Application of hemin as a labeling reagent in mimetic enzyme immunoassay for hepatitis B surface antigen

A novel mimetic enzyme immunoassay method has been developed for the determination of hepatitis B surface antigen (HBsAg) in solution. Hemin, a horseradish peroxidase substitute, was used as a labeling reagent to catalyze the reaction of p-hydroxyphenyl acetic (HPA) and hydrogen peroxide in alkaline medium. In the sandwich immunoassay, anti-HBsAg antibody was coated on a 96-well plate (polystyrene), which first reacted with standard HBsAg solution or HBsAg in the test blood serum 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 HPA and hydrogen peroxide. The fluorescence intensity was directly proportional to the concentration of HBsAg. The calibration graph for HBsAg was linear over the range 0-450 ng/well with a detection limit of 0.9 ng/well. The method is sensitive enough for the determination of HBsAg level in hepatitis B blood serum.