Fluorescence immunoassay of α-1-fetoprotein with hemin as a mimetic enzyme labelling reagent

A novel mimetic enzyme immunoassay to determine α-1-fetoprotein (AFP) in solution was developed. Hemin, a horseradish peroxidase substitute, was used as a labelling reagent to catalyze the reaction of p-hydroxyphenylacetic (HPA) and hydrogen peroxide in alkaline media. In the competitive immunoassay, monoclonal anti-AFP antibody was coated on a 96-well plate (polystyrene) and a constant amount of hemin-labelled AFP and a known volume of test solution were added. Non-labelled and hemin-labelled AFP compete for binding to the plate-bound antibody. After the immunoreaction, the immunochemically adsorbed hemin-AFP conjugate moiety was determined by measuring the fluorescence produced in a solution containing HPA and hydrogen peroxide. The calibration graph for AFP was linear over the range 0 ∼ 380 ng/ well with a detection limit of 1.0 ng/well. The method has been applied to determine the AFP in human blood serum with satisfactory results.