Hi,
I am trying to develop a competitive lateral flow assay to detect a protein. I coat the protein on the test line and use gold nanoparticle-antibody as the conjugate that binds to the test line and gives color.
As we understand, in a competitive lateral flow assay, the color at the test lie should decrease in intensity with the increase in concentration of the analyte in the sample. But, in my case, the color at the test line remains the same intensity when there is no protein in the sample and when the concentration of protein in the sample is very high (0.1mg/ml).
Did anyone of you face a similar problem ? what could be the reason for this observation?
Any help would be appreciated. Thank you for your time.
Hi Gangula,
This not a very unusual problem with competitive lateral flow assays. There are two possible reasons for the problem I can imagine: 1) Your analyte concentration is not high enough to saturate the binding capacity of your reporter particle bioconjugates or 2) There is a high degree of non-specific binding your reporter particles to the test line.
First, I would check if there is a similar binding to the test line with a reporter particle conjugated with some non-target IgG. If you see binding with this the solution could be to adjust the binder line protein amount and adjusting the pH, salinity and detergents of your reaction.
Decreasing the binding capacity of your reporter can be done by conjugating your reporters with a mixture of the target-specific antibody and some non-target binding IgG.
I hope this helps you forwards.
-Etvi
Hello everyone,
sir I want to assemble all pads on lateral flow assay please give appropriate suggestion to proceed this process.
Hi Etvi,
Thank You Very Much for your two suggestions. They look very apt. Will try these two out and hopefully find a way out.
Hi Yachana,
I can help you out with the assembly. It is a bit detailed process. you can email me at [email protected] with your specific questions so that I can answer them.
Thank You
Helo sir i am also working on lateral flow sandwich assay I wantyour suggestions regading test line which protocol i have to follow to make test line on nitrocellulose membrane
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