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Lateral Flow Assay

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Lateral Flow Assay

Postby knclayton » Wed Mar 07, 2012 11:03 pm

Hi Everyone,

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

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

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

However, I don't get one.

I have a couple of questions that could solve this problem if answered.
1. Does the entire nitrocellulose pad need to be pre-treated? (i.e. treated before aptamers are spotted onto the paper for fixation) And if so, with what?
2. I was recommended to pre-treat my conjugate pad with either 10% sucrose, or a sucrose/borate solution. 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 I cannot tether this VEGF aptamer since it is only 28 NT long? A friend did IgE aptamer that was under 50 NT and got it to work (she used FITC with her antibodies though).

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

Thanks :) :D :)
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Re: Lateral Flow Assay

Postby jonmoulton » Thu Mar 08, 2012 6:55 pm

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

Don't know, sorry.

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

The most common use of borate is for a salt of boric acid.

3. Is it possible that I cannot tether this VEGF aptamer since it is only 28 NT long? A friend did IgE aptamer that was under 50 NT and got it to work (she used FITC with her antibodies though).

Hopefully you are assessing the conjugation before you start the assay. Can you check how the tethering went?
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