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

Autores
Abud, Julián Elías; Luque, Enrique Hugo; Ramos, Jorge Guillermo; Rodriguez, Horacio Adolfo
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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.
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
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
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
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
Materia
Glutathione S-Transferase
Immuno-Polymerase Chain Reaction
Monoclonal Antibodies
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/56067
Acceder
id CONICETDig_00c87c8a875eefcb64dde307d5e0265a
oai_identifier_str oai:ri.conicet.gov.ar:11336/56067
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferaseAbud, Julián ElíasLuque, Enrique HugoRamos, Jorge GuillermoRodriguez, Horacio AdolfoGlutathione S-TransferaseImmuno-Polymerase Chain ReactionMonoclonal Antibodieshttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3GST-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.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; ArgentinaFil: 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; ArgentinaFil: 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; ArgentinaFil: 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; ArgentinaAcademic Press Inc Elsevier Science2017-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/56067Abud, 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; 16-231046-5928CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2017.04.014info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046592817301419info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:32:33Zoai:ri.conicet.gov.ar:11336/56067instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:32:33.533CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
title Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
spellingShingle Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
Abud, Julián Elías
Glutathione S-Transferase
Immuno-Polymerase Chain Reaction
Monoclonal Antibodies
title_short Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
title_full Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
title_fullStr Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
title_full_unstemmed Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
title_sort Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase
dc.creator.none.fl_str_mv Abud, Julián Elías
Luque, Enrique Hugo
Ramos, Jorge Guillermo
Rodriguez, Horacio Adolfo
author Abud, Julián Elías
author_facet Abud, Julián Elías
Luque, Enrique Hugo
Ramos, Jorge Guillermo
Rodriguez, Horacio Adolfo
author_role author
author2 Luque, Enrique Hugo
Ramos, Jorge Guillermo
Rodriguez, Horacio Adolfo
author2_role author
author
author
dc.subject.none.fl_str_mv Glutathione S-Transferase
Immuno-Polymerase Chain Reaction
Monoclonal Antibodies
topic Glutathione S-Transferase
Immuno-Polymerase Chain Reaction
Monoclonal Antibodies
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv 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.
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
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
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
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
description 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.
publishDate 2017
dc.date.none.fl_str_mv 2017-07
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/56067
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
1046-5928
CONICET Digital
CONICET
url http://hdl.handle.net/11336/56067
identifier_str_mv 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
1046-5928
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2017.04.014
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046592817301419
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Academic Press Inc Elsevier Science
publisher.none.fl_str_mv Academic Press Inc Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1844612993831141376
score 13.070432

Publicaciones similares