Epitope Determination for Antibodies Raised Against Recombinant Human Interferon-Gamma

Abstract

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-γ) 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 BFN-γ 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-γ demonstrated that there was no difference in antibody binding by ELISA. To confirm this result, IFN-γ was digested with trypsin and the resulting peptides separated by reverse-phase high-performance liquid chromatography, the isolated peptides being identified by matrix-assisted laser desorption/ionisation mass spectrometry. Glycopeptides containing the Asn₂₅ and ASH₉₇ glycosylation sites were not recognised by anti-IFNγ 20B8 in ELISA studies. Its epitopes were located on non-glycosylated regions of IFN-γ, 20B8 recognising protein epitopes rather than the carbohydrate moieties. The purification and quantification of IFN-γ by ELISA with this antibody are therefore independent of the glycosylation status and are not selecting a sub-population of IFN-γ molecules.