N-Acetyl-glucosaminidase Assay Kit Insert March HAEMOSCAN BV
COMPANY WITH
QUALITY MANAGEMENT SYSTEM
CERTIFIED BY DNV GL
= ISO 9001 =
N-acetyl-glucosaminidase
Assay
Kit Insert
Version: March 2017
Introduction
N-acetyl-glucoseaminidase (NAG) is excreted into the urine following injury to
the tubular system of the kidney. This injury may occur after renal ischemia
or may be induced by an inflammatory reaction. NAG is determined by means
of a substrate conversion assay. Under appropriate conditions, NAG present
in a urine sample converts a chromogenic substrate. After development of
the color the optical density is determined. The concentration (U/L) is
quantified by applying standards with known concentrations of the enzyme. A
quantitative measure of tubular damage can be obtained after simultaneous
measurement of urea, to correct for dilution.
Principle of the Test
This standard operating procedure describes the determination of NAG in
urine samples. The samples are incubated with a chromogenic substrate.
After one hour of incubation at low pH, a basic stop solution is added and the
optical density at 400 nm is measured. Because the samples may contain
components which can result in a high background signal, for every sample a
sample blank value is determined in which the stop solution is added prior to
the reaction, instead of after the reaction.
The concentration of NAG is expressed in units/liter (U/L). One unit converts
1 mmol of substrate per minute at a pH of 4.25 and a temperature of 25°C.
Precautions
• Keep the kit in an as cold as possible freezer. The shelf life of one year
is based on -20 ºC.
• The kit is intended for research use only.
• The kit should not be used beyond its expiry date.
• Do not combine reagents from kits with different lot numbers.
• Chemicals and reagents have to be treated as hazardous waste
according to biohazard safety guidelines or regulations.
• Wear disposable (latex) gloves when handling specimens and
reagents.
• Never pipette by mouth and avoid contact of skin and mucous
membranes
• Use disposable pipette tips throughout the procedure to avoid
contamination of reagents.
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Contents of the Kit
• NAG Substrate 11 mL 1 bottle
• Stop Solution 11 mL 1 bottle
• NAG Standard (25 U/L) 500 µL 1 tube
• Dilution Buffer 2 mL 1 bottle
• Control 1, normal 250 µL 1 tube
• Control 2, high 250 µL 1 tube
Additional Materials and Equipment
The following materials and equipment are required but are not provided with
the kit:
• (Calibrated) adjustable pipettes with disposable tips.
• (Micro-centrifuge) tubes.
• Incubator at 37 °C.
• Transparent 96-well flat-bottom microplate
• Plate shaker.
• Spectrophotometer capable of measuring at 400 nm.
Test Procedure
Reagent Preparation
• Substrate: warm to 37°C before use.
• Calibrators: The NAG standard is used stepwise (1:1) diluted with
Dilution Buffer in separate vials. Use these dilutions as standard curve
(Table 1).
Table 1. Preparation of NAG Calibrators.
NAG Concentration (U/L)
CAL1 100 µL NAG Standard + 100 µL Dilution Buffer 12.5
CAL2 100 µL CAL1 + 100 µL Dilution Buffer 6.25
CAL3 100 µL CAL2 + 100 µL Dilution Buffer 3.13
CAL4 100 µL CAL3 + 100 µL Dilution Buffer 1.56
CAL5 100 µL CAL4 + 100 µL Dilution Buffer 0.78
CAL6 100 µL CAL5 + 100 µL Dilution Buffer 0.39
CAL7 Dilution Buffer 0
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Assay Procedure
1. Pipette 100 µL Stop Solution in all Blank wells of a transparent 96-
well flat-bottom microplate (please refer to the example plate layout
[Table 2]).
2. Pipette 20 µL of calibrators, controls and samples
3. Warm the plate at 37°C for 5 minutes
4. Add 100 µL prewarmed Substrate to each well
5. Shake the plate for 20 seconds on a plate shaker
6. Incubate the plate at 37°C for 60 minutes
7. Add 100 µL Stop Solution to each well except the Blank wells (please
refer to the example plate layout [Table 2])
8. Shake the plate for 20 seconds on a plate shaker
9. Measure the optical density at 400 nm
Table 2. Suggested 96-well template for the NAG Assay
Calculations
1. Subtract the mean optical density of the ‘CAL7 Blank’ (NAG
concentration = 0 U/L) from all the optical density values
2. Construct a calibration curve (y=a·x+b) with the calibrators. The NAG
concentration should go on the y-axis and the OD values of the
calibrators on the x-axis.
3. Calculate the NAG concentration in the Blank wells, the control
samples and the other samples by means of interpolation on the
calibration curve.
4. Subtract the values of the Blank wells from the corresponding
samples
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1 2 3 4 5 6 7 8 9 10 11 12
A CAL1 CTRL1 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
B
CAL2
C CAL3 CTRL2 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
D
CAL4
E CAL5 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
F
CAL6
G CAL7 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
H
CTRL1
Blank
Sample
Blank
Sample
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Sample
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Sample
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Sample
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Sample
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CTRL2
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Sample
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Sample
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Sample
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Sample
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CAL7
Blank
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Assay Criteria
• The correlation coefficient of the calibration curve should be ≥0,98.
Reference values
In a group (n=8) of healthy volunteers, an average NAG concentration
of 1.04 U/l (SD = 0.40 U/l, Range: 0.4 – 1.7 U/l) was found.
Characteristics
Figure 1. Example of a NAG calibration curve.
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0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700
0
2
4
6
8
10
12
14
f(x) = 21.03x - 0.32
R² = 1
OD-BLK (400 nm)
N-Acetyl-glucosa
minidase (U/L)
N-Acetyl-glucosaminidase Assay Kit Insert March HAEMOSCAN BV
N-Acetyl-glucosaminidase Assay Kit Insert March HAEMOSCAN BV
N-Acetyl-glucosaminidase Assay Kit Insert March HAEMOSCAN BV