Alkali treatment stabilizes fluctuations of urine AQP2 values measured by ELISA

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. 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. Urine samples stored at 4 or -80 A degrees C did not show significant AQP2 values, whereas those stored at -25 A degrees 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 A degrees C. 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.