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

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.

1. Reagent preparation​
   1. 1x phosphate-buffered saline (PBS)
      1. Prepare 1 L of 10x PBS by dissolving a packet in 1 L of deionized (DI) water.
      2. Autoclave solution for 30 min.
      3. Prepare a 1x solution by diluting the above solutio....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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