1Type I Collagen C-Terminal Telopeptide (CTX-I) Detection ELISA Kit
Catalog # 6033
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA Kit to quantify CTX-I fragments/peptides

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Competitive ELISAASSAY TIME:

3 hours

STANDARD RANGE:

500 - 8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Urine, Serum, and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:100 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (1.6-2.2%)/Inter-Assay (5.5-10%)/Spiking Test (108-113%)

NOTES:© 2022 Chondrex, Inc. All Rights Reserved, 6033 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2Type I Collagen C-Terminal Telopeptide (CTX-I) Detection ELISA Kit
Catalog # 6033
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Collagen is the most abundant protein in the mammalian body and lends structural integrity to tissues as the primary component of theextracellular matrix (1). Type I collagen is the main component of bone, tendon, skin, and other tissues (2). In fact, type I collagen makes up
20% of bones by mass, which accounts for more than 90% of the organic components. As a result, degradation products of type I collagencan be detected in serum and urine in stages of bone loss or metabolism and can be potential markers of bone metabolism (3).
Proteinases mediate resorption of type I collagen from bone and generate specific peptide fragments of degraded collagen. For example,matrix metalloproteinases (MMPs) exclusively produce C-terminal degraded fragments (ICTP) from type I collagen, while cathepsin K
produces CTX-I fragments from the C-terminus and NTX-I fragments from the N-terminus of type I collagen (4). Because the proteinase
activities differ among diseases and degraded fragments reflect the metabolism of type I collagen, immunoassays have been developed tomonitor the levels of these degraded fragments in biological fluids (5).
Patients with osteoporosis display reduced bone mass. During disease progression, degraded peptides of type I collagen are observed inserum as well as urine. Therefore, ICTP and NTX-I have been used as markers of osteoporosis (6-8). In addition, it was reported that CTXI levels in urine correlate with disease activity of osteoarthritis (9). Furthermore, cancers which metastasize to bone can affect the metabolismof type I collagen. Serum CTX-I levels correlate with prognosis of these cancers, especially prostate, lung, breast, and urinary bladdercancers (10-13).
Thus, degraded type I collagen fragments are very useful tools for evaluating disease not only in humans, but also in mice, leading to thedevelopment of many mouse disease models for cancer, osteoporosis, osteoarthritis, and rheumatoid arthritis. Chondrex, Inc. has developed
a CTX-I Detection ELISA kit (Cat # 6033) for mouse and human samples using a competitive assay system with a monoclonal antibody
which recognizes conserved peptide sequences in mouse and human (13).

KIT COMPONENTS
Item

Quantity

AmountStorage

Capture Antibody (60332)

1 vial100 µl-20°C

Standard C-Telopeptide, CTX-I (60331)

1 vial100 µl-20°C

Biotinylated C-Telopeptide, CTX-I (60333)

1 vial100 µl-20°C

Solution A - Coating Buffer (9052)

1 bottle

10 ml

-20°C

Solution B - Sample/Standard Dilution Buffer (67015)

1 bottle

50 ml

-20°C

Solution D - Streptavidin Peroxidase Dilution Buffer (9055)

1 bottle

20 ml

-20°C

Streptavidin Peroxidase (9029)

2 vials

50 µl-20°C

TMB Solution (90023)

2 vials

0.2 ml-20°C

Chromogen Dilution Buffer (90022)

1 bottle

20 ml

-20°C

Stop Solution - 2N Sulfuric Acid (9016)

1 bottle

10 ml

-20°C

Wash Buffer, 20X (9005)

1 bottle

50 ml

-20°C

96-well ELISA Plate

1 each

96-well, (8-well strips x 12)-20°C

Flexible 96-well Plate

1 each

96-well-20°C© 2022 Chondrex, Inc. All Rights Reserved, 6033 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3ASSAY OUTLINE
96-well ELISA plate
Add 100 ml of diluted capture antibody solution to wellsIncubate at 4 C overnight.
Wash plates.

Flexible 96-well plate
Add 75 ml of diluted standards and samples to wells

Add 75 ml of diluted biotinylated CTX-I to wells
Mix the solution in the flexible 96-well plate.
Then, transfer 100 ml of the mixture into theCapture Antibody coated ELISA plate wells.

Incubate at room temperature for 2 hours.
Wash plates.
Add 100 ml of Streptavidin Peroxidase
Incubate at room temperature for 30 minutes.
Wash plates.
Add 100 ml of TMB solution
Incubate plate at room temperature for 25 minutes.
Add 50 ml of Stop Solution

Read plates at 450 nm/630 nm

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placing
the bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.
NOTE 6: For partial reagent use, please see the assay protocol’s corresponding step for the appropriate dilution ratio. For example, if theprotocol dilutes 50 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 25 µl of the stock solution in 5 ml of buffer.
Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.
NOTE 7: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and fordisposal.

© 2022 Chondrex, Inc. All Rights Reserved, 6033 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4

PLATE MAPPING
123456789101112A

500

500

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B250

250

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C125125

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D6363

S4S4S12S12

S20

S20

S28

S28

S36

S36E3131

S5S5S13

S13S21

S21

S29

S29

S37

S37F1616S6S6S14S14

S22

S22S30

S30

S38

S38

G88

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

HBB

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples

ASSAY PROCEDURE
1.

Add Capture Antibody: Dilute one vial of Capture Antibody with 10 ml of Coating Buffer (Solution A). Alternatively, dilute according tothe table below. Add 100 µl of capture antibody solution to each well and incubate at 4°C overnight. Any leftover Capture Antibody
Stock Solution may be stored at -20°C for future assays.
Strip #246810122.

Capture Antibody (µl)
1733506682100

Solution A (ml)
1.7
3.3
5.0
6.6
8.2
10.0

Prepare Standard Dilutions: The recommended CTX-I standard range is 8 - 500 ng/ml. Add 900 µl of Sample/Standard Dilution Buffer(Solution B) to one vial of Standard (500 ng/vial) and keep it as a 500 ng/ml standard stock. Then, serially dilute it with Solution B. Forexample, mix 250 µl of the 500 ng/ml solution with an equal volume of Solution B to make a 250 ng/ml solution, and then repeat it five
more times for 125, 63, 32, 16, and 8 ng/ml standard solution.
900 ml
250 ml

250 ml

250 ml

250 ml

250 ml23456781000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml500 ng/ml

250 ng/ml

125 ng/ml

63 ng/ml

31 ng/ml

16 ng/ml

8 ng/ml

Blank

13.

250 mlPrepare Sample Dilutions: Sample dilution (serum or urine) varies (1:100 or more) depending on the disease and timing of serum
collection. In general, no CTX-I is observed in normal serum at a 1:100 dilution.

© 2022 Chondrex, Inc. All Rights Reserved, 6033 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

54.

Prepare Biotinylated CTX-I: Dilute one vial of biotinylated c-telopeptide with 10 ml of Sample/Standard Dilution Buffer (Solution B).
Strip #24681012

Biotinylated CTX-I (µl)
17335066
82100Solution B (ml)
1.7
3.3
5.0
6.6
8.2
10.0

5.

Mix Standards, Samples, and Biotinylated CTX-I: Using the Plate Mapping figure as a guide, mix 75 µl of standards, diluted sample,and Solution B (blank) with 75 µl of diluted biotinylated C-telopeptide in the flexible plate provided.

6.

Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer). Wash the plate with 1X wash buffer
at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blotting on a paper
towel to remove excess liquid. Do not allow the plate to dry out.

7.

Transfer Mixtures: Transfer 100 µl from the wells of the flexible plate to the corresponding wells of the washed capture antibody coatedplate. Incubate at room temperature for 2 hours.

8.

Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

9.

Add Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Peroxidase Dilution Buffer (SolutionD). Add 100 µl of streptavidin peroxidase solution to each well and incubate at room temperature for 30 minutes.
Strip #24681012Streptavidin Peroxidase (µl)
81725334250

Solution D (ml)
1.7
3.3
5.0
6.6
8.2
10.0

10. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.
11. Add TMB: Use new tubes when preparing TMB. Dilute one vial of TMB with 10 ml Chromogen Dilution Buffer just prior to use. Add 100µl of TMB solution to all wells immediately after washing the plate and incubate for 25 minutes at room temperature.
Strip #
2468
1012TMB (µl)
3466
100
132
164
200

Chromogen Dilution Buffer (ml)
1.7
3.3
5.0
6.6
8.2
10.0

12. Stop: Add 50 µl of 2N sulfuric acid (Stop Solution) to each well.

© 2022 Chondrex, Inc. All Rights Reserved, 6033 2.116928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

613. Read Plate: Read the OD values at 450 nm (a 630 nm filter can be used as a reference).
NOTE: This is a competitive assay. Re-assaying samples may be necessary if the samples show the following results.
A.

If the OD values of samples are lower than the OD values of the highest standard, re-assay the samples at a higher dilution.

B.

If the OD values of samples are higher than the OD values of the lowest standard, re-assay the samples at a lower dilution.

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank, standards, and test samples.

2.

Calculate the ratio of OD values of each standard or sample against the OD values of the blank (B).
Ratio = OD values of standard/samples / OD value of blank.

3.

Plot the OD values of standards against the concentration of C-telopeptide standard (ng/ml). Using a log/log plot will linearize the data.
Figure 1 shows a representative experiment where the standard range is 8 - 500 ng/ml.

4.

The ng/ml of C-telopeptides in test samples can be calculated using regression analysis. Multiply the ratio of the sample OD values by
the sample dilution factor to obtain the CTX-I concentration (ng/ml) in original sample specimens. For additional assistance, please
download a sample calculation worksheet from www.chondrex.com.

Figure 1 - A Typical Standard Curve for the CTX-I Detection ELISA Kit© 2022 Chondrex, Inc. All Rights Reserved, 6033 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

7ASSAY VALIDATION
Table 1 - Reproducibility Data for the CTX-I Detection ELISA Kit
Test
16 ng/ml
63 ng/ml
250 ng/ml
Intra-Assay CV (%)
1.8
2.2
1.6
Inter-Assay CV (%)
7.2
5.5
10.0
Spike Test* (%)
108%
113%
109%
* Known amounts of CTX-I were added to samples and then diluted with Sample/Standard/Detection Antibody Dilution Buffer (SolutionB) to assay CTX-I by ELISA.

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s ELISA FAQ for more information.

REFERENCES
1.

E. Hohenester, J. Engel, Domain structure and organisation in extracellular matrix proteins. Matrix Biol 21, 115-28 (2002).

2.

K. Gelse, E. Pöschl, T. Aigner, Collagens--structure, function, and biosynthesis. Adv Drug Deliv Rev 55, 1531-46 (2003).

3.

E. Eriksen, K. Brixen, P. Charles, New markers of bone metabolism: clinical use in metabolic bone disease. Eur J Endocrinol 132, 25163 (1995).

4.

G. Wheater, M. Elshahaly, S. Tuck, H. Datta, L. van, The clinical utility of bone marker measurements in osteoporosis. J Transl Med
11, 201 (2013).

5.

J. Risteli, I. Elomaa, S. Niemi, A. Novamo, L. Risteli, Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptideof type I collagen: a new serum marker of bone collagen degradation. Clin Chem 39, 635-40 (1993).

6.

M. Bonde, C. Fledelius, P. Qvist, C. Christiansen, Coated-tube radioimmunoassay for C-telopeptides of type I collagen to assess boneresorption. Clin Chem 42, 1639-44 (1996).

7.

K. Lee, M. Lee, C. Chung, W. Seong, S. Lee, M. Park, et al., Measurement of urinary N-telopeptides and serum C-telopeptides fromtype I collagen using a lateral flow-based immunoassay. Sensors (Basel) 13, 165-74 (2012).

8.

J. Clemens, M. Herrick, F. Singer, D. Eyre, Evidence that serum NTx (collagen-type I N-telopeptides) can act as an immunochemicalmarker of bone resorption. Clin Chem 43, 2058-63 (1997).

9.

S. Ok, S. Lee, H. Park, S. Jeong, C. Ko, Y. Kim, et al., Concentrations of CTX I, CTX II, DPD, and PYD in the urine as a biomarker forthe diagnosis of temporomandibular joint osteoarthritis: A preliminary study. Cranio 36, 366-372 (2018).

10. A. Ferreira, I. Alho, S. Casimiro, L. Costa, Bone remodeling markers and bone metastases: From cancer research to clinical implications.
Bonekey Rep 4, 668 (2015).
11. A. Zissimopoulos, K. Stellos, D. Matthaios, G. Petrakis, V. Parmenopoulou, et al., Type I collagen biomarkers in the diagnosis of bonemetastases in breast cancer, lung cancer, urinary bladder cancer and prostate cancer. Comparison to CEA, CA 15-3, PSA and bone
scintigraphy. J BUON 14, 463-72 (Jul-).
12. A. Franjević, R. Pavićević, G. Bubanović, ICTP in bone metastases of lung cancer. Coll Antropol. 35, 43–47 (2011).
13. A. Srivastava, S. Bhattacharyya, G. Castillo, N. Miyakoshi, S. Mohan, D. Baylink, et al., Development and evaluation of C-telopeptide
enzyme-linked immunoassay for measurement of Bone resorption in mouse serum. Bone 27, 529-33 (2000).

© 2022 Chondrex, Inc. All Rights Reserved, 6033 2.116928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com