Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay

Published: January 31, 2024

Abstract

Source: Lin, Y., et al. Optimization of a Quantitative Micro-neutralization Assay. J. Vis. Exp. (2016)

This video demonstrates the imaging-based micro-neutralization assay to analyze the antigenic relations between influenza A and B viruses. It provides a quantitative and visual way to assess the effectiveness of antibodies or other treatments in preventing viral infection.

Protocol

1. Virus Titration NOTE: Depending on the number of viruses and the number of duplicates, a well plate can be set up with the following flexibilities: (1) The viral dilution can be arranged along either the rows or the columns (Figure 1). Each virus should occupy a separate row/column. (2) The viral dilution increment is flexible, but it should start with the highest viral concentration at the top-left corner. (3) There is no restriction on the …

Representative Results

Figure 1: Example of a well-plate setup in a virus titration experiment. Test viruses are assigned to separate rows (A to H). Columns are designed for different viral dilutions (1 to 12). Two columns were used as duplicates for each viral dilution. Scale bar = 10 mm. …

Disclosures

The authors have nothing to disclose.

Materials

VGM Sigma D6429, P0781 500 ml DMEM+5 ml Pen/Strepb
PBS A Nature pH Phosphate-buffered saline: NaCl 10 g, KCl 0.25 g, Na2HPO4 1.437 g, KH2PO4 0.25 g, and Dist. Water 1 L
Avicell FMC RC-581F 2.4 g in 100 ml distilled water dissolved by agitation on a magnetic stirrer for 1 hr. Sterilize by autoclaving
2x DMEM Gibco 21935-028
Trypsin Sigma T1426
Overlay (10 ml/plate): 5 ml 2x DMEM, Trypsin 2 μg/ml final concentration, Avicell 5 ml
Triton X- 100 e Sigma T8787 Permeabilization buffer: 0.2% in PBS A (v/v)
Tween 80 Sigma P5188 Wash Buffer: 0.05% Tween 80 in PBS A (v/v)
Horse serum PAA Labs Ltd B15-021 ELISA Buffer: 10% in PBS A (v/v) + 0.1% Tween 80
Mouse MAb against influenza type A Biorad MCA 400 1 st Antibody: 1:1,000 in ELISA Buffer
Goat anti-mouse IgG (H+L) HRP conjugate Biorad 172-1011 2 nd Antibody: 1:1,000 in ELISA Buffer
True Blu peroxidase substrate KPL 50-78-02 Substrate: True blue +0.03% H2O2 g (1:1,000 of 30% solution)
96-well flat-bottom microtitre plates Costar 3596
8 channel multiwall-plate washer and manifold Sigma M2656
Perfection Plate Scanner Epson V750 Pro The imaging software can be downloaded freely from http://www.epson.com/cgi-bin/ Store/support/supDetail.jsp? oid=66134&infoType=Downloads.
Software operational environment: LabVIEW National Instruments Corporation Window based with NI Vision builder Version: LabVIEW2012 or above

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Cite This Article
Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay. J. Vis. Exp. (Pending Publication), e21924, doi: (2024).

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