RANSOD
αβχ
Superoxide dismutase
MANUAL
RX MONZA
INTENDED USE
For the quantitative in vitro determination of Superoxide
dismutase in whole blood. This product is suitable for
manual use and on the Rx Monza.
SAFETY PRECAUTIONS AND WARNINGS
For in vitro diagnostic use only. Do not pipette by mouth.
Exercise the normal precautions required for handling
laboratory reagents.
Cat. No.
SD 125
5 x 20 ml
Health and Safety data sheets are available on request.
R1a.
R1b.
R2.
CAL
Mixed Substrate
Buffer
Xanthine Oxidase
Standard
5 x 20 ml
1 x 105 ml
3 x 10 ml
5 x 10 ml
ASSAY PRINCIPLE
The role of superoxide dismutase (SOD) is to accelerate the
•
dismutation of the toxic superoxide radical (02 ), produced
during oxidative energy processes, to hydrogen peroxide
and molecular oxygen.
This method employs xanthine and xanthine oxidase (XOD)
to generate superoxide radicals which react with
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium
chloride (I.N.T.) to form a red formazan dye. The
superoxide dismutase activity is then measured by the
degree of inhibition of this reaction. One unit of SOD is that
which causes a 50% inhibition of the rate of reduction of
INT under the conditions of the assay.
XOD
•
Xanthine
•
I.N.T.
formazan dye
OR
•.
+
02 + 02 + 2H
SOD
02 + H202
SAMPLE PREPARATION
Use heparinized or EDTA whole blood samples. It is
recommended that erythrocytes should be washed four
times with 0.9% NaCl solution.
Centrifuge 0.5 ml of whole blood for 10 minutes at
3000 rpm and then aspirate off the plasma. Then wash
erythrocytes four times with 3 ml of 0.9% NaCl solution
centrifuging for 10 minutes at 3000 rpm after each wash.
The washed centrifuged erythrocytes should then be made
up to 2.0 ml with cold redistilled water, mixed and left to
stand at +4°C for 15 minutes. The lysate is diluted with 0.01
mol/l Phosphate Buffer pH 7.0, so that the % inhibition falls
between 30% and 60%.
A 25 fold dilution of lysate is recommended for human
samples (Final dilution factor = 100) and a 50 fold dilution
for bovine samples (Final dilution factor = 200).
REAGENT COMPOSITION
Contents
STABILITY AND PREPARATION OF REAGENTS
R1a. Mixed Substrate
Reconstitute the contents of one vial of Mixed
Substrate R1a with 20 ml of Buffer R1b. Stable for 10
days when stored at +2 to +8°C.
R1b. Buffer
Contents ready for use. Stable up to the expiry date
when stored at +2 to +8°C.
R2.
Xanthine Oxidase
Reconstitute one vial of Xanthine Oxidase R2 with
10 ml of redistilled water. Stable for 2 weeks when
stored at +2 to +8°C.
Uric acid + 02
02
•.
The reagents must be used only for the purpose
intended by suitably qualified laboratory personnel,
under appropriate laboratory conditions.
Initial Concentration of Solutions
R1a. Mixed Substrate
Xanthine
0.05 mmol/l
I.N.T.
0.025 mmol/l
R1b. Buffer
CAPS
40 mmol/l, pH 10.2
EDTA
0.94 mmol/l
R2. Xanthine Oxidase
80 U/l
CAL Standard See assigned value on Lot Specific Insert
CAL Standards
Reconstitute one vial of Standard (CAL) with 10 ml of
redistilled water. Subsequent dilutions of this standard
should be prepared with Ransod sample diluent.
R1 = Mixed Substrate
R2 = Xanthine Oxidase
It is recommended that all reagents and samples are
equilibrated to room temperature prior to use.
MATERIALS PROVIDED
Mixed Substrate
Buffer
Xanthine Oxidase
Standard
MATERIALS REQUIRED BUT NOT PROVIDED
Ransod Diluent (Cat. No. SD 124) (0.01 mol/l Phosphate
Buffer, pH 7.0)
Ransod Control (Cat. No. SD 126)
MANUAL/ RX MONZA - RANSOD - SD 125
PAGE 2 OF 3
PROCEDURE
Select SOD in the Run Test screen and carry out a water
blank as instructed.
Pipette into a cuvette:
Standard
S1
Ransod Sample Diluent
30 µl
Standard
Diluted Sample
Diluted Control
Mixed substrate (R1)
1000 µl
Standards
S2 TO S6
30 µl
1000 µl
Samples
Control
30 µl
1000 µl
30 µl
1000 µl
150 µl
150 µl
150 µl
CALCULATION
A2 - A1
= −A/min of standard or sample
3
Sample diluent rate (S1 rate) = rate of uninhibited reaction
= 100 %
All standard rates and diluted sample rates must be
converted into percentages of the sample diluent rate, and
subtracted from 100 % to give a percentage inhibition.
Mix well and add :(−Astd/min x 100)
Xanthine Oxidase (R2)
150 µl
100 -
= % inhibition
(−AS1/min)
Mix well, insert the cuvette into the Rx Monza cell holder
and press read.
(−Asample/min x 100)
100 -
CALIBRATION
Calibrate this assay using the standard supplied with the kit.
It is recommended that the following dilutions are made of
the Standard CAL (or S6) to produce a standard curve:Standard
S6
S5
S4
S3
S2
Volume of
Standard Soln
Sample Diluent
Undiluted Standard
5 ml of S6
5 ml of S5
5 ml of S4
3 ml of S3
5 ml
5 ml
5 ml
6 ml
S1 = Sample Diluent (0.01 mol Phosphate buffer pH 7.0)
All diluted standards are stable for 2 weeks at +2 to +8°C.
A fresh calibration is required for each run.
FOR MANUAL USE
Wavelength:
Cuvette:
Temperature:
Measurement:
505 nm
1 cm path length
37°C
against air
Pipette sample and R1 into a cuvette and mix well. Add R2,
mix and read initial absorbance A1 after 30 seconds and
start timer simultaneously read final absorbance A2 after 3
minutes.
= % inhibition
(−AS1/min)
Plot percentage inhibition for each standard against Log10
(standard conc. in SOD units/ml)
Use percentage inhibition of sample to obtain units of SOD
from standard curve.
SOD units/ml of whole blood =
SOD units/ml from std curve x dilution factor
Converting to SOD units/g Haemoglobin
SOD units /ml
= SOD units/g Haemoglobin
g Haemoglobin/ml
ILLUSTRATION
(a) A bovine sample was diluted 1 in 300 times with Ransod
sample diluent. The diluted sample gave an inhibition of
33 %.
From the standard curve:
Number of SOD units in sample = 0.575
(b) Converting to SOD units/ml of whole blood
0.575 x 300 = 172.5 SOD units/ml
(c) Converting to SOD units/g Haemoglobin
Sample haemoglobin value = 0.118 g/ml
172.5
(SOD units/ml)/(g Haemoglobin/ml) =
= 1461.9
0.118
QUALITY CONTROL
Ransod Control is recommended for daily quality control. The
control should be assayed at least once a day. Values
obtained should fall within a specified range. If these values
fall outside the range and repetition excludes error, the
following steps should be taken:
1. Check instrument settings and light source.
2. Check cleanliness of all equipment in use.
3. Check water, contaminants i.e. bacterial growth may
contribute to inaccurate results.
4. Check reaction temperature.
5. Check expiry date of kit and contents.
6. Contact Randox Laboratories Customer Technical
Support, Northern Ireland (028) 94422413.
RANDOX Laboratories Ltd., 55 Diamond Road, Crumlin, Co. Antrim, United Kingdom, BT29 4QY
Tel: +44 (0) 28 9442 2413 Fax: +44 (0) 28 9445 2912
Email: applications@randox.com Website: www.randox.com
ΑΒΧ
MANUAL/ RX MONZA - RANSOD - SD 125
PAGE 3 OF 3
NORMAL RANGES
1102 - 1601 U/g Hb
164 - 240 U/ml
It is recommended that each laboratory establish its own
reference range to reflect the age, sex, diet and
geographical location of the population.
SPECIFIC PERFORMANCE CHARACTERISTICS
The following performance data were obtained using an Rx
Monza analyser in cuvette mode at 37ºC.
LINEARITY
Samples should be diluted to give an inhibition between
30 % and 60 % of the sample diluent rate (i.e. the
uninhibited reaction).
SENSITIVITY
The minimum detectable concentration of SOD less than
standards should be reported as <S1 standard value.
PRECISION
Within run precision
Mean
SD
CV(%)
n
Level 1
101.0
4.88
4.64
20
Level 2
131.5
4.30
3.58
20
Level 1
101.0
6.27
5.96
20
Level 2
131.5
8.50
7.07
20
Between run precision
Mean
SD
CV(%)
n
CORRELATION
The Randox method on the Rx Monza (Y) was compared to
the Rx Daytona (X) and the following linear regression
equation was obtained:
Y = 0.9417 X + 0.1004
and a correlation coefficient of 0.965
41 patient samples were analysed spanning the range 23 318U/ml.
REFERENCES
1. Woolliams JA, Wiener G, Anderson PH, McMurray
CH Research in Veterinary Science 1983, 34: 253-256.
2. Suttle NF, The Veterinary Record 1986, 119: 519-522.
3. Suttle NF, McMurray CH Research in Veterinary
Science 1983; 35: 47-52.
4. Arthur JR, Boyne R Life Sciences 1985 36: 1569-1575.
Revised 07 Oct 09 ck
RANDOX Laboratories Ltd., 55 Diamond Road, Crumlin, Co. Antrim, United Kingdom, BT29 4QY
Tel: +44 (0) 28 9442 2413 Fax: +44 (0) 28 9445 2912
Email: applications@randox.com Website: www.randox.com
ΑΒΧ