in ELISA studies. Its epitopes were located on non-glycosylated regions of IFN-gamma, 20B8 recognising protein epitopes rather than the carbohydrate moieties. The purification and quantification of IFN-gamma by ELISA with this antibody are therefore independent of the glycosylation status and are not selecting a sub-population of IFN-gamma molecules.
|Item Type:||Conference or workshop item (Paper)|
Q Science > QR Microbiology
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||M.A. Ziai|
|Date Deposited:||08 Jun 1914 10:10|
|Last Modified:||08 May 2012 13:44|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/18109 (The current URI for this page, for reference purposes)|
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