Application of a mimetic enzyme for the enzyme immunoassay for α-1-fetoprotein

A new mimetic enzyme immunoassay (MEIA) method for α-1-fetoprotein (AFP) was developed using a mimetic enzyme Mn(III)-tetra(sulfophenyl)porphine (Mn-TPPS4) as a labelling reagent to catalyze the fluorescence reaction of 4-hydroxy-phenylacetic acid (HPA) and hydrogen peroxide. In the sandwich assay standard AFP solution or AFP containing umbilical blood serum first reacts with antibody (anti-AFP)coated on a 40-well plate (polystyrene), and then further reacts with Mn-TPPS4-labelled anti-AFP; the Mn-TPPS4 on the bound fraction, after removing the free fraction was determined by measuring the fluorescence intensity as a result of the reaction between HPA and H2O2, catalyzed by bound Mn-TPPS4 and anti-AFP conjugate, which was proportional to the concentration of AFP. AFP in the range 0.01–10 μg per well can be determined with a detection limit of 1 ng per well. The method has sufficient sensitivity to be applied to the determination of AFP in umbilical blood serum.