Dear Melika Gorgani

Based on Tsai et al., (https://www.nature.com/articles/s41598-018-35694-9), you need to do the following steps;

For cleaning the cellulose pad, immerse the cellulose pad in a cleaning solution composed of 50% methanol, 30% 2-propanol, and 20% ethanol. Use a volume of cleaning solution sufficient to cover the cellulose pad completely. Let it soak for 10 minutes with occasional gentle agitation. Then, prepare the AuNP-anti-Protein conjugates separately. Mix 1 mL of 30 nm diameter AuNPs with 1 μg of the antibody in 0.01 M amine-free PBS solution at pH 8.4. Incubate the mixture for 45 minutes with gentle stirring to allow antibody binding to the AuNPs. Terminate the reaction by adding 0.1 M tris-buffered saline (TBS) with 0.1% Tween 20. Remove excess antibodies by centrifugation at 8000 g for 35 minutes and reconstitute the conjugates in 0.01 M TBS containing 2% bovine serum albumin (BSA). Store the conjugates at 4°C until use.

For conjugation, dilute the AuNP-antibody conjugates to an optical density of 0.060 in conjugate buffer (2 mM borate buffer with 5% sucrose). Soak the cellulose pad in the diluted conjugate solution for 1 minute, then dry it at 37°C for 5 hours. Finally, for assembly, cut the treated cellulose pad to a suitable size, for example, 4 mm × 4 mm, to serve as the stacking pad. Assemble the lateral flow test strip in the following order: adhesive backing pad, sample pad, stacking pad, conjugate pad (with AuNP-antibody conjugates), and nitrocellulose membrane-based test pad. Ensure a 1 mm overlap between the conjugate pad and the test pad.

-I have written this protocol based on the mentioned paper, you may set-up yours, or use other antibody conjugates.

Thanks a lot Alireza Mohebbi