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

A novel mimetic enzyme immunoassay to determine alpha-1-fetoprotein (AFP) i n solution was developed. Hemin, a horseradish peroxidase substitute, was u sed as a labelling reagent to catalyze the reaction of p-hydroxyphenylaceti c (HPA) and hydrogen peroxide in alkaline media. In the competitive immunoa ssay, monoclonal anti-AFP antibody was coated on a 96-well plate (polystyre ne) 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 bindin g to the plate-bound antibody. After the immunoreaction, the immunochemical ly adsorbed hemin-AFP conjugate moiety was determined by measuring the fluo rescence produced in a solution containing HPA and hydrogen peroxide. The c alibration graph for AFP was linear over the range 0 similar to 380 ng/well with a detection limit of 1.0 ng/well. The method has been applied to dete rmine the AFP in human blood serum with satisfactory results.