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 Human 2,3-DPG. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Human 2,3-DPG. 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 Human 2,3-DPG, 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 Human 2,3-DPG in the samples is then determined by comparing the OD of the samples to the standard curve.