A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

Fournier, Carole; Duverlie, Gilles; François, Clément; Schnuriger, Aurélie; Dedeurwaerder, Sarah; Brochot, Étienne; Capron, Dominique; Wychowski, Czeslaw; Thibault, Vincent; Castelain, Sandrine

doi:10.1186/1743-422x-4-35

Cited by 19 publications

(21 citation statements)

References 36 publications

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“…Absolute quantification of infectious particles can be obtained by TCID 50 (50% tissue culture infectious dose) [6,191] and FFU (focus forming unit) assays [7,192] which are both applicable for all infectious HCV constructs and rely on immunolabelling of HCV-infected cells. An alternative is the use of a reporter system, with 14 either a reporter cell line (see Table 1) or a reporter virus (see Figure 3).…”

Section: Virus Titration Methodsmentioning

confidence: 99%

“…A resembling protocol is routinely used in our laboratory, with some modifications in the immunolabelling procedure and TCID 50 Next, the FFU assay corresponds to the quantification of HCV-infected cell foci [7,192]. As for the TCID 50 Last, the use of reporter viruses, and in particular the luciferase reporter viruses, is certainly the most convenient and time-saving alternative [63].…”

Section: Virus Titration Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Entry and replication of recombinant hepatitis C viruses in cell culture

Vièyres

Pietschmann

2013

Methods

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Section: Virus Titration Methodsmentioning

confidence: 99%

Section: Virus Titration Methodsmentioning

confidence: 99%

Entry and replication of recombinant hepatitis C viruses in cell culture

Vièyres

Pietschmann

2013

Methods

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“…The serum IgG fraction was purified using protein G-Sepharose (GE Healthcare), as previously described [8]. The IgG concentration was determined using a Bradford assay (BioRad).…”

Section: The Hepatitis C Virus (Hcv) Neutralizing Antibody (Nab) Respmentioning

confidence: 99%

“…The HCV focus reduction neutralization assay was performed in 96-well microtiter plates, as previously described [8]. Serial dilutions (ranging from 1:10 to 1:1280) of purified IgG (10 g) were established.…”

Section: The Hepatitis C Virus (Hcv) Neutralizing Antibody (Nab) Respmentioning

confidence: 99%

Low Levels of Hepatitis C Virus (HCV) Neutralizing Antibodies in Patients Coinfected with HCV and Human Immunodeficiency Virus

et al. 2008

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The hepatitis C virus (HCV) neutralizing antibody (nAb) response in 37 subjects with HCV monoinfection and 37 HCV-infected subjects with well-controlled human immunodeficiency virus (HIV) infection was evaluated using a focus reduction neutralization assay. HCV nAb levels were retrospectively studied in both groups of patients, who were matched on the basis of sex, age, and HCV genotype. The mean HCV nAb level (+/- standard deviation) among coinfected patients (1.613+/-0.416) was significantly less than that among monoinfected patients (1.912+/-0.578) (P= .013). Lower HCV nAb titers in coinfected patients could help worsen the outcome of HCV infection. These results favor starting HCV therapy as soon as possible in coinfected patients.

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“…The objective of our study was to examine the role of anti-HCV antibodies using the newly discovered infectious HCV culture system ( JFH-1), and to use this technique to examine the anti-HCV antibody titers in the sera of HCV patients, including IL-10-therapy recipients (20)(21)(22). Our study shows the presence of broad cross-reactive neutralization of antibodies from different HCV genotypes, with wide variation in the titers among individuals.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Anti-HCV Antibodies in IL-10-Treated Patients

et al. 2010

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There is limited information on the direct role of the neutralizing antibody responses against hepatitis C virus (HCV) infection or methodologies to study them. Previously we have demonstrated that interleukin-10 (IL-10) administered to chronic hepatitis patients led to a decrease in disease activity, but an increase in HCV viral burden. The mechanism behind this is unknown. The objective of this study was to examine the antibody response in IL-10-treated patients. To establish a neutralization antibody assay, HCV-positive and HCV-negative sera were collected and incubated with HCV strain JFH-1 particles before culture with Huh 7.5 cells. Viral replication was measured a week later by either indirect immunofluorescence assay (iIFA) or real-time reverse transcriptase polymerase chain reaction (RT-PCR). After validation of the methodology, the sera from 30 previously-described subjects of a group previously treated with IL-10 were tested for the neutralization capacity of their antibodies. The amount of total anti-HCV antibody in the sera was also measured by direct staining of HCV full-length replicon cells. With this validated neutralization assay for anti-HCV antibodies we found that HCVneutralizing antibodies are universally present, but with significantly different titers. In patients who were treated with IL-10, the total anti-HCV antibody titers appear to be constant, but with significantly decreased antibody neutralization activity. Our study validates an assay to quantitatively determine the presence and strength of HCV-specific neutralizing antibodies. We have found that IL-10-treated patients have significantly lower HCV antibodies, but maintain the total anti-HCV antibody titer, suggesting a novel mechanism by which IL-10 treatment increases viral load in patients.

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A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

Cited by 19 publications

References 36 publications

Entry and replication of recombinant hepatitis C viruses in cell culture

Entry and replication of recombinant hepatitis C viruses in cell culture

Low Levels of Hepatitis C Virus (HCV) Neutralizing Antibodies in Patients Coinfected with HCV and Human Immunodeficiency Virus

Characterization of Anti-HCV Antibodies in IL-10-Treated Patients

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