UREA
Enzymatic UV Method
*principle
Enzymatic determination of Urea according to the
following reactions:
Urease
Urea + H2O + H2
CO2 + 2 NH4+
GLDH
2 NH4+ + 2 - α - Ketoglutarate + 2 NADH
2 - L - Glutamate + NAD + H2O
The Rate of NADH consumption, determined
photometrically at 340 nm, is direct
proportional to Urea concentration in the sample
Reagents composition
Reagent (R1)
Good Buffer
pH 7.8 80 mmol/l
ADP
0.16 mmol/l
Urease
= 8000 U/l
GLDH
= 1500 U/l
Sodium Azide
< 0.1%
Reagent (R2)
NADH
1.4 mmol/l
a - Ketoglutarate
6.7 mmol/l
Sodium Azide
< 0.1%
standard (ST)
Urea
50 mg/dl
According to the present laws the kit does not
contain substances classified as dangerous
Storage and Stability of Reagents
Store the kit at 2 - 8° C
All the components are stable until the stated
expiration date if stored tightly closed,refrigerated
and protected from light.
It seldom occurs bacterial defilement that is pointed
out from the presence of filaments in the reagents.
In that case it is necessary discard the reagent
Preparation and Stability of working solution
Reagents liquid and ready to use
Mix 4 volumes of Reagent 1 with 1 volume of
Reagent 2
Working solution stability: 10 days at 2 - 8oC and
protected from light
Bring reagents at Room Temperature before use
Safety precautions
For in vitro diagnostic use only
Do not pipette by mouth
Exercise the normal precautions required for
handling laboratory reagents.
*Procedure
1. Wavelength:
340 nm
2. Cuvette light path:
1 cm
3. Temperature
30/37. C
4. Adjust the instrument to zero against distilled
water
Pipette into cuvettes
Sample
Calibrator
Sample
10 µl
Calibrator
10 µl
Working Solution
1 ml
1 ml
Mix, wait 30 seconds, read the Absorbance of
Calibrator (A1Cal) and Sample (A1Sample). After 1
minute read the second Absorbances (A2Cal) and
(A2Sample).
Calculate the differences of Absorbance A = A1 - A2
*Calculation
Serum/Plasma
A Sample
UREA (mg/dl) =
x standard Value
A standard
Urine
A Sample
UREA (mg/dl) =
x standard Value x 20
A standard
Urine/24 h
Urea in urine (mg/dl)
Urea (g/24 h) =
x Urine Volume 24 h (lt)100
Reference values (as Urea)
Serum, plasma:
17 - 50 mg/dl
Urine:
20 - 35 g/24 h
These values should only be used as a guideline.
Each laboratory should establish its Normal
Reference Range
Performance Characteristics
A. LINEARITY LIMIT
The reaction is linear up to 300 mg/dl
For higher value dilute sample 1:2 with normal
saline, repeat the test and multiply
the value by 2
B. DETECTION LIMIT
Values less than 2.2 mg/dl give non - reproducible
results
C. INTRA - ASSAY PRECISION (N = 20 replicates)
Mean (mg/dl)
SD
CV%
Control 1
42.05
1.658 3.94
Control 2
141.05
2.655 1.88
D. INTER - ASSAY PRECISION (20 replicates for 3
days)
Mean (mg/dl)
SD
CV%
Control 1
41.72
1.82
4.35
Control 2
140.90
2.35
1.67
E. ACCURACY
Comparation between this method (y) and another
commercial one (x), gave the following results:
N = 20 r = 0.993041 y = 1.03x + 1.35
F. INTERFERENCES
1.Haemoglobin up to100 mg/dl does not interfere
2. Bilirubin up to 20 mg/dl does not interfere
3.Triglycerides up to 1000 mg/dl do not interfere
Quality Control
Control sera are recommended to monitor the
performance of manual and automated assay
procedures.
Notes
Conversion:
BUN = UREA x 0.47 (1 mg BUN = 0.47 mg UREA)
UREA = BUN x 2.13 (1 mg UREA = 2.13 mg BUN)
Bibliography
Talke H., Schubert G. E.: Klin Wchers 43, 174
(1965)
For in vitro diagnostic use only.
The following symbols are used on labels
For in vitro diagnostic use
Use by (last day of the month)
Temperature limitation
Batch Code
www.betalab-eg.com