Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti-vaccinia-neutralizing antibodies in human sera

Eyal, Osnat; Olshevsky, Udy; Lustig, Shlomo; Paran, Nir; Halevy, M.; Schneider, Paula A.; Zomber, Gil; Fuchs, Pinhas

doi:10.1016/j.jviromet.2005.05.027

Cited by 23 publications

(16 citation statements)

References 7 publications

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“…We analysed a number of parameters (such as practicability, reproducibility and specificity) and tested the effect of a range of variables (viral inoculum size, incubation time, fixation and permeabilization methods, blocking and revelation reagents) on these parameters (data not shown). Overall, the neutralization assay described in this study performs similarly to standardized neutralization assays for many other viruses [26][27][28].…”

Section: Discussionmentioning

confidence: 99%

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

Fournier

Duverlie

François

et al. 2007

Virol J

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Background/Aim: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies.

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Section: Discussionmentioning

confidence: 99%

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

Fournier

Duverlie

François

et al. 2007

Virol J

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“…For plaque reduction neutralization test (PRNT) 34 , Vero E6 cells were seeded overnight (as detailed above) at a density of 0.5 × 10 6 cells/well in 12-well plates. Antibody samples were 2-fold serially diluted (ranging from 100 to 0.1 μg/ml) in 400 μl of MEM supplemented with 2% FBS, MEM non-essential amino acids, 2 nM L-Glutamine, 100 Units/ml Penicilin, 0.1 mg/ml streptomycin and 12.5 Units/ml Nystatin (Biological Industries, Israel).…”

Section: Methodsmentioning

confidence: 99%

A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

et al. 2020

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The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

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“…In recent years, there have been several assays developed that are high-throughput, semi-automated, and do not rely on plaque formation and manual counts. Some of these assays detect aggregate cell infection as indicated by enzyme immunoassay [17] or the expression of recombinant reporter genes, such as b-galactosidase (b-gal) and green fluorescent protein (GFP) [18,19]. Perceived difficulties of these assays may include the use of cell suspension cultures for GFP assays, which may be laborious to maintain, and a lower dynamic range observed with enzymatic (BGZ) assays.…”

Section: Introductionmentioning

confidence: 99%

A Novel High-Throughput Vaccinia Virus Neutralization Assay and Preexisting Immunity in Populations from Different Geographic Regions in China

et al. 2012

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Background Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. Methodology/Principal Findings A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30–55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. Conclusion A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

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Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti-vaccinia-neutralizing antibodies in human sera

Cited by 23 publications

References 7 publications

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

A Novel High-Throughput Vaccinia Virus Neutralization Assay and Preexisting Immunity in Populations from Different Geographic Regions in China

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