I am trying to detect anti-influenza antibodies in cell culture of human B cells using hemagglutination assay inhibition. I am wondering if FBS in my media may already have non-specific inhibitors or bovine antibodies that could lead to false positives in this assay.
Hello,
It really is possible that you have some unwanted proteins in FBS.
Try to do tests with different concentrations of FBS, can help to understand this.
A hug
Eduardo
Hi Girija,
I think you can quite easily verified if your medium with FBS has inhibitory activity.
In control experiment you should perform the HI assay using the medium with FBS instead of culture supernate - the relevant result should be "no inhibition"
It is also important to work with subhemagglutination titre of the virus. Otherwise if you have too few viral particles , you will always have the lack of agglutination indicating the false positive reaction of Ab-dependent neutralization.
The FBS in your culture media almost certainly contains sialic acid bearing proteins which are "inhibiting" agglutination of the influenza virus independent of your mAb. If you want to confirm that this is the case, perform your virus backtiter in PBS or your culture media.
This phenomena is precisely why RDE (receptor destroying enzyme) is used to treat serum prior to testing in HAI assays. I would suggest either treating your culture supernatants with RDE or purifying the mAb before evaluating them for HAI activity. Best of luck!
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