The test principle of the Bone Sialoprotein ELISA (BSP ELISA) is based on interaction of the antigen in the samples or standards with the antibody coated on the Wells of the microplate. A peroxidase-conjugated secondary antibody is used for detection and quantification, and tetramethylbenzidine (TMB) as a peroxidase substrate. The enzymatic reaction is terminated by an acidic stop solution. A dose response curve of absorbance unit (optical density, OD at 450 nm) vs. concentration is generated using the values obtained from the standards. The quantity of Bone Sialoprotein present in the samples is determined directly from this curve.