Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein

dc.contributorUniversidade Estadual Paulista (UNESP)
dc.creatorTaghavini, S. A.
dc.creatorMikawa, A. Y.
dc.creatorYamanaka, H.
dc.creatorHenrique-Silva, F.
dc.creatorCosta, P. I.
dc.date2014-05-20T15:31:39Z
dc.date2016-10-25T18:07:33Z
dc.date2014-05-20T15:31:39Z
dc.date2016-10-25T18:07:33Z
dc.date2008-04-15
dc.date.accessioned2017-04-06T00:23:17Z
dc.date.available2017-04-06T00:23:17Z
dc.identifierTalanta. Amsterdam: Elsevier B.V., v. 75, n. 2, p. 461-465, 2008.
dc.identifier0039-9140
dc.identifierhttp://hdl.handle.net/11449/40724
dc.identifierhttp://acervodigital.unesp.br/handle/11449/40724
dc.identifier10.1016/j.talanta.2007.11.036
dc.identifierWOS:000255270700021
dc.identifierhttp://dx.doi.org/10.1016/j.talanta.2007.11.036
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/883473
dc.descriptionIn this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L-1 of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and continued by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1: 10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1: 1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application. (c) 2007 Elsevier B.V. All rights reserved.
dc.languageeng
dc.publisherElsevier B.V.
dc.relationTalanta
dc.rightsinfo:eu-repo/semantics/closedAccess
dc.subjecthepatitis C virus
dc.subjectcore protein
dc.subjectrecombinant antigen
dc.subjectsiloxane-poly(propylene oxide) discs
dc.titlePolysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein
dc.typeOtro


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