I am developing nucleic acid based lateral flow immunoassay. I am getting false positive while running buffer (borate, pH 8.5, 1 % BSA, 0.05 % Tween 20) only. My system consists of gold nanoparticles and tagged PCR products, and monoclonal antibodies. I have tried with different buffers including tris-cl, PBS but of no use. Kindly suggest solutions for the same. Thanks in advance.
AuNPs are very unstable and aggregate in presence of salts, maybe that's why you are getting false positive with buffers. First check for stability of AuNPs in the buffer you will be using for assay.
Hi Smita, Thanks for your reply. Particles are stable in the buffer and they run properly without any stain or aggregation seen on the membrane except for Ab immobilized area. So i think there is some other problem i am facing. Kindly suggest other solution if you have any.
Dear,
Please share your complete protocol, so we will help to remove false positive.
Nalin,
What is your test line antibody? I am assuming an anti-FITC/DIG etc or some other reagent like streptavidin? What is your PCR tag? What is your conjugate blocking buffer and protocol, what is your conjugate diluent? Did you performing the necessary reaction sweeps? (pH/buffer, blocking, incubation times etc).
This assay is relatively straightforward with the right reagents.
I was able to reduce NSB of a LAMP-Lateral flow assay just by incorporating 1% tween in my running buffer. But there could be a large number of reasons why you would see NSB.
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