Ns were excised, and the splenocytes were fused with Sp2/0 mouse myeloma cells using polyethylene glycol 4000 (Merck). Hybridoma were selected on HAT (Hypoxanthine-aminopterin-thymidine medium) supplemented by IMDM subsequently. Hybridoma obtained were tested for specific antibody production using ELISA and subcloned to obtain single cells. Monoclonal antibodies (mAbs) were purified from culture supernatant by affinity chromatography on a protein A-Sepharose column by following standard procedures [31].Purification of HCV-LPsSf21 cells were infected with recombinant baculovirus at a moi of 5?0, and cells were harvested 72 h post infection. Cell pellets were washed with phosphate buffered saline (PBS: 50 mM phosphate buffer pH 7.2 containing 150 mM NaCl) thrice and were resuspended using a tissue homogenizer in a lysis buffer (50 mM Tris, 50 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 0.1 NP40 and 0.25 protease inhibitors). The lysate was centrifuged at 15006g for 15 min at 4uC and the supernatant was pelleted 18325633 over a 30 sucrose cushion. The pellet was resuspended in 20 mM Tris and 150 mM NaCl which was then applied on a 20 to 60 sucrose gradient in SW41 rotor (Beckman). After 22 h of ultracentrifugation at 30,000 rpm at 4uC, fractions (1 ml) were collected and tested for E1 and E2 by enzyme-linked immunosorbent assay (ELISA) and western blotting. Anti E1 2 polyclonal antibody raised in rabbit was used for the above assays. Fractions containing HCV-LPs were diluted with 10 mMImmunoassays. (i) ELISAMicrotiter ELISA plates (Nunc) were coated overnight with antigen (HCV-LP) (5 mg/ml in PBS) followed by blocking of unoccupied sites with 0.5 gelatin in PBS. The plates were incubated with different culture supernatant samples. After three washes with PBS containing 0.05 Tween 20, the plates were incubated with rabbit anti-mouse Ig-HRP conjugate (DAKO, Glostrup, Denmark) for 1 h. The bound-peroxidase activity was detected using tetramethylbenzidine (TMB) and 0.03 H2O2. The reaction was stopped with 1 M H2SO4, and absorption at 450 nm was measured in an ELISA plate reader (Spectramax; Molecular Devices).(ii) Western BlottingHCV-LPs were electrophoresed on 10 polyacrylamide gel under reducing conditions and transferred onto nitrocellulose membranes. After blocking the non-specific sites with 0.5 BSAMonoclonal Antibodies Inhibiting HCV 6R-Tetrahydro-L-biopterin dihydrochloride chemical information InfectionFigure 2. Tubastatin A site Inhibition of HCV-LP and Huh7 cell binding by mAbs. HCV-LPs of both genotypes 1b and 3a were incubated with increasing concentrations of mAbs as indicated. The Y-axis depicts the percentage activity representing both the percent binding (dark grey) and percent inhibition HCV-LP attachment (light grey). doi:10.1371/journal.pone.0053619.gin PBS, the membranes were incubated with mouse antibodies specific to the HCV-LP, followed by rabbit anti-mouse Ig-HRP conjugate. The blot was developed with diaminobenzidine (1 mg/ ml in citrate buffer, pH 5.0, containing 0.05 H2O2) or Enhanced Chemiluminescence.Inhibition of Binding of HCV-LP to Huh7 Cells by Monoclonal Antibodies against Genotypes 1b and 3aHCV-LPs were pre-incubated with different concentrations of purified individual monoclonal antibodies and the complexes were allowed to react with Huh 7 cells. The binding of the labeled VLPs was monitored by flow cytometry analysis as described above.Monoclonal Antibodies Inhibiting HCV InfectionTable 2. Percentage inhibition of HCV-LP genotype 3a binding to Huh 7 cells using monoclonal antibodies.Table 3. Per.Ns were excised, and the splenocytes were fused with Sp2/0 mouse myeloma cells using polyethylene glycol 4000 (Merck). Hybridoma were selected on HAT (Hypoxanthine-aminopterin-thymidine medium) supplemented by IMDM subsequently. Hybridoma obtained were tested for specific antibody production using ELISA and subcloned to obtain single cells. Monoclonal antibodies (mAbs) were purified from culture supernatant by affinity chromatography on a protein A-Sepharose column by following standard procedures [31].Purification of HCV-LPsSf21 cells were infected with recombinant baculovirus at a moi of 5?0, and cells were harvested 72 h post infection. Cell pellets were washed with phosphate buffered saline (PBS: 50 mM phosphate buffer pH 7.2 containing 150 mM NaCl) thrice and were resuspended using a tissue homogenizer in a lysis buffer (50 mM Tris, 50 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 0.1 NP40 and 0.25 protease inhibitors). The lysate was centrifuged at 15006g for 15 min at 4uC and the supernatant was pelleted 18325633 over a 30 sucrose cushion. The pellet was resuspended in 20 mM Tris and 150 mM NaCl which was then applied on a 20 to 60 sucrose gradient in SW41 rotor (Beckman). After 22 h of ultracentrifugation at 30,000 rpm at 4uC, fractions (1 ml) were collected and tested for E1 and E2 by enzyme-linked immunosorbent assay (ELISA) and western blotting. Anti E1 2 polyclonal antibody raised in rabbit was used for the above assays. Fractions containing HCV-LPs were diluted with 10 mMImmunoassays. (i) ELISAMicrotiter ELISA plates (Nunc) were coated overnight with antigen (HCV-LP) (5 mg/ml in PBS) followed by blocking of unoccupied sites with 0.5 gelatin in PBS. The plates were incubated with different culture supernatant samples. After three washes with PBS containing 0.05 Tween 20, the plates were incubated with rabbit anti-mouse Ig-HRP conjugate (DAKO, Glostrup, Denmark) for 1 h. The bound-peroxidase activity was detected using tetramethylbenzidine (TMB) and 0.03 H2O2. The reaction was stopped with 1 M H2SO4, and absorption at 450 nm was measured in an ELISA plate reader (Spectramax; Molecular Devices).(ii) Western BlottingHCV-LPs were electrophoresed on 10 polyacrylamide gel under reducing conditions and transferred onto nitrocellulose membranes. After blocking the non-specific sites with 0.5 BSAMonoclonal Antibodies Inhibiting HCV InfectionFigure 2. Inhibition of HCV-LP and Huh7 cell binding by mAbs. HCV-LPs of both genotypes 1b and 3a were incubated with increasing concentrations of mAbs as indicated. The Y-axis depicts the percentage activity representing both the percent binding (dark grey) and percent inhibition HCV-LP attachment (light grey). doi:10.1371/journal.pone.0053619.gin PBS, the membranes were incubated with mouse antibodies specific to the HCV-LP, followed by rabbit anti-mouse Ig-HRP conjugate. The blot was developed with diaminobenzidine (1 mg/ ml in citrate buffer, pH 5.0, containing 0.05 H2O2) or Enhanced Chemiluminescence.Inhibition of Binding of HCV-LP to Huh7 Cells by Monoclonal Antibodies against Genotypes 1b and 3aHCV-LPs were pre-incubated with different concentrations of purified individual monoclonal antibodies and the complexes were allowed to react with Huh 7 cells. The binding of the labeled VLPs was monitored by flow cytometry analysis as described above.Monoclonal Antibodies Inhibiting HCV InfectionTable 2. Percentage inhibition of HCV-LP genotype 3a binding to Huh 7 cells using monoclonal antibodies.Table 3. Per.
