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.
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 number of duplicates.
2. Virus Neutralization
NOTE: Calculate the number of plates required for the neutralization assay. Each plate can accommodate one virus and a number of antisera.
NOTE: Depending on the number of antisera and the number of duplicates, a well plate can be set up with the following flexibilities: (1) The serum dilution can be arranged along either a row or a column (Figure 2). Each serum should occupy a separate row or column. (2) The increment of serum dilution is flexible, but it should start with the highest serum concentration at the top-left corner of a plate. (3) The number of duplicates balances the number of antisera. More duplicates can help to smooth out experimental variations. (4) The number of the VC and the number of the cell control (CC) duplicates are flexible, but they should also be in separate rows or columns. It is expected that the number of VC duplicates is significantly greater than that of the antisera.
3. Neutralization Quantification
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.
Figure 2: Schematics of a sample scanning system. (a) A 96-well plate on the imaging position of a flatbed scanner and (b) dimensions of the L-shape position limit shown in (a).
Figure 3: Desktop diagram of the "Wellplate Reader" quantitation software.
Figure 4: Example of a well-plate setup in a neutralization experiment. Each antiserum is assigned to two columns with duplicates (1 to 10). Rows are designed for different serum dilutions (A to H). The virus control takes Column 11, with eight duplicates (A11 to H11). The cell control is in Column 12, with eight duplicates (A12 to H12). Scale bar = 10 mm.
The authors have nothing to disclose.
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 |