Zhu Q Z, Yang H H, Li D H, Chen Q Y, Xu J G
Key Laboratory of Analytical Sciences of MOE and Department of Chemistry, Xiamen University, Xiamen 361005, China.
Analyst. 2000 Dec;125(12):2260-3. doi: 10.1039/b005748g.
Iron tetrasulfonatophthalocyanine (FeTSPc), a peroxidase mimic, was used as a labeling reagent and poly(N-isopropylacrylamide) (PNIP) as the separation support of the immune complex for the mimetic-enzymatic immunoassay of hepatitis B surface antigen (HBsAg). PNIP was precipitated from aqueous solution when the ambient temperature was higher than its lower critical solution temperature of 31 degrees C. In a sandwich immunoassay, the antigen (HBsAg) first reacted with mouse anti-human HBsAg antibody immobilized on PNIP (PNIP-antibody) and then further reacted with FeTSPc-labeled mouse anti-HBsAg antibody (antibody-FeTSPc) at room temperature in a homogeneous format. After changing the temperature to separate the PNIP-antibody-HBsAg-antibody-FeTSPc conjugate moiety, it was re-dissolved and determined by coupling with the fluorogenic reaction of hydrogen peroxide and p-hydroxyphenylpropionic acid. The sensitivity of this method (3 ng mL-1) was close to that of the traditional ELISA using the same reactants. However, the assay was much faster (the assay time decreased from 100-120 to 45 min). This method was applied to determine HBsAg in human serum with satisfactory results.
四磺酸基酞菁铁（FeTSPc）作为一种过氧化物酶模拟物，被用作标记试剂；聚（N-异丙基丙烯酰胺）（PNIP）作为免疫复合物的分离支持物，用于乙型肝炎表面抗原（HBsAg）的模拟酶免疫测定。当环境温度高于其31℃的低临界溶液温度时，PNIP会从水溶液中沉淀出来。在夹心免疫测定中，抗原（HBsAg）首先与固定在PNIP上的小鼠抗人HBsAg抗体（PNIP-抗体）反应，然后在室温下与FeTSPc标记的小鼠抗HBsAg抗体（抗体-FeTSPc）以均相形式进一步反应。改变温度以分离PNIP-抗体-HBsAg-抗体-FeTSPc共轭部分后，将其重新溶解，并通过与过氧化氢和对羟基苯丙酸的荧光反应偶联来进行测定。该方法的灵敏度（3 ng mL-1）与使用相同反应物的传统ELISA相近。然而，该测定速度更快（测定时间从100 - 120分钟减少到45分钟）。该方法应用于测定人血清中的HBsAg，结果令人满意。
