Sample
Fresh serum free of haemolysis, or heparinized Plasma1.
Stability is 7 days at 2 - 8°C protected from light
Note: CK - MB activity decreases a 10% after 24 hours at
4°C or 1 hour at 25°C
CK MB
Kinetic UV Method
Principle
The procedure involves measurement of CK activity in the
presence of an antibody to CK - M monomer. The antibody
completely inhibits the activity of CK - MM and half of the
activity of CK - MB while not affecting the B subunit activity
of CK - MB and CK - BB. Then it’s used the CK method to
quantitatively determine CK - B activity1,2. The CK - MB
activity is obtained by multiplying the CK - B activity by two
.
Reagents composition, Contents and Safety warnings
1. Buffer/enzyme reagent
Anti human polyclonal CK - M antibody (sheep) sufficient to
inhibit up to 2000 U/L of CK - MM
Imidazole
pH 6.7
125 mmol/L
D - Glucose
25 mmol/L
N - Acetil - L - Cysteine
25 mmol/L
Magnesium Acetate
12.5 mmolL
NADP
2.52 mmol/L
EDTA
2.02 mmol/L
Exokinase
≥ 6800 U/L
Sodium Azide
< 0.1%
2. Coenzyme
ADP
15.2 mmol/L
AMP
25 mmol/L
di - adenosine - 5 - pentaphosphate
103 mmol/L
Creatine phosphate
250 mmol/L
Sodium Azide
< 0.1%
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, protected from light
The Reagents are stable until the stated expiration date If
stored tightly closed, refrigerated and protected from light.
*Preparation and Stability of Working solution
Reagent 1
liquid and ready to use
Reagent 2
liquid and ready to use
*Prepare the working solution mixing 4 volumes of Reagent
1 with 1 volume of Reagent 2
Stability 2 weeks at 2 - 8° C or 24 hours at room t
emperature
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. Wavelenght
340 nm
2. Cuvette
light path: 1 cm
3. Temperature
37° C.
4. Adjust the instrument to zero against distilled water
Sample
50 μL
Working solution
1 ml
Mix, incubate 5 minutes, read initial Absorbance (A1) and
start the stopwatch. Read absorbance at 1minutes intervals
thereafter for 3 minutes .
Mean (U/L)
25
74
CV %
9.80
2.62
E. ACCURACY
Comparation between this method (y) and another
commercial one (x) gave the following results:
r = 0.99 y = 1.0183
x + 0.308
The results of the performance characteristics depend on
the analyzer used
F. INTERFERENCES
1. Glucose till to 7 g/L does not interfere
2. Haemoglobin till to 6 g/L does not interfere
3. Triglycerides till to 8 mmol/L do not interfere
4. A list of drugs and other interfering substances with CK
determination has been reported by Young et al 3,4
Quality Control
Control serum is recommended to monitor the performance of
assay procedures.
Limitation of the procedure
Calculate the difference between absorbances A = A2 - A1
A macro form of BB (immunoglobulin complexed) has been
observed wich will be measured as B in the assay. If the measured
CK - B activity exceeds 20 % of the total CK activity, the presence
of macro BB should be suspected.
*Calculation
Bibliography
CK - MB (U/L) = A x 1651
One international unit (IU) in the amount of enzyme that
transforms 1 μmol of substrate per minute, in standard
conditions. The concentration is expressed in units per litre
pf sample (U/L).
*Reference values
CK - MB
TOTAL CK
(Men)
(Women)
up to 24 U/L
up to 195 U/L
up to 170 U/L
CK - MB Activity
———————— x 100 6 - 25 % CK - MB Activity in the sample
CK Total Activity
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 till to 600 U/L
For higher value dilute sample 1:10 with normal saline,
repeat the test and multiply the value by 10
B. DETECTION LIMIT
Values less than 1 U/L give non - reproducible results
C. INTRA - ASSAY PRECISION
Mean (U/L)
CV %
24.95
10.36
66
4.59
D. INTER - ASSAY PRECISION
1. Abbot B. et al: Creatinine Kinase. Kaplan A. et al - Clin. Chem.
The C.V. Mosby Co St. Louis. Toronto Princeton 1984 : 1112 - 1116
2. Gerhardt W. Et et al: Creatine Kinase B - subunit activity in serum
after Immunohinhibition of M - subunit activity. Clin Chem 1979
(25/7) : 1274 - 1280
3. Young D.S. Effects of drugs on Clinical Lab Tests, 4th ed. AACC
Press, 1995 4. Young D.S. Effects of dlsease on Clinical Lab Test,
4th ed. AACC 2001
5. Burtis A. et al. Tietz. Texbook of Clinical Chemistry, 3rd ed.
AACC 1999
6. Tietz N.W. et al, Clinical Guide to Laboratory Tests, 3rd ed.
AACC 1995
7. Mathieu M. et coll.- Reccomandation pour la mesure de la
concentration catalytique de la creatinine kinase dans la serum
human. Ann Biol. Clin. 40 (1482), 87
8. Neumeier D.Preliwtz W., Wurzburg U. et coll. - Determination of
creatine kinase isoenzyme MB activity in serum using
immunological inhibition of creatine kinase M subunit activity.
Activity kinetics and diagnostic significance in myocardial infarction.
Clin. Chim. Acta, 73 (1976) 445
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