Hello everyone,
I've recently purchased 1 ml of concentrated H1N1 A/California/7/2009 that is stored in allantoic fluid. I'd like to use the virus in an ELISA to detect strain specific antibodies from mucus supernatant and serum samples.
What is the best method purification to get a high yield?
I've seen other posts regarding this requestion but seem to address purification for large volumes of viral supernatant, such as labs that grown their own virus. This is my first time working with influenza and purifying virus, so any advice is much appreciated.
Dear Felix Lam,
A simple procedure which enables the concentration and purification of influenza virus,
rising an angular rotor, is described. Virus is concentrated over a sucrose step gradient. The
same gradients are reused and volumes up to 4 liters are concentrated in 1 day. The concentrated
virus is further purified by a simplified density-gradient technique. No host cell protein is
detectable in the final product. The technique offers a broad application potential for
concentrating and purifying other viruses
please find attached files,
Thanks and regards.
Rajkoti.K
Felix - do you know the titre of the stuff you bought? I'm afraid you're going to struggle to work with only 1 ml. You can try pelleting the virus through a sucrose pad in a benchtop ultra and your recoveries will be reasonable, but I very much doubt that there's enough material to make the next stage of density gradient separation feasible. The technique relies on there being enough virus loaded to be able to see the virus band because of light scattering - we would typically start with 100 ml of allantoic fluid from a poor-growing virus like Cal/7 to be able to see it. Starting with this much also accounts for the inevitable (50%?) losses of material.
If you need purified virus, my advice is to either invest the time in growing up larger stocks yourself in eggs (if this is feasible in your lab) or to find a collaborator who can supply you with material. We have lots of semi- and gradient-purified Cal/7 and related viruses as byproducts of our own experiments for instance, but if you can find someone more local to you than the UK the shipping and paperwork would be easier...
Paul
Small column chromatography (anion exchangers) is a possibility as well. You can find an example in the link. The smallest column is 0.1 mL and the elution could be done in less than 1 mL.
But it is not easy to predict the yield with very low loading of virus (far below the capacity of the column).
http://www.biaseparations.com/applications/viruses-vlps/product/771-purification-of-influenza-a-and-b-virus-using-ion-exchange-monolith-chromatography
Using a column is an option. The one Urh posted was for virus from Vero cells. There is a lot more protein in allantoic fluid so you might want to consider a column that deals with that (link below). But you still will have some loss.
http://virocyt.com/shop/viroprep-plus/
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