Sample
Fresh serum free of haemolysis, heparinized or EDTA
Plasma
AST (GOT)
Kinetic UV Method
Principle
Reaction principle
Kinetic determination of AST according IFCC
recommendation:
AST
α - Ketoglutarate + L - Alanine
L - Glutamate + Pyruvate
MDH
+
Pyruvate + NADH + H
L - Malate + NAD+
The Rate of NADH consumption, determined
photometrically, is direct proportional to ALT activity
Reagents composition, Contents and Safety warnings
1. Buffer /Enzyme Reagent
Good Buffer
pH 7.5
80 mmol/l
L - Aspartate
220 mmol/l
LDH
≥ 2100 U/l
MDH
≥ 1100 U/l
EDTA
3 mmol/l
Sodium Azide
0.09%
2. Coenzyme Reagent
α - Ketoglutarate
71 mmol/l
NADH
≥ 1.4 mmoll l
Sodium Azide
0.09%
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 and refrigerated.
*Preparation and Stability of Working solution
REAGENTS
liquid and ready to use
1 - REAGENT PROCEDURE
Mix 4 volumes of. Buffer /Enzyme Reagent with 1 volume
of Coenzyme Reagent
Working solution stability: 10 days at 2 - 8°C.
2 - REAGENTS PROCEDURE
See Procedure
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. Curette
light path: 1 cm
3. Temperature
30/37°C.
4. Adjust the instrument to zero against air or distilled water
One Reagent Procedure
Sample
100 µl
Working Solution
1 ml
Two Reagents Procedure
Buffer
0.8 ml
Sample
100 µl
Coenzyme
200 µl
Reagent
Mix, wait 1 minute, read the Absorbance and start the
stopwatch simultaneously.
Read again after 1, 2 and 3 minutes.
Calculate the average absorbance difference per minute
(A/min)
*Calculation
AST (U/I) = A/min x 1746
Reference values
Male
Female
30°C
< 30 U/l
< 24 U/l
37°C
< 46 U/l
< 38 U/l
C. INTRA - ASSAY PRECISION (N = 20 replicates)
Control 1
Control 2
Mean (U/l)
37.15
136.45
SD
0.853
1.596
D. INTER - ASSAY PRECISION (20 replicates for 3 days)
Mean (U/l)
SD
CV%
Control 1
36.48
1.670
4.58
Control 2
137.02
2.670
1.95
E. ACCURACY
Comparation between this method (y) and another
commercial one (x), gave the following results:
N = 20
r = 0.98321
y = 0.88
x - 1.48
F. INTERFERENCES
1. Haemoglobin up to 50 mg/dl does not interfere
2. Bilirubin up to 30 mg/dl does not interfere
3.Triglycerides up to 1000 mg/dl do not interfere
4. Ascorbic Acid up to 25 mg/dl does not interfere
Quality Control
Control sera are recommended to monitor the performance
of manual and automated assay procedures.
Bibliography
1. EPE/IFCC Provisional recommendation on IFCC
methods for the measurement of catalytic activity
concentrations of enzymes. Part 2 (revised 1977): IFCC
method for Aspartate aminotransferase L - aspartate: 2 oxo glutarate aminotransferase EC 2.6.1.1.: Clin. Chim.
Acta 80, F21 (1977)
2. Redefinition of conditions previously published as part 2
in Clin. Chim. Acta 70: F19 (1976)
For in vitro diagnostic use only.
The following symbols are used on labels
For in vitro diagnostic use
These values should only be used as a guideline.
Each laboratory should establish its Normal Reference
Range
Use by (last day of the month)
Performance Characteristics
Batch Code
A. LINEARITY LIMIT
The reaction is linear up to 400 U/I
For higher value dilute sample1:2 with normal saline,repeat
the test and multiply the value by 2
B. DETECTION LIMIT
Values less than 2 U/I give non - reproducible results
CV%
2.30
1.17
Temperature limitation
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