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).