Abstract: Anovel mimetic enzyme immunoassay method was developed for the determina-tion of hepatitis Bsurface antigen (HBsAg) in solution. Hemin, a horseradish peroxidase substitute, was used as a labelling reagent to catalyze the reaction of thiamine, a new fluoro-genic substrate, with hydrogen peroxide at pH 8.5. In the sandwich immunoassay,anti-HBsAg antibody was coated on a 96-well plate(polystyrene), which first reacted with stan-dard HBsAg solution, and then further reacted with the fixed amount of hemin-labelled anti-HBsAg. After the two-step immunoreaction, the immunochemically adsorbed hemin-anti-HBsAg conjugate moiety was determined by measuring the fluorescence produced in a solu-tion containing thiamine and hydrogen peroxide. The flourescence intensity was directly pro-portional to the concentration of HBsAg. The calibration graph for HBsAg was linear over the range 2.5-500ng/well with a detection limit of 2.5ng/well.

Key words: Mimetic enzyme flourescence immunoassay analysis, Hemin, HBsAg, Thiamine