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化学学报
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化学学报 ›› 2008, Vol. 66 ›› Issue (1): 73-78. 上一篇    下一篇

研究论文

4-羟基苯乙烯基吡啶为HRP底物的酶荧光免疫传感体系测定布氏杆菌抗体

龚福春*，湛雪辉，龙姝，曹忠，谭淑珍，谭亚非   

  1. (长沙理工大学化学与环境工程学院 长沙 410076)
  • 投稿日期:2007-05-01 修回日期:2007-06-28 发布日期:2008-01-14
  • 通讯作者: 龚副春

A Fluorometric Enzyme-linked Immunosensing System Based on New Substrate 4-Hydroxylstyrylpridine for HRP for Brucella melitensis Antibody Assay

GONG Fu-Chun* ZHAN Xue-Hui LONG Shu CAO Zhong TAN Shu-Zhen TAN Ya-Fei   

  1. (School of Chemistry and Environmental Engineering, Changsha University of Science and Technology, Changsha 410076)
  • Received:2007-05-01 Revised:2007-06-28 Published:2008-01-14
  • Contact: GONG Fu-Chun

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本文合成了一种新型辣根过氧化物酶（HRP）荧光底物—4-羟基苯乙基吡啶（pHSP）,并首次将它运用于酶联荧光免疫传感体系。对pHSP化学性质的研究证实，pHSP在空气中较稳定，对HRP、H2O2的荧光响应性能优于传统HRP荧光底物如对羟苯乙酸、Amplex Red和佳味醇等。pHSP本身只有极弱的荧光，在HRP催化下可被 H2O2氧化成二聚体产物，该二聚体在300 nm的激发光下能发射波长为437 nm的强荧光，并且反应体系的荧光增加与HRP量在一定浓度范围内成线形相关。根据此原理，建立了兔布氏杆菌抗体的酶联荧光传感分析新方法。运用制备的传感装置测定兔布氏杆菌抗体的线形范围为110-5 1.6 10-3 g/L，抗体检出限为110-5 g/L，相对标准偏差为4.1%（n=11）。 pHSP的二聚体产物水溶性很低，利用设计的装置较好地解决了传统测定溶液体系方法灵敏度打折的问题。

关键词: 4-羟基苯乙烯基吡啶, 辣根过氧化物酶荧光底物, 酶联免疫传感, 布氏杆菌抗体

A novel substrate, 4-hydroxylstyrylpridine (pHSP) was synthesized and used in horseradish peroxidase-enzymatic reaction. A fluorometric enzyme-linked immunosensing device, which was based on pHSP for HRP enzymatic reaction for Brucella melitensis antibody (BrAb) assay, has been developed. In pH 5.8 Britton-Robinson(B-R) buffer solution, HRP-antibody conjugate(HRP-BrAb)can catalyze the oxidation reaction of pHSP by H2O2, and the pHSP is converted to a fluorescent species. The increase of the fluorescence intensity (Emission: 437 nm) of the HRP enzymatic product is proportional to the concentration of HRP-BrAb binding to the Brucella melitensis antigen modified sol-gel matrix. The linear range of determination is 110-5~1.610-3g/L with the relative standard deviation of 4.1%. The detection limit is 110-5 g/L. The proposed method has been successfully used for analysis of commercial formulation and plasma sample with satisfactory results.

Key words: 4-(p-hydroxystyryl)pyridine, horseradish peroxidase fluorogenic substrate, immunosensing, Brucella melitensis antibody