The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Rat NPHN. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Rat NPHN. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Rat NPHN, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Rat NPHN in the samples is then determined by comparing the OD of the samples to the standard curve.