Hemin as a peroxidase substitute in mimetic enzyme immunoassay for hepatitis B surface antigen

A novel mimetic enzyme immunoassay method was developed for the determination of hepatitis B surface antigen (HBsAg) in solution. Hemin, a horseradish peroxidase substitute, was used as a labelling reagent to catalyze the reaction of thiamine, a new fluorogenic 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 standard 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 solution containing thiamine and hydrogen peroxide. The flourescence intensity was directly proportional to the concentration of HBsAg. The calibration graph for HBsAg was linear over the range 2. 5∼500 ng/well with a detection limit of 2. 5 ng/well.