These results are in agreement with those of previous studies in which the putative nucleocapsid protein (C) and non-structural proteins (NS3) were found to contain the most immunodominant epitopes (15C19). Several studies have indicated that a peptide spanning aa 2C120 of the C gene (C22) is a major component of the commercially available second-generation anti-HCV tests. 94.33%. In conclusion, our new Elbasvir (MK-8742) testing method is rapid, simple, sensitive and specifically detects the presence of anti-HCV antibodies in human serum or plasma. Therefore, it may be used for monitoring HCV. Keywords: hepatitis C virus, recombinant protein, double antibody sandwich-lateral flow immunoassay, colloidal gold Introduction It is estimated that approximately 180 million people have been infected with the hepatitis C virus (HCV) and approximately 130 million people are chronic HCV carriers (1). HCV is a common cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide (2C4). Currently, there are two main methods for detecting an HCV infection: one detects viral RNA by RT-PCR (5C8) and the other detects HCV antibodies by immunoassay [enzyme-linked immunosorbent assay (ELISA)] in serum (9,10). The sensitive ELISA assay uses recombinant viral proteins corresponding to multiple polypeptides from different viral regions, including structural proteins and non-structural polypeptides (11C13). It can be used as a confirmation test. However, its usage is limited in the clinical setting as the procedure requires sophisticated laboratory equipment and there Elbasvir (MK-8742) is Elbasvir (MK-8742) a high probability of contamination. Therefore, the purpose of our study was to develop an affordable and reliable rapid lateral flow test to detect the presence of HCV antibodies in blood samples by screening for HCV antigens, which would help decrease the chances of HCV infection from blood transfusions. Materials and methods Plasmids and bacterial strains The p90/HCVFLlongpU plasmid carrying full-length coding sequences of HCV was a Elbasvir (MK-8742) generous gift from Professor Charles M. Rice from the Center for the Study of Hepatitis C, Rockefeller University, New York, NY, USA. The (strains, Jm109, DH5a and BL21 (DE3), were used as the cloning and expression hosts. Reagents and instruments A panel of 23 standard positive sera, 8 standard negative sera, a set of quality control references for anti-HCV detection that contain known amounts of anti-HCV antibodies (Artron BioResearch Inc., Burnaby, BC, Canada) and 300 clinical sera were used for the antigenicity assessment of HCV proteins. Other reagents and instruments included goat anti-mouse HCV IgG polyclonal antibody, 30C60 nm colloidal gold particles (from Artron BioResearch Inc.), HCV-ELISA (KHB, Shanghai, China), a NanoDrop? ND-1000 Spectrophotometer, a Bio-Rad BioLogic LP, ZQ4000 test strip cutter, and XYZ-3000 Bio-Dot (all from Bio-Rad, Shanghai, China). Construction and expression of recombinant HCV antigens To obtain the HCV antigens, the sequences encoding the desired regions in the HCV genome were amplified by RT-PCR, and cloned into the prokaryotic expression vectors, pQE30 (Qiagen, Hilden, Germany), pET32a(+) (Novagen, Darmstadt, Germany), or pGEX-4T-2 IL22R (GE Healthcare Life Sciences, Chalfont St. Giles, UK), in-frame downstream of the 6-His-tag or glutathione S-transferase (GST)-tag coding sequence. Primers used for the HCV PCR amplification are listed in Table I and the structures of the plasmids are illustrated in Fig. 1. BL21 (DE3) cells harbouring the HCV gene fragment were grown at 37C in LB medium containing 50 g/ml of ampicillin to OD600 = 0.8. The expression of the antigens was induced by adding isopropyl–D-thiogalactopyranoside (IPTG) to a final concentration of 1 1 mmol/l. The cells were harvested 4C6 h later by centrifugation at 10,000 rpm for 15 min and stored at ?20C. Solubility analyses of expression products were performed as previously described (14). Briefly, harvested bacteria were re-suspended in phosphate-buffered saline (PBS; containing 140 mmol/l NaCl, 2.7 mmol/l KCl, 10 mmol/l Na2HPO4, 1.8 mmol/l KH2PO4, pH 7.3), sonicated on an ice-bath, and centrifuged at 10,000 rpm for 20 min at 4C. After centrifugation, the soluble and insoluble fractions were analyzed for the presence of expression products. Open Elbasvir (MK-8742) in a separate window Figure 1. HCV genome and recombinant proteins. (A) The HCV genome contains a single major open reading frame (ORF) flanked by untranslated regions (UTRs). The 10 proteins encoded within the main ORF are indicated by alternated shading..