Description
This ELISA is designed for the quantitative determination of nitrotyrosine. It utilises the sandwich technique with two polyclonal antibodies against nitrated proteins. Standards, controls and diluted samples which are assayed for nitrotyrosine are ad-ded into the wells of a microtiter plate coated with polyclonal anti-nitrotyrosine an-tibody. During the first incubation step, nitrated proteins are bound by the immobi-lised primary antibody. Then a peroxidase-conjugated polyclonal anti-nitrotyrosine antibody is added into each microtiter well and a sandwich of primary antibody – nitrated protein – peroxidase-conjugate is formed. Tetramethylbenzidine is used as peroxidase substrate. Finally, an acidic stop solution is added to terminate the reaction. The colour changes from blue to yellow. The intensity of the yellow colour is directly proportional to the concentration of nitrotyrosine. A dose response curve of the absorbance unit (optical density, OD at 450 nm) vs. concentration is generated, using the values obtained from the standards. Nitrotyrosine, present in the patient samples, is determined directly from this curve