Description
Principle of the Assay: The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the NGAL present in samples reacts with the anti-NGAL antibodies which have been adsorbed to the surface of polystyrene microtitre wells. After the removal of unbound proteins by washing, the Detection Antibody, biotin conjugated anti-NGAL, is added and complexes are formed. Following a wash step, the horseradish peroxidase (HRP) conjugated Streptavidin is added and complexes are formed. After another washing step, the complexes are assayed by the addition of a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB). The quantity of bound enzyme varies directly with the concentration of NGAL in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of NGAL in the test sample. The quantity of NGAL in the test sample can be interpolated from the standard curve constructed from the standards, and corrected for sample dilution