艾狄斯®细胞培养I型胶原C端肽检测试剂盒

CrossLaps for Culture ELISA
Enzymeimmunoassay for the quantitative
determination of fragments of type I collagen
released into bone cell culture supernatants
during bone resorption in vitro
For Research Use Only. Not for use in diagnostic procedures.
R EF AC-07F1  96
2
INTRODUCTION
Intended use
The test is an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of
degradation products of C-terminal telopeptides of type I collagen in bone cell culture supernatants. It
represents a technically improvement of the assay originally described in 1996 (6).
Summary and explanation of the test
Type I collagen accounts for more than 90% of the organic matrix of bone (1). During renewal of the
skeleton bone matrix is degraded and consequently fragments of type I collagen is released into
circulation. The resorption process can be studied in vitro by culturing bone cells on devitalised slices of
bone or dentin.
The CrossLaps® for Culture ELISA is based on the observation that certain C-telopeptide degradation
products from type I collagen released during osteoclastic bone resorption occur in the circulation as
modi! ed di-peptides (9). These modi! ed ("-isomerised) and cross-linked di-peptides (Glu-Lys-Ala-
His-Asp-"-Gly-Gly-Arg) must be covalently cross-linked through the lysine residue for signal in the
CrossLaps® for Culture ELISA. This epitope is present in type I collagen of many species, including
human, bovine, elephant and chicken (2-5, 7, 8, 10). However, not in rat and mouse.
Principle of the procedure
The assay is based on the simultaneous binding of the collagen fragments by two monoclonal
antibodies, one of which will subsequently mediate the binding to the solid surface of the microtitre plate.
Standards, control, or unknown samples are pipetted into the appropriate well of a microtitre plate coated
with streptavidin, and subsequently a mixture of a biotinylated antibody and a peroxidase-conjugated
antibody is added. Then a complex between the collagen fragments and the two antibodies is generated,
and the complex will bind to the streptavidin surface via the biotinylated antibody. Following this one step
incubation at room temperature the wells are washed and a chromogenic substrate added. The colour
reation is stopped with sulphuric acid and the absorbance is measured.
PRECAUTIONS
Storage
Store the CrossLaps® for Culture ELISA kit upon receipt at 2-8#C. Under these conditions the kit is stable
up to the expiry date stated on the box.
Warnings
The CrossLaps® for Culture ELISA is for research-use-only and is not for internal use in humans or
animals. This product must be used strictly in accordance with the instructions set out in the Package
Insert. IDS Limited will not be held responsible for any loss or damage (except as required by statute)
howsoever caused, arising out of non-compliance with the instructions provided.
CAUTION: this kit contains material of animal origin. Handle kit reagents as if capable of transmitting an
infectious agent.
Appropriate precautions and good laboratory practices must be used in the storage, handling and
disposal of the kit reagents. Disposal of kit reagents should be in accordance with local regulations..
Do not use reagents beyond their expiration date and do not mix reagents from different lots of kits.
MATERIAL
Specimen collection
The determination should be carried out using culture supernatants harvested from bone cells cultured
on surfaces of bone or dentin. The culture supernatants preferably should be tested on the same day as
they are harvested, but current date indicates that culture supernatants can be stored for 2 weeks at 4#C.
Fetal and newborn bovine serum in a concentration up to 10% (v/v) in the culture supernatant does not
interfere with the test.
In order to measure the background release of collagen fragments, three types of control specimens are
recommended for each experiment:
3
Medium control specimens
Medium on plastic surface (culture dishes/microwells) under culture conditions. At least 2 specimens are
recommended for each experiment.
Cell control specimens
Bone cells in medium on plastic surface (culture dishes/microwells) under culture conditions. At least 2
specimens are recommended for each experiment.
Slice control specimens
Slices of bone or dentin without cells in medium under culture conditions. At least 4 specimens are
recommended for each experiment.
All specimens, except those delivered with the kit (i.e. standards and control) should be pre-diluted 1+4
in Standard Diluent prior to testing.
Materials supplied
Before opening the kit, please read the section on Precautions. The kit contains reagents suf! cient for 96
determinations.
Streptavidin coated microtitre plate  MICROPLAT
Microwell strips (12x8 wells) precoated with streptavidin. Supplied in a plastic frame.
Standard Diluent  CAL   0 
One vial (min. 9 mL) of ready-for-use PBS-buffered solution with protein stabiliser and preservative.
Standard  CAL   1 
One vial (min. 1 mL) containing CrossLaps standard (desalted urinary antigens) in a PBS-buffered
solution with protein stabiliser and preservative. The exact concentration is stated on the bottle label.
Serial dilutions of the Standard in the Standard Diluent must be made prior to performing the ELISA.
Control  CTRL
One vial (min. 0.5 mL) of ready-for-use control reagent, containing desalted urinary antigens of human
origin in a PBS-buffered solution with protein stabiliser and preservative. Please refer to the enclosed QC
Report for concentration range.
Biotinylated Antibody  Ab  B IOTIN
One vial (min. 0.25 mL) of a concentrated solution containing a biotinylated monoclonal antibody speci! c
for degradation products of C-terminal telopeptides of Type I collagen in a buffered solution with protein
stabiliser and preservative.
Peroxidase Conjugated Antibody  E N Z Y MCON J
One vial (min. 0.25 mL) of a concentrated solution containing a peroxidase conjugated monoclonal
antibody speci! c for degradation products of C-terminal telopeptides of Type I collagen in a buffered
solution with protein stabiliser and preservative.
Incubation Buffer  B U F  
One vial (min. 19 mL) of a ready-for-use buffered solution with protein stabiliser, detergent and
preservative.
Substrate Solution  S U B S 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 slightly bluish.
Stopping Solution  H 2 S O4
One vial (min. 12 mL) of ready-for-use 0.18 M sulphuric acid.
Washing Buffer  W AS H B U F 5 0x 
One vial (min. 20 mL) of a concentrated washing buffer with detergent and preservative.
4
Sealing tape
Adhesive ! lm for covering wells during incubation.
Materials required – not supplied
• Microtubes (or similar) for preparation of serial dilutions of the Standard
• Containers for preparing the Antibody Solution and the Washing Solution
• Precision micropipettes to deliver 20-250 µL
• Distilled water
• Precision 8-or 12-channel multipipette to deliver 100 µL and 150 µL
• Microwell mixing apparatus (300 rpm)
• ELISA plate reader with both 450 nm and 650 nm ! lters
ASSAY PROCEDURE
Prior to use, equilibrate all solutions to room temperature. The assay should be performed at room
temperature (18-22ºC).
Determine the number of strips needed for the entire experiment. It is recommended to test all samples
in duplicate. In addition, for each ELISA plate 16 wells are recommended for standards and 2 wells are
recommended for the Control. Furthermore, for each experiment (but independently of the number of
ELISA plates used), a total of 16 wells are recommended for the 2 Medium control specimens (4 wells),
the 2 Cell control specimens (4 wells) and the 4 Slice control specimens (8 wells).
Prepare Standards (recommended dilutions)
Prepare a two-fold dilution row of the Standard  CAL   1   in the Standard Diluent  CAL   0  . For each dilution
2x50 "L will be needed for the ELISA. E.g., pipette 300 "L of the Standard CAL   1   into the ! rst microtube
(I) and 150 "L of Standard Diluent  CAL   0   into each 7 other microtubes (II-VIII), transfer 150 "L from I
to II and mix, transfer 150 "L from II to III and mix, continue until VII. Leave VIII with only the Standard
Diluent  CAL   0  . Of the 150 "L in each microtube, 2x50 "L are used in the ELISA. The rest (50 "L) is
discarded.
1. Pre-dilution of test specimens
All specimens, except those delivered with the kit (i.e. standard and control) must be pre-diluted 1+4 in
Standard Diluent prior to testing (e.g. 30 "L (specimen) +120 "L CAL   0  ).
2. Preparation of the Antibody Solution
ATTENTION: prepare the following Antibody Solution maximum 30 minutes before starting the test;
Mix the solutions of Biotinylated Antibody  Ab  B IO T IN , Peroxidase Conjugated Antibody  E N Z Y M CO N J and
Incubation Buffer  B U F    in the volumetric ratio 1+1+100 in an empty container. Mix carefully and avoid
formation of foam. Prepare a fresh solution before each test.
3. One Step incubation
Pipette 50 "L of each Standards, Control CT R L , or unknown samples into appropriate wells followed
by 150 "L of the Antibody Solution. Cover the immunostrips with sealing tape and incubate for 120±5
minutes at room temperature (18-22ºC) on a microtitre plate mixing apparatus (300 rpm).
4. Washing
Wash the immunostrips 5 times manually with 300 "L diluted Washing Buffer ( W AS H B U F 5 0x   diluted
1+50 in distilled water). Make sure that the wells are completely emptied after each washing cycle. When
using an automatic plate washer, follow the instructions of the manufacturer or the guidelines of the
laboratory. Usually 5 washing cycles are adequate.
5. Incubation with chromogenic substrate solution
Pipette 100 "L of the Substrate Solution S U B S T M B    into each well and incubate for 15±2 minutes at
room temperature (18-22ºC) in the dark on the mixing apparatus (300 rpm). Use sealing tape.
Do not pipette directly form the vial containing TMB substrate but transfer the needed volume to a clean
reservoir. Remaining substrate in the reservoir should be discarded and not returned to vial TMB.
6. Stopping of colour reaction
Pipette 100 "L of the Stopping Solution H 2 S O 4 into each well.
5
7.  Measurement of absorbance
The absorbance is measured within two hours at 450 nm. It is recommended to use the reading at 650
nm as reference.
RESULTS
Calculation of results
• Calculate the mean of the duplicate absorbance determinations. Construct a standard curve by
plotting the mean absorbances of the eight Standards I-VIII (ordinate) against the corresponding
concentrations (abscissa). Draw the best ! tting curve.
Alternatively, a quadratic curve ! t can be used.
• Determine the CrossLaps® for Culture ELISA concentration of the Control, Medium, Cell and Slice
control Specimens and each of the Test Specimens by interpolation on the curve.
• CrossLaps® for Culture ELISA concentration determined for the Control should be within the range
giving at the enclosed QC Report.
• As a consequence of the pre-dilution the CrossLaps® for Culture ELISA concentration of the Medium,
Cell and Slice control Specimens as well as the Test Specimens should be multiplied by 5 to obtain
the true concentration.
Example:
Standards/
Controls/
Specimen
CrossLaps
conc.
(nM)
A450-650
(nM)
Mean
A450-650
(nM)
CrossLaps
conc. in
prediluted
specimen
(nM)
CrossLaps
conc. in
undiluted
specimen
(nM)
Zero Std (VIII)
Std VII
Std VI
Std V
Std IV
Std III
Std II
Std I
Control
Medium co
Medium co
Cell co
Cell co
Slice co
Slice co
Slice co
Slice co
Sample I
Sample II
Sample III
0.00
1.32
2.65
5.29
10.59
21.18
42.35
84,70
0.041/0.042
0.067/0.071
0.096/0.100
0.147/0.155
0.249/0.266
0.447/0.507
0.897/0.973
1.844/1.915
0.291/0.281
0.041/0.045
0.041/0.042
0.046/0.044
0.041/0.047
0.049/0.051
0.053/0.055
0.048/0.052
0.053/0.052
0.064/0.061
0.181/0.181
0.521/0.512
0.042
0.069
0.098
0.151
0.258
0.477
0.935
1.880
0.286
0.043
0.042
0.045
0.044
0.050
0.054
0.050
0.053
0.063
0.184
0.571
11.88
0.05
0.01
0.15
0.10
0.40
0.59
0.40
0.54
1.01
6.80
22.91
0.25
0.05
0.75
0.50
2.00
2.95
2.00
2.70
5.05
34.00
114.55
Please note: The data above were calculated from a quadratic curve ! t of the standard curve and are
for illustration only. They should not be used to calculate the results of tests.
For all bone cell culture supernatants the results obtained by interpolations must be corrected for the
6
medium, cell and slice background effects. Using the value from the table below:
Medium control : 0.15 nM (Mean of 0.25 & 0.05)
Cells control : 0.48 nM (Mean of 0.75 & 0.50) minus 0.15
Slice control : 2.26 nM (Mean of 2.00, 2.95, 2.00 & 2.70) minus 0.15
Total Background effect: 2.89 nM (0.15 nM + 0.48 nM +2.26 nM)
Corrected values for :
Sample I : 2.16 nM (5.05 - 2.89 nM)
Sample II : 31.11 nM (34.00 - 2.89 nM)
Sample III : 111.66 nM (114.55 - 2.89 nM)
Performance characteristics
Detection limit: 0.44 nM CrossLaps
This is the concentration corresponding to three standard deviations above the mean of 21
determinations of the Standard 0.
Precision
The precision of the CrossLaps® for Culture ELISA was evaluated for three serum samples. The results
are summarised in the table below:
InterAssay Variation (n=10) IntraAssay Variation (n=10)
Mean
(nM)
SD
(nM)
CV
(%)
Mean
(nM)
SD
(nM)
CV
(%)
1.5 0.2 11.1 1.5 0.1 7.7
20.0 0.9 4.8 20.0 0.4 2.0
60.4 2.0 3.1 60.4 0.8 1.2
Measuring Range
The measuring range for the CrossLaps® for Culture ELISA is between 0.44 nM and 112.7 nM
CrossLaps.
SPECIAL INSTRUCTIONS
Limitations of the procedure
1. The content of antigenic collagen fragments in fetal calf serum (FCS) varies from product to
product and from lot to lot. However, when used as additive to the bone cell culture medium in ! nal
concentrations up to 10% (v/v), all of the more than 20 commercially available fetal and newborn
bovine serum products tested until now have shown a CrossLaps® for Culture ELISA concentration
below 1nM and therefore do not cause a problem for the analysis. If a serum additive is used for the
bone cell culture it is, however still recommended to:
a) Check its concentration of CrossLaps® for Culture ELISA and if necessary choose another product
with a lower CrossLaps for Culture concentration.
b) Reduce the concentration of serum in the culture medium. Most osteoclast preparations are growing
well at 5% (v/v) or even lower concentration of serum additive and in one study (1) their ability to
resorb bone was found to be invariable at serum concentrations form 0.04 to 5%.
7
REFERENCES
1. Burgeson RE. New collagens, new concepts. Annu Rev Cell Biol (1988);4:551-77.
2. Breuil et al. Human osteoclast formation and activity in vitro: effects of alendronate. J Bone Miner Res
(1998);13:1721-1729
3. Chabaud,M. & Miossec,P. The combination of tumor necrosis factor alpha blockade with interleukin-1
and interleukin-17 blockade is more effective for controlling synovial in! ammation and bone resorption
in an ex vivo model. Arthritis Rheum (2001);44:1293-1303
4. Fisher et al. Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate
pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro.
Proc Natl Acad Sci USA (1999);96:133-138
5. Foged et al. Quanti" cation of the collagenolytic activity of isolated osteoclasts by enzyme-linked
immunosorbent assay. J Bone Miner Res (1996);11:226-237
6. Hollberg et al. Osteoclasts from mice de" cient in tartrate-resistant acid phospahtase have ruf! ed
borders and distributed intravesicular transport. Exp Cell Res (2002);279(2):227-38
7. James et al. Development and characterization of a human in vitro resorption assay: demonstration of
utility using novel antiresorptive agents. Journal of Bone and Mineral Research (1999);14:1562-1569
8. Pedersen BJ. et al. Type I Collagen C-Telopeptide Degradation Products as Bone Resorption Markers
Clin Ligand Assay (1998);21:118-127
9. Stroup et al. Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption
in vivo in a nonhuman primate. J Bone Miner Res (2001);16:1739-1746
Document Number: AC-07PL
Issue: 3 
Date: 21 November 2008
 
EXP
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
Use By
Verwendbar bis
Fecha de caducidad
Utilizzare entro
Utiliser jusque
Houdbaar tot
Holdbar til
Použitelné do
Použitené do
!"#$ %&’!(
Prazo de validade
Felhasználható
Använd före
U)y* przed
LOT
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
Batch code
Chargenbezeichnung
Código de lote
Codice del lotto
Code du lot
Lot nummer
Lotnummer
+íslo šarže
+íslo šarže
,-./( 0$1#2$(
Código do lote
Sarzsszám
Lot nummer
Kod partii
REF
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
Catalogue number
Bestellnummer
Número de catálogo
Numero di catalogo
Référence du catalogue
Catalogus nummer
Katalognummer
Katalogové 3íslo
Katalógové 3íslo
,-./( 4$1$%/56
Referência de catálogo
Katalógusszám
Katalognummer
Numer katalogowy
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
Manufacturer
Hersteller
Fabricante
Fabbricante
Fabricant
Fabrikant
Producent
Výrobce
Výrobca
7$1$846$81&(
Fabricante
Gyártó
Tillverkare
Producent
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
Contains suf9 cient for tests
Inhalt ausreichend für Prüfungen
Contenido su9 ciente para ensayos
Contenuto suf9 ciente per “n” saggi
Contenu suf9 sant pour “n”tests
Inhoud voldoende voor “n” testen
Indeholder tilsttrækkeligt til “n” test
Lze použít pro test:
Obsah posta3uje na stanovení
0-;/" =$4?( 5-$ «"» ’1@8-(
Conteúdo su9 ciente para “n” ensaios
A doboz tartalma vizsgálat
elvégzéséhez elegendD
Räcker till “n” antal tester
W ystarczy na wykonanie testów
IVD
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
In Vitro Eiagnostic Medical Eevice
InJVitroJEiagnostikum
Producto sanitario para diagnóstico in vitro
Eispositivo medicoJdiagnostico in vitro
Eispositif médical de diagnostic in vitro
Medisch hulpmiddel voor inJvitro diagnostiek
Medicinsk udstyr til in vitroJdiagnostik
In Vitro diagnostický zdravotnický prostQedek
Zdravotnícka pomocka in vitro
In Vitro X-$5"Y81-4/ [$11;"%5-4/ =\/"
Eispositivo médico para diagnóstico in vitro
In vitro diagnosztikum
Medicintekniska produkter för in vitro diagnostik
W yrób do diagnistyki In Vitro
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
Temperature limitation
Temperaturbegrenzung
Límite de temperatura
Limiti di temperatura
Limites de température
Temperatuurlimiet
Temperaturbegrænsning
Teplotní rozmezí od do
Teplotné rozmedzie od do
0--8# .4$8#$(
Limites de temperatura
HDmérséklettartomány
Temperaturbegränsning
Przestrzega* zakresu temperatury
GB
DE
ES
IT
FR
NL
DK
CZ
SK
GR
PT
HU
SE
PL
Consult Instructions for Use
Gebrauchsanweisung beachten
Consulte las instrucciones de uso
Consultare le istruzioni per l’uso
Consulter les instructions d’utilisation
Raadpleeg de gebruiksaanwijzing
Se brugsanvisning
Viz návod k použití
Vi] návod na pužitie
^6_6%61#1 1-( 2!5#( ;&8!(
Consulte as instruções de utilização
Nézze meg a Használati utasítást
Se handhavandebeskrivningen
Sprawd‘ w instrukcji obsxugi
Immunodiagnostic Systems Ltd (IDS Ltd).
UK Immunodiagnostic Systems Ltd (IDS Ltd), 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD
Tel: +44 (0) 191 519 0660 • Fax: +44 (0) 191 519 0760 • e-mail: info.uk@idsplc.com • www.idsplc.com
USA Immunodiagnostic Systems Inc (IDS Inc.), P.O. Box 17063, Fountain Hills, AZ 85269-7063
Tel: 480-836-7435 • Fax: 480-836-7437 • e-mail: info.us@idsplc.com • www.idsplc.com
Germany Immunodiagnostic Systems GmbH (IDS GmbH), Mainzer Landstrasse 49, 60329 Frankfurt am Main
Tel: +49 (0) 69 3085-5025 • Fax: +49 (0) 69 3085-5125 • e-mail: info.de@idsplc.com • www.idsplc.com
France Immunodiagnostic Systems EURL (IDS EURL), 55 rue Sainte Anne, 75002 PARIS
Tel: +33 (0)1 42 44 12 63 • Fax: +33 (0)1 42 44 40 76 • e-mail: info.fr@idsplc.com • www.idsplc.com
Scandinavia Immunodiagnostic Systems Nordic a/s (IDS Nordic a/s), Marielundvej 30, 2. Sal, 2730 Herlev, Denmark
Tel:+45 44 84 0091 • Fax:+45 44 84 0092 • email: info.nordic@idsplc.com • www.idsplc.com