I am working on heterologous sandwich lateral flow assay.
I conjugate one antibody with carboxyl-gold, then print the other antibody on the nitrocellulose membrane.So the antigen should bind to the first antibody, then immobilize by the second antibody.
Even the two antibodies do not bind to each other, I always get two positive bands at the test line and control line when there is no antigen.
Different blocking condition(BSA%) and running buffer was tried, but I still see false positive band.
Is there any explanation or solution to this problem?
you need to provide more information! what species is your capture and detector antibodies? are they monoclonal or polyclonal antibodies? are your reagents pure antibodies or IgG fraction? what is the buffer that you use for spraying your capture antibodies?
The antibodies are monoclonal nanobodies purified from E.coli.
The capture antibodies are 1.5mg/ml in 8mM phosphate buffer, then blocked by 8mM phosphate buffer(pH 8.0) with 0~1% BSA.
The running buffer is 20mM Tris, pH 8.0 with 0.5%BSA and 0.5% Triton X-100.
Exactly the same conditions were tried with different antibodies, some of the antibodies just give false positive regardless of the blocking condition.
is it is a strong signal, I would say that you should change the antibodies.
If it is a very faint signal, the blocking the NC or reducing the concentration of the gold conjugate could help.
If it is not really cross reaction or non-specific interaction, then explore the possibility that you have a stability problem in the conjugate that is damaging the antibody
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