The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the FETUIN present in serum sample reacts with the anti-FETUIN antibodies which have been adsorbed to the surface of polystyrene microtitre wells. After the removal of unbound serum proteins by washing, anti-Fetuin antibodies conjugated with horseradish peroxidase (HRP) are added. These enzyme-labeled antibodies form complexes with the previously bound serum Fetuin. Following another washing step, the enzyme bound to the immunosorbent is 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 Fetuin in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of Fetuin in the test sample. The quantity of Fetuin in the test sample can be interpolated from the standard curve constructed from the standards, and corrected for serum dilution.
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