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Related Experiment Video

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Optimization of a Quantitative Micro-neutralization Assay
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Development of a microneutralization assay for HSV-2.

Melanie S Horton1, Michael Minnier2, Scott Cosmi3

  • 1Infectious Diseases and Vaccines Discovery, Merck & Co., Inc., West Point, PA, USA.

Journal of Virological Methods
|August 26, 2021
PubMed
Summary

A new microneutralization assay (MNA) offers a faster, high-throughput alternative to the traditional Plaque Reduction Neutralization Test (PRNT) for measuring Herpes simplex virus type-2 (HSV-2) neutralizing antibodies, crucial for drug and vaccine development.

Keywords:
Antibody characterizationAssay developmentHSV-2NeutralizationSerology

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Area of Science:

  • Virology
  • Immunology
  • Assay Development

Background:

  • The Plaque Reduction Neutralization Test (PRNT) is the gold standard for quantifying neutralizing antibodies against Herpes simplex virus type-2 (HSV-2).
  • However, the PRNT is recognized as a laborious, time-consuming, and low-throughput method.
  • Developing a high-throughput microneutralization assay (MNA) is essential for accelerating drug and vaccine development for HSV-2.

Purpose of the Study:

  • To develop and validate a high-throughput microneutralization assay (MNA) for quantifying HSV-2 neutralizing antibodies.
  • To compare the performance of the developed MNA with the traditional Plaque Reduction Neutralization Test (PRNT).

Main Methods:

  • Generation of a microneutralization assay (MNA) using anti-HSV monoclonal antibodies (mAbs) for virus detection in HSV-2 infected Vero cells.
  • Antibody selection and characterization via ELISA, infected cell binding assays, and Western Blot.
  • Determination and comparison of anti-HSV-2 neutralizing titers in immune sera using both MNA and PRNT, analyzed by linear regression.

Main Results:

  • The developed 96-well MNA demonstrated strong correlation with the traditional PRNT for determining HSV-2 neutralization titers.
  • Correlation coefficients (R²) were 0.8250 in the absence of complement and 0.7075 in the presence of complement.
  • The MNA significantly reduces assay time, saving approximately 90% of the time compared to the PRNT.

Conclusions:

  • A microneutralization assay (MNA) has been successfully developed as a viable, high-throughput alternative to the PRNT for assessing anti-HSV-2 neutralizing activity.
  • The MNA offers substantial time savings and increased throughput, facilitating rapid evaluation of large sample sets in antiviral research.
  • This advancement supports accelerated drug and vaccine development efforts for Herpes simplex virus type-2 infections.