Journal: PLoS ONE

Article Title: Patient-derived monoclonal antibody neutralizes HCV infection in vitro and vivo without generating escape mutants

doi: 10.1371/journal.pone.0274283

Figure Lengend Snippet: The neutralization effects of anti-E2 antibodies were assessed using an infection system with chimeric HCVcc, including (A) H77/JFH-1, (B) TH/JFH-1, (C) J6/JFH-1 and (D) S310/JFH-1. A total of 200 focus forming unit of these viruses were mixed with IgG or scFv at the designated concentrations and the mixtures were incubated at room temperature for 30 minutes. The antibody-HCVcc mixture was added to naïve Huh-7.5.1 cells (2 x 10 4 cells/well, 48-well plate) at 100 μL/well and plates were incubated in a 5% CO 2 incubator at 37°C for 3 hours. The antibody-HCVcc mixture then was removed and replaced with 500 μL of DMEM containing 10% FBS, and the cells were cultured in a 5% CO 2 incubator at 37°C for a further 72 hours. The cells then were washed with PBS, and Passive Lysis Buffer (Promega) was added at 100 μL/well to generate a cell lysate. HCV core protein in the collected cell lysate was quantified using a Lumipulse Ortho HCV Ag (Ortho Clinical Diagnostics, Tokyo, Japan). The efficiency of neutralization was calculated and presented as the % neutralization normalized to the amount of HCV core protein in the HCVcc-PBS mixture-inoculated cell lysate. Assay were performed in triplicate and infection rate are expressed as mean ± SEM. Statistical significance of difference was analyzed using one-way ANOVA with a Williams’ test (*P < 0.025, **P < 0.005, ***P < 0.0005 vs PBS).

Article Snippet: The culture supernatant again was removed and this time replaced with 200 μL of DMEM containing 10% FBS and 1% MEM nonessential amino acid solution (Thermo Fisher Scientific); the resulting cultures were incubated at 37°C for 48 hours.

Techniques: Neutralization, Infection, Incubation, Cell Culture, Lysis