Validation of New Micro-spectrophotometric Method to Determine Diamine Oxidase Activity in Serum

Food allergy and histamine intolerance (HI) are prevalent worldwide. Diamine oxidase (DAO) activity determination in serum is a promising approach for diagnosing HI. Despite the numerous methods that have been published for determining diamine. oxidase activity in serum, most of them have not been validated and have inherent limitations, such as interference and multi-step analysis. Our objective was to perform the analytical validation of a novel micro-spectrophotometric method for the determination of DAO activity in a routine clinical laboratory setting. In this study we determined DAO activity in serum using a 4-aminoantypirine (4-AAP) and 2,4,6 tribromo-3-hidroxybenzoic acid (TBHBA) combination as chromogen for the first time. Validation steps included determination of accuracy (recovery and linearity) and precision (within-run and between-run repeatability) following the Clinical and Laboratory Standards Institute protocols and the other guidelines. Reagent stability assessment at both 4 °C temperature and frozen (- 20 °C) after 4 h, 24 h, 72 h and a week storage were conducted. The optimum test conditions were determined as pH = 6.0, 37 °C, 55 mM Putrescine and 60 min. LOD and LOQ levels were found as 10.08 U/L and 30.56 U/L respectively. Coefficients of variation (CVs) for within-run and between-run precisions were 5.2%, 9.4% for the normal level and 2.6%, 2.2% for the high range, respectively. This reliable method for quantifying DAO activity in serum provides an optimal approach, offering rapid and straightforward performance in laboratories for clinical use.