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An automated high-throughput enterovirus D68 microneutralization assay platform.

Eric E Rhoden1, Bernardo A Mainou1, Jennifer L Konopka-Anstadt1

  • 1Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

Journal of Virological Methods
|July 25, 2022
PubMed
Summary

A new automated 384-well enterovirus microneutralization assay increases throughput for enterovirus D68 (EV-D68) seroprevalence studies. This high-density assay maintains reproducibility and reduces sample volume, supporting large-scale serological screening.

Keywords:
Acute flaccid myelitis (AFM)CCID(50)Cell culture 50 % infectious doseEnterovirus D68 (EV-D68)Viral cytopathic effect (CPE)Virus neutralization assay

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Area of Science:

  • Virology
  • Immunology
  • Assay Development

Background:

  • Virus neutralization assays are crucial for enterovirus serology.
  • Conventional 96-well assays assess enterovirus D68 (EV-D68) seroprevalence.
  • Higher-density assays can improve efficiency for large-scale serological studies.

Purpose of the Study:

  • To develop and validate an automated 384-well microneutralization assay for EV-D68.
  • To increase testing throughput and reduce sample volume compared to 96-well assays.
  • To ensure assay reproducibility and accuracy for large population-based serology.

Main Methods:

  • Automation of reagent dispensing, serial dilutions, and luminescence measurement.
  • Development of a 384-well microneutralization assay using EV-D68 strains.
  • Comparison of the 384-well assay with the conventional 96-well assay using human sera and control antisera.

Main Results:

  • The automated 384-well assay demonstrated high throughput, reproducibility, and reduced sample volume.
  • Coefficients of variation (CV) for accuracy were well below 20% (7.35%, 5.97%, 2.85%).
  • High concordance (0.955 correlation) was observed between 96-well and 384-well assay results.

Conclusions:

  • The automated 384-well microneutralization assay is a robust and efficient tool for EV-D68 serology.
  • This assay supports large-scale serological screens for EV-D68 neutralizing antibodies.
  • The validated assay enhances the capacity for population-based enterovirus surveillance.