I am developing and optimizing an ELISA and I was jsut wondering how you determine what the best concentrations of antibody and antigen are based off the OD values. I have performed checkerboard titration, so what should I look for in the values to determine the best combination?
You want to use the least amount of antibody that will give you OD values around the middle of your standart curve, to make the experiment cost-effective.
You want the expected analyte concentration to be well between upper and lower limit of quantitation, which in photometric ELISA are less than an order of magnitude appart. Measuring chemiluminescence instead will increase the working range of the assay.
Example: If the analyte concentration in the serum of healthy people is between 10 and 20 pM, and in people with disease X between 50 and 100 pM, you have two options: either develope an assay that covers this entire range, or you could develop an assay with a LLOQ of 30 pM, to get a disease present/absent answer. The former would be good for research purposes, the latter for clinical application.
If your ELISA is for diagnosis, then serial dilutions of antigen and sera were make, you must select those where OD between positive and nagative is greater.
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