CONICET | Buscador de Institutos y Recursos Humanos

The Human Granulocyte Macrophage Colony Stimulating Factor (hGM CSF) is a cytokine that regulates the proliferation and differentiation of hematopoietic progenitor cells. The epitopes of hGM-CSF were evaluated by the SPOT Synthesis technique using monoclonal antibodies (MAbs). MAb interactions with cellulose-bound overlapping peptide scans, spanning the GM-CSF sequence, were analysed. MAb CC1H7 recognised the continuous epitope A1PAR4 representing the N terminal sequence of the protein. Combinatorial hexapeptide library approaches were carried out to describe linear peptide sequences that are recognised by antibodies and mimic a conformational epitope (mimotope). Dual-positional scanning of a combinatorial hexapeptide library and subsequent iterative searches with two defined positions identified that MAb CC5B5 bound peptides AERRF and RERW, probably mimicking the conformational dependent epitopes A18E21R23R24F119 and R24E21N17W13 on GM-CSF. MAb M1B8 primarily bound peptides PFEWE, FFEWE and WFEWE. These peptides might mimic the nonlinear epitope P118F119W13E14 in the GM-CSF molecule. We observed that epitopes bound by these MAbs were very closely located on the native protein surface. An overlapping peptide scan followed by electroblotting of bound antibodies showed that the sequence L61YKQGLRGSLTK72 was recognised by MAb M7E10 and could probably describe part of the continuous epitope bound by this antibody. Finally, MAb affinity constant measurements, using glycosylated and nonglycosylated rhGM CSF, demonstrated that MAb CC1H7, CC5B5 and M1B8 are able to bind regions in the proximity of glycosylation sites. Contrarily, MAb M7E10 should recognise a site not involved in the protein glycosylation.

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