1Type I Collagen N-Terminal Telopeptide (NTX-I) Detection ELISA Kit
Catalog # 6040
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify NTX-I fragments/peptides

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

4 hours

STANDARD RANGE:

20 ng/ml to 0.31 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Urine, Serum, and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:1 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C

VALIDATION DATA:

Intra-Assay (0.8-7.4%)/Inter-Assay (4.8-6.9%)/Spiking Test (92-102%)

NOTES:© 2020 Chondrex, Inc. All Rights Reserved, 6040 2.0
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 N-Terminal Telopeptide (NTX-I) Detection ELISA Kit
Catalog # 6040
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). Among the many types of collagen, 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, accounting for more than 90% of the organic components. As a result, degradedtype I collagen peptides appear in serum and urine in early stages of bone loss or metabolism, and indicate bone turnover levels (3, 4).
Proteinases mediate resorption of type I collagen from bone and generate specific peptides of degraded collagen. For example, matrix
metalloproteinases (MMPs) exclusively produce C-terminal degraded peptides, ICTP, from type I collagen, while cathepsin K produces CTXI peptides from the C-terminus and NTX-I peptides from the N-terminus of type I collagen (3).
Patients with osteoporosis display reduced bone mass, indicating that bone cell destruction is greater than bone cell production. In fact, boneloss is increased by lower levels of estrogen after menopause. During the progression of osteoporosis, ICTP and NTX-I are observed inserum as well as urine, and are useful as markers of osteoporosis (4-6). Additionally, bone metastasized cancers, such as breast cancer,
prostate carcinoma, and lung cancer, show increased NTX-I levels in serum and urine due to high levels of degraded type I collagen (7, 8).
Thus, degraded peptides of type I collagen are useful tools for evaluating bone metabolism levels in many diseases not only in humanpatients, but also in mouse disease models of cancer, osteoporosis, osteoarthritis, and rheumatoid arthritis. Chondrex, Inc. provides a mouse
NTX-I Detection ELISA Kit (Cat # 6040) as well as a CTX-I Detection ELISA Kit (Cat # 6033) and a Creatinine Assay Kit (Cat # 6041) which
is useful for normalizing results among urine samples. Please contact support@chondrex.com or visit www.chondrex.com for more
information.

KIT COMPONENTS
Item

Quantity

AmountStorage

NTX-I Standard (60401)
Detection Antibody (60403)

1 vial1 vial20 ng, lyophilized
100 µl-20°C-20°C

Solution B - Sample/Standard/Detection Antibody Dilution Buffer (67015)Solution D – Streptavidin Peroxidase Dilution Buffer (9055)
Streptavidin Peroxidase (9029)
TMB (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)

1 bottle
1 bottle
2 vials
2 vials
1 bottle
1 bottle

50 ml
20 ml
50 µl
0.2 ml
20 ml
10 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Wash Buffer, 20X (9005)
Capture Antibody Coated 96-Well ELISA Plate (Pink)

1 bottle
1 each50 ml
8-well Strips x12-20°C-20°C© 2020 Chondrex, Inc. All Rights Reserved, 6040 2.016928 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

3

ASSAY OUTLINE
Add 100 µl of diluted standards and samples into wells

Incubate at room temperature for 2 hours.
Wash plate.
Add 100 µl of diluted Detection Antibody

Incubate at room temperature for 1 hour.
Wash plate.
Add 100 µl of diluted Streptavidin Peroxidase

Incubate at room temperature for 30 minutes.
Wash plate.
Add 100 µl of TMB solution

Incubate at room temperature for 25 minutes.

Add 50 µl of Stop Solution

Read plates at 450 nm/630 nm

PLATE MAPPING
123456789101112A20

20

S1S1S9S9S17

S17S25

S25

S33

S33B

1010

S2S2

S10

S10

S18

S18

S26

S26

S34

S34

C

55

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

2.52.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

1.25

1.25

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

0.63

0.63

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G0.31

0.31

S7

S7

S15

S15

S23

S23

S31S31S39S39HBB

S8S8S16S16S24

S24

S32

S32S40S40StandardsSamples© 2020 Chondrex, Inc. All Rights Reserved, 6040 2.016928 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

4NOTES 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.

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 0.31 - 20 ng/ml. Dissolve one vial of NTX-I standard in 1 ml ofSample/Standard/Detection Antibody Dilution Buffer (Solution B) for the 20 ng/ml standard. Then serially dilute it with Solution B. Forexample, mix 250 µl of the standard (20 ng/ml) with an equal volume of Solution B to make a 10 ng/ml solution, and then repeat it five
more times for 5, 2.5, 1.25, 0.63, and 0.31 ng/ml solutions. The remaining 20 ng/ml standard stock may be stored at -20°C for use in asecond assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 ml
250 ml

250 ml

250 ml

250 ml

250 ml

250 ml23456781000 ml

250 ml250 ml

250 ml

250 ml

250 ml

250 ml

250 ml20 ng/ml

10 ng/ml

5 ng/ml

2.5 ng/ml

1.25 ng/ml

0.63 ng/ml

0.31 ng/ml

Blank

12.

Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids. Dilute thesupernatants with an equal volume of Solution B. For example, take 100 µl of supernatant, and mix with 100 µl of Solution B. If the OD
values of samples are higher than the OD values of the 20 ng/ml standard, re-assay the sample at a higher dilution.
NOTE: Dilute samples at least 1:1 with Solution B depending on the estimated NTX-I level in the samples. Two to three different sample
dilutions are recommended if the NTX-I levels in the samples are unknown.

3.

Add Standards and Samples: Add 100 µl of Solution B (blank), standards, and samples to designated wells. Incubate at room
temperature for 2 hours.

4.

Dilute Wash Buffer: Dilute 50 ml of Wash Buffer, 20X 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.

© 2020 Chondrex, Inc. All Rights Reserved, 6040 2.0
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

55.

Add Detection Antibody Solution: Prepare detection antibody solution with Solution B as shown in the following table. Add 100 µl ofdetection antibody solution to each well and incubate at room temperature for 1 hour.
Strip #24681012Detection Antibody (µl)
1725506682100

Solution B (ml)
1.7
3.3
5.0
6.6
8.2
10.0

6.

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.

7.

Add Streptavidin Peroxidase Solution: Prepare streptavidin peroxidase solution with Streptavidin Peroxidase Dilution Buffer (SolutionD) as shown in the following table. Add 100 µl of streptavidin peroxidase solution to each well and incubate at room temperature for 30minutes.
Strip #24681012Streptavidin Peroxidase (µl)
8.51725334150

Solution D (ml)
1.7
3.3
5.0
6.6
8.2
10.0

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 TMB Solution: Use new tubes when preparing TMB solution. Just prior to use, prepare TMB solution with Chromogen Dilution
Buffer as shown in the following table. Add 100 µl of TMB solution to each well immediately after washing the plate and incubate for 25
minutes at room temperature.
Strip #2
46810
12

TMB (µl)
3466
100132
164
200

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

10. Stop: Stop the reaction with 50 µl of 2N Sulfuric Acid (Stop Solution) to each well.
11. Read Plate: Read the OD values at 450 nm. If the OD values of samples are greater than the OD values of the highest standard, reassay the samples at a higher dilution. A 630 nm filter can be used as a reference.

© 2020 Chondrex, Inc. All Rights Reserved, 6040 2.0
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

6

CALCULATING RESULTS
1.

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

2.

Subtract the “blank” (B) values from the averaged OD values in step 1.

3.

Plot the OD values of standards against the concentration of NTX-I (ng/ml). Using a log/log plot will linearize the data. Figure 1 showsa representative experiment where the standard range is 0.31 - 20 ng/ml.

4.

The ng/ml of NTX-I in test samples can be calculated using regression analysis.

Figure 1 - A Typical Standard Curve for the NTX-I Detection ELISA Kit

10.0
R² = 0.9995

OD 450 nm

1.00.1

0.00.1110100NTX-I Concentration (ng/ml)

VALIDATION DATA
Table 1 – Reproducibility Data for the NTX-I Detection ELISA Kit
Test

0.63 ng/ml

2.5 ng/ml

10 ng/ml

Intra-Assay CV (%)

7.4

1.8

0.8

Inter-Assay CV (%)

4.8

5.6

6.9

Spike Test* (%)
92%
97%
102%
* Known amounts of NTX-I were added to samples and then diluted with Sample/Standard/Detection Antibody Dilution Buffer (Solution B).

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

© 2020 Chondrex, Inc. All Rights Reserved, 6040 2.016928 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

7

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.

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).

4.

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

5.

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).

6.

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).

7.

M. Koizumi, E. Ogata, Bone metabolic markers as gauges of metastasis to bone: a review. Ann Nucl Med 16, 161-8 (2002).

8.

K. Jung, M. Lein, Bone turnover markers in serum and urine as diagnostic, prognostic and monitoring biomarkers of bone metastasis.
Biochim Biophys Acta 1846, 425-38 (2014).

© 2020 Chondrex, Inc. All Rights Reserved, 6040 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373Fax: 425.882.3094

www.chondrex.com
support@chondrex.com