Hi!
I've been trying to perform the haemagglutination assay in my lab using Guinea Pig erythrocytes. The problem we are having is that we don't get to observe the "button" of RBC in the no haemagglutinated controls. All wells seem to be haemaglutinated.
We are using Sterile Flat-bottom Microtitre 96-well plate (Sterilin 611F96). Could be the shape (maybe it should be U or V-shapped?) of the wells be the cause?
The Guinea Pig erythrocytes we are using are this ones: https://www.fitzgerald-fii.com/guinea-pig-red-blood-cells-88r-1047.html
We have tried different final concentrations of RBC in the wells, diluted in sterile PBS: 5%, 2.5%, 1.25% and 0.5%.
Could anybody suggest if we are doing something wrong or provide me a detailed protocol of their haemagglutination assay?
Thanks!
Joan
Please look at the following below links which may help you in your analysis:
https://consise.tghn.org/site_media/media/articles/2_Wood_HI_protocol_comparison_Sept_2013. https://www.aphis.usda.gov/animal_health/vet_biologics/publications/124.pdf
https://en.wikipedia.org/wiki/Hemagglutination_assay
https://microbeonline.com/hemagglutination-inhibition-test-hai-principle-procedure-result-interpretations/
Thanks
Thanks Isam,
I've finally realized that our problem was as simple as the shape of the wells. Indeed, the wells have to be U or V-shapped, because the red button is the consequence of the precipitation of the RBC at the bottom of the U or V-shapped well. If it's flat, there's no button to observe.
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