Hi everyone,

I’m currently developing a lateral flow immunoassay (LFIA) and encountering an issue with non-specific binding at the test line, even when applying only the running buffer (no analyte). The test line consistently shows a faint to moderate signal, suggesting potential protocol flaws.

Here is a brief outline of my process:

• AuNP–antibody conjugation: 20-OD AuNPs coupled with antibody (final 2.5 µg/mL), blocked with 1 mg/mL casein, followed by centrifugation and resuspension in a conjugate pad buffer containing PBS, 5% sucrose, 0.5% casein, and 0.05% Tween-20.
• Conjugate pad: Soaked with 12 µL conjugate solution and dried at 37 °C for 1 h.
• Sample pad: Pre-treated in PBS buffer with 2.5% sucrose, 0.5% casein, and 0.05% Tween-20 and dried at 37 °C for 2 h.
• Test line: 0.5 µg antibody manually striped on nitrocellulose; buffer includes 2.5% sucrose and 0.05% Tween-20.
• Running buffer: PBS + 0.05% Tween-20 (no analyte added).

Could you please review this process and share your thoughts on what might be causing this non-specific signal?

Any feedback or suggestions would be highly appreciated!