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Artículo

Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase

Abud, Julián ElíasIcon ; Luque, Enrique HugoIcon ; Ramos, Jorge GuillermoIcon ; Rodriguez, Horacio AdolfoIcon
Fecha de publicación: 07/2017
Editorial: Academic Press Inc Elsevier Science
Revista: Protein Expression and Purification
ISSN: 1046-5928
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Otras Biotecnologías de la Salud

Resumen

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 μg/ml. To develop the rGST-IPCR assay, we selected “Universal-IPCR” format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.
Palabras clave: Glutathione S-Transferase , Immuno-Polymerase Chain Reaction , Monoclonal Antibodies
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
Identificadores
URI: http://hdl.handle.net/11336/56067
DOI: https://dx.doi.org/10.1016/j.pep.2017.04.014
URL: https://www.sciencedirect.com/science/article/pii/S1046592817301419
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Articulos(ISAL)
Articulos de INSTITUTO DE SALUD Y AMBIENTE DEL LITORAL
Citación
Abud, Julián Elías; Luque, Enrique Hugo; Ramos, Jorge Guillermo; Rodriguez, Horacio Adolfo; Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase; Academic Press Inc Elsevier Science; Protein Expression and Purification; 135; 7-2017; 16-23
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