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

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 solution 1:10 in DI water.
      4. Filter sterilize the solution by passing it through a 0.22 μm filter and storing it at room temperature.
   2. 5% BSA
      1. Add 5 g of bovine serum albumin (BSA) powder to 100 mL of PBS to generate a 5% (w/v) solution.
      2. Mix the solution by vortexing or using a metal stir bar on a stir plate until clumps have dissolved.
      3. Filter sterilize using a 0.22 μm filter.
      4. Store the solution at 4 °C.
   3. Flow cytometry stain buffer (FCSB)
      1. Filter purchased FCSB (see Table of Materials) through a 0.22 μm filter.
      2. Store the remaining solution at 4 °C.
   4. 5% Blocking Buffer (BB)
      NOTE: Prepare fresh each time.
      1. Add 0.5 g of BSA to 10 mL of sterile FCSB.
      2. Vortex to thoroughly mix the sample and leave on ice until use.
2. Antibody conjugation​
   1. Conjugate human norovirus GII antibody (see Table of Materials) with r-phycoerythrin (PE) using an R-PE antibody labeling kit per the manufacturer's instructions (see Table of Materials).
   2. Store the conjugated antibody in the dark at 4 °C for future use in VLP-bacterium attachment assay analysis.
      NOTE: The primary antibody should be titrated before use in the VLP-bacteria attachment assay to determine the proper concentration necessary for flow cytometry analysis. Antibody titration should be performed every time the conjugation reaction is performed and for each bacterium used in the attachment assay.
3. Antibody titration
   1. Dilute the conjugated anti-human norovirus GII antibody 100-fold, with a starting dilution of 1:100 and an ending dilution of 1:600 (six dilutions in total).
   2. Perform a virus attachment assay as described below with the following exception: in step 1.5.7, resuspend the bacterial pellet in 350 µL of 5% BB.
   3. Divide the sample into seven 50 µL aliquots.
   4. Add 50 µL of each antibody dilution to one tube.
   5. Add 50 µL of 5% BB to the seventh tube as an unstained control.
   6. Perform flow cytometry on all samples as described below.
   7. Compare diluted antibody samples to unstained controls and to each other. The lowest antibody concentration that does not result in a decrease or loss of positive signal should be chosen for subsequent assays.
4. Preparation of bacteria
   1. Grow the bacteria in 40 mL of appropriate liquid medium under proper atmospheric conditions until the bacteria are in the stationary phase.
      1. Grow Enterobacter cloacae cultures aerobically overnight in Luria Broth at 37 °C, shaking at 200 rpm to reach the stationary phase.
      2. Grow Lactobacillus gasseri cultures in De Man, Rogosa, and Sharpe (MRS) broth in black screw cap tubes without shaking in a water bath set to 37 °C for 18 h to reach the stationary phase.
   2. Transfer stationary phase cultures to clean 50 mL conical tubes and centrifuge samples at 2,288 x g for 10 min to pellet the bacteria.
   3. Remove the supernatant and resuspend it in 13 mL of sterile 1x PBS. Repeat this wash step for a total of two washes.
   4. Centrifuge the samples again, remove the supernatant, and resuspend the samples in 20 mL of 1x PBS.
      NOTE: If the cell pellet is small, the resuspension volume can be decreased.
   5. Measure the OD₆₀₀ of the culture.
   6. Using a standard curve, determine the colony-forming unit (CFU) per mL of the washed culture.
   7. Dilute the bacteria with 1x PBS to adjust the culture concentration to 1 x 10⁸ CFU/mL.
   8. Transfer 1 mL of the 1 x 10⁸ CFU/mL bacterial culture to the required 1.5 mL centrifuge tubes.
   9. Centrifuge the tubes at 10,000 x g for 5 min.
   10. Resuspend the bacterial pellet in 1 mL of sterile 5% BSA.
   11. Incubate the tubes for 1 h at 37 °C with constant rotation.
5. Virus attachment​
   1. Working inside a BSL-2 biosafety cabinet, add 10 µg of human norovirus (HuNoV) VLPs (see Table of Materials) to each tube containing bacteria resuspended in PBS and mix thoroughly by pipetting.
      NOTE: VLP concentration differs with each VLP preparation. Therefore, the volume added to bacteria will vary between preparation batches and should be adjusted accordingly so that 10 µg is added to each tube in the experiment. For bacteria-only controls (e.g., samples without VLP), add a volume of PBS that equals the volume of VLP added to experimental samples.
   2. Incubate tubes for 1 h at 37 °C with constant rotation.
      ​NOTE: A tube revolver (see Table of Materials) set to 40 rpm is used during incubation.
   3. After incubation, centrifuge the samples at 10,000 x g for 5 min.
   4. Discard the supernatant and resuspend the bacterial pellet in 1 mL of PBS.
   5. Repeat the wash steps 1.5.3 and 1.5.4.
   6. Centrifuge the samples at 10,000 x g for 5 min.
   7. Discard supernatant and resuspend bacterial pellet in 150 µL of 5% BB.
6. Antibody staining
   1. Prepare fresh 5% BB.
      ​NOTE: All work using the fluorescently tagged antibody should be performed in the dark, and the antibody, BB, and FCSB should be kept on ice.
   2. Dilute human norovirus GII antibody 1:125 for E. cloacae samples and 1:150 for L. gasseri samples in 5% BB, preparing 50 µL of diluted antibody per sample.
   3. Dilute the isotype antibody in the same way the human norovirus GII antibody was diluted for each bacterium in 5% BB, preparing 50 µL of diluted isotype control per sample.
   4. Divide each attachment assay sample from step 1.3.7 into three 50 µL aliquots by transferring them into clean 1.5 mL centrifuge tubes.
   5. To the first set of samples, add 50 µL of BB. This set will be the unstained (Uns) controls.
   6. Add 50 µL of the GII antibody dilution to the second set. This results in a final antibody concentration of 1:250 for E. cloacae and 1:300 for L. gasseri. This sample set will be the stained (AB) samples.
   7. Add 50 µL of the diluted isotype antibody to the third set. This set will be the isotype controls (IC). Mix well by pipetting. Incubate the samples on ice and in the dark for 30 min.
   8. Centrifuge all samples at 10,000 x g for 5 min.
   9. Discard the supernatant and resuspend the samples in 100 µL of FCSB.
   10. Centrifuge all samples at 10,000 x g for 5 min. Discard the supernatant and resuspend the samples in 100 µL of FCSB.
   11. Centrifuge all samples at 10,000 x g for 5 min.
   12. Discard the supernatant and resuspend the samples in 150 µL of FCSB.
   13. Transfer each sample to an FCSB tube containing 400 µL of FCSB buffer for a total volume of 550 µL.
   14. Keep samples at 4 °C until they are analyzed by flow cytometry.
       NOTE: Flow cytometry is performed within 4 h of antibody staining.