optimization of a competitive ELISA assay. I need to add labeled antigen and an unknown sample (or standard) simultaneously to the well which is pre-coated with capture antibody.
labeled antigen : competes with the unknown sample or standard for binding to the capture antibody
Usually, it is better (more sensitive) to first add the sample or standard to the plate and incubate and shake for e.g. 10 min. Then you add your labeled antigen to the plate and incubate and shake again. The concentration of the labeled antigen is not related to any standard. It is simply optimized empirically (test of different dilutions) for optimal signal and low background.
http://link.springer.com/article/10.1007/BF01240369
A competition ELISA is a very precise test that requires the use of well-calibrated parameters, in particular for the amount of antigen that determines the sensitivity of the test.
To calibrate the antigen, we perform a cascade dilution, and we determine the dilution that gives between 50 and 100% of maximum signal, for example 1.000 O.D. corresponding to the point of inflection of the dilution curve.
In order to carry out the competition test, the antigen is first mixed at the optimum concentration with the test sample, the positive control, and the negative control, and then the mixture is transferred to the plate containing the capture antibody .
These two procedures are essential to have a good sensitivity, and reproducibility of the assay.
@michael G. Weller and @Jean-Jacques Pin thanks you for your answering and good points that you have shown me.
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