Nonspecific primary antibody in ELISA? - (May/11/2015 )
I bought the components for Upstate 17-289 (the HAT-ELISA assay kit), which is no longer being manufactured together. The primary antibody is a rabbit anti-acetyllysine antibody also from Upstate (catalog number 06-933. The data sheet doesn't say it's rated for ELISA, but EMD Millipore (the new parent company of Upstate) told me it is). Even if I dilute the antibody to a 1:10000 dilution (the recommended dilution is 1:250), I get a heavy nonspecific signal (with secondary concentrations as low as 1:50000). ThermoFisher, which manufactured the streptavidin plates, told me that I should check with another primary antibody to see if could be an antibody issue (I am a little unsure of whether an antibody manufactured for Western blots can be used in ELISA). Sure enough, I got no signal this time. Could it be possible for a primary antibody to have an affinity for streptavidin? (Note: I did not block because the plates were preblocked during manufacture and ThermoFisher told me blocking wasn't necessary. Maybe I should do antibody incubation with block? But if that were the case, then why didn't I see a signal with the other primary antibody?) And if blocking does nothing, then what do I do? I am not sure that
are the primaries poly or monoclonal?
the recognized epitopes may include part of the blocking agent. polyclonals may recognize several epitopes and different monoclonals may be to different epitopes.
we always include blocking agent in our antibody solutions.
Antibody is polyclonal. I'm going to try blocking the plate and incubating with block (even though it may not be necessary with the plate I'm using), but I doubt it will make a difference.
Blocking did nothing. Millipore sent me an alternate antibody that was rated for ELISA through Chemicon, but that too gave a nonspecific signal. I still find it really hard to understand, unless there's something about anti-acetyllysine antibodies that give them some sort of affinity for BSA or streptavidin. If it's BSA, then I'm hoping that plates pre-blocked in a non-BSA block like SuperBlock can do the trick. If it's the streptavidin, well, has anyone ever seen that before?
Could you give a detailed info about your exp? I am little bit confused your exp. If you use streptavidin coated plate, you may need biotin-labeled Ab. Blocking usually can minimize the non-specific binding , but it can not get rid of. I guess polyclonal Ab is the issue. If you tell me more, I can help you more.