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elisa background - (Apr/12/2008 )

I have problems of background with my sandwish ELISA: i use a mouse monoclonal antibody for coating the plate then I block with PBS/BSA 1% and use a rabbit polyclonal serum against my protein (then anti-rabbit biotinylated then streptavidine peroxidase and finally TMB). Perhaps the serum polyclonal antibody contains antibodies against BSA (i've read something like this but i'm not sure i've understood correctly)?
The fact is that the lysis buffer has a value important (the same as when I put 1600pg of the protein in the well) and more intriguingly; cells lysates without the protein of interest (I'm sure because it's a viral protein and my cells are not infected by the virus) have the same value as 12500pg of the protein alone (in the same buffer)
I don't know what i can change in the protocol...if someone can help


Use 3-5% mlik to block instead of BSA
I had the sam problem and corrected when I used milk instead of BSA. I had background with FBS,BSA, ..etc


ok thanks I will try


Milk, casein, etc may work for you. Just remember any protein...any protein may react with the abs in your system. Mouse against cow, rabbit against mouse etc etc.

Search 'heterophile antibody' and you will learn more on the topic.