1Hydroxyproline Assay Kit
Catalog # 6017

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to quantify total collagen content (regardless of type or species)

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Colorimetric assay

ASSAY TIME:

1 hour

STANDARD RANGE:

400 µg/ml to 6.3 µg/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) colorless samples/plate and up to 20 (duplicate) colored samples/plate

SAMPLE TYPES:

Tissue homogenate and cell culture media

RECOMMENDED SAMPLE DILUTIONS:

Not required

CHROMOGEN:

N/A (read at 530-560 nm)

STORAGE:-20°C

VALIDATION DATA:

Intra-assay (3.4-6.3%)/Inter-assay (5.1-7.6%)/Spiking Test (91-95%)

NOTES:

Select samples may require a 24-hour hydrolysis step© 2020 Chondrex, Inc. All Rights Reserved, 6017 7.0
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

2

Hydroxyproline Assay Kit
Catalog # 6017

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTIONCollagen is the major structural protein of the extracellular matrix in many tissues. Hydroxyproline, a major component of collagen, makes
up about 13.5% of its amino acid composition. Due to its highly restricted distribution in collagen, the hydroxyproline content accuratelyreflects the amount of collagen. Therefore, quantitating hydroxyproline has been utilized for evaluating tissue fibrosis or collagen deposition(1 - 3). However, conventional hydroxyproline assays are not useful because they require cumbersome procedures and special tools.
Chondrex, Inc. offers a hydroxyproline assay kit (Cat # 6017) which employs an improved assay system that can be operated with ease andprecision using 96-well plates. This kit can quantify the total collagen content in any tissue specimen or tissue homogenate, regardless ofcollagen type or species. Chondrex, Inc. also provides additional collagen detection kits for different purposes. Firstly, the Sirius Red Total
Collagen Detection Kit (Cat # 9062 or 9062P) can be used to quantify solubilized collagen in samples. Secondly, the collagen detection
ELISA kits specific to type I or II collagen of various species are used for distinguishing collagen types and species in samples. Lastly, theSirius Red/Fast Green Collagen Staining Kit (Cat # 9046) is a semi-quantitative assay to determine the total collagen content in tissue
sections. For more information, please visit www.chondrex.com or contact support@chondrex.com.

KIT COMPONENTS
Item

Quantity

AmountStorage

Hydroxyproline Standard (60171)
Solution A- Chloramine T Dilution Buffer (60172)
Solution B - DMAB Dilution Buffer (60173)

1 vial
1 bottle
1 vial4 mg/ml x 0.5 ml
10 ml
5 ml

-20°C
-20°C
-20°C

10X Chloramine T Concentrate (60174)
1 vial
1 ml-20°C
2X DMAB (dimethylaminobenzaldehyde) Concentrate (60175)
1 vial5 ml-20°C
96-well (8-well strips x 12)
ELISA Plate
1 each-20°C
Not included: concentrated HCl (10N) and glass screw-thread vials (1-2 ml) with Teflon caps (Example: National Scientific B7999-1)

ASSAY OUTLINE
Add 10 µl of diluted standards and samples to wells

Add 100 µl of 1X Chloramine T solution to wells (Solution A for sample blanks)

Mix and incubate at room temperature for 20 minutes.

Add 100 µl of 1X DMAB solution to wells

Mix and incubate at 60 degrees C for 30 minutes.

Read plates at 530-560 nm© 2020 Chondrex, Inc. All Rights Reserved, 6017 7.0
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

3PLATE LAYOUT
Colorless (clear) samples

Colored (turbid) samples
123456789101112A

400

400

S1S1

SB1

SB1S9S9SB9SB9S17SB17S34B

200200S2S2SB2SB2S10S10SB10 SB10S17SB17

S35

S35

C100

100

S3S3SB3SB3S11

S11

SB11 SB11

S18

SB18

S28

S36

S36

D5050

S4S4SB4SB4S12S12

SB12 SB12

S18

SB18

S29

S29

S37

S37

E2525

S5S5SB5SB5S13

S13

SB13 SB13

S19

SB19

S22

S30

S30

S38

S38

F12.512.5

S6

S6

SB6

SB6

S14

S14

SB14 SB14

S19

SB19

S23

S23

S31

S31

S39

S39

G6.3

6.3

S7

S7

SB7

SB7

S15

S15

SB15 SB15

S20

SB20

S24

S24

S32

S32

S40

S40

HBB

S8

S8

SB8

SB8

S16

S16

SB16 SB16

S20

SB20

123456789101112A400

400

S1S1S9S9S17

S17S25

S25

S33S33

B

200200S2S2S10S10S18S18S26

S26

S34C100100

S3S3S11S11S19S19S27

S27

D5050

S4S4

S12S12S20

S20

S28

E2525

S5S5S13

S13

S21

S21

F

12.5

12.5

S6S6

S14

S14

S22

G6.3

6.3

S7

S7

S15

S15H

B

B

S8

S8

S16

S16

Standard

SamplesStandard

Samples

SampleBlanksNOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: 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 3: If there are precipitates in the bottles/vials, it is necessary to warm up the bottles/vials in warm water until they completely dissolve.

SAMPLE HYDROLYSIS
1.

Weigh 10 mg of a tissue sample in a glass screw-thread vial.

2.

Add 100 µl of distilled water.

3.

Mash the tissue sample with a small spatula.
NOTE: 100 µl of a sample homogenate can be used. Skip steps 1-3 and directly add 100 µl of the sample homogenate to the vial.

4.

Add 100 µl of concentrated HCl (10N) and tightly screw on the teflon cap.

5.

Incubate at 120°C for 24 hours. Mix the sample periodically during incubation.
NOTE: A heat block or dry bath can be used for incubation.

6.

Cool down. Do not open the cap before cooling down.

7.

Use the supernatant for the assay.
NOTE: Black residue is occasionally produced from tissue samples in the hydrolysis process. However, sample hydrolyzation should
be complete by the end of the incubation period. If hydrolyzed black residue is still present in the sample, transfer to a microcentrifuge
tube and spin at 10,000 rpm for 3 minutes. Collect supernatant.

© 2020 Chondrex, Inc. All Rights Reserved, 6017 7.0
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

4ASSAY PROCEDURE
1.

Prepare Standard Dilutions: Take 100 µl of the 4 mg/ml Hydroxyproline Standard and add to 900 µl of distilled water to make a 400µg/ml HP standard solution. Then serially dilute it with distilled water. For example, mix 500 µl of the standard (400 µg/ml) with an equalvolume of distilled water to make a 200 µg/ml solution, and then repeat it five more times to make 100, 50, 25, 12.5, and 6.3 µg/ml
standard solutions.

2.

Prepare Sample Dilutions: The hydrolyzed samples can be used undiluted. If necessary, the samples can be diluted with 5N HCl. Ifthe samples have color (are not clear), Sample Blank wells should be prepared due to the potential for high background color. See
steps 4 and 5 for this process.

3.

Prepare Chloramine T solution: Mix 10 µl of the 10X Chloramine T solution with 90 µl of Solution A for each well. For example, 10samples, 7-point standard, one blank (all in duplicate) will require 3.6 ml of the 1X Chloramine T solution. Mix 360 µl of the 10X
Chloramine T solution with 3.24 ml of Solution A.
NOTE: Prepare the 1X solution just before use. Do not store and reuse the mixed solution for the next assay.

4.

Add Standards and Samples: Choose 4-1 or 4-2 depending on your samples.
4-1. Colorless Samples: Add 10 µl of standards, distilled water (blank, B) into the purple wells, and samples into the orange wellsin duplicate. For example, add 10 µl of sample 1 into the S1 wells, then add 10 µl of sample 2 into the S2 wells. Proceed to Step
5-1.
4-2. Colored Samples: Add 10 µl of standards, distilled water (blank, B) into the purple wells, and samples into the orange andgreen wells in duplicate. For example, add 10 µl of sample 1 into the S1 and SB1 wells, then add 10 µl of sample 2 into the S2and SB2 wells. Proceed to Step 5-2.

5.

Add 1X Chloramine T Solution:
5-1. Colorless Samples: Add 100 µl of the 1X Chloramine T solution into all wells. Incubate at room temperature for 20 minutes.
5-2. Colored Samples: Add 100 µl of the 1X Chloramine T solution into the purple and orange wells and add 100 µl of Solution A
into the green wells. Incubate at room temperature for 20 minutes.

6.

Prepare DMAB solution: Mix 50 µl of 2X DMAB solution with 50 µl of Solution B for each well. For example, 10 samples, 7-point
standard, one blank (all in duplicate) will require 3.6 ml of the 1X DMAB solution. Mix 1.8 ml of the 2X DMAB solution with 1.8 ml ofSolution B.
NOTE: Prepare the 1X solution just before use. Do not store and reuse the mixed solution for the next assay.

7.

Add 1X DMAB solution: Add 100 µl of 1X DMAB solution into all wells and incubate at 60°C for 30 minutes.
NOTE: Mix the plate by tapping gently or using a plate shaker.

8.

Read Plate: Read the OD values at 530-560 nm. If the OD values of samples are greater than the OD values of the highest standard,
re-assay the hydrolyzed samples at a higher dilution using 5N HCl.

© 2020 Chondrex, Inc. All Rights Reserved, 6017 7.0
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

5

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank, standards, test samples, and sample blanks (if used).

2.

Subtract the “blank” (B) values from the averaged OD values in step 1. If sample blanks were used, also subtract the averaged sample
blank values from their corresponding averaged sample values.

3.

Plot the OD values of standards against the concentration of the standards (µg/ml). Using a log/log plot will linearize the data. Figure 1
shows a representative experiment where the standard range is 6.3 - 400 µg/ml.

4.

The concentration of Hydroxyproline (µg/ml) in the hydrolyzed samples can be calculated using regression analysis. Multiply the resultsby the dilution factors if the hydrolyzed samples were diluted.

5.

Choose one of the following equations depending on the sample type:5-1 Solid Samples: Hydroxyproline level in a tissue sample (µg/mg) is determined by the following equation:
Hydroxyproline (µg/ml) x (Distilled Water Volume (ml)+ HCl Volume (ml))
Sample Weight (mg)

= Hydroxyproline level (µg/mg) in the sample

5-2 Solution or Homogenate: Hydroxyproline level in a solution sample (mg/ml) is determined by the following equation:
Hydroxyproline (µg/ml) x (Sample Volume (ml)+ HCl Volume (ml))= Hydroxyproline level (µg/ml) in the sample

Sample Volume (ml)6

Hydroxyproline levels can be converted into collagen levels with the following equation (4):Hydroxyproline level (µg/mg or µg/ml) x100
13.5

= Collagen level (µg/mg or µg/ml)

Note: Hydroxyproline accounts for 13.5% of the collagen amino acid composition.
Figure 1 - A Typical Standard Curve for the Hydroxyproline Assay Kit

10.00

R² = 0.9975

OD 530 nm

1.00

0.10

0.01
1101001000

Hydroxyproline Concentration (µg/ml)© 2020 Chondrex, Inc. All Rights Reserved, 6017 7.016928 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

6ASSAY VALIDATION
Table 1 - Reproducibility Data for the Hydroxyproline Assay Kit
Test
Mouse Kidney
Hydroxyproline 200 µg/ml
Intra-Assay CV (%)
5.1
6.3
Inter-Assay CV (%)
5.1
6.1
Spike Test* (%)
91-95%*Standard was mixed with known amounts of mouse kidney samples or hydroxyproline solution

Hydroxyproline 12.5 µg/ml
3.4
7.6-TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

REFERENCES
1.

N. Blumenkrantz, G. Asboe-Hansen, A Quick and Specific Assay for Hydroxyproline. Anal Biochem 55, 288-91 (1973).

2.

G. Reddy, C. Enwemeka, A Simplified Method for the Analysis of Hydroxyproline in Biological Tissues. Clin Biochem 29, 225-9 (1996).

3.

C. Rogers, J. Kimmel, M. Hutchin, H. Harper, A Hydroxyproline Method of Analysis for a Modified Gelatin in Plasma and Urine. J Biol
Chem 206, 553-9 (1954).

4.

R. Neuman, M. Logan, The Determination of Hydroxyproline. J Biol Chem 184, 299-306 (1950).

© 2020 Chondrex, Inc. All Rights Reserved, 6017 7.0
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