Optimizing an ELISA assay

[moleculardude]

Hey,
does anyone have experience in setting up and optimizing an ELISA assay?

thanks in advance for your contributions

[admin]

Hey Moleculardude,

I think I can start. I would definitely want to optimize the antibody:antigen ratios. I guess you could start doing this with some standards.

Wash conditions as well would need to be optimized. Wash solution and wash times.

[MicroKiller]

I have always found the checkerboard titration to be a good and fast way to optimize your elisa. Not only does it optimize 3 factors at one go but it gives you a good and clear picture of what dilutions and concentration to use for your desired readings...

[ruby]

use dot blot (known ag conc) and first standardize the antibody dilution .

once u know the best ag-ab combination, u can try different blocking reagents, inacse u see a high background level. the best way to reduce background is to use 1% detergent.

incubate o/n with primary ab

wash thoroughly (4X) after conjugated secondary ab and develop the ELISA

hope this helps.
ruby