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dc.contributor.author
Abud, Julián Elías
dc.contributor.author
Luque, Enrique Hugo
dc.contributor.author
Ramos, Jorge Guillermo
dc.contributor.author
Rodriguez, Horacio Adolfo
dc.date.available
2018-08-17T13:37:14Z
dc.date.issued
2017-07
dc.identifier.citation
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
dc.identifier.issn
1046-5928
dc.identifier.uri
http://hdl.handle.net/11336/56067
dc.description.abstract
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.
dc.format
application/pdf
dc.language.iso
eng
dc.publisher
Academic Press Inc Elsevier Science
dc.rights
info:eu-repo/semantics/openAccess
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.subject
Glutathione S-Transferase
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Immuno-Polymerase Chain Reaction
dc.subject
Monoclonal Antibodies
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Otras Biotecnologías de la Salud
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Biotecnología de la Salud
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CIENCIAS MÉDICAS Y DE LA SALUD
dc.title
Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
dc.type
info:eu-repo/semantics/article
dc.type
info:ar-repo/semantics/artículo
dc.type
info:eu-repo/semantics/publishedVersion
dc.date.updated
2018-08-16T17:20:22Z
dc.journal.volume
135
dc.journal.pagination
16-23
dc.journal.pais
Países Bajos
dc.journal.ciudad
Amsterdam
dc.description.fil
Fil: Abud, Julián Elías. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina
dc.description.fil
Fil: Luque, Enrique Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina
dc.description.fil
Fil: Ramos, Jorge Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
dc.description.fil
Fil: Rodriguez, Horacio Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina
dc.journal.title
Protein Expression and Purification
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/https://dx.doi.org/10.1016/j.pep.2017.04.014
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046592817301419
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