Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm–Horsfall protein in human urine

• Published in: Annals of clinical biochemistry 2016-01, Vol.53 (1), p.75-84
• Main Authors: Akimoto, Masaru, Hokazono, Eisaku, Ota, Eri, Tateishi, Takiko, Kayamori, Yuzo
• Format: Article
• Language: English
• Subjects: Analytic Sample Preparation Methods; Chromatography, High Pressure Liquid - methods; Chromatography, Reverse-Phase - methods; Humans; Limit of Detection; Reproducibility of Results; Salts - chemistry; Urinalysis - methods; Uromodulin - isolation & purification; Uromodulin - urine
• ISSN: 0004-5632; 1758-1001
• DOI: 10.1177/0004563215583698

Background 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. Methods 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. Results 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. Conclusions 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.