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

• Published in: Talanta (Oxford) 2008-04, Vol.75 (2), p.461-465
• Main Authors: Tagliavini, S.A., Mikawa, A.Y., Yamanaka, H., Henrique-Silva, F., Costa, P.I.
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Chemistry; Core protein; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli - genetics; Exact sciences and technology; Hepatitis C Antibodies - blood; Hepatitis C virus; Miscellaneous; Polymers - chemistry; Propylene Glycols - chemistry; Recombinant antigen; Recombinant Proteins - chemistry; Sensitivity and Specificity; Siloxanes - chemistry; Siloxane–poly(propylene oxide) discs
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2007.11.036

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 × 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 confirmed 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.