氯化血红素作为模拟酶荧光免疫分析乙肝表面抗原

乙肝表面抗原的测定在临床诊断上是一项很重要的指标．现在一般采用酶联吸附免疫分析技术测定，但是酶本身性质不稳定且价格昂贵、操作繁琐；更重要的是大分子的酶作为标记物，由于空间位阻效应而阻碍抗原-抗体的免疫反应．所以，用小分子催化剂代替大分子酶的研究显得日益重要1，2]．近年来有关模拟酶在免疫分析中的应用已有报道3，4]．本文提出了以氯化血红素作为辣根过氧化物酶（HRP）的模拟酶来标记抗体，以盐酸硫胺素（维生素B1）作为供氢体，成功地实现了乙肝表面抗原（HBsAg）的夹心法荧光免疫分析．测定范围是2．5～500ng／wel…

Hemin as a Peroxidase Substitute in Mimetic Enzyme Immunoassay for Hepatitis B Surface Antigen

A novel mimetic enzyme immunoassay method was developed for the determina-tion 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 fluoro-genic substrate, with hydrogen peroxide at pH8.5. 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.