Blocking small molecules - (Sep/23/2009 )

I am having trouble with non-specific binding of a small biotinylated molecule as part of a competitive ELISA. I have found that the small molecule binds non-specifically to a variety of blocking agents. I have tried serum protein based blockers as well as a protein-free blocking buffer. In each case, the small molecule is detected more strongly in a blocked well than in a well not blocked at all. This suggests that the blocking agents are interacting with the conjugated molecule and are counter-effective. Below is the molecule that I cannot seem to block, that I am using to bind to coating antibody and detecting with HRP-streptavidin:

5-methoxytryptamino/PEG4/biotin

Any insight would be greatly appreciated.

In a "non-blocked at all well" your SA-HRP conjugate does not bind to the naked plastic surface?

There are also fish based blockers and peptide based materials if your background is truly blocker analyte related.

Do you have no background issue with ab coated, blocked plate and hrp conjugate or with blocked plate with conjugate only?

I wonder if instead of using the biotinylated molecule to compete with the analyte you used an HRP conjugated molecule?