In any biochemical assay it is paramount to maintain the highest achievable purity of the sample. And since you have concentrated the virus from the allantoic fluid, you should consider further purification of your sample.

I would suggest using ultracentrifugation. We in our lab follow a routine of 30% sucrose cushion purification followed by a sucrose gradient (10-40%), depending upon your need. Further purification is done using buffer exchange.

traces of yolk proteins are still present after ultracentrifugation, so in the vaccine preparations. It's good to have a sucrose gradient purified virus for HAI. Yolk should not affect HAI, but have controls.

Here's a dissenting answer...  I would say quite possibly not.

I've certainly never used purified virus in an HAI assay. It's a few years since I've done one so my memory may be a little off on the numbers, but say you use 4HAU of virus target and your virus stock in allantoic fluid is a good titre (512 HAU would have been low for us) then the dilution factor is likely to render allantoic fluid contaminants irrelevant. Also depending on the source and titres of the antisera you're interested in testing of course. If you can avoid the hassle, losses and damage to the virus involved in multiple centrifugation steps, life gets a lot quicker and easier.

Paul

PS - just seen your other query re Cal7/09... If this is the virus you're asking about above, then I expect the titre to be relatively low. I would still recommend trying an HAI without attempting purification because if you only have 1ml of stock to play with, you don't have enough to attempt the sucrose gradient methods suggested above.

Paul

Thank you everyone for their insight! It has been invaluable.

HA protein of the virus is what gives you haemagglutination, so egg contaminants do not count, but you can always use a negative control (non infected allantoid extract. If HAI titers are small, i would first amplify the virus on MDCK cell culture for 2-3 days and use the supernatant in HAI.