sandwich elisa - how to get rid of non-specific binding (Jun/24/2007 )

Hi all,
i am doing sandwich elisa between two monoclonal antibodies to know whether they belong to same or overlapping epitopes or do they belong to distinct epitopes. for this i am coating with mab A first, then add ag and then detect the antigen by a mab B.
as both of them are of mouse monoclonals, i have to block the mab A sites with anti-mouse IgG without any conjugate. then while adding secondary to detect mabB, i need to preincubate the secondary with mouse IgG after incubating with mabB. but i am not getting any positive result for both the different mabs which were checked in different combinations and were also shown by western that they belong to different fragments of the protein. can someone suggest me a way out of it?
thanking you all in advance
leelaram

I think a part of your problem could be steric hindrance of the anti-IgG stopping the second mAb from binding (which implies you could well have epitopes that are close to each other). By blocking the first antibody with an IgG, you may be crowding the second antibody out.

Why not conjugate the enzyme (HRP, I presume) to the second mAb? That way you don't have to block the first mAb with anti-IgG. If the two mAbs are against the same or an overlapping epitope, you won't get a result; if, on the other hand, they bain to different epitopes, you will get a signal. By carrying the experiment out in both forms (A bound, B conjugate and B bound, A conjugate) you should be able to see if the sites overlap.

Another variant is to coat the plate with a polyclonal IgG, then conjugate the HRP to each of the mAbs in turn and try to compete. Bind your antigen to the polyclonal, then add mAb A (non-conjugated), incubate, then add mAb B (conjugated) and see if your get a signal. Try the other way around, too, for completeness.

Hi L,

I agree with Swanny, your problem may be steric hinderance.

My suggestion is that you coat with mAB A, block with casein (make blocking buffer), add ag, and then add mAB B. Then add anti mouse IgG that is labelled.

I have the protocol for making a blocking buffer, let me know if you need it.

Good luck


Best regards,
Olabelle
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Hi O,
thanks for the advice
could u please give me the composition for the blocking buffer
according to what you told how could i prevent anti-mouse IgG binding to mAb A
thanking you in advance
Leelaram