A single monoclonal antibody as probe to detect the entire set of native and partially unfolded rhEPO glycoforms.
Human erythropoietin (hEPO) is a highly heterogeneous glycosylated protein that requires well-characterised immunochemical reagents to evaluate the glycoform profile along its biotechnological production as a recombinant hormone. These reagents should be suitable for several assay conditions (like those used for immunoblotting analysis, liquid or solid-phase quantitative assays, immunoaffinity purification) with no glycoform selectivity. Five anti-recombinant hEPO monoclonal antibodies (mAbs) were characterised with the aim of selecting the appropriate reagent. These 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 denatured and the native form of the protein. Isoelectric focusing analysis followed by immunoblotting confirmed that all the mAbs, herein described, were able to bind to each glycoform, recognising amino acid sequences of the hEPO. Nevertheless, only mAb 2B2 preserved the ability to bind to soluble recombinant human erythropoietin (rhEPO) when it was coated to polystyrene plates or immobilised on CNBr-activated Sepharose matrix. Besides, mAb 2B2 was able to bind to the complete set of soluble rhEPO glycoforms, showing the same affinity for the glycosylated and deglycosylated cytokine. Thus, mAb 2B2 was useful as a capture antibody to develop a sandwich enzyme-linked immunosorbent assay (ELISA), performing a simple, specific and fast assay 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 and single reagent to perform a variety of immunochemical approaches, where the binding of each glycoform in the native or partially unfolded form of rhEPO is required.
Amadeo
Ignacio
I
Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje El Pozo, C.C. 242, S3000ZAA Santa Fe, Argentina.
Oggero
Marcos
M
Zenclussen
María Laura
ML
Robles
Lucía
L
Pereira
Dardo
D
Kratje
Ricardo
R
Etcheverrigaray
Marina
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004921:Erythropoietin / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D004921:Erythropoietin