WinterVomitingLab:Protocols/HBGA binding with NeutrAvidin plates

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Contents

Overview

Histo-blood group antigen (HBGA) binding of human norovirus using NeutrAvidin-coated wells and a stool suspension.

Materials

  • NeutrAvidin-coated (Pierce) 96-well plates or 8-well break away rows
  • PBS-T (PBS containing 0.05% Tween 20)
  • biotinylated HBGA (Glycotech)
  • blocking buffer of choice, such as;
    • SuperBlock (Pierce)
    • 5% Blotto
    • 5% Fetal Bovine Serum
    • 5% BSA
  • stool suspension containing human norovirus

Procedure

  1. Wash NeutrAvidin-coated (Pierce) 96 well plates or 8-well break away rows with PBS-T 3X.
  2. Coat wells with 10 µg of synthetic biotinylated HBGA (Glycotech) in 100 µl blocking buffer (we use SuperBlock). Be sure to leave one row/well uncoated (negative control).
  3. Incubate for 1.5 hr at RT.
  4. Wash 3X with PBS-T.
  5. Add 98 µl blocking buffer to each well. Add 2 µl of virus (or virus dilution) to both coated and uncoated wells. (Dilution series can be performed on the plate).
  6. Incubate for 2-2.5 hr at RT.
  7. Wash 5X with PBS-T. Note: using a plate washer for this step is not recommended since infectious virus is present. Be sure to discard spent wash solution in biohazard waste container.
  8. Wash 3X with PBS. See note in step above.
  9. Add 100 µl of lysis buffer to each well for viral RNA extraction method of choice. Alternatively, add 100 µl of RNase-free water to each well and heat-release viral RNA.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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References

Relevant papers and books

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