I am trying to develop an ELISA to measure antibodies to Hepatits B surface antigen in human serum.My detection antibody is an anti-human IgG-HRP. I getting a proper sigmoidal standard curve.
However I have tried coating with vaccine from 5 ug/ml to as low as 1 pg/ml concentration. I am getting the same absorbance and the standard curve looks more or less the same.
Can anyone please help me out in answering what is happening?
So your problem is that you reduce coating concentration to levels that are not normally used for coating (too low) and the result is the same, is that right?
I think you should also coat with a non-relevant antigen to see if the signal is related to specific recognition of your coating antigen or just non-specific background? In the latter case you should try to improve blocking on non-specific interactions by diluting the standard/samples in a proper blocking solution.
I would advise you to run a SDS PAAGe first to see if any other (abundant) protein(s) are present in the vaccine. Consider using a capture antibody (sandwich ELISA)
You can also use a monoclonal (anti HBSag) to check for specificity (most likely you would need a anti mouse conjugate)
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