Development of diagnostic methods and study of the immunoreactivity of a mixture of recombinant core and E2 proteins fused to GST with control serum positive for hepatitis C

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dc.contributor.authorKenfe, Flavia Regina [UNESP]
dc.contributor.authorUrbaczek, Ana Carolina [UNESP]
dc.contributor.authorSilva, Juliana Cristina [UNESP]
dc.contributor.authorNeo, Thalita Athie [UNESP]
dc.contributor.authorSilva, Flavio Henrique da
dc.contributor.authorCosta, Paulo Inácio da [UNESP]
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.contributor.institutionUniversidade Federal de São Carlos (UFSCar)
dc.date.accessioned2014-12-03T13:09:12Z
dc.date.available2014-12-03T13:09:12Z
dc.date.issued2013-06-15
dc.description.abstractThe hepatitis C virus (HCV) is an enveloped virus that is about 50-70 nm in diameter, has positive-strand RNA, and belongs to the genus Hepacivirus and the family Flaviridae. The detection and quantification of the core antigen, HCV nucleocapsid protein, has been successful in many trials and is considered a marker of viral replication since it presents a sequence of highly conserved amino acids, giving it high sensitivity and specificity. The E2 protein is an envelope glycoprotein of HCV with 11 glycosylation sites; most of these are well-conserved, making it a target antigen. The aim of this study is to develop high-sensitivity, low-cost diagnostic methods for HCV, which could be used for serological screening. The genomic regions encoding the core (part 136 aa) and E2 proteins of HCV were expressed in Escherichia coli Rosetta strain, cloned in expression vector pET-42a, and induced with 0.4 mmolL(-1) IPTG, producing recombinant proteins that were fused to glutathione S-transferase (GST) protein, which was then purified by affinity chromatography. The immunoreactivity was assessed by Western blot, Slot Blot, and developed and improved diagnostic methods (capture, indirect, and immunoblotting enzyme-linked immunosorbent assay (ELISA)). After applying the results to the formulas for determining the quality parameters, obtained for immunoblotting method 100% sensitivity and specificity and for ELISA 100% sensitivity and 87.5% specificity. The methods developed were more sensitive and specific using the mixture of the recombinant proteins fused to GST (core+E2). (C) 2012 Elsevier B.V. All rights reserved.en
dc.description.affiliationUniv Estadual Paulista, Sao Paulo State Univ, UNESP, Dept Biosci & Biotechnol Appl Pharm, BR-14802470 Araraquara, Brazil
dc.description.affiliationUniv Fed Sao Carlos, UFSCAR, Dept Genet & Evolut, Sao Paulo, Brazil
dc.description.affiliationUnespUniv Estadual Paulista, Sao Paulo State Univ, UNESP, Dept Biosci & Biotechnol Appl Pharm, BR-14802470 Araraquara, Brazil
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.sponsorshipFUNDECIF, Araraquara-SP, Brazil
dc.description.sponsorshipIdFAPESP: 08/58612-3
dc.format.extent32-38
dc.identifierhttp://dx.doi.org/10.1016/j.talanta.2013.02.017
dc.identifier.citationTalanta. Amsterdam: Elsevier Science Bv, v. 110, p. 32-38, 2013.
dc.identifier.doi10.1016/j.talanta.2013.02.017
dc.identifier.issn0039-9140
dc.identifier.lattes6720223715917381
dc.identifier.orcid0000-0002-3350-8308
dc.identifier.urihttp://hdl.handle.net/11449/112060
dc.identifier.wosWOS:000319089500006
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relation.ispartofTalanta
dc.relation.ispartofjcr4.244
dc.relation.ispartofsjr1,186
dc.rights.accessRightsAcesso restrito
dc.sourceWeb of Science
dc.subjectHepatitis C virusen
dc.subjectGST-core proteinen
dc.subjectGST-E2 proteinen
dc.subjectCapture ELISAen
dc.subjectIndirect ELISAen
dc.subjectImmunoblottingen
dc.titleDevelopment of diagnostic methods and study of the immunoreactivity of a mixture of recombinant core and E2 proteins fused to GST with control serum positive for hepatitis Cen
dc.typeArtigo
dcterms.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dcterms.rightsHolderElsevier B.V.
unesp.author.lattes6720223715917381[6]
unesp.author.orcid0000-0002-3350-8308[6]
unesp.campusUniversidade Estadual Paulista (Unesp), Faculdade de Ciências Farmacêuticas, Araraquarapt

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