An enzyme-catalyzed reaction system puerarin-horseradish peroxidase (HRP)-hydrogen peroxide, which used puerarin as substrates for HRP, was developed. In enzymatic reaction procedure, HRP catalyze the polymerization of puerarin by H₂O₂, and the puerarin is partly converted to dimers, a significantly fluorescent species. The increase of the fluorescence of the HRP-enzymatic reaction solution at emission of 478 nm (excitation: 315 nm) is proportional to the concentration of HRP. The enzyme-linked fluoroimmunoassay based on the principle mentioned above and competitive immunoassay model for the determination of Brucella melitensis antibodies (BrAb) was established. The results of the property investigation of puerarin demonstrate the advantages in stability and reactivity with HRP over conventional substrates such as p-hydroxyphenylacetic acid (p-HPA), Amplex red and homovanillic acid. The experimental parameters such as BrAb-HRP, temperature were optimized. The proposed method has been successfully used for analysis of BrAb in rabbit plasma sample. The linear range of determination for BrAb is 1.3～120 ng/mL with the relative standard deviation of 3.8%. The detection limit is 1.3 ng/mL (3σ).

Key words: puerarin, HRP fluorogenic substrate, Brucella melitensis antibody, enzyme-linked fluoroimmunoassay