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Serum Pre-Clinical CartiLaps®
ELISA
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For the quantification of degradation products of C-terminal telopeptides
of type II collagen (CTX-II) in animal serum.
The Serum Pre-Clinical CartiLaps®
ELISA kit is for in vitro use only.
Nordic Bioscience Diagnostics is not responsible for any other use of the kit
or consequence hereof than the one specified above. Neither
for misuse, e.g. use deviating from the procedure described
in this manual.
Furthermore, Nordic Bioscience Diagnostics A/S is not to be made responsible
for any diagnoses or conclusions made by the user or third
party based on the results obtained with the Serum Pre-Clinical
CartiLaps® ELISA
kit nor for any consequences such interpretations may cause.
Nordic Bioscience Diagnostics A/S
Herlev Hovedgade 207
DK - 2730 Herlev
Denmark
0805A
INTRODUCTION
Intended use
The Serum Pre-Clinical CartiLaps®
ELISA detects degradation products of C-terminal telopeptides
of type II collagen (CTX-II) in animal serum. The test is
intended For Research Use Only. Not for use in diagnostic
procedures.
Limitations
The use of the test has not been established for determination of the
level of cartilage destruction.
Background
Disruption of the structural integrity of cartilage is the major histological
finding in osteoarthritis and rheumatoid arthritis. Type
II collagen is the major organic constituent of cartilage
and fragments of type II collagen (CTX-II) are being released
into circulation and subsequently secreted into urine following
degradation of cartilage. In non-human serum, the CTX-II
fragments can be quantified by Serum Pre-Clinical CartiLaps®
ELISA. The
Serum Pre-Clinical CartiLaps® ELISA
has been reported to be useful for rat, mice and rabbit
specimen.
Principle of the procedure
Serum Pre-Clinical CartiLaps®
ELISA is based on binding of two identical monoclonal antibodies
to cross-linked serum fragments of type II collagen. Initially,
biotinylated monoclonal antibody are bound to the surface
of streptavidin-coated wells of the microtitre plate. After
washing, standards, controls, serum samples and Incubation
buffer are pipetted into the wells. After incubation the
wells are washed, and a solution of peroxidase-conjugated
monoclonal antibody is added to the wells. Following the
third washing step, a chromogenic substrate is added to
all wells and the colour reaction is stopped with sulphuric
acid and the absorbance is measured.
PRECAUTIONS
The following precautions should be observed in the laboratory:
- • Do not eat, drink or smoke where immunodiagnostic materials are
being handled.
- • Do not pipette by mouth.
- • Wear gloves when handling immunodiagnostic materials.
- • Do not use reagents beyond their expiration date and do not mix reagents
from different lots of kits.
- • Always use clean containers.
Warnings
For in vitro use only.
- • All reagents and laboratory equipment should be handled and disposed of
as if they were infectious.
- • Do not use kit components beyond the expiry date and do not mix reagents
from different lots.
Storage
Store the Serum Pre-Clinical CartiLaps®
ELISA kit at 2-8°C upon receipt. Under these conditions
the kit is stable up to the expiry date stated on the box.
MATERIALS
Specimen collection
The Serum Pre-Clinical CartiLaps®
ELISA is intended for use with serum samples. However in-house
data has verified that CTX-II can be detected in synovial
fluid and EDTA plasma as well. Collect blood taking care
to avoid haemolysis. Separate the serum from the cells within
3 hours after collection of blood. It is recommended to
freeze (< -18°C) samples immediately.
Materials supplied
Before opening the kit, read the section on Precautions. The kit
contains reagents sufficient for 96 determinations. For
reconstitution of lyophilized material add appropriate volume
of distilled water and leave for 10 minutes before mixing.
Make sure to avoid foam.
Streptavidin coated microtitre plate (MTP)
Microwell strips (12x8 wells) pre-coated with streptavidin. Supplied in
a plastic frame.
Serum Pre-Clinical CartiLaps®
Standard (Vial A)
One
vial (min. 9.0 mL/vial) of ready-for-use PBS-buffered solution
with protein stabiliser and preservative.
Serum Pre-Clinical CartiLaps®
Standard (Vial B)
One
vial (lyophilized) containing Foetal Calf Serum (FCS) in
a PBS-buffered solution with protein stabilizer and preservative.
Reconstitute with 0.5 mL of distilled water. The exact concentration
is stated on each vial. The standards must be stored below
-18°C after use, and should only be frozen and thawed
twice.
Control (Vial CO)
One vial (lyophilized) containing FCS in a PBS-buffered solution with
protein stabilizer and preservative. Reconstitute with 0.5
mL of distilled water. The exact concentration is stated
on the Technical data sheet provided with the kit. The control
must be stored below -18°C after use, and should only
be frozen and thawed twice.
Biotinylated Antibody (Vial no. 1)
One vial (min. 0.20 mL) of a concentrated solution of a biotinylated monoclonal
murine antibody specific for degradation products of C-terminal
telopeptides of type II collagen (CTX-II). Prepared in a
buffered solution with protein stabiliser and preservative.
Peroxidase Conjugated Antibody (Vial no. 2)
One vial (min. 0.20 mL) of a concentrated solution of a peroxidase conjugated
murine monoclonal antibody specific for degradation products
of C-terminal telopeptides of type II collagen (CTX-II).
Prepared in a buffered solution with protein stabiliser
and preservative.
Incubation Buffer (Vial no. 3 )
One vial (min. 25 mL) of a ready-for-use buffered solution with protein
stabiliser, detergent and preservative.
Biotinylated Antibody Buffer (Vial no. 4 )
One vial (min. 12.5 mL) of a ready-for-use buffered solution with protein
stabiliser, detergent and preservative.
Substrate Solution (Vial TMB)
One vial (min. 12 mL) of a ready-for-use tetramethylbenzidine (TMB) substrate
in an acidic buffer.
Please note that the chromogenic substrate might appear sligthy blueish.
Stopping Solution (Vial ST)
One vial (min. 12 mL) of ready-for-use 0.18 mol/L sulfuric acid.
Washing Buffer (Vial W)
One vial (min. 20 mL) of a concentrated washing buffer with detergent and preservative.
Sealing tape
Adhesive film for covering wells during incubation.
Materials required - not supplied
• Containers for preparing the Antibody Solutions and the Washing
Solution
• Precision micropipettes to deliver 25-200 µL
• Distilled water
• Precision 8- or 12-channel multipipette to deliver 100 µL
• Microwell mixing apparatus
• Microtiter plate reader
ASSAY PROCEDURE
Assay Procedure
Mix all reagents and samples before use (avoid foam). Prior to use, prepare and
equilibrate all solutions to room temperature. Perform
the assay at room temperature (18-22°C).
Determine the number of strips needed for the assay. It is recommended
to test all samples in duplicate. In addition, for each
run a total of 14 wells are needed for standards and control.
Place the appropriate number of strips in the plastic frame.
Store unused immunostrips in the tightly closed foil bag
with desiccant capsules.
Assay procedure
1 Preparation of standards
Standards covering the appropriate measuring range are prepared by dilution
of Standard B in Standard A. Usually four
2.5 times dilutions of Standard B, in addition to
Standard A and Standard B, will provide a
suitable measuring range for most purposes.
Example:
|
Standard |
Preparation |
Calculated CTX-II conc.
(pg/mL) |
STD.B |
Ready-for-use |
247.6 |
STD.C |
50µL STD.B + 75µL STD.A |
99.0 |
STD.D |
50µL STD.C + 75µL STD.A |
39.6 |
STD.E |
50µL STD.D + 75µL STD.A |
15.8 |
STD.F |
50µL STD.E + 75µL STD.A |
6.3 |
STD.A |
Ready-for-use |
0.0 |
2 Pre-incubation
Prepare the following Biotinylated Antibody Solution before starting the
assay. Mix the solutions in vial no. 1 (Biotinylated Antibody)
and vial no. 4 (Biotinylated Antibody Buffer) in the volumetric
ratio 1+100 in an empty container. Mix carefully and avoid
formation of foam. Prepare a fresh solution before each
run of the assay.
Add 100 µL of Biotinylated Antibody Solution to each well,
cover with sealing tape, and incubate for 30±5 minutes
at room temperature (18-22°C) on a microtitre plate mixing
apparatus (300 rpm).
3 Washing
Wash the immuno strips 5 times manually with Washing Solution (vial
W) diluted 1 + 50 in distilled water. Using an automated plate
washer, follow the instructions of the manufacturer or the
guidelines of the laboratory. Usually 5 washing cycles are
adequate. Make sure that the wells are completely emptied
after each manual or automated washing cycle.
4 Incubation of samples and standards
Pipette 25 µL of Standards (vial A-F), Control (vial
CO) or unknown samples into appropriate wells followed by
100 µL Incubation Buffer (vial no. 3). Cover
the immunostrips with sealing tape and incubate for 60±5
minutes at room temperature (18-22°C on a microtitre plate
mixing apparatus (300 rpm).
5 Washing
See step 3.
6 Secondary incubation
Prepare the following Peroxidase Conjugated Antibody Solution before
use. Mix the solutions in vial no. 2 (Peroxidase Conjugated
Antibody) and vial no. 3 (Incubation Buffer) in the volumetric
ratio 1+100 in an empty container. Mix carefully and avoid
formation of foam. Prepare a fresh solution before each
run of the assay.
Add 100 µL of Peroxidase-Conjugated Antibody to each well,
cover with sealing tape, and incubate for 60±5 minutes
at room temperature (18-22°C) on a microtitre plate mixing
apparatus (300 rpm).
7 Washing
See step 2.
8 Incubation with chromogenic substrate solution
Pipette 100 µL of the Substrate Solution (vial TMB) into
each well, cover the immunostrips with sealing tape and incubate
for 15±2 minutes in the darkness at room temperature
(18-22°C) on a microtitre plate mixing apparatus (300
rpm).
9 Stopping of color reaction
Pipette 100 µL of the Stopping Solution (vial ST) into each
well.
10 Measurement of absorbance
Measure the absorbance at 450 nm with 650 nm as reference within two
hours.
Limitations of the procedure.
If the absorbance of a sample is higher than Standard B, it is recommended
that the sample be diluted in Standard A.
QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens
in each series of assays in order to check the performance
of the assay. Controls should be treated as unknown samples,
and the results analysed with appropriate statistical methods.
RESULTS
Calculation of results
A
4-parametric linear curve fit can be used.
Alternatively, calculate the mean of the duplicate absorbance determinations.
Construct a standard curve on graph paper by plotting the
mean absorbances of the six standards A-F (ordinate) against
the corresponding CartiLaps®
concentrations (abscissa). Determine the CartiLaps®
concentration of the controls and each patient sample by interpolation.
Example of results obtained:
|
Standards/
Controls/
Samples |
CartiLaps®
conc.
(pg/mL) |
A450-650
(nm)
Obs 1/ Obs 2 |
Mean A450-650
(nm) |
Interpolated CartiLaps®
conc. (pg/mL) |
Standard A
Standard F
Standard E
Standard D
Standard C
Standard B
Control CO
Sample I
Sample II
Sample III |
0.0
6.9
17.3
43.3
108.2
270.5 |
0.121 / 0.112
0.174 / 0.168
0.253 / 0.252
0.428 / 0.446
0.946 / 0.924
2.011 / 1.964
0.684 / 0.687
0.230 / 0.229
0.473 / 0.467
0.880/ 0.848 |
0.117
0.171
0.253
0.437
0.935
1.988
0.686
0.230
0.470
0.864 |
74.8
15.2
46.5
98.8 |
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Please note: The data above are for illustration only and should not be
used to calculate the results of any run.
Performance characteristics
Detection limit: 1.2 pg/mL
This is the concentration corresponding to three standard deviations above the
mean of 21 determinations of the blank ("CartiLaps®
Standard A").
Precision <5.8%
The precision was determined using ten analytical runs, each with duplicate
determinations of samples.
|
Sample |
Mean
(pg/ml) |
Intraassay <7.8% |
Interassay <12.2% |
SD
(pg/ml) |
CV
(%) |
SD
(pg/ml) |
CV
(%) |
LOW
MEDIUM
HIGH |
11.67
49.09
107.44 |
0.96
1.83
5.40 |
5.8
3.7
5.0 |
0.62
0.47
2.61 |
3.7
1.0
2.4 |
Dilution/Linearity
The Serum Pre-Clinical CartiLaps® ELISA
is linear in the range 1.2 pg/mL to 300.0 pg/mL.
Serum samples with the concentration of 38.8-57.0 pg/mL
CartiLaps® were diluted with
standard A and the concentration of CartiLaps®
were determined with Serum Pre-Clinical CartiLaps®
ELISA. The serum neat sample is set to 100%.
The data below is calculated from 1 run:
|
|
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Sample 1
|
Sample 2
|
Sample 3
|
Overall RC |
Serum (%) |
Standard A (%) |
RC% |
RC% |
RC% |
101 |
100
80
60
40
20
10 |
0
20
40
60
80
90 |
100
106
103
90
101
82 |
100
99
106
115
110
97 |
100
105
101
104
98
no data |
|
|
DF: Dilution Factor; RC: Recovery
CLINICAL DATA
Expected values
It is advisable for each laboratory to establish its own reference
ranges. As an example, the mean values for various species
are given below.
|
Species |
Mean CTX-II value
(pg/mL) |
Rats, 8 weeks normal |
26.7 |
Mice, 8 weeks normal |
24.0 |
Mice, 10 weeks normal |
11.3 |
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REFERENCES
- 1. Christgau S, Tankó LB, Cloos PAC, Mouritzen U, Christiansen C, Delaissé
J-M, Høegh-Andersen P. Suppression of Elevated
Cartilage Turnover in Postmenopausal Women and in Ovariectomized
Rats by Estrogen and a Selective Estrogen Receptor Modulator
(SERM). Menopause (2004); 11:508-518.
- 2. Christgau S, Garnero P, Fledelius C, Moniz
C, Rosenquist C, Ensig M, Gineyts E, Rosenquist C, Qvist
P. Collagen type II degradation products in urine as an
index of cartilage degradation. Bone (2001); 29: 209-215.
- 3. Høegh-Andersen P, Tankó LB, Andersen T, Vingsbo C, Heegaard
A-M, Delaissé J-M, Christgau S. Ovariectomized
Rats as a Model of Postmenopausal Osteoarthritis. Validation
and Application. Annals of Rheum Dis. (2004); 6(2): R169-80.
- 4. Reijman M, Hazes JMW, Bierma-Zeinstra SMA, Koes BW, Christgau S, Christiansen
C, Uitterlinden AG, Pols HAP. A new marker for osteoarthritis:
cross-sectional and longitudinal approach. Arthritis &
Rheum. (2004); 50:2471-2478.
- 5. Roy-Beaudry M, Martel-Pelletier J, Pelletier J-P, M’Barek KN, Christgau
S, Shipkolye F, Moldovan F. Entothelin-1 promotes osteoarthritic
cartilage degradation via mmp-1 and mmp-13 induction.
Arthritis & Rheum (2003); 48:2855-2864.
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