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