Akimoto Masaru, Hokazono Eisaku, Ota Eri, Tateishi Takiko, Kayamori Yuzo
Clinical Chemistry, Division of Biochemical Science and Technology, Department of Health Sciences, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
Clinical Chemistry, Division of Biochemical Science and Technology, Department of Health Sciences, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan Department of Medical Laboratory Science, Faculty of Health Sciences, Junshin Gakuen University, Fukuoka, Japan.
Ann Clin Biochem. 2016 Jan;53(Pt 1):75-84. doi: 10.1177/0004563215583698. Epub 2015 Apr 2.
Tamm-Horsfall protein (also known as uromodulin) is the most abundant urinary protein in healthy individuals. Since initially characterized by Tamm and Horsfall, the amount of urinary excretion and structural mutations of Tamm-Horsfall protein is associated with kidney diseases. However, currently available assays for Tamm-Horsfall protein, which are mainly enzyme-linked immunosorbent assay-based, suffer from poor reproducibility and might give false negative results.
We developed a novel, quantitative assay for Tamm-Horsfall protein using reversed-phase high-performance liquid chromatography. A precipitation pretreatment avoided urine matrix interference and excessive sample dilution. High-performance liquid chromatography optimization based on polarity allowed excellent separation of Tamm-Horsfall protein from other major urine components.
Our method exhibited high precision (based on the relative standard deviations of intraday [≤2.77%] and interday [≤5.35%] repetitions). The Tamm-Horsfall protein recovery rate was 100.0-104.2%. The mean Tamm-Horsfall protein concentration in 25 healthy individuals was 31.6 ± 18.8 mg/g creatinine. There was a strong correlation between data obtained by high-performance liquid chromatography and enzyme-linked immunosorbent assay (r = 0.906), but enzyme-linked immunosorbent assay values tended to be lower than high-performance liquid chromatography values at low Tamm-Horsfall protein concentrations.
The high sensitivity and reproducibility of our Tamm-Horsfall protein assay will reduce the number of false negative results of the sample compared with enzyme-linked immunosorbent assay. Moreover, our method is superior to other high-performance liquid chromatography methods, and a simple protocol will facilitate further research on the physiological role of Tamm-Horsfall protein.
Tamm-Horsfall蛋白（也称为尿调节蛋白）是健康个体中最丰富的尿蛋白。自最初由Tamm和Horsfall鉴定以来，Tamm-Horsfall蛋白的尿排泄量和结构突变与肾脏疾病相关。然而，目前可用的Tamm-Horsfall蛋白检测方法主要基于酶联免疫吸附测定，重现性差，可能会给出假阴性结果。
我们开发了一种使用反相高效液相色谱法测定Tamm-Horsfall蛋白的新型定量检测方法。沉淀预处理避免了尿液基质干扰和过度的样品稀释。基于极性的高效液相色谱优化使得Tamm-Horsfall蛋白与其他主要尿液成分能够得到出色的分离。
我们的方法具有高精度（基于日内[≤2.77%]和日间[≤5.35%]重复的相对标准偏差）。Tamm-Horsfall蛋白回收率为100.0 - 104.2%。25名健康个体中Tamm-Horsfall蛋白的平均浓度为31.6±18.8 mg/g肌酐。高效液相色谱法和酶联免疫吸附测定法获得的数据之间存在很强的相关性（r = 0.906），但在低Tamm-Horsfall蛋白浓度下，酶联免疫吸附测定值往往低于高效液相色谱法值。
与酶联免疫吸附测定法相比，我们的Tamm-Horsfall蛋白检测方法具有高灵敏度和重现性，将减少样品的假阴性结果数量。此外，我们的方法优于其他高效液相色谱法，并且一个简单的方案将有助于进一步研究Tamm-Horsfall蛋白的生理作用。
