Lateral Flow Assay

[knclayton]

Hi Everyone,

 

 

So, I am making a lateralflow assay using gold colloidal particles as my visual signal for the test.

 

 

To explain a little more, Ihave a small nitrocellulose membrane, and I want to detect VEGF in sample, byusing a anti-VEGF aptamer to capture it.

 

 

My setup has an input whereI place in VEGF sample and it wicks up and through a conjugate pad (made offiber glass) that is saturated in 30 nm gold colloidal particle conjugated toanti-VEGF antibody. These should be moving together, and should bind to anaptamer that is immobilized on the nitrocellulose. The gold would aggregate andshow a signal..

 

 

However, I don't get one.

 

 

I have a couple ofquestions that could solve this problem if answered.

 

1. Does the entirenitrocellulose pad need to be pre-treated? (i.e. treated before aptamers arespotted onto the paper for fixation) And if so, with what?

 

2. I was recommended topre-treat my conjugate pad with either 10% sucrose, or a sucrose/boratesolution. I tried the 10% sucrose to no avail. Also... what does 'borate' mean?This seems to be quite a general term.

 

3. Is it possible that Icannot tether this VEGF aptamer since it is only 28 NT long? A friend did IgEaptamer that was under 50 NT and got it to work (she used FITC with herantibodies though).

 

 

If any of this is confusingor needs further clarification, please let me know! I am ultra confused by thispoint.

 

 

Thanks