UREA
Enzymatic, Colorimetric
principle
Urea is hydrolyzed by urease forming ammonia and
carbamic acid. Carbamic acid spontaneously
decomposes into ammonia and carbon dioxide.
The released ammonium, in the presence of
salicylate and nitroferricyanide reacts in alkaline
solution of sodium hypochlorite, to form a green dye
compound.
The intensity of the green color produced is directly
proportional to the amount of urea concentration.
Reagents composition
R1
Urea Standard
50mg/dl
Urease
>5000 U/l
R2
(Urease)
R3
Phosphate Buffer pH 8.0
50 mmol/l
(buffer reagent)
Sodium Salicylate
52 mmol/l
Sodium nitroprusside
1.0 mmol/l
Sodium Hydroxide
20 mmol/l
R4
Reagent
1000 L
1000 L
Drop
Drop
Drop
50u/l
50 u/l
50 u/l
Sodium Hypo-chlorite
CORROSIVE
10 L
Standard(R1)
10 L
Sample
Mix and incubate for 3 min at 37°C or 5 min at 20-25o C.
Reagent (R4)
(color reagent)
1000 L
(R3)
(Standard)
R2
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
All reagents are ready for use and stable up to expiry
date given on label when stored at 2-8oC
Safety precautions
For in vitro diagnostic use only
Do not pipette by mouth
Exercise the normal precautions required for
handling laboratory reagents.
Procedure
Wave length :
578 nm ( 578-630 )
Optical Path :
1 cm light path
Temperature :
20-25/37oC
Reading :
Against reagent blank
Assay type
End point
BLANK
STANDARD
SAMPLE
200 L
200 L
10 mmol/l
Min, incubate for 5 min at 37°C or 10 min at 25oC and
read sample and standard extinction against blank.
*Calculation
Storage and Stability of Reagents
200 L
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
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