Hi
I am currently working on lateral flow assay (LFA). I found that my negative control gave false-positive results in my test. I have checked using ELISA and did not face this problem. I blocked my LFA assay with BSA, but the false-positive still occurred. I am using PBS as my negative control.
Has anyone experienced the same issue?
Blocking the membrane with BSA and using PBS is not sufficient to remove non-specific binding. you need detergent/polymer/proteins either in the chase buffer or incorporated into the gold conjugate and/or the sample pads. Proteins: BSA or casein, Polymers PVP, PVA, Detergents Tweens or Tritons, etc.
Ahmed Jehanli thank you for your suggestion. I treated the nitrocellulose with 1% BSA after lining the capture antibodies. My conjugated quantum dot-detection antibody contained 1% BSA too.
Is it because the concentration of the
detection antibody is too high that caused non-specific happened?
It can be. You will need to titrate how much antibody you need to use and maybe use detergent too like Tween-20 or Tween-80. You will also need to tirate the concentration of the detergent. I have no experience working with quantum dot detection antibody.
Ahmed Jehanli I will try to titrate the concentration for both antibody and detergent.
Thank you so much.
1. You can change your antibodies.
2.if you have false positive after 10 min, it is not problem. Usually reading the result after 10 min is not recommended.
3. Use the reagents like tween, triton, or PVA with a proper concentration in sample pad buffer.
Shafieeh Mansoori Thank you for your suggestion.
1. I tried new antibodies, but still could not solve the problem.
2. The false positive happened immediately after the flow of the running buffer/PBS.
3. We have used blockers such as casein, skimmed milk, BSA etc. But they did not work. The false positive still occurred.
Thank you for all the suggestions.
After changing the antibody, I successfully overcome the problem.
In my exeprience there is a limit to how much you can fix a system where the antibody is the cause of the problem. If blocking the membrane, blocking the conjugate or reducing the concentration of the conjugate does not work, then the use of detergents will not be as effective as you expect. There is a stability or non-specific interaction problem
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