ABSTRACT
Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at -25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20-100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at -80 °C, but were severely damaged at -25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at -25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs.
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. CONCLUSION: 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.
Subject(s)
Alkalies/chemistry , Aquaporin 2/urine , Enzyme-Linked Immunosorbent Assay , Exosomes/chemistry , Sodium Hydroxide/chemistry , Specimen Handling/methods , Urinalysis/methods , Biomarkers/urine , Cold Temperature , Enzyme-Linked Immunosorbent Assay/standards , Healthy Volunteers , Humans , Hydrogen-Ion Concentration , Reproducibility of Results , Time Factors , Urinalysis/standardsABSTRACT
Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs.
Subject(s)
Chromatography, Affinity , Nasopharynx/metabolism , Otitis Media with Effusion/diagnosis , Pneumococcal Infections/diagnosis , Rhinitis/diagnosis , Sinusitis/diagnosis , Streptococcus pneumoniae/isolation & purification , Teichoic Acids/metabolism , Acute Disease , Adolescent , Adult , Bacterial Proteins/genetics , Case-Control Studies , Child , Child, Preschool , Ear, Middle/microbiology , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Otitis Media with Effusion/microbiology , Pneumococcal Infections/microbiology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Rhinitis/microbiology , Sensitivity and Specificity , Sinusitis/microbiology , Streptococcus pneumoniae/immunology , Teichoic Acids/immunology , Young AdultABSTRACT
We tested for four kinds of allergic substances (egg, milk, wheat and peanuts) in 52 imported processed foods using immunochromatographic test kits (ITK). ELISA was also employed to confirm the effectiveness of the ITK. Among 92 data from 23 samples, allergic substances were detected in 9 cases with one kind of ITK, but not with the ELISA test. Among 116 data from 29 samples, 6 were negative with one kind of ITK and but positive with the other ITK. These results suggested that these 4 kinds of allergic substances in imported foods can be detected by using a double-check method with two kinds of ITK.
