We use a primary amine surface to bind our antibodies. Thereafter we use BSA and other protein blockers for blocking. The results are not really great, and vary quite a bit on repeats.
We wanted to improve the system and use other non-protein blocking strategies that can reduce non-specific binding (NSB) arising because of the primary amine reacting with the detection reagents we use. We have used PVP and PEG but they were not very effective.
We feel that aldehyde based blocking may work, but it is also possible that it can bind to the amine on the antibody reducing signal. What do you think?
Would highly appreciate the inputs on the same.
Rohan, having lots of amino groups on your surface means you have lots of positive charges at neutral pH. Basically, you have created an ion exchanger. Increasing salt concentration (NaCl should do) might do the trick. If not, you might have a real problem. If you're activating the amino surface with a cross-linker in order to bind the antibodies, you could try to block remaining amino groups with a reactive small molecule (Acetic anhydride, acetyl chloride, methyl iodide oder dimethyl sulphate might do) before adding the antibody.
An alternative approach (which is frequently used in industrial ELISA) is to coat an antibody that will bind your capture antibody (e.g., if your primary/capture antibody is from mouse, do a first coat with goat anti mouse).
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