Rational selection of an antibody probe to detect the heterogeneous collection of CHO-derived rhGM-CSF glycoforms.
Six anti-E. coli rhGM-CSF monoclonal antibodies were generated using hybridoma technology. One of these showed identical affinity for the CHO and E. coli-derived cytokine (dissociation constants of 0.14 +/- 0.01 nM and 0.13 +/- 0.01 nM, respectively), mapping an epitope that is not hindered by carbohydrates. The antibody was used to develop a simple, specific and sensitive competitive ELISA to quantify the entire set of rhGM-CSF glycoforms (detection limit of 780 pg/ml) and it was successful as an affinity ligand to purify them. Therefore, this particular antibody is a useful, reliable and reagent for most immunochemical purposes with the aim of detecting, quantifying and purifying the highly heterogeneous collection of the CHO-derived rhGM-CSF isoforms.
Oggero
Marcos
M
Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Santa Fe, Argentina. moggero@fbcb.unl.edu.ar
Forno
Guillermina
G
Kratje
Ricardo
R
Etcheverrigaray
Marina
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000378:metabolism; D000915:Antibody Affinity; D000917:Antibody Formation; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli; D005260:Female; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis* / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011499:Protein Processing, Post-Translational / Q000276:immunology*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor