I'm developing a lateral flow assay. My gold-antibody conjugate is stable according to UV-Vis measurements. The nitrocellulose membrane was striped/spotted on the same week as conjugation and testing; the membrane is unblocked. I'm running the strip in a "wet assay" format. I load conjugate then chase with buffer (negative) or sample+buffer. I'm using a novel camera-based reader to measure signal intensities.
Over 7 days the conjugate is increasing in sensitivity across all antigen loading conditions INCLUDING the negative condition (this clears over time leading me to believe it's not actual non-specific binding). Because the conjugate is stable, I plan on drying down conjugate and running the assay "dry". I will be applying sugars and possibly look into blocking the conjugate pad and the membrane.
I am just curious what are some possible reasons for the increase in signal over time in this specific format. Could there be some membrane curing effects? (membrane has been kept at low humidity). Mind you, my dose response curve has maintained with the exception of the upward shift.
Thank you for the response.
The antigen is a a bacteria surface cell protein, the sample is inactivated bacteria. I thought about sample stability, however that still doesn't explain why my negative condition would see an equivalent increase in test line intensity. The negative condition is just PBS (no antigen).
I'm going to cross multiple conjugates with multiple striping events to see if I can pin down the location of this effect.
Vasily, thank you for the response.
The GNP-conjugate appears to be stable. The abs max wavelength for these particles is 800nm. I've been tracking the max abs everyday. These particles are 80nm silica core-gold nano-plated shells (GNS). These are not traditional GNPs. My OD has stayed stable at 20.
Hi Angel,
have you got solution for membrane curing affect ? I am aslo facing the same problem.
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