Choice of the adequate quantification method for recombinant human GM-CSF produced in different host systems

Mariela Bollati Fogolín
Laboratorio de Cultivos Celulares
Facultad de Bioquímica y Ciencias Biológicas
Universidad Nacional del Litoral
Ciudad Universitaria - C.C. 242
S3000ZAA Santa Fe
Argentina
Tel: 54 342 4575 214
Fax: 54 342 4575 214
E-mail: mrb@gbf.de

Marcos Oggero Eberhardt
Laboratorio de Cultivos Celulares
Facultad de Bioquímica y Ciencias Biológicas
Universidad Nacional del Litoral
Ciudad Universitaria - C.C. 242
S3000ZAA Santa Fe
Argentina
Tel: 54 342 4575 214
Fax: 54 342 4575 214
E-mail: moggero@fbcb.unl.edu.ar

Ricardo Kratje
Laboratorio de Cultivos Celulares
Facultad de Bioquímica y Ciencias Biológicas
Universidad Nacional del Litoral
Ciudad Universitaria - C.C. 242
S3000ZAA Santa Fe
Argentina
Tel: 54 342 4575 214
Fax: 54 342 4575 214
E-mail: rkratje@fbcb.unl.edu.ar

Marina Etcheverrigaray*
Laboratorio de Cultivos Celulares
Facultad de Bioquímica y Ciencias Biológicas
Universidad Nacional del Litoral
Ciudad Universitaria - C.C. 242
S3000ZAA Santa Fe
Argentina
Tel: 54 342 4575 214
Fax: 54 342 4575 214
E-mail: marina@fbcb.unl.edu.ar

* Corresponding author

Finanacial support: This work was supported by a grant from the Agencia Nacional de Promoción Científica y Tecnológica (Argentina), BID 802, PID Nr. PMT-SID 187.

Keywords: bioassay, ELISA, GM-CSF, monoclonal antibody, quantification.

Abbreviations: rhGM-CSF: recombinant human granulocyte-macrophage colony stimulating factor; ELISA: enzyme-linked-immunosorbent assays; MAb: monoclonal antibody; RIA: radioimmnunoassay; PAb: polyclonal antibody; FCS: foetal calf serum; PBS: phosphate buffered saline; LB: Luria-Bertani medium; HRP: horseradish peroxidase; OPD: o-phenylenediamine; CV: coefficients of variation; CHO: Chinese hamster ovary; SEM: standard error of the mean.

Three enzyme-linked-immunosorbent assays (ELISA) were developed and compared with a bioassay to quantify the recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF). These assays were suitable to quantify the non-glycosylated rhGM-CSF present in mixtures with variable protein content, and therefore useful for determining concentrations of the cytokine in production processes. Among these assays, the competitive ELISA, developed with a MAb that recognises an epitope not involved in glycosylation, was the only one suitable to quantify the glycosylated form of rhGM-CSF produced in mammalian cell cultures.