This problem caused by defusio of the conjugate to the adges of the pad, you should apply the conjugate by spraying instead of deeping, increase humidity conditions, dry it at 37C , add blocking & detergent to the conjugate solution.

I assume that this is a variant of the coffee ring effect:

https://en.wikipedia.org/wiki/Coffee_ring_effect

It is caused by liquid flows from the center to the rim during the drying process. You may change the drying conditions, e.g., by freeze-drying.

Ngan Nguyen I can suggest to add blocking reagent to the conjugate pad treatment buffer. As, suggested you can spray and dry it at 37C.

It is common problem for LFIA, GNP-Ab can aggrigate on the conjugate pad, in this paper writen the conjugate pad treatment buffer https://doi.org/10.3390/bios11070231

Reduce usage of detergent in conjugate pad

Thank you so much everyone. The ingredient of conjugate treatment buffer I used are: Tris 50 mM pH 8, BSA 2.5%, sucrose 5%, tween 20 0.5%. I tested this buffer with some GNP-Ab, the results are ok, only this GNP-Ab gives bad results. So I wonder if the cause is antibody.

Remove Tween 20, Use tris at 10 mM, add BSA at 0.1%, rest ok

Thank Prakash Rana. But the pad I use made of polyester fibers, hydrophobic polyester, requires pre-treatment with Tween-20. Before I didn't use Tween 20 for treatment but the conjugate can not dispense onto the pad because hydrophobic.

Anyway, I will try it one more.

Try with 10mM Tris pH 7.6 (set pH of protein buffer system).

Addition of following stabilizer and detergent may give false positive results but have to be optimised.

0.1% BSA

0.1 % Tween20,

10% Sucrose,

5% Trehalose.