Development, Optimization and Validation of a Sandwich ELISA for Detection of Recombinant Human Factor VII
| Autor: | Sheila Sarial, Behrouz Vaziri, Sara Rezaeiravesh, Mohammadreza Abolhassan, Rozhan Sonboli, Shayan Maleknia, Fatemeh Ashori, S. Zomorrod Nouri, Fereidoun Mahboudi |
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| Rok vydání: | 2015 |
| Předmět: |
Detection limit
Chromatography biology Factor VII Chemistry medicine.drug_class Monoclonal antibody Molecular biology Epitope law.invention chemistry.chemical_compound Linear range law hemic and lymphatic diseases biology.protein Recombinant DNA medicine cardiovascular diseases Antibody Quantitative analysis (chemistry) |
| Zdroj: | South Asian Journal of Experimental Biology. 5:26-31 |
| ISSN: | 2230-9799 |
| DOI: | 10.38150/sajeb.5(1).p26-31 |
| Popis: | Recombinant human factor VII (rh-FVII) is produced by engineered BHK cells at low dose. Accordingly, establishment of a precise method is crucial to reliably measuring expression level of this protein during manufacturing pro-cesses. We developed and established a reproducible sandwich enzyme-linked immunosorbent assay (ELISA) method for measuring amount of FVII during biopharmaceutical in upstream and downstream processes of Aryo-Seven. A sandwich ELISA was designed using two different high affinity mon-oclonal antibodies (mAb1 and mAb2) against h-FVII. The bounded FVII to the first antibody was revealed by the use of a second mouse anti-FVII monoclo-nal antibody (1F1-B11), labeled with HRP that binds to another antigenic determinant of the FVII. Then, validation was done by determination of spec-ificity, linearity, accuracy, precision, reproducibility, detection and quantifica-tion limit and robustness according to ICH Q2 (R1) guideline. In developed ELISA, no interference was found between FVII and proteins derived from BHK which commonly exist in the supernatant. Linear range of detection was from 25-1.56 ng/mL with P-Value |
| Databáze: | OpenAIRE |
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