2. Test condition (basic parameters).

5’-NT Temperature 37℃ Reagent 1 Volume 180μL

5’-NUCLEOTIDASE REAGENT KIT（ENZYME METHOD） Main Wavelength 550nm Sample Volume 5 μL
Absorbance Range 0~2 A Reagent 2 Volume 90 μL
◆Product Name Optical Path 1.0 cm Delay Time 120 s
5’-NUCLEOTIDASE REAGENT KIT（ENZYME METHOD） Test Time 120 s Reaction mode Rate assay
◆Intended Use 3.Testing procedure
The reagent is used for the in vitro quantitative measurement of 5’- nucleotidase activity in human serum or plasma. Sample volume: 5μL Reagent 2 volume: 90μL Temperature: 37℃
5’-nucleotidase is a enzyme that catalyze nucleotide -5’- monophosphate, and hydrolyze into nucleotide and inorganic Reagent 1 volume: 180μL Main wavelength: 550nm
phosphate. It is widely distributed in a variety of organizations in humans and animals. 5’-nucleotidase in serum is got from
the release of liver cell membrane, under the action of bile salts. It has been used as a diagnostic marker for liver disease.
Record absorbance change △A/min
5’-nucleotidase elevation in serum is found in certain liver diseases, such as the liver, extrahepatic obstruction. The diagnostic
value of 5’-nucleotidase is superior to other liver enzymes. 300s 120s 120s
◆Principle
5’-nucleotidase hydrolyze hypoxanthine riboside -5’- monophosphate (5’-IMP), generating hypoxanthine riboside. In the
presence of purine nucleoside phosphorylase (PNP), the latter turns into hypoxanthine. And then by the action of xanthine
oxidase (XOD), hypoxanthine turned into uric acid and H2O2. In the presence of peroxidase (POD), H2O2 react with EHSPT 4. Calibration
and 4 - amino-antipyrine (4-AA) to generate colored quinine. Continuously monitor the generation rate of colored quinine at
550nm, 5’-NT activity can be calculated. The whole process can be expressed with the following reaction. It is suggested to use supplementary calibrator as instructed. When lot number is changed or QC is invalid, calibration shall be
conducted again.
IMP+ H2O 5'-NT
  Hypoxanthine Riboside +Pi (Inorganic Phosphorus) Please input K factor if calibrator is not used.
5. QC
Hypoxanthine Riboside +Pi PNP
 Hypoxanthine + Ribose -1- Phosphoric Acid It is suggested to use QC products produced by Dirui. The laboratory shall establish its own QC area and limit. If QC value is

Hypoxanthine +2 H2O +2O2 

 Uric Acid+2 H2O2
XOD out of control, correction measures shall be taken.
6. Calculation
2 H2O2+4-AA+EHSPT 
 4 H2O+ Colored Quinone
POD
Sample Tube A / min
Sample Concentration（ U / L）   Calibrator Concentration（ U / L）
Calibration Tube A / min
◆Reagent Composition
Reagent 1 Calculation method without using calibrator（550nm）:
HEPES 100 mmol/L Sample concentration（U/L）= △A/min×K factor = △A/min×3036
4- amino-antipyrine 2 mmol/L For getting more accurate result, it is suggested to use calibrator for tests.
Purine Nucleoside Phosphorylase 100U/L
◆Reference Range
Xanthine Oxidase 200U/L
Adult：≤10U/L
Peroxidase 600U/L
The reference range applied is the expected value for this method, which is only for reference. It is recommended for all
Reagent 2
laboratories to do relevant tests to validate such range or establish their own reference ranges.
HEPES 100 mmol/L
5’- hypoxanthine riboside monophosphate 10 mmol/L ◆Explanation of Results
EHSPT 2 mmol/L 1. If 5’-nucleotidase activity＞200U/L，the sample should be tested after dilution with distilled water. Multiply the result by
Component of kits with different lot numbers are not interchangeable. the dilution times.
◆Storage Conditions and Shelf Life 2. The test of 5’-nucleotidase activity in serum and plasma is only one of the indicators of clinical diagnosis for patients, and
When stored at 2~8℃ and protected from light, the reagent is stable for 12 months. It is stable for 30 days at 2~8℃ after clinicians will conduct a comprehensive diagnosis according to the patient’s body、history diagnosis, as well as other items
opened. and diagnostic methods.
◆Suitable Device ◆Limit
This reagent is suitable for all kinds of semi-auto and full automatic chemistry analyzers. The verification is needed if it is 1. The accuracy of results relies on the control of calibration of the instrument, testing temperature and time.
used in other instrument. All kinds of application parameter of automatic chemistry analyzers are prepared for reference. 2. When hemoglobin is＞2.5g/L, triglyceride is ＞50mmol/L, the test result may be affected.
◆Sample Requirements ◆Specifications
1. The best sample is fresh non-hemolytic serum or plasma. 1. Linearity: up to 200U/L
2. For plasma, heparin can be used as anticoagulant. Please centrifuge the plasma right after the blood collection, or centrifuge 2. Blank absorbance：A≤0.500
within 1 hour under cold condition.
Blank absorbance change rate: 丨△A 丨/5min≤0.02
3. The plasma sample is stable for 7 days at 4℃. 3. The minimum test limit: test normal saline 20 times repeatedly, and the minimum test limit is determined as 2U/L by
◆Method average +2 times SD.
1. Reagent preparation: Reagent 1 and Reagent 2 are liquid reagent, which is ready to use. 4. Precision: test two samples with different concentration on the same test system within 20 work days.
 Precision of the same lot number n=20 Precision between days n=20 Main wavelength 546 546 546 546 546 546 546 546 546 546
QC serum
X ( U/L) SD CV% X (U/L) SD CV% Sub wavelength 660 660 660 660 660 660 660 660 660 660

Sample 40.40 0.74 1.83 3.98 0.04 1.01 Reagent R1/T1 180 180 180 180 180 180 180 180 180 180

5.Method comparison: Conduct tests upon 200 samples with reagent y of the company and the on the market reagent x, the R2/T2 90 90 0 90 0 90 90 90 90 0
relevance between our reagent (y) and approved market reagent (x) is: y=0.8509x+3.6871，r=0.9836 R3/T3 —— —— 90 —— 90 —— —— —— —— 90

◆Standardization Traceability R4/T4 —— —— 0 —— 0 —— —— —— —— 0

The constant value of calibrator can be traced to method of Dirui. Normal volume
5 5 5 5 5 5 5 5 5 5
of serum sample
◆Matters Need Attention Absorbance limit 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2
1. Cautions for operation Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
Reaction type
1.1 The product is only for in vitro diagnosis. reaction reaction reaction reaction reaction reaction reaction reaction reaction reaction
1.2 Do not add reagent during the test. Prozone check
-3.3 lower -3.3 lower -3.3 lower -3.3 lower -3.3 lower -3.3 lower -3.3 lower -3.3 lower -3.3 lower -3.3 lower
limit limit limit limit limit limit limit limit limit limit
1.3 Volume of reagent and sample can be changed proportionally in accordance with the requirements of instrument.
Calibration 1-point 1-point 1-point 1-point 1-point 1-point 1-point 1-point 1-point 1-point
1.4 If the reagent is turbid, or the absorbance value is greater than 0.500, then it can not be used. method linearity linearity linearity linearity linearity linearity linearity linearity linearity linearity
1.5 The calculated result—3036 is the K-value for the 550nm dominant wavelength, different models of automatic analyzer K factor 3036 3036 3036 3036 3036 3036 3036 3036 3036 3036
will be various in the measurement of the dominant wavelength, K values are also different. Dominant wavelength λ = 546nm, Discreteness
K = 3364; dominant wavelength λ = 560nm, K = 3128. 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3
check
2. Cautions for safety Discreteness
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
2.1 Consider the product as dangerous materials that may cause HIV, HBV, HCV and other infections. To avoid the risk, use check

single-use gloves. Sensitivity check 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

2.2 Avoid contact with skin, clothes, and eyes. Once in contact with skin or clothes, rinse the contact part with plenty of water, Blank horizontal
-3.3~3.3 -3.3~3.3 -3.3~3.3 -3.3~3.3 -3.3~3.3 -3.3~3.3 -3.3~3.3 -3.3~3.3 -3.3~3.3 -3.3~3.3
check
and go to see a doctor.
Linearity range 2~200 2~200 2~200 2~200 2~200 2~200 2~200 2~200 2~200 2~200
2.3 The samples and waste liquid have potential infectious risk, and the user should manage them according to the laboratory
safety operation rule, local laws and regulations.
◆Reference
1. Dinkov L,Krainkova M,Brailski KH;Serum 5’-nucleotidase activity inchronic hepatobiliary diseases.Vutr
BOLES.1979;18(1):39～46.
2. NCCLS.Interference Testing in Clinical Chemistry;Approved Guideline,2005.
◆Date of Approval and Revision: 11/2018

◆Packaging Specification
No. Specifications Type

232014402002 R1:1×50mL R2:1×25mL Dirui CS-400/600/800/1200/1300/1600/6400 Package

232014402001 R1:1×50mL R2:1×25mL Dirui CS-240/300 Package

232014402009 R1:1×40mL R2:1×20mL Dirui CS-T Series Package

232014402010 R1:1×40mL R2:1×20mL Dirui CS-T Series Package

232014402013 R1:2×150mL R2:1×150mL Dirui CS-1600/6400 Package

◆P.S.：CS Series Auto-Chemistry analyzer parameters

Model CS-240 CS-300 CS-400 CS-600 CS-1200 T240 T300 CS-6400 CS-1600 CS-1300

Item 5'-NT 5'-NT 5'-NT 5'-NT 5'-NT 5'-NT 5'-NT 5'-NT 5'-NT 5'-NT

Unit U/L U/L U/L U/L U/L U/L U/L U/L U/L U/L

Method Rate A Rate A Rate A Rate A Rate A Rate A Rate A Rate A Rate A Rate A

Time 20 20 10 10 10 13 10 9 12 9
Photometric
22~27 22~27 23~31 26~36 23~30 29~37 31~41 21~28 19~24 23~31
point