Protocol to Detection Antiviral Antibodies

Antiviral antibodies are promising candidates for the prevention and treatment of severe viral diseases. It is necessary to characterize the antiviral activity of antiviral antibodies in vitro and in vivo. Antiviral testing is done to reveal the antiviral properties of drugs, biomolecules, and other small molecules. Creative Biolabs provides antiviral drug testing services to help our clients analyze antiviral activity that inhibits the replication of viruses in vitro and in vivo models.

Antiviral Assays In Vitro

For many human viruses, there are cell culture systems or animal hosts in which the infectious agent can undergo a complete virus life cycle. It is important to obtain antiviral activity data using primary human target cells, if possible. Specific antiviral activity can use a quantitative assay to measure virus replication in the presence of increasing concentrations of the product compared to replication in the absence of the product. There are several ways to run an antiviral test.

Antiviral testing of antiviral substances and antibodies in vitro. Fig.1 Antiviral testing of antiviral substances and antibodies in vitro. (Aoki-Utsubo, 2018)

  • CPE-based Antiviral Assays
  • The antiviral activity of the product is evaluated based on the ability of the product to prevent the virus from causing viral CPE in mammalian cell culture. CPE is determined by microscopic observation of cell culture monolayers as well as the uptake of neutral red dye.

  • Plaque Reduction Assays (PRA)
  • This assay applies diluted doses of a possibly antiviral drug into multiwell plates to suppress the formation of plaques. The sample is incubated with the virus and the mixture is then added to monolayers of cells. Agar or carboxymethyl cellulose is overlaid on the cell monolayer to prevent the spreading of the virus which leads to plaque formation. After incubation with or without antibodies for a certain number of days, plaques are visualized with immunostaining.

  • Microneutralization or Virus Neutralization Assay
  • Equal volumes of virus and the tested dilutions of antibody samples are mixed and incubated for 1 h at 37 ℃. After the incubation period, the antibody/virus mixture is added to 96-well microplates containing cells susceptible to virus infection. The cell's plaque number is counted. The reciprocal of the lowest antibody dilution that can prevent the CPE effect is calculated as the virus-neutralizing (VN) titer.

  • Virus Yield Reduction Assays
  • Susceptible cells are infected with the virus and allowed to stand at 37 . After 30 min incubation, the test antibody at varying dilutions is added to the cells. The cultures are incubated for 3 days until a complete cytopathic effect is observed in untreated infected cells. The virus load of the supernatant is measured by a standard plaque assay.

Virus yield reduction assays. Fig.2 Virus yield reduction assays.

Antiviral Assays In Vivo

In cases where no in vitro cell culture or replicon system is predictive of antiviral activity in humans, measurement of viral titers after treatment in animal model systems can be used to assess the antiviral activity of the investigational product. Creative Biolabs routinely evaluates antiviral efficacy using in vivo models. We have capabilities for testing candidates in a wide range of viral infection models in rodents and non-human primates. Our experienced scientists can evaluate antiviral efficacy, immune responses to viral infection, pharmacokinetics (PK) studies, cytokine and biomarkers of viral disease, and maximum tolerated dose (MTD) studies. Please contact us for specific details since available models change periodically.

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Because of long development timeframes, antiviral antibodies are limited in rapid deployment and use. Virologists at Creative Biolabs are experienced with antiviral testing, including propagating viruses, determining viral titers, and testing the efficacy of antiviral drugs. Please feel free to contact us for further information.

Reference

  1. Aoki-Utsubo, C.; et al. Virucidal and neutralizing activity tests for antiviral substances and antibodies. Bio-protocol. 2018, 8(10): e2855-e2855.
All products and services are intended for Research Use Only, and NOT to be used in diagnostic or therapeutic procedures.

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