1Type I Collagen Detection ELISA Kits
Catalog # 6012, 6013, 6014, 6015, 6016, 6019, and 6021
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify Type I Collagen6012: Mouse Type I Collagen Detection ELISA Kit
6013: Rat Type I Collagen Detection ELISA Kit
6014: Bovine Type I Collagen Detection ELISA Kit
6015: Porcine Type I Collagen Detection ELISA Kit
6016: Rabbit Type I Collagen Detection ELISA Kit
6019: Canine Type I Collagen Detection ELISA Kit
6021: Human Type I Collagen Detection ELISA Kit

FORMAT:

96-well ELISA plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

5.5 hours

STANDARD RANGE:

6012/6013/6015/6021: 5 µg/ml to 0.08 µg/ml
6014/6016/6019: 10 µg/ml to 0.16 µg/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Solubilized collagen and cell culture media

RECOMMENDED SAMPLE DILUTIONS:

1:1 - 1:1000

CHROMOGEN:

OPD (read at 490 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

6021 (only): Intra-assay (1.4-3.9%)/Inter-assay (2-6.6%)/Spiking Test (99-106%)NOTES:

These kits are intended to detect native type I collagen and will only weakly detect denatured
collagen© 2022 Chondrex, Inc. All Rights Reserved, Type I ELISA 1.1
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 Detection ELISA Kits
Catalog # 6012, 6013, 6014, 6015, 6016, 6019, and 6021
For Research Use Only - Not Human or Therapeutic Use
INTRODUCTION
Type I collagen is one of several interstitial fibrillar collagens consisting of two identical α1(I) chains and one α2(I) chain. It is the mostabundant collagen type and is found in most connective tissues such as skin, bone, tendon, ligament, and heart. Many tissues containheterotypic fibrils meaning that two or more distinct collagen types coexist. For example, most connective tissues (except for bone) containheterotypic fibrils of type I and III collagen, although the type III collagen content is minor compared to Type I collagen. Chondrex, Inc.
provides a variety of species-specific Type I Collagen Detection ELISA Kits designed to quantify the amount of species-specific type I collagenin cultured cells and tissue specimens.

ANTIBODY SPECIFICITY
Monoclonal antibodies are used as the capture and detection antibodies in these Type I Collagen Detection ELISA Kits. All clones are highly
specific to the native conformation of their respective type I collagens and poorly cross-react with denatured type I collagen. Chondrex, Inc.
recommends carefully selecting kits to determine species-specific type I collagen in samples containing other species of type I collagen ortype II collagen based on the specificity of the capture antibodies in the table below. Some capture antibodies are highly specific to theirrespective type I collagens and do not cross-react with other species of type I or type II collagen.
Collagen Type
Mouse I
Mouse Denatured I
Mouse II
Mouse III
Rat I
Rat Denatured I
Rat II
Rat III
Bovine I
Bovine Denatured I
Bovine II
Bovine III
Porcine I
Porcine Denatured I
Porcine II
Porcine III
Rabbit I
Rabbit Denatured I
Rabbit II
Rabbit III
Canine I
Canine Denatured I
Canine II
Canine III
Human I
Human Denatured I
Human II
Human III
Chick I
Chick Denatured I
Chick II
Chick III

Mouse
CADA
100% 100%5%3%0%0%
ND
ND
0%
5%0%
24%
0%
56%
0%
0%0%0%-*CA: Capture Antibody**DA: Detection Antibody (Biotinylated)***ND: No Data

CA
0%
100%
2%0%
ND
0%
0%
0%
50%-

Rat

DA
0%
100%
3%
0%
ND
100%
75%
0%0%-

BovineCADA
31%
0%100% 100%3%
3%0%0%
ND
ND
100%
0%0%0%-

PorcineCADA
31%
0%100% 100%
100%
75%
3%
4%
0%0%
ND
ND
100%0%0%0%-Rabbit
CADA
31%0%100% 100%100%75%100%70%2%
1%
ND
ND
100%
0%0%0%-Canine
CADA
31%
0%
0%
100%
100% 100%
100%
75%
100% 100%5%5%0%0%
ND
ND
100%
0%0%0%-

Human
CADA
0%
1%
0%
1%
0%
0%
0%
1%
100% 100%
1%
6%
0%
33%
3%
100%
0%1%-© 2022 Chondrex, Inc. All Rights Reserved, Type I ELISA 1.1
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.comsupport@chondrex.com

3

KIT COMPONENTS
Item

Quantity

Mouse (60121)
Rat (60131)
Bovine (60141)
Standard
Porcine (60151)
Rabbit (60161)
Canine (60191)
Human (60081)
Mouse (60122)
Rat (60132)
Bovine (60142)
CapturePorcine (60152)
Antibody
Rabbit (60162)
Canine (60192)
Human (60212)
Mouse (60123)
Rat (60133)
Bovine (60143)
Detection
Porcine (60153)
Antibody
Rabbit (60163)
Canine (60193)
Human (60213)
Solution A - Coating Buffer (9052)
Solution B - Sample/Standard Dilution Buffer (9053)

1 vialAmount100 µl (Mouse, Rat, Porcine, Human)
150 µl (Bovine, Rabbit, Canine)

Storage-20°C

100 µg/ml

1 vial100 µl, 1 mg/ml-20°C

1 vialLyophilized-20°C

1 bottle
1 bottle

10 ml
50 ml

-20°C
-20°C

Solution C - Detection Antibody Dilution Buffer (9054)
Solution D - Streptavidin Peroxidase Dilution Buffer (9055)
Streptavidin Peroxidase (9029)
OPD (90021)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)

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

10 ml
20 ml
50 µl
Lyophilized
20 ml
10 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Wash Buffer, 20X (9005)
ELISA Plate

1 bottle
1 each

50 ml

-20°C
-20°C

96-well (8-well strips x 12)© 2022 Chondrex, Inc. All Rights Reserved, Type I ELISA 1.1
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

4ASSAY OUTLINE
Add 100 ml of capture antibody solution into wells

Incubate at 4 degrees C overnight.
Wash plate.
Add 100 ml of diluted standards and samples into wells

Incubate at room temperature for 2 hours.
Wash plate.

Add 100 ml of diluted detection antibody solution into wells

Incubate at room temperature for 2 hours.
Wash plate.
Add 100 ml of diluted streptavidin peroxidase solution into wells

Incubate at room temperature for 1 hour
Wash plate.
Add 100 ml of OPD solution into wells

Incubate at room temperature for 30 minutes.

Add 50 ml of Stop Solution into wells

Read plates at 490 nm/630 nm

PLATE MAPPING
123456789101112A

1010

S1S1S9S9S17S17S25

S25S33S33S34B

55

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

S35

S35

C

2.52.5

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

S28

S36

S36

D

1.3

1.3

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

S29

S29

S37

S37

E

0.63

0.63

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

S22

S30

S30

S38

S38

F

0.31

0.31

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

S23

S23

S31

S31

S39

S39

G

0.16

0.16

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

S24

S24

S32

S32

S40

S40HB

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

123456789101112A

55

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33B

2.5

2.5

S2

S2

S10

S10

S18

S18

S26

S26

S34

C

1.25

1.25

S3

S3

S11

S11

S19

S19

S27

S27

D

0.63

0.63

S4

S4

S12

S12

S20

S20

S28

E

0.31

0.31

S5

S5

S13

S13

S21

S21

F

0.16

0.16

S6

S6

S14

S14

S22

G

0.08

0.08

S7

S7

S15

S15

H

B

B

S8

S8

S16

S16

Standards

Samples

ORStandards

Samples© 2022 Chondrex, Inc. All Rights Reserved, Type I ELISA 1.1
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

5SOLUBILIZING COLLAGEN
For determining collagen content in cultured cell layers and tissues by ELISA, solubilizing collagen is required. For tissue specimens, alimited digestion with pepsin is highly recommended for native collagen preparation as other neutral proteinases, such as pronase andpapain, digest collagen into small peptides. Pepsin only digests telopeptides located on both the N- and C-terminals of the collagen moleculeand is not capable of digesting the helical conformation region of the collagen molecule itself. Please inquire with Chondrex, Inc. customer
service for “Tips on Solubilization of Collagen”.
Solubilizing collagen from tissues by limited pepsin digestion (generally collagen to pepsin ratio is 100:1) depends on the types of tissues
and the contents of the intra- and inter-molecular cross-linkages. For example, bone and Achilles tendon are resistant to pepsin digestionand only 10-20% of the collagen tissue will be solubilized. Young calf skin collagen will be completely solubilized by pepsin digestion within
24-48 hours, while it takes 7-9 days to solubilize adult skin. Pepsin resistant insoluble collagen might be solubilized by alkaline treatment.
Suspend insoluble collagen in cold 0.1N NaOH solution containing 10% Na2SO4 and 0.1M Amine such as Tris and incubate at 4°C for 1-2weeks. After treatment of collagen with alkaline, neutralize the pH to 5.0 with HCl, and then dilute it with 0.05M acetic acid or neutral buffer
such as 0.1M Tris-0.3M NaCl, pH 7.5.
Therefore, the optimum solubilization condition for individual samples should be determined before processing samples. Collagen can beanalyzed by 6% SDS-gel under non-reducing conditions, using authentic type I collagen as a standard. If samples contain bands larger thanthe -chain (MW = 300 Kd), the samples must be further digested by pepsin or elastase which will digest the intra- and inter-collagen crosslinkages. On the other hand, if smaller bands or smear bands are observed under the α-chain (MW = 100 Kd), the samples might be overdigested. Therefore, it is critical to understand the biological and physico-chemical properties of individual collagen samples.

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, Type I ELISA 1.1
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

6

ASSAY PROCEDURE
1.

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

Capture Antibody (µl)
1733
5066
82
100Solution A (ml)
1.7
3.3
5.0
6.6
8.2
10.0

Prepare Standard Dilutions: Please see the figure below for each assay’s recommended standard range. Dilute the standard stock
accordingly to get the first stock solution. Then serially dilute it with Solution B. For example, mix 250 µl of the first stock solution withan equal volume of Solution B to make the second stock solution, and then repeat it five more times. The original standard stock solution
may be stored at -20⁰C for use in a future assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
Mouse/Rat/Porcine/Human: 25 µl + 475 µl
Bovine/Rabbit/Canine: 50 µl + 450 µl

250 µl1250 µl

250 µl250 µl250 µl250 µl2345678250 µl

250 µl250 µl250 µl250 µl250 µl250 µl100 µg/ml

3.

Mouse/Rat/Porcine/Human:

5 µg/ml

2.5 µg/ml

1.25 µg/ml

0.63 µg/ml

0.31 µg/ml

0.16 µg/ml

0.08 µg/ml

Blank

Bovine/Rabbit/Canine:

10 µg/ml

5 µg/ml

2.5 µg/ml

1.25 µg/ml

0.63 µg/ml

0.31 µg/ml

0.16 µg/ml

Blank

Prepare Sample Dilutions: Dilute tissue samples 1:1-1:1000 with Solution B depending on the estimated collagen content in thesamples. The samples must be diluted with Solution B to maintain optimal assay conditions.
NOTE: Cell samples and culture media can be assayed at a 1:1 dilution with Solution B.

4.

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.

5.

Add Standards and Samples: Add 100 µl of standards, Solution B (blank), and samples to appropriate wells in duplicate. Incubate atroom temperature for 2 hours.

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.

© 2022 Chondrex, Inc. All Rights Reserved, Type I ELISA 1.1
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

77.

Add Detection Antibody: Dissolve one vial of Detection Antibody in 10 ml Detection Antibody Dilution Buffer (Solution C). Alternatively,dissolve one vial of Detection Antibody in 50 µl of Solution C and dilute accordingly. Add 100 µl of detection antibody solution to eachwell and incubate at room temperature for 2 hours.
Strip #24
68
1012Detection Antibody (µl)
8172533
4250

Solution C (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 Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Dilution Buffer (Solution D). Add 100µl of streptavidin peroxidase solution to each well and incubate at room temperature for 1 hour.
Strip #2
4681012Streptavidin 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 OPD: Dissolve one vial of OPD with 10 ml of Chromogen Dilution Buffer just prior to use. Add 100 µl of OPD solution to each wellimmediately after washing the plate and incubate for 30 minutes at room temperature.
12. Stop: Add 50 µl of 2N sulfuric acid (Stop Solution) to each well.
13. Read Plate: Read the OD values at 490 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.

CALCULATING RESULTS
1.

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

2.

Subtract the averaged blank OD values from the averaged OD values of the standards and test samples.

3.

Plot the OD values of standards against the concentration of standards (µg/ml). Using a log/log plot will linearize the data.

4.

The µg/ml of collagen in test samples can be calculated using regression analysis. Multiply it by the sample dilution factor to obtain thecollagen concentration (µg/ml) in the original sample specimens.

© 2022 Chondrex, Inc. All Rights Reserved, Type I ELISA 1.1
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

8Figure 1 - Typical Standard Curves for the Type I Collagen Detection ELISA Kits
Mouse Type I Collagen Detection ELISA Standard

Rat Type I Collagen Detection ELISA Standard

Bovine Type I Collagen Detection ELISA Standard
101010R² = 0.9978

R² = 0.9931

R² = 0.9994
11OD 490 nm

OD 490 nm

OD 490 nm

10.1

0.1

0.01

0.1

0.01
0.01

0.1

1100.01
0.01

0.1

1100.1

Rat Type I Collagen Concentration (µg/ml)

Mouse Type I Collagen Concentration (µg/ml)

Rabbit Type I Collagen Detection ELISA Standard

Porcine Type I Collagen Detection ELISA Standard

10.0

R² = 0.9963

R² = 0.9962

R² = 0.9964

1.01.0OD 490 nm

OD 490 nm

OD 490 nm

10.1

0.1

0.01

0.1

0.0
0.01

0.1

110Porcine Type I Collagen Concentration (µg/ml)

10Canine Type I Collagen Detection ELISA Standard

10.0101Bovine Type I Collagen Concentration (µg/ml)

0.00.1110Rabbit Type I Collagen Concentration (µg/ml)

0.1110Canine Type I Collagen Concentration (µg/ml)

Human Type I Collagen Detection ELISA Standard
10.0

R² = 0.9971

OD 490 nm

1.00.1

0.00.00.1

1.010.0

Human Type I Collagen Concentration (µg/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the Human Type I Collagen Detection ELISA KitTest
0.16 μg/ml
0.63 μg/ml
2.5 μg/ml
Intra-Assay CV (%)
1.4
3.9
3.2
Inter-Assay CV (%)
6.6
2.0
4.3
Spike Test* (%)
106%
99%
100%
* Known amounts of Human Type I collagen were added to samples and then diluted with Solution B for assaying Human Type I collagen.

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

© 2022 Chondrex, Inc. All Rights Reserved, Type I ELISA 1.1
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