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Purpose 1. Establish a simple, sensitive, economic PVDF membrane carrier dye binding assay (PVDF film - dye binding) Determination of trace amounts of albumin in the urine. 2 to establish a simple, rapid size exclusion high performance liquid chromatography (SE-HPLC) determination of trace amounts of albumin in the urine. The method PVDF membrane - a dye binding method: PVDF film before adding methanol, plus albumin standards and urine samples to be tested, with Coomassie Brilliant Blue staining solution dyeing, bleaching, after drying, the film forming a sample spots. Gel imaging systems for to spot density scanning of the dot density - albumin concentration of the standard standard curve, and then calculate the concentration of albumin in the urine sample to be tested. SE-HPLC method: clear standard protein or urine samples to 200 ul 0.2mol / L phosphate buffer (pH 7.0) 400μ whirlpool after mixing, automatic injector 20 ul determination; molecular sieve column Agilent ZORBAX GF -250 column (250mm × 4.6mmi.d, 4 um); mobile phase: solvent A 0.1% (v / v) aqueous solution of formic acid and solvent B was acetonitrile, 85:15 volume ratio of solvent A and solvent B; flow rate 1.0 ml / min; column temperature was 30 ° C; detection wavelength was 205nm peak area; using an external standard method for quantitative analysis of urine microalbumin. Results PVDF membrane - the dye-binding method: intra-and inter-assay CV were 9.1% and 10.0% (50 mg / L), 5.6% and 9.6% (100 mg / L) and 5.0% and 7.4% (200 mg / L). 63 patients to be tested sample testing, immunization rate scattering nephelometry and PVDF film - dye binding test results mean 179.30 ± 187.01 mg / L and 237.63 ± 157.81mg / L, the two laws, there is a good correlation ( r2 = 0.8552), but the the PVDF film - dye binding results higher than the immunization rate of scattering nephelometry (P = 0.000). The method detection limit to 15 mg / L, the linear range of up to 600 mg / L. Hb, bilirubin, IgG positive interference. The urine pH no significant effect on the results. PVDF film sensitivity is higher than with a cellulose acetate membrane urine albumin. SE-HPLC method: albumin retention time of approximately 1.73 min, intra-and inter-assay CV were 3.98% and 4.05% (20 mg / L) and 4.65%, 3.55% and 3.60% (200 mg / L) and 4.74% (2000 mg / L), the average recovery was 99.2%. SE-HPLC method with the immune rate nephelometry compared their test results mean normal albuminuria group were 40.0 ± 24.6 mg / L, 8.4 ± 4.3 mg / L (P = 0.000; n = 40), trace the albuminuria group were 260.2 ± 138.8 mg / L, 57.9 ± 37.3 mg / L (P = 0.000; n = 30), in the proteinuria group were 1127.9 ± 622.1 mg / L, 1212.7 ± 696.3 mg / L (P = 0.001; n = 30). Test results of the first two sets of SE-HPLC method was significantly higher than that of immune rate nephelometry, about 7% of the test results in the the proteinuria group of SE-HPLC method than the immune Rate scattering nephelometry. SE-HPLC method detection limit for the 2 mg / L and 2000 mg / L concentration range of linear r2 = 0.9969) Conclusion This study established a PVDF membrane - dye binding assay urine microalbumin concentration, the high precision of the method is simple, low cost, high sensitivity, is an economic and simple urine albumin detection method suitable for the rapid screening of the MAU. (2) This study established a SE-HPLC method detection urine microalbumin concentration, the precision of the method, accuracy, recovery rate, lower limit of detection, linear range, and other indicators are in line with the requirements of the diagnostic tests, especially the mobile phase simple preparation, albumin retention time is short, simple, rapid and sensitive, large quantities of automatic detection can be achieved, suitable for clinical routine quantitative detection of urine microalbumin.
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