ELISA - ELISA with polyclonal Ab (Feb/26/2005 )

I'm trying to set up an ELISA with the polyclonal Abs against a protein.
I want Abs for coating the plate and for the capture. Is it make any difference to make Ab in two different animals ( rabbit and rat ) instead of one (rabbit ) ? which one should work better ?

I personally prefer to use polyclonal ABs from different animal species, since then you can work with an anti-Species AB for detection, and don't have to label one of you ABs youself.

Example:

Coating with Horse Anti-(Your Protein)
Incubation with (Your Protein)
Incubation with Mouse Anti-(Your Protein)
Detection with Goat Anti-Mouse Fc

Another point is, that you have to ensure that your detection an your capture AB recognise different epitopes, because if both recognise the same immunodominant epitope, steric inhibition can and will occcur, which will give you no signal whatsoever. While it can happen that two different organisms produce antibodies that recognise the same epitope, this is less likely than if you use just one polyclonal serum from one single animal.

Which one will work better can't be said in advance, that has to be done experimentally.

mike