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My question is with regards to the usability of the indirect ELISA in measuring protein concentration. Suppose we want to quantify the concentration of a protein. The indirect ELISA method would require one to coat the bottom of the well with protein antigen and wash off unbound antigens.

My question is that wouldn't washing off unbound proteins mean you are 'diluting' the actual protein concentration? Your resulting colour intensity will not reflect the actu protein concentration. Thanks!

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The method is based on the principle that the amount of antigen bound to the plastic will be proportional to the concentration in the sample being tested. And of course an ELISA will usually be run with a calibration in parallel so that the signal from your sample is compared to that from standards.

The wells will be rinsed anyway in subsequent steps to remove unbound antibody molecules: if you didn't wash off unbound antigen it would simply compete for binding the antibody then disappear later in the procedure.

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