Hi Dr. Mohamed, 

To my knowledge, the problem is the agg adapted virus against the anti-chicken conjugate. You can start cultivating your virus on non-avian cell line MDCK for example. This will give you more purified virus and very little background. 

I hope that you find my answerhelpful.

All the love, all the success,

Allah with you

Basem

l

Hi Mohamed,

I have been measuring influenza specific antibodies (IgA, IgG & IgM) in human subjects vaccinated with TIV (Kurupati et al. B cell responses to the 2011/12-influenza vaccine in the aged. Aging (Albany NY). 2013 Mar;5(3):209-26). The protocol should be the same in chickens too. We do purify the virus in the eggs by similar method as you described but with minor modifications. 

Points to consider:

Hope this helps.

Raj

http://www.ncbi.nlm.nih.gov/pubmed/23674565

Article B cell responses to the 2011/12-influenza vaccine in the aged

Couple of suggestions:

1. Change your blocking buffer.  You can try 1% BSA in your blocking buffer instead of using nonfat dry milk.

2. Add some tween to you blocking buffer.  You can try and add a little tween (0.05%) to your blocking buffer as well to reduce the background.

3. What is your substrate that you are using?  The problem could also be there and changing it could increase your signal to noise ratio and therefore reduce your background.

Thanks Thomas Rowe for your suggestions.

1. I tried 1% BSA  as blocking buffer. It didn't show any difference, but it also increased the background.

2. I am using TMP substrate (KPL) as my secondary antibody is goat anti-chicken HRP labelled. Do you have another suggestion or alternative?

Other substrates to try:

OPD

ABTS

Thanks Thomas. I will try it and inform you with the results.

The other important aspect which may help is purity of your coating antigen. 

Thomas,

Can you please clarify what you mean by the purity of coating antigen?