title: Alkali treatment stabilizes fluctuations of urine AQP2 values measured by ELISA.
abstract: BACKGROUND
  Aquaporin-2 (AQP2) in urine is now measured in many water-balance disorders and regarded as a useful biomarker for diagnosis and prognosis. An enzyme-linked immunosorbent assay (ELISA) method has been developed for measurement of large numbers of clinical samples. However, fluctuations in the measured values were sometimes observed depending on storage conditions. Urine AQP2 is present in exosome membranes and we speculated that this structural organization causes the fluctuations.
  
  
  METHODS
  Human urine samples from healthy subjects were measured by ELISA. Effects of maneuvers to disrupt the exosome membrane mechanically (freezing and thawing at different temperatures) and chemically (treating with alkali and detergents) prior to ELISA were examined.
  
  
  RESULTS
  Urine samples stored at 4 or -80 °C did not show significant AQP2 values, whereas those stored at -25 °C for more that 2 weeks provided the values. Urine samples treated with 0.4 N NaOH and 0.5 % Triton X-305 showed the consistent and comparable values to those stored at -25 °C.
  
  
  CONCLUSIONS
  Pretreatment with alkali (0.4 N NaOH) to disrupt exosome membranes allowed consistent ELISA measurements of urinary AQP2. This simple method is applicable to ELISA of other membrane proteins included in exosomes.
authors.affiliation: Department of Nephrology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8519, Tokyo, Japan. ssasaki.kid@tmd.ac.jp.
authors.forename: Sei
authors.initials: S
authors.lastname: Sasaki
authors.affiliation: Diagnostic Division, Department of Research and Development, Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Saijo
authors.affiliation: Institute of Biomedical Innovation, Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan.
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Ohmoto
authors.affiliation: Institute of Biomedical Innovation, Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan.
authors.forename: Fusako
authors.initials: F
authors.lastname: Iwata
authors.affiliation: Diagnostic Division, Department of Research and Development, Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Koga
authors.affiliation: Diagnostic Division, Department of Research and Development, Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan.
authors.forename: Kiyonori
authors.initials: K
authors.lastname: Katsuragi
mesh_terms: D000468:Alkalies / Q000737:chemistry*; D051399:Aquaporin 2 / Q000652:urine*; D015415:Biomarkers / Q000652:urine; D003080:Cold Temperature; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D055354:Exosomes / Q000737:chemistry*; D064368:Healthy Volunteers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015203:Reproducibility of Results; D012972:Sodium Hydroxide / Q000737:chemistry*; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D016482:Urinalysis / Q000379:methods* / Q000592:standards
chemical_list: C496023:AQP2 protein, human; D000468:Alkalies; D051399:Aquaporin 2; D015415:Biomarkers; D012972:Sodium Hydroxide
keywords: Aquaporin-2; Biomarker; Exosome; Kidney; Membrane protein; Water balance