NAG  Principle

N-ACETYL-β-D-GLUCOSAMINIDASE (NAG) reagent adopts the MPT-NAG substrate method, using

_____________ N-ACETYL-β-D-GLUCOSAMINIDASE REAGENT KIT (MPT-NAG METHOD) 6-methyl-2-pyridinoyl-N-acetylamino-β-D-glucosaminide (MPT-NAG) as the substrate. Under the effect of NAG in a
 Product Name sample, the substrate is broken down to generate methyl-2-pyridinethiol (MPT). NAG activity can be obtained by
1. Common name: N-ACETYL-β-D-GLUCOSAMINIDASE REAGENT KIT (MPT-NAG METHOD) determining the absorbance increase rate at 340nm.
2. English abbreviation: NAG
MPT-NAG + H2O  N-acetyl-β-D-glucosamine + MPT
NAG

 Package Specification
 Main Components
Product Specification
Reagent Kit Main Components Concentration/Content
Calibration Sample: 1mL (optional) Citrate buffer 200mmol/L
R1: 1×18mL R2: 1×6mL
Control: 2×1mL (optional) Reagent 1（R1） Sodium chloride 50mmol/L
Calibration Sample: 1mL (optional) EDTA 2mmol/L
R1: 1×45mL R2: 1×15mL
Control: 2×1mL (optional) Citrate buffer 10mmol/L
Calibration Sample: 1mL (optional) Reagent 2（R2） MPT-NAG 10mmol/L
R1: 1×30 mL R2: 1×10mL
Control: 2×1mL (optional) PEG6000 2%
Calibration Sample: 1mL (optional) N-ACETYL-β-D-GLUCOSAMINIDASE CALIBRATION SAMPLE See the label
R1: 3×20 mL R2: 1×20mL
Control: 2×1mL (optional) N-ACETYL-β-D-GLUCOSAMINIDASE CONTROL See the label
Calibration Sample: 1mL (optional) Note: Each component of the reagent kits with different lot numbers is not interchangeable. The fixed value of
R1: 3×35mL R2: 1×35mL
Control: 2×1mL (optional) Calibration Sample can be traced to the self-established method of the company. The fixed values of every batch of
Calibration Sample: 1mL (optional) Calibration Sample differ slightly from others’. For detailed fixed values refer to the label.
R1: 4×30 mL R2: 4×10mL
Control: 2×1mL (optional)  Storage Conditions and Shelf Life
Calibration Sample: 1mL (optional) 1. The shelf life is 12 months, if kept airtight in dry, dark places at 2℃—8℃.
R1: 2×60 mL R2: 1×40mL
Control: 2×1mL (optional) 2. Stable for 30 days at 2℃—8℃ after opened.
Calibration Sample: 1mL (optional)  Date of Manufacture: See the label.
R1: 2×45L R2: 1×30L
Control: 2×1mL (optional)
 For Instrument
Calibration Sample: 1mL (optional)
R1: 4×60L R2: 2×40L DIRUI CS-300A, CS-300B, CS-400A, CS-400B, CS-600A, CS-600B, CS-800A, CS-800B, CS-620, CS-1200,
Control: 2×1mL (optional) CS-1300A, CS-1300B, CS-1600, CS-1600A, CS-6400, CS-400C, CS-800C, CS-800D, CS-T240A, CS-T240B,
Calibration Sample: 1mL (optional) CS-T300A, CS-T300B, CS-400, CS-800, CS-380, CS-1400, CS-4000, CS-T240, CS-T300, CS-T180, CS-480, CS-680,
R1: 3×500L R2: 1×500L
Control: 2×1mL (optional) CS-9200, CS-T120 Auto-Chemistry Analyzer; DR-7000D Semi-automatic Chemistry Analyzer; Hitachi 7180, 7600
Calibration Sample: 1mL (optional) automatic biochemical analyzer; Toshiba TBA-40FR, TBA-120FR, TBA-2000FR automatic biochemical analyzer;
R1: 3×1L R2: 1×1L
Control: 2×1mL (optional) Abbott C8000, C16000 automatic biochemical analyzer; Backman DxC 600, DxC 800, AU480, AU680, AU2700,
Calibration Sample: 1mL (optional) AU5400, AU5800 automatic biochemical analyzer. The company has application parameters of each type of automatic
R1: 2×60mL R2: 2×20mL biochemical analyzer for the user to refer to.
Control: 2×1mL (optional)
Calibration Sample: 1mL (optional)  Sample Requirement
R1: 3×50mL R2: 2×25mL
Control: 2×1mL (optional) 1. Urine sample: take fresh urine to be tested;
Calibration Sample: 1mL (optional) 2. Can be stable for a week, if kept at 2℃—8℃; one month if at -20℃. If urine pH is smaller than 4 or larger than 8,
R1: 4×60mL R2: 4×20mL long-term storage may make NAG lose activity.
Control: 2×1mL (optional)
 Detection Method
 Intended Use
1. Reagent preparation: dual reagents; can be used directly.
The reagent kit is applicable to the in vitro quantitative detection of the activity of
2. Detection conditions:
N-ACETYL-β-D-GLUCOSAMINIDASE (NAG) in a human urine sample.
Detection temperature 37℃ R1 consumption 210μL
NAG (E.C.3.2.1.30) is a kind of hydrolytic enzyme found in Cytolysosome, and NAG in urine is mainly from the renal
Main wavelength 340nm R2 consumption 70μL
proximal tubule epithelium, but healthy human urine has little of it. Urinary NAG activity increase is a sensitive indicator
Sub-wavelength 700nm Sample consumption 14μL
of early renal damage and diabetic micro-vascular disease. More and more detailed research data have confirmed the
Optical path of cuvettes 1.0cm Delay time 71s
clinical value of urinary NAG detection, which has been widely used for the early renal damage in diabetes, diabetic
Detection mode Rate assay Reaction time 229s
microangiopathy, shock wave therapy of kidney stones, and renal toxicity detection in antibiotic therapy. The function
cannot be replaced by microalbuminuria detection. Absorbance range 0A—3.2A Reaction direction Positive reaction
3. Operation procedure
 Matters Needing Attention
Detection temperature: 37℃
Main wavelength: 340nm
1. Matters needing attention regarding use
Sample consumption：14μL
Add R2：70μL Sub-wavelength: 700nm 1.1 The product is only used for in vitro diagnosis.
R1 consumption：210μL 1.2 Avoid adding reagents during testing, and avoid direct sunlight during operation; because pigment is very sensitive
to temperature change, the temperature for testing should be kept constant.
Record absorbance change △A/min 1.3 According to the requirements of different instruments, the dosage of reagent and sample can be changed
proportionally.
1.4 Screw tight the bottle cap of the reagent after use immediately.
300s 1.5 The reagent cannot be frozen.
71s 229s
2. Matters needing attention regarding safety
2.1 Please dispose of the sample as hazardous materials possibly infected with HIV, HCV, HBV, etc. To avoid or reduce
4. Calibration
the risk of infection, use disposable gloves.
It’s recommended to use the matching calibration sample to execute calibration according to the instrument instructions.
2.2 The reagent is toxic and corrosive; if it comes into contact with the eyes, mouth, or skin, immediately wash with
In the case of reagent lot number change or QC out of control, calibration should be done again.
water; if necessary, please go to see a doctor.
5. QC
2.3 Samples, waste liquid, etc. are potentially infectious, so the operator should comply with laboratory safety operation
It’s advised to use the control products made by DIRUI for quality control. The lab shall establish control ranges and
regulations, and dispose of them according to the local medical waste, infectious waste, industrial waste and other
limited values by itself, if control values are out of control, proper measures shall be taken.
treatment regulations.
6. Detection result calculation
/  Reference
NAG concentration(U/L)＝ ×calibration concentration(U/L) 1. Synthesis of novel substrate for N-acetyl-β-D-glucosaminidase and its value in diagnosis of renal tubular lesions 2012：
/
32（5）：684-688 Xu Lei; Yao Liyun; Liu Huizhong; Zhang Jianhua; Yang Wanhua
Note: There is no matter that one must pay attention to during testing.
2. NCCLS. Interference Testing in Clinical Chemistry; Approved Guideline, 2005.
 Reference Range
 Specifications Approval Date and Modification Date:11/2018
0.3U/L — 11.5U/L
The reference range is only for reference. It’s recommended that each laboratory consider the applicability of the
reference values to the exposed population group, and define a reference range by itself if necessary.
 Detection Results Explanation
1. The accuracy of the test results depends on the instrument calibration, test temperature, and time control.
2. Detection of N-ACETYL-β-D-GLUCOSAMINIDASE activity in urine is only one of the indexes for clinicians to
diagnose patients’ disease. Clinicians need to make a comprehensive judgment according to patients’ symptoms,
medical history, and other diagnostic items and methods.
 Limitations of Detecting Method
1. According to the requirements of different instruments, the dosage of reagent and sample can be changed
proportionally.
2. When the concentration of NAG exceeds 200U/L, the sample should be re-determined after dilution.
3. When ascorbic acid is more than 1mg/dL, it may have an effect on the detection.
 Product Performance Characteristics
1. Reagent blank:
Reagent blank absorbance: A≤0.200.
Reagent blank absorbance change rate: |△A| /min≤0.010.
2. Analysis sensitivity: when testing 1U/L analyte, absorbance change rate △A/min≥0.0002.
3. Linear range: 2U/L—200U/L, linear correlation coefficient r ≥0.990; within [2，20]U/L, absolute deviation should
not exceed ±3U/L; within (20，200]U/L, relative deviation should not exceed ±15％.
4. Accuracy: 85%≤rate of recovery≤115%.
5. Measurement precision:
Repeatability: CV≤5%.
Between-run difference: R≤7%.
6. Appearance: R1 should be colorless transparent liquid, and can contain a small amount of insoluble substances that
don’t affect testing; R2 should be colorless or slight yellow transparent liquid, and can contain a small amount of
insoluble substances that don’t affect testing.