I conjugated gold nanoparticles with a primary antibody and mixed it with the corresponding antigen. I ran the solution over a nitrocellulose membrane with two dots on it (one with the same primary antibody that I conjugated the gold nanoparticles with and one with the secondary antibody). The gold nanoparticles avoided the dot with the primary antibody, but binded to the second dot with the secondary antibody. What might be causing this? I expected both dots to be visible with the antigen on the first dot bound to the primary antibody on the gold nanoparticle and the primary antibody on the gold nanoparticle bound to the secondary antibody on the second dot.
If I got your experiment right, the reason is that most antigens only have one epitope (binding area) of a kind. Therefore, if the antigen is bound by your (monoclonal) primary antibody on your gold nanoparticles this binding area is blocked, and you would need a different primary antibody to bind a second, different epitope. Such two antibodies are called matching pairs and are used in "sandwich" immunoassays.
Most secondary antibodies are polyclonal ones (mixtures) and can bind many epitopes. Therefore, they nearly always bind.
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