Artículo
Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor
Fuselli, Antonela
; Bürgi Fissolo, María de Los Milagros
; Kratje, Ricardo Bertoldo
; Prieto, Claudio
Fecha de publicación:
12/2022
Editorial:
Springer
Revista:
Applied Microbiology and Biotechnology
ISSN:
0175-7598
Idioma:
Inglés
Tipo de recurso:
Artículo publicado
Clasificación temática:
Resumen
Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses.
Palabras clave:
HEK-293
,
MONOCLONAL ANTIBODY
,
PROTEIN GLYCOSYLATION
,
STEM CELL FACTOR
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Identificadores
Colecciones
Articulos(CCT - SANTA FE)
Articulos de CTRO.CIENTIFICO TECNOL.CONICET - SANTA FE
Articulos de CTRO.CIENTIFICO TECNOL.CONICET - SANTA FE
Citación
Fuselli, Antonela; Bürgi Fissolo, María de Los Milagros; Kratje, Ricardo Bertoldo; Prieto, Claudio; Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor; Springer; Applied Microbiology and Biotechnology; 106; 24; 12-2022; 8121-8137
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