A single monoclonal antibody as probe to detect the entire set of native and partially unfolded rhEPO glycoforms

OGGERO EBERHARDT, M.; AMADEO, G.; ZENCLUSSEN, M. L.; ROBLES, L.; KRATJE, R. B.; ETCHEVERRIGARAY, M.

San Carlos de Bariloche. Pcia. de Río Negro. Argentina

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) – Simposio Internacional de Proteínas; 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Human erythropoietin (hEPO) is a highly heterogeneous glycosylated protein that requires well characterised-immunochemical reagents to evaluate its glycoform profile. Five anti recombinant hEPO monoclonal antibodies (mAbs) were analysed in terms of their abilities to bind to rhEPO, with the aim of selecting the appropriate mAb to develop several immunochemical approaches with no glycoform selectivity. The antibodies mapped two spatially distinct epitopes and neutralised the in vitro biological activity of the cytokine. All of them were able to bind to both, the partially folded and the native form of the protein. Isoelectric focusing analysis followed by immunoblotting confirmed that all the mAbs were able to bind to each glycoform. Nevertheless, only mAb 2B2 preserved the ability to bind to the complete set of soluble rhEPO glycoforms when it was immobilised onto polystyrene or chromatographic matrixes. Therefore, mAb 2B2 was useful as a capture antibody to perform an accurate, specific and fast sandwich ELISA to quantify rhEPO with a detection limit of 7 ng.ml-1. mAb 2B2 was also satisfactorily employed as affinity ligand to purify rhEPO. Our work led us to find a suitable reagent to perform a variety of immunochemical approaches where the binding of native and partially unfolded rhEPO glycoforms is required.