ssDNA Lateral Flow Assay
Posted 07 March 2012 - 03:15 PM
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.
Posted 08 March 2012 - 12:56 PM
borate is a buffer in the range of ~8.2 to 10.2 (pK is 9.2).
genius does what it must
i do what i get paid to do
Posted 15 March 2012 - 11:17 AM
The particles should be free of unbound antibody and demonstrate binding to your VEGF...do you have a QC test. This is critical before going forward!
The aptamer should be in high concentration and applied as a straight line perpendicular to the flow path.
After adsorption the nitrocellulose should be blocked with BSA and tween (maybe some sugar) and dried.
<<alternatively....instead of nitrocellulose you can use glass fiber and in place of attaching aptamer couple the aptamer to 0.1 latex and apply this as a line to the fiber material...this would give you a high concentration of ligand that the particles have to flow through. Methodology similar to old First Response pregnancy tests>>
Sucrose, dextrans, trehalose, etc etc are only used as preservatives for the ligands upon drying or lyophilizing.
Buffer is not that critical but you have to have the correct pH for binding and the buffer is dried in the sample introduction pad.
Tween/surfactant is a critical component to allow the colloid to flow from the pad to the nitrocellulose or filter/capture material and up to the wick at the top (suggest you look at material similar to cigarette filters)
To test the binding to the aptamer PRE mix and incubate the colloid/VEGF then apply it and allow that to wick up the nitrocelluose to the capture line.
To QC your colloid-ab react with VEGF and see if color change OR react with aptamer-latex and gently centrifuge some of the colloid should be pulled out of solution.
Finally, if all reagents work in QC independant tests their should be sufficient distance between the conjugate/colloid pad and the capture ligand, the sample should be in excess (to wash all the colloid past the capture line)
Another way to QC your colloid is to replace the aptamer/capture material with VEGF and see if the colloid will bind to it.
your 'device' configuration should be something like:
sample absorbant pad with buffer/surfactant(dry)----colloid/tween/bsa/sugar(dry)----capture ligand/bsa/sugar-----absorbant wick/reservoir
If I am right are you duplicating a test by some company in Sarasota Florida for an eye disease?
For further information there are many many patents in this field.