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Optimizing an ELISA assay

Optimizing an ELISA assay - Protein Science

Optimizing an ELISA assay - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 09-13-2006, 09:25 PM
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Question Optimizing an ELISA assay



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

thanks in advance for your contributions

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Old 10-05-2006, 12:01 AM
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Default Re: Optimizing an ELISA assay

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.
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Old 01-29-2007, 10:58 PM
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Default Re: Optimizing an ELISA assay

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...
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Old 01-31-2007, 03:38 AM
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Default Re: Optimizing an ELISA assay

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
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