In general to overcome the nonspecific binding you should add some protein (BSA, casein 0.05-2%) and detergent (tween-20, 0.01-0.2%) into running buffer. Try to moderate the pH of running buffer. You can block your NC membrane by soaking in blocking buffer (consist of protein and detergent).

Can you provide more detailed information about the conditions of the analysis? Specify the analysis protocol, name the reagents and concentration that you use to apply to the membranes, and so on.

Dear Ivan Maksin ,

Thank you for your reply. I am using commercial lateral flow dipsticks, make#hybridetect (MGHD 1, https://www.milenia biotec.com/en/product/hybridetect/), and for running buffer, I use the assay buffer supplied with the dipsticks. I applied 10ul of product and 40ul of assay buffer on the sample application area. I tried to reduce the reaction time from 30 minutes to 15 minutes to avoid non-specific cleavage of the reporters, which just reduced the intensity of the non-specific band but did not remove it completely.

Do you use the assay buffer from a commercial kit, or is it a homemade solution? Generally, it is not recommended to use homemade solutions with commercial kit reagents.

Try running the test strip without the amplified product (blank), using only the assay buffer. When running a positive sample, is the test line significantly brighter compared to the strip run with just the buffer and no amplified product? Please share pictures if possible.

If you observe any nonspecific binding when runing blank (buffer) sample, I strongly recommend contacting the distributor or manufacturer of the test strip kit.

Ivan Maksin , I am using the commercial assay buffer supplied with the kit. I have also checked the blank run. The strip is fine. I think I have to optimize the sample volume to be applied on the strip according to my assay.