Abstract: Objective To establish a continuous monitoring method to detect the activity of diamine oxidase(DAO) with double-reagent, and to evaluate methodologically. Methods The method was used to identify the optimum reaction conditions about the specific absorption peak,the optimum detection time,a reasonable ratio of reagents,the optimum substrate concentration, the best pH value of reaction buffer system and the concentration of the buffer. The sensitivity,accuracy and anti-interference capability were evaluated by linear range,precision,recovery, interference test and stability of reagents and samples. The reference interval and diagnostic accuracy were identified by detecting the activity of DAO in 150 healthy subjects and 30 patients who were severely burnt and combined with gastrointestinal mucosa barrier dysfunction or failure. Results The final reaction buffer system was based on the 100 mmol/L Tris(hydroxy-methyl)-aminomethane hydrochloride (Tris-HCl) buffer (pH 8.0). The optimum substrate was 3.2 mmol/L 1,4-tetranethylenediamine, and the ratio of reagent Ⅰ and reagent Ⅱ was 4∶1 . The activity of DAO was calculated by the continuous monitoring of the lowering speed of nicotinamide adenine dinucleotide(NADH) absorbance under the wavelength of 340 nm during 200-300 s. The linear range was 0-100 U/L, the imprecision was < 5%, the recovery rate was 95.8% and 98.0%,the method was not interfered when bilirubin＜150 μmol/L,hemoglobin＜4 g/L,lipid＜4 g/L,NH₄⁺＜60 μmol/L and monoamine oxidase(MAO)＜120 U/L. The reagents were stable in 2 weeks. The normal activity of serum DAO was (3.90 ± 2.96)U/L,and according to 95% confidence interval, the diagnostic reference range was is 0 - 9.7 U/L. The area under receiver operating characteristic(ROC) curve was 0.960, and the diagnostic accuracy was good. Conclusions The continuous monitoring method to detect the activity of DAO with double-reagent is sensitive and reproducible,and has strong ability of anti-interference and good diagnostic accuracy. The stability of reagent can meet the daily needs of automatic biochemical analyzer for detection. It may become a routine clinical detection method for the diagnosis of intestinal mucosa injury.

Key words: Diamine oxidase, Enzyme activity, Continuous monitoring method