WinterVomitingLab:Protocols/HBGA binding with NeutrAvidin plates
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Histo-blood group antigen (HBGA) binding of human norovirus using NeutrAvidin-coated wells and a stool suspension.
- 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
- Wash NeutrAvidin-coated (Pierce) 96 well plates or 8-well break away rows with PBS-T 3X.
- 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).
- Incubate for 1.5 hr at RT.
- Wash 3X with PBS-T.
- 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).
- Incubate for 2-2.5 hr at RT.
- 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.
- Wash 3X with PBS. See note in step above.
- 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.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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