This nitrotyrosine (3-nitrotyrosine, 3NT) competitive ELISA is used to determine the level of nitrotyrosine modified proteins in a sample. The provided microplates have been evenly coated with a nitrotyrosine containing antigen. The competitive ELISA is performed by adding the test sample mixed with the provided HRP conjugated anti-3NT antibody. If no 3NT modified proteins are present in the sample, all of the anti-3NT detector antibody is available to bind to the immobilized 3NT containing protein coating the wells, generating a maximum (100%) binding signal. In contrast, if soluble 3NT modified protein is present in the sample, it will compete for binding by the anti-3NT detector antibody. The degree of competition is proportional to the concentration of soluble 3NT modified proteins in the sample. Therefore the signal in each well has an inverse relationship to the amount of 3NT in each sample. When performing each assay a standard curve is generated from the provided standard to allow the accurate quantitation of the 3NT content in the test samples.

The assay is followed by monitoring the HRP dependant color change in each well at 600 nm. Alternatively, the assay can be terminated, at a user-defined time, by the addition of 1N HCl (not supplied) and the assay performed as an end-point measurement at 450 nm

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