Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein
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Data
2008-04-15
Autores
Taghavini, S. A. [UNESP]
Mikawa, A. Y. [UNESP]
Yamanaka, H. [UNESP]
Henrique-Silva, F.
Costa, P. I.
Título da Revista
ISSN da Revista
Título de Volume
Editor
Elsevier B.V.
Resumo
In 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.
Descrição
Palavras-chave
hepatitis C virus, core protein, recombinant antigen, siloxane-poly(propylene oxide) discs
Como citar
Talanta. Amsterdam: Elsevier B.V., v. 75, n. 2, p. 461-465, 2008.