I have modified the paper with sodium periodate for covalent immobilization of my target protein. However, even in the absence of that protein, I get high-intensity colour by just adding the Enzyme-tagged detection antibody and its substrate. I wash the detection antibody with 1X PBST. My washing procedure is similar to that mentioned in most literature sources. I drop 10 uL of PBST on the test area (6mm diameter) and then remove excess water using a blotting paper on the bottom side of the paper. The paper I am currently working on is Type-1 Chromatography paper.