Hi,
I am trying to do a lateral flow immunoassay with antibodies (1mg / mL) immobilized in nitrocellulose (pore of 0.45 um). My line of antibodies is 4 mm x 1 mm, however I can not obtain a visible signal when running antibodies conjugated with gold nanoparticles. The concentration of the dried nanoparticles in the pad conjugate is 40 nm is 1 OD (7x10 ^ 10 particles). Does anyone know what concentration is necessary to generate a visible signal? At 4 OD I also can not get a visible signal.
Is this a "standard" sandwich assay? Ensure the antigen concentration is high in initial tests, e.g. @ 100 ng/ml or higher.
Try to swap the antibodies from the test line to the gold: often the assay only works one way round.
Have you performed a pH / concentration, (Protection Point) optimisiation on your gold conjugate, i.e. @ which pH and concentration of antibody best "protects" the gold - antibody conjugate from NaCl challenge?
http://www.diagnostics1.com/MANUAL/LFIA_Book[1].pdf
A more quantitative general test of "How much gold can be detected on the test line" may be derived from:
1. Prepare test sticks with a polystreptavidin, PSA test line.
2. Prepare gold - biotin conjugate, e.g. attach biotin - PEG - lipoic acid your gold: http://www.nanocs.net/Lipoic-acid-PEG-Biotin-2k.htm
You can also buy 40nm gold - biotin (likely cheaper to make you own).
3. Flow various amounts of wet gold - biotin up the PSA test sticks to find the minimum gold detection limit.
John Clarkson Indeed, I changed the system, that is, the immobilized antibody and the mobile antibody and it worked. I have not investigated much about it but I imagine that it must be the kinetics of the affinity reaction. Thanks for the help!
Article Quantifying the Numbers of Gold Nanoparticles in the Test Zo...
- Badges
- Science method
- Similar topics
- Biological Science
- Immunology
- Immunology Techniques
- Immunoassay