Double coating in Sandwich ELISA - Problems - (Sep/05/2012 )
Something strange happened to me just recent,
I have two antibodies that bind to different epitopes and is quite strong in their reactivity alone with a specific type of detection mab.
But when I double coat some wells with these mabs and detect with the mab that workes together with both of them, I get no signal.
In the same plate I coated wells with the mabs alone and I get a normal signal.
I ran a competitve ELISA with these mabs and have seen that they do not block each other.
Can these two coating mabs somehow create some kind of complex that blocks the binding region of the coating mabs?
Anyone who has seen this before?
So I'm assuming that this is some sort of sandwich ELISA with mAb capture reagents and an mAb detection regent for the determination of some other molecule.
If this is the case, the double coating may capture the 'other molecule' in an orientation that is not accessible to the detection reagent whereas the individual capture reagent coatings leave the detection epitope on the 'other molecule' exposed.
Ben Lomond on Thu Sep 6 01:33:00 2012 said:
So I'm assuming that this is some sort of sandwich ELISA with mAb capture reagents and an mAb detection regent for the determination of some other molecule.
If this is the case, the double coating may capture the 'other molecule' in an orientation that is not accessible to the detection reagent whereas the individual capture reagent coatings leave the detection epitope on the 'other molecule' exposed.
I see what you mean. If the epitope of the molecule is "between" the epitopes of the two capture mabs, this area will not be visible to the detection mab, and no signal will shown. It sounds logical.
Thanks a lot for the help!