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TECHNOZYM
vWF:CBA ELISA
PRODUCT DESCRIPTION
INTENDED USE
The von Willebrand Factor (vWF) is a large, multifunctional glycoprotein, occupying a key position in
primary haemostasis. It has a multiple structure with several functions:
-
It is the carrier protein for Factor VIII in plasma; it forms a complex and thus protects Factor VIII
from early proteolytic decomposition.
-
It acts as a mediator for platelet aggregation by attaching itself to platelet membrane receptors (GP
Ib and GP lIb/IIIa) following previous platelet activation.
-
It plays a part in primary haemostasis by acting as a mediator between adhesioned platelets and
the subendothelium (lesioned vascular wall).
In order to analyze the adhesive properties, as a rule the platelet aggregation is measured (measuring
system = ristocetin-dependent platelet aggregation). However, this does not reflect the physiological
setting nor the function of the vWF. For determining the adhesive properties of the vWF, its binding
capacity to collagen serves as a parameter which corresponds to the physiological function of the vWF
COMPOSITION
1.
ELISA test strips (12) with 8 wells each, coated with human collagen Typ III; the drying agent is supplied in an
aluminium bag.
2.
Washing buffer concentrate: (PBS; pH 7.3); containing detergent; 0.01% merthiolate; 1 bottle, 80 mL.
3.
Incubation buffer: (PBS; pH 7.3); contains stabiliser protein; Proclin 300 and dye 1 bottle, 90 mL, ready for
use.
4.
Calibrators (Standards) numbered; lyophilised; 1 bottle each.
Concentrations are lot-dependent; consult label on the vial.
5.
Control plasmas "low level" and "high level" for checking purposes, lyophilised; 1 bottle each.
Concentrations are lot-dependent; consult the label on the vial.
6.
Conjugate polyclonal Anti-vWF-POX; dyed blue; 1 bottle, 0.3 mL.
7.
Chromogen TMB (tetramethylbenzidine); 1 bottle, 12 mL; ready to use.
8.
Stopping solution: sulphuric acid 1.9 mol/L; 1 bottle 12 mL; ready for use.
9.
Adhesive film: for ELISA test strips (2).
MATERIAL REQUIRED
(but not supplied with the kit)
1. Distilled water
2. Test tubes for diluting standard and samples
3. Measuring cylinder (1000 mL)
4. Precision pipettes (10, 100 and 1000 µL)
5. Variable pipette (1000 µl)
6. Multichannel and/or dispensing pipettes (100 and 200 µL)
7. ELISA washer or multichannel pipette
8. ELISA reader with 450 nm filter, with a 620 nm reference filter if available.
STABILITY AND STORAGE
All components contained in the kit may be used until the expiry date as indicated. The bench stability of the
components after opening, reconstitution and/or dilution may be inferred from the table below:
When necessary the samples, controls and calibrators can be frozen/thawed up to 5 times. But making aliquots is
recommended.
Material/Reagent
State
Calibrators,
control
after reconstitution
plasmas
ELISA test strip
after opening
Washing
buffer
after opening
concentrate
1+11.5
dilution
Washing buffer
concentrate
Incubation buffer
after opening
after opening
Conjugate
working solution
Chromogenic TMB
after opening
TEST PROCEDURE
PREPARATION OF SAMPLES
Material: plasma
Obtaining plasma: mix 9 parts venous blood with 1 part sodium citrate solution (0.11 mol/L)
and centrifuge for 15 minutes at a minimum of 2500 g (DIN 58905). The plasma sample may
be stored for 3 hours at room temperature; otherwise the sample ought to be frozen
immediately after centrifugation. Stable at -20°C for 6 months.
PREPARATION OF REAGENT
1. Before starting the test, all the required components are to be brought to room temperature.
2. Preparing the washing buffer: Dilute 1 part by volume washing buffer concentrate with 11.5 parts
by volume distilled water (1+11.5). Mix well! (Diluted washing buffer concentrate = washing buffer).
There may be crystalline precipitations which will dissolve at 37°C within 10 minutes.
3. Reconstituting calibrators and control plasmas:
Calibrators and control plasmas are reconstituted with 500 µL distilled water and mixed for 10
seconds after a reconstitution time of 15 minutes (vortex mixer). Reconstituted components are
clear to slightly turbid.
4. Diluting calibrators, control plasmas and samples (1+25): Dilute 20 µL samples, 20 µL calibrators
and/or 20 µL controls with 500 µL each of incubation buffer. Mix for 10 seconds!
5. Preparing the conjugate working solution (1+50): Dilute 1 part by volume conjugate with 50 parts by
volume incubation buffer:
For 8 test wells: Mix 20 µL conjugate with 1000 µL incubation buffer.
PERFORMANCE OF THE TEST
Pipette
diluted
SAMPLE INCUBATION
plasmas and diluted samples into test wells.
cover test strips with film.
(reference 1, 2)
incubate at room temperature
WASHING (reference 1,3,4)
washing buffer
pipette conjugate working solution into wells, cover
CONJUGATE REACTION
test strip with film
(reference 1,2)
incubate at room temperature
WASHING (reference 1,3,4)
washing buffer
pipette substrate solution
SUBSTRATE REACTION
into test wells cover test strips with film
(reference 1,2)
Incubate at room temperature
STOP SOLUTION
pipette stopping solution into wells
(reference 1,2)
MEASURING (reference 5, 6)
ELISA-Reader, 450 nm
Storage
Stability
room temperature
8 hours
-20°C
6 months
2 ... 8 °C with adhesive film
in plastic bag with drying
expiry date
agent
2 ...8°C
6 months
of
2 ... 8 °C
3 weeks
2 ... 8 °C
2 months
2 ... 8 °C
6 months
room temperature
60 minutes
2 ... 8 °C
expiry date
calibrators,
diluted
control
100 µL
45 minutes
3 x 200 µL
100 µL
45 minutes
3 x 200 µL
100 µL
15 minutes
100 µL
References
1. Reagents of different lots must not be combined
2. Precision and performance, among others, essentially depend on the following factors:
Thorough mixing of all substances used for dilution
Test calibrators, controls and samples in duplicates.
Incubation to be done at correct temperatures (Room temperature is 20 ... 25°C)
Strict observance of the order of pipetting and of the time element as indicated:
The time for sample incubation, conjugate and substrate reaction as indicated starts after pipetting
the last sample. Incubation times should not vary by more than ±10%.
During sample incubation and conjugate reaction, the time for pipetting the diluted
calibrators/samples/control plasmas and/or conjugate solutions must not exceed 60 seconds per
ELISA test strip (8 wells).
During substrate reaction and at stopping, the time needed for pipetting the substrate and/or the
stopping solution must not exceed 10 seconds per ELISA test strip. Short pipetting times may be
secured by using multichannel- and dispensing pipettes.
3. Label/number strips with a water resistant pen in case the strips accidentally fall out of the frame
during testing.
4. After the last washing, wells must be aspirated thoroughly, turned upside down and positioned on a
blotting paper; by gentle tapping, the last remnants must be removed.
5. Measuring the difference in wave lengths at 450 and 620 nm or at 450 and 690 nm, the precision of
the test is increased.
6. Shake 10 sec., measure within10 minutes
LIMITATION OF THE TEST
Reduced levels of vWF:CBA are associated with blood group 0.
vWF:CBA is also affected by physical exercise, pregnancy, use of contraceptive pill, ethnic group and
the antigen increases with age.
WARNING AND PRECAUTIONS
-
All human blood or plasma products as well as samples must be considered as potentially
infectious. They have to be handled with appropriate care and in strict observance of safety
regulations. The rules pertaining to disposal are the same as applied to disposing hospital waste.
-
Calibrators and control plasmas are made from human blood and any individual plasma involved in
the procedure is HBsAg, HIV 1/2 Ab and HCV-Ab-negative (see labels on kit and/or bottles).
-
Stopping solution (sulphuric acid) may irritate the skin. Should acid get into your eyes, wash out
immediately with water and consult a doctor.
-
The reagents sometimes contain preserving agents (merthiolate). Beware of swallowing! Avoid
contact with skin or mucous membranes
ANALYSIS RESULTS
CALCULATION OF THE RESULTS
Setting up a reference curve:
X axis: Concentration vWF:CBA U/ml (1U/ml = 100%)
Y axis: Extinction
Graph plot is linear-linear with a point to point or cubic spline
Assessment of reference curve
• The extinction coefficient of the highest calibrator should be between 1.0 and 2.5.
• The validity of the test may be checked on the basis of the calculated control values.
Example of standard curve.
2,5
2
1,5
1
0,5
0
0
0,2
0,4
0,6
Measuring concentration of samples
• Read off the concentration from the reference curve.
• If there are samples with extinction coefficients higher than that of the highest point on the curve, they
have to be prediluted with incubation buffer (1+1). The measured concentration then has to be
multiplied with the dilution factor 2.
REFERENCE RANGE
Normal range for vWF:CBA is between 0,4 – 2,5 U/mL (40-250%) (Fig 1)
It is recommended that individual laboratories establish their own normal range.
STANDARDIZATION
The calibration material used is the WHO International standard for Blood coagulation Factor VIII and
von Willebrand factor in plasma (human)
PERFORMANCE CHARACTERISTICS
Performance data are given below. Results obtained in individual laboratories may differ.
PRECISION
Reproducibility was determined with different samples (in series and day to day).
The following results were obtained:
Intra assay
Sample 1
N
24
Mean (U/mL)
1,372
SD (U/mL)
0,020
CV (%)
1,47%
COMPARISON OF METHODS OR CORRELATION
The TECHNOZYM vWF:CBA (Technoclone) was correlated with two assays measuring 113 samples
(24 samples of normal individuals and 89 samples of patients with VWD classified as type 1 (n=26),
type 2A/2B (n=11), type 2M (n=45), type 2N (n=4) and type 3 (n=3)).
With the Asserachrom vWF:CB of Stago (using human collagen type III) a correlation of r=0.98,
slope=0.90 was observed. With the Collagen Binding Assay of Life Diagnostics (using equine collagen
95% type I, 5% type III), a correlation of r=0.99, slope = 0.95 was observed.
ASSAY RANGE:
0.01 – 1.70 U/mL
However the effective range of each test run will depend on the assayed value of calibrator.1.
DETECTION LIMIT:
0.01 U/mL
LITERATURE
1) Blood 69; 1691 – 1695, 1987. The effect of ABO blood group on the diagnosis of vWD. Gill et al.
2) Thromb Haemost 2000; 83: 127 – 35. Collagen Binding Assay for von Willebrand Factor (VWF:CBA) Detection
of VWD and discrimination of VWD Subtypes, Depends on Collagen Source. E J Favaloro
3) Haemophilia (Suppl. 3), 1998, 15 – 24. The determination of von Willebrand factor activity by collagen binding
assay. Siekmann et al.
2 / 8
0,8
1
1,2
1,4
1,6
vWF:CBA U/ml
Inter assay
Sample 2
Sample 1
24
10
1,630
1,366
0,029
0,074
1,75%
5,39%
GB
Sample 2
10
0,306
0,013
4,13%
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