Hello Everyone,
I faced a problems when I run my sandwich LFIA.
I’m doing spot test in my half LFIA.
1. When I applied antigen, It’s getting colored in the test line but faint intensity. In the control line, the color intensity is good.
2. When I didn’t apply any antigen/sample, both of them (test line and control line) are getting colored but, in the test line the color intensity is weak.
It’s showed False-Positive-Result on my Sandwich LFIA system.
For the conjugation antibody, I used Chemically conjugation.
I know so many people are strugling with this problem too. but any suggestion is very helpful for me.
Thank You.
Best Regards,
Wahyu Ramadhan
More blocking:BSA, serum
Add detergent like tween20 or triton x-100.
Sometime PEG or PVA helps too
Increase blocking time and blocking protein concentration. The blocking protein and the antigen should not have cross reactivity.
I agree, try differnt blocking methods. (BSA, OVA, Gelantine....) or use buffer components, that reduce non-specific adsorbtion (also try for PVP, PAA, detergents etc)
Also, there is the possibility, that your nitrocellulose is not suitable for your assay, simply try out some of them.
Its also possible, that your labbeled antibody binds the capture antibody. Sometimes that happens. Check that with ELISA if possible (use the same concentrations). If that is the case, cross adsorb your labeld antibody before labeling. There is a kit from Thermo for that. Especially, if you use polyclonal Abs.
Best regards
Peter
Perhaps there is a cross reactivity between antibody and the blocking protein.
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