An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

Published: April 30, 2024

Abstract

Source: Madrigal, J. L., et al. Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry. J. Vis. Exp. (2020).

This video demonstrates an assay for quantifying human norovirus virus-like particles (VLPs) binding to human intestinal bacteria. The bacterial suspensions are mixed with the VLPs and incubated to allow the formation of VLP-bacteria complexes. The complexes are then labeled with fluorophore-tagged VLP-specific antibodies, followed by flow cytometry-based visualization and quantification to identify the percentage of VLP-bound bacteria.

Protocol

1. VLP-bacteria Attachment Assay CAUTION: Human norovirus VLPs are a biosafety level (BSL)-2 hazard, and all work involving VLPs should be performed in a biosafety cabinet. Preparation of the bacterial cultures before the attachment assay should be performed using safety conditions appropriate for the organism. Reagent preparation​ 1x phosphate-buffered saline (PBS) Prepare 1 L of 10x PBS by dissolvi…

Divulgaciones

The authors have nothing to disclose.

Materials

5ml Polystrene Round-Bottom Tubes with Cell-Strainer Cap Corning 352235 After antibody staining, sample are transferred into tubes for flow cytometry analysis.
AnaeroPack Thermo Scientific R681001 Anaerobic gas pack used for culture of Lactobacillus gasseri
BD FacsDiva software
BD LSR Fortessa flow cytometer
Bovine Serum Albumin Fisher Bioreagents BP1605 Used for flow cytometry
Flow Cytometry Stain Buffer (FCSB) BD Biosciences 554657 Used for flow cytometry
Mouse IgG2b kappa Isotype Control (eBMG2b), PE, eBioscience Thermo Fisher Scientific 12473281 Isotype control. This antibody is purchased in the conjugated form from the manufacturer.
MRS Powder BD Biosciences 288130 Used for media preparation and to culture Lactobacillus gasseri.
Norovirus capsid G2 Monoclonal Antibody (L34D) Thermo Fisher Scientific MA5-18241 Norovirus GII antibody. This antibody is only available in the unconjugated form and thus must be fluorescently conjugated prior to use in the outlined flow cytometry assays. In this protocol, PE was the chosen fluor, however, other fluorescent molecules can be chosen as best suits the flow cytometer being used by the researcher.
Norovirus GII.4 VLP Creative Biostructure CBS-V700 Human norovirus virus like particle, VLPs were generated using the baculovirus system and resuspended in phosphate buffered saline with 10% glycerol. The authors performed independent nanosight tracking analysis to determine the particle concentration of the VLPs. The concentration is approximately 1011 VLPs per milliliter. Based on the protein concentration of the VLPs, approximately 200 particles are added per bacterium in VLP attachment assays.
PBS 10X Fisher Bioreagents BP665 Dilute to 1X prior to use.
SiteClick R-PE Antibody Labeling Kit Thermo Fisher Scientific S10467 Conjugation kit used for labling of unconjugated antibody.
Sodium Chloride Fisher Scientific S271 Used for media preparation
Tryptone Oxoid LP0042 Used for media preparation
Tube Revolver ThermoFisher Scientific 88881001 Used in virus:bacterium attachment assay. Set to max speed (40 rpm).
Yeast Extract BD Biosciences 212750 Used for media preparation
Agar Sigma A7002 Used for media preparation

Tags

Play Video

Citar este artículo
An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria. J. Vis. Exp. (Pending Publication), e22132, doi: (2024).

View Video