I need to use viral antigen from enveloped virus as the coated antigen in ELISA. should I extract these viral antigen from the cell culture supernatant or use the entire cell culture? Any detergent? I tried to add NP-40 into the entire cell culture after freezing and thawing,yet failed. the color in ELISA is too weak. are there any better methods to tackle this problem? Hope u suggest relevant papers. thank u.
Hi, do you know the yield of viral Ag production? did you transfect cells or infect them (use a high MOI arround MOI 3 to 10)?
If you can not purified your viral antigen then you can fixed your cells in monolayer and performed your ELISA. Then if the viral Ag is expressed at the cell surface you don't need to permeabilized cells with PFA 4% (10min).
If the signal is too weak , you can track your viral Ag by ELISA but you need a specific Ab against your viral Ag.
good like
Anne
Have you tried ppting the viral particles/antigen with either PEG or Zinc-chloride, thereby concentrating the antigen that goes on the ELISA plate?
Avoid detergents, if possible, if you want the antigens to stick to the well of the your ELISA plate. If you are using a whole virus preparation then, depending on the virus, you need to understand the relative amount of a given viral antigen to the rest of the stuff that comes along with the envelope. The other stuff will be lipids, and cellular antigens. Not all viral antigens stick equally well to ELISA plates. The other stuff in the mix can also inhibit the binding of the antigens you are interested in.
If you are purifying viral antigen then that will be on a case by case basis for the optimal purification system. Is your viral antigen glycosylated, phosphorylated, myristalated, etc.? Starting with a decent viral titer you should be able to purify your target antigen with the appropriate HPLC methodology.
In the end try to avoid detergents in the final preparation as these will tend to inhibit binding to the plate.
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