I'm using 0.1 ug/ml of biotinylated antibody conjugated with the 40 nm streptavidin gold nanoparticles, 100 ug/ml as monoclonal antibody for the test line and 0.25 mg/ml of Biotinylated BSA. I have the control line, but I can´t obtain the test line.
Your description is not clear!! where is the antigen in your test format? you have a mouse monoclonal antibody as the test (capture) line and gold labelled streptavidin-biotin-antibody as the detector. Why not first gold conjugate the detector antibody first before attempting to increase test sensitivity with biotin-avidin. Also, for sandwich tests you need to maximise the amount of capture antibody so use 1mg/mL as test line.
You did not mention whether you have verified the antibody pair in some way. The setup of a new sandwich immunoassay is not trivial. We usually test our antibodies in a microtitration plate format.
Ahmed Jehanli, so I put the antigen on the sample pad at a concentration of 5 μg/ml. Yes, I have a human monoclonal antibody as the test line and a biotinylated antibody conjugated with streptavidin gold nanoparticles. The concentration of the biotinylated antibody is 0.1 μg/ml. I can´t increase this concentration because the particles start to aggregate. I used the streptavidin nanoparticles because, for the control, I was biotinylated BSA.
Michael G. Weller: Yes, I already made an ELISA to see if the sandwich is working. And through the ELISA I can obtain a signal so apparently the sandwich works.
Something may be wrong with your gold particles. It is strange that they aggregate with more antibody. What is the buffer of your antibody solution?
What is the molecular weight of your antigen? Have you carried out titration of the antigen? you are using 5ug/mL antigen versus 0.1ug/mL of the detector antibody. This seems to me a huge amount of antigen to antibody. Need to know the molecular weight of the antigen to work out the molar ratios of detector antibody to antigen. With sandwich tests there is the risk of "hook effect" where you have huge excess of antigen with respect to the detector antibody leading to loss of signal. Also, I still think that you may get an answer by directly gold labelling your detector antibody and at least check its ability to bind to the antigen by doing dot test.
Michael G. Weller Thank you for your answer. The buffer is 20 mM Sodium borate (ph 8), 1%BSA, 2% Sucrose, 0.02% sodium azide
The BSA and the sucrose in your buffer can contain traces of biotin, which inhibit the binding of the antibody. Use only PBS for conjugation, and perform the blocking later.
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