Boehm Thomas, Pils Sophie, Gludovacz Elisabeth, Szoelloesi Helen, Petroczi Karin, Majdic Otto, Quaroni Andrea, Borth Nicole, Valent Peter, Jilma Bernd
Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
Department of Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
Clin Biochem. 2017 May;50(7-8):444-451. doi: 10.1016/j.clinbiochem.2016.12.011. Epub 2016 Dec 29.
Diamine oxidase (DAO) is essential for extracellular degradation of histamine. For decades activity assays with inherent limitations were used to quantify the relative amounts of DAO. No reference DAO standard is available. Absolute DAO amounts cannot be determined. Controversy exists, whether DAO is circulating or not in non-pregnant individuals. The role of DAO as biomarker in various diseases is ambiguous. It is not clear, whether precise quantification of human DAO antigen using commercially available enzyme-linked immunosorbent assays (ELISAs) is possible. The objective was to develop a precise and robust ELISA to quantify DAO in various biological fluids.
A research prototype ELISA was established using a mouse monoclonal antibody for capturing and a polyclonal rabbit serum IgG fraction for the detection of human DAO. The limit of blank (LoB), limit of detection (LoD) and estimated limit of quantification (eLoQ) and normal DAO concentrations in serum and plasma were determined.
The LoB, LoD and eLoQ derived from 42 standard curves are 0.27, 0.48 and 0.7ng/mL respectively. The detection range using the LoD as the lower and the highest DAO standard as the upper boundary is 0.5 to 450ng/mL. Serum and plasma mean/median concentrations are between 0.5 and 1.5ng/mL in healthy volunteers (n=58) and mastocytosis patients (n=19) and plateau at approximately 145ng/mL (n=16) during pregnancy. Accurate quantification was not influenced by heparin (DAO is a heparin-binding protein), lipaemic or hemolytic serum. The measured DAO antigen concentrations are in close agreement with published enzymatic activity data using radioactive putrescine as substrate.
This research prototype ELISA is able to reliably and accurately quantify human DAO in different biological fluids. The potential of DAO as biomarker in various diseases can be evaluated.
二胺氧化酶（DAO）对组胺的细胞外降解至关重要。几十年来，人们使用存在固有局限性的活性测定法来量化DAO的相对含量。目前尚无参考DAO标准品。无法确定DAO的绝对含量。对于非妊娠个体中DAO是否循环存在争议。DAO作为各种疾病生物标志物的作用尚不明确。使用市售酶联免疫吸附测定（ELISA）是否能够精确定量人DAO抗原尚不清楚。目的是开发一种精确且可靠的ELISA，用于量化各种生物体液中的DAO。
建立了一种研究用ELISA原型，使用小鼠单克隆抗体进行捕获，多克隆兔血清IgG组分进行人DAO的检测。确定了空白限（LoB）、检测限（LoD）和估计定量限（eLoQ）以及血清和血浆中的正常DAO浓度。
从42条标准曲线得出的LoB、LoD和eLoQ分别为0.27、0.48和0.7ng/mL。以LoD为下限、最高DAO标准品为上限的检测范围为0.5至450ng/mL。健康志愿者（n = 58）和肥大细胞增多症患者（n = 19）的血清和血浆平均/中位数浓度在0.5至1.5ng/mL之间，孕期（n = 16）时稳定在约145ng/mL。准确的定量不受肝素（DAO是一种肝素结合蛋白）、脂血或溶血血清的影响。测得的DAO抗原浓度与使用放射性腐胺作为底物发表的酶活性数据密切一致。
这种研究用ELISA原型能够可靠且准确地量化不同生物体液中的人DAO。可以评估DAO作为各种疾病生物标志物的潜力。
