I used sandwich assay on a test strip. When I allowed only PBS to flow, it still gave signals on nitrocellulose membrane where I immobilized the antibody. I tried to use a blocking agent (BSA and skim milk) on the gold nanoparticle surface after conjugation of protein but they cross reacted with the immobilized antibody and gave a red signal. I also tried to block the nitrocellulose membrane but nothing is worked. Any suggestion or theory would be very helpful.