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1 N-acteyl-β-D-glucosaminidase (NAG) assay kit PreparationAim:To prepare liquid NAG assay kit for clinical testing.Methods:Use VRA-NAG as substrate, select appropriate buffer system, concentration of substrate, and surface active agent, preparation Liquid NAG assay kit and evaluated its linearity, precision, accuracy, stability and anti-jamming and other properties.Results:NAG activity is maximum in 100 mmol/L pH 5.8 citric acid buffer, there is little difference between pH 5.0-6.6, and taking into accout of the stability of the substrate, we choose pH 5.0 as the best value of NAG determination. According to Michaelis-Menten equation, Km value is 0.32 mmol/L, therefore, We choose 10 times of the Km value 3.2 mmol/L as the best substrate concentration of the NAG assay kit. Add 0.1% Tween 20 and 0.1% BSA to substrate solution helps VRA-NAG to dissolve and maintain it stability. NAG activity of 0-360 U/L range is in good linearity. Intraassay CV of low sample is 1.74%, while high sample is 0.86%; between CV of low sample is 3.21%, median sample is 1.65% and high sample is 1.08%. The average recovery was 103.7%. Reagent stays stability if opened for 7 days or kept for one year. The concentration of vitamin C<50 mg/L not interfere the assay, while a higher concentration will cause negative interference. Lactate<0.9 g/L，pyruvate <20 mg/L. tartaric acid<17 mg/L, uric acid <0.13 g/L, glucose <9g/L, bilirubin <4.6 mg/L show no significant effect on measurement.Conclusion:This study developed a liquid NAG assay kit which can sensitively. accurately and fastly measure NAG activity in human urine. The linearity, sensitivity，precision, stability and anti-interference performance of the NAG assay kit meet the requirements of clinical application.2 Clinical application of NAG kit in the diagnosis of diabetic nephropathyObjective:To study the application of urinary NAG in the diagnosis of diabetic nephropathy.Methods:Assay the NAG activity of diabetic patients without nephropathy group, diabetic nephropathy group and the control group,Results:The NAG value of diabetic patients without nephropathy is 20.5±16.6 U/L, diabetes nephropathy group is 40.6±13.4 U/L, while control group is 7.2±2.8 U/L. There are significant difference in NAG concentration of diabetic patients without nephropathy, and diabetes nephropathy, compared with control group (P<0.05). There are significant difference in NAG concentration of diabetes nephropathy compared with diabetic patients without nephropathy (P<0.05).Conclusion:Urinary NAG detection play a certain part in the diagnosis of the renal damage in early diabetes.Summary1. NAG assay kit can sensitively, accurately and fastly measure NAG activity in human urine.2. The linearity, sensitivity, precision, stability and anti-interference performance of the NAG assay kit meet the requirements of clinical application.3. The preliminary clinical testing proved that NAG assay kit can sensitively and accurately measure NAG activity, which might be a sensitive early indicator of diabetic nephropathy.
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