Just wondering how I can stick the 2 antibodies to the wall of the plate? as I have them in separate aliquots.
Should I incubate the first Ab O/N then next day incubate the other for another night before blocking?
Or incubate both together?
I tried to google similar protocols, got nothing.
I think that if you coat with one of the antibodies first you would be giving all the advantage to that one. I would coat with the mixture of both antibodies instead. Their relative amounts depend on whether their coating efficiencies are the same or not. Are them of the same origin (species) and isotype? If so, they will probably coat the plate in a similar way.
Just in case, I would check first whether they are able to coat the plate separately in your coating conditions.
If they are not of the same origin or there is a simple way to distinguish them based on their binding properties, you could check the separate coating first, then coat with the mixture and check that both antibodies are on the plate with the specific reagents.
To avoid all of these complications, is it really necessary to use both antibodies?
Usually one antibody is used for capture and the other for detection. It sounds like you are trying to use two different antibodies for capture (and a third antibody for detection?). For coating the plate, I think I would mix them together in the same molar ratio as their epitopes, if they are monoclonals or epitope-specific polyclonals. If they are polyclonal (not epitope-specific), they probably share many epitopes and there may be no advantage to mixing them as opposed to using more of one of them.
Hi Zahran, you need to explain in more detail. Why do you want to coat with two antibodies? If you really want to coat with two antibodies, then it really depends on what you want to do with the assay. The two antibodies could have varying affinities, and mixing them 1:1 could therefore bias binding of one epitope. Most likely, you are looking to bind one Ab and detect with the second, as this is the common use of ELISA.
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