1Rat Anti-Type I and Type II Collagen IgG Antibody ELISA Kits with TMB
Catalog # 1021T - 1025T, 2041T - 2045T

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to quantify rat anti-collagen antibodies

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.25 hours

STANDARD RANGE:

16 units/ml to 0.25 units/ml

NUMBER OF SAMPLES:

Up to 39 (duplicate) samples/standard plate (will vary for custom kits)

SAMPLE TYPES:

Serum and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:1000 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C

VALIDATION DATA:N/ANOTES:© 2022 Chondrex, Inc. All Rights Reserved, ELRG-TMB 6.1
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

2Rat Anti-Type I and Type II Collagen IgG Antibody ELISA Kits with TMB
Catalog # 1021T - 1025T, 2041T - 2045T

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Heterologous type II collagen is widely used as an immunogen for the collagen-induced arthritis (CIA) model. In CIA-susceptible rat, theserum antibody levels to the type II collagen used for immunization are very high. Furthermore, these antibodies cross-react to various
species of type II collagen including autologous type II collagen, due to the conserved amino acid sequences of type II collagen. It is
important to note that although type I collagen shares about 80% of its amino acid sequence with type II collagen, it is not capable ofinducing autoimmune-mediated diseases. This indicates that epitope specificity of antibodies and T-cells are important for establishingautoimmunity and subsequent development of autoimmune diseases. Therefore, type I collagen might be useful as a control for studying
the pathogenesis of autoimmune mediated arthritis and B and T-cell epitope specificity.
This kit is designed to assay type I and type II collagen antibodies in rat serum. Chondrex Inc.’s ELISA systems incorporate uniqueblocking agents to reduce non-specific reactions. These agents reduce the background levels by inhibiting the hydrophobic binding of
immunoreactive serum components in sample specimens onto plastic surfaces. Chondrex, Inc. offers various species of type I and type
II collagen-coated strips as shown below.

KIT COMPONENTS
ItemQuantity

AmountStorage

Standard Antibody (2046)
Secondary Antibody (Peroxidase-Conjugated Goat Anti-Rat IgG) (10214)

1 vial2 vials

20 units, lyophilized
50 µl

-20°C
-20°C

Solution A - Blocking Buffer (2071)
Solution B - Sample/Standard Dilution Buffer (2072)
Solution C - Secondary Antibody Dilution Buffer (2073)
TMB (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)

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

12 ml
50 ml
20 ml
0.2 ml
20 ml
10 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Wash Buffer, 20X (9005)
Type I or Type II Collagen-Coated 8-Well Strips
Reference Standard Strips (two strips per run)

1 bottle
10 each4 each50 ml
8-well strips
8-well strips

-20°C
-20°C
-20°C

© 2022 Chondrex, Inc. All Rights Reserved, ELRG-TMB 6.1
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

3

PLATE COATING AND SETUPSpeciesChickBovine
PorcineRat

Type I Collagen Color Coding – Catalog #(CI) Gold – 1021T(BI) Dark Blue – 1022T(PI) Brown – 1023T(RI) Lime Green – 1024T

Type II Collagen Color Coding – Catalog #(CII) Yellow – 2041T(BII) Green – 2042T(PII) Pink – 2043T(RII) Purple – 2044T

HumanStandard(HI) Silver – 1025T
Red(HII) Blue – 2045T
RedStandard ELISA Kit with One Species of Type I or Type IICollagenA standard ELISA kit consisting of ten 8-well strips coated with
one species of type II collagen and two 8-well strips for reference
standards. “B” represents blank wells to determine backgroundvalues caused by the secondary antibody. Standards andsamples (1-39) are run in duplicate.

A custom kit for assaying antibody levels to the immunizing type
II collagen and their cross-reactivity to homologous rat type IIcollagen. “B” represents blank wells to determine backgroundvalues caused by the secondary antibody. Standards andsamples (1-19) are run in duplicate.
Immunizing Type II Collagen

Standards

Rat Type II Collagen

123456789101112AB

B

S8

S8

S16

S16

S24

S24

S32

S32

B

B

AB

S4S8

S12

S16B

S4

S8B

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

0.25

0.25

B

B

S4S8

S12

S16B

S4C

S2

S2

S10

S10

S18

S18

S26

S26

S34

S340.50.5

C

S1

S5

S9

S13

S17

S1

D

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

11D

S1

S5

S9S13

S17

E

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

22ES2S6S10S14F

S5S5

S13

S13S21

S21

S29

S29

S37

S3744FS2S6S10GS6S6S14S14S22S22S30

S30

S38

S3888GS3S7H

S7S7S15

S15

S23

S23

S31

S31

S39

S391616

H

S3S7

1SamplesSamplesSingle Species of Collagen

Custom ELISA Kit with Multiple Species of Collagen23456789Standards

101112

S12

S16B

B

S8

S12

S160.25

0.25

S5

S9

S13

S170.50.5

S1S5S9S13

S1711S18

S2S6

S10

S14S1822S14S18

S2S6S10S14S1844S11S15S19S3S7S11S15S1988S11S15S19S3S7S11S15S191616NOTES 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, ELRG-TMB 6.1
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 blocking buffer into wells

Incubate at room temperature for 1 hour.
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 secondary antibody solution into wells

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

Incubate at room temperature for 15 minutes.

Add 50 ml of Stop Solution into wells

Read plates at 450 nm/630 nm

ASSAY PROCEDURE
1.

Add Blocking Buffer: Add 100 µl of Blocking Buffer (Solution A) to all wells. Incubate for 1 hour at room temperature.

2.

Prepare Standard Dilutions: Dissolve one vial of standard (20 units/vial) in 1.25 ml of Sample/Standard Dilution Buffer (Solution B) tomake a 16 units/ml solution. Prepare serial dilutions of the standard by mixing 250 µl of the 16 units/ml standard with 250 µl of Solution
B - 8 units/ml. Then repeat this procedure to make five more serial dilutions of standard: 4, 2, 1, 0.5 and 0.25 units/ml solutions. The 16units/ml standard may be stored at –20°C for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for eachassay.
1.25 ml
250 ml

250 ml

250 ml

250 ml

250 ml

250 ml23456781.25 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml16 units/ml

8 units/ml

4 units/ml

2 units/ml

1 units/ml

0.5 units/ml

0.25 units/ml

Blank

1

© 2022 Chondrex, Inc. All Rights Reserved, ELRG-TMB 6.1
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

53.

Prepare Sample Dilutions: Centrifuge serum samples at 10,000 rpm at room temperature for 3 minutes to remove insoluble materials
and lipids. Dilute samples 1:1000 or more with Solution B. For example, dilute 10 µl of sample with 0.99 ml of Solution B (1:100). Keepthis as a stock solution for future assays. If it is necessary to assay antibodies at a low dilution (less than 1:200) due to low antibody
levels, please contact Chondrex, Inc. customer service at support@chondrex.com. In the CIA model, anti-collagen antibody levels mayvary from zero (naïve rodents) to 1,000,000 units/ml or higher (arthritic rodents) depending on the immune response, arthritis severity,and time of sample collection. With unknown anti-collagen antibody levels, Chondrex, Inc. recommends testing several dilutions, suchas 1:1000, 1:10,000, and 1:100,000, in order to find the appropriate dilution for the samples.

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 wells in duplicate. Incubate at room
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.

7.

Add Secondary Antibody: Dilute one vial of secondary antibody in 10 ml Secondary Antibody Dilution Buffer (Solution C). Add 100 µlof secondary antibody solution to each well and incubate at room temperature for 1 hour.
Strip #24681012

Secondary Antibody (µl)
81725334250

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.

TMB: Use new tubes when preparing TMB. Dilute one vial (200 µl) of TMB in 10 ml of Chromogen Dilution Buffer just prior to use. Add100 µl of TMB solution to each well immediately after washing the plate. Incubate for 15 minutes at room temperature.
Strip #24681012TMB (µl)
3466100132164200Chromogen Dilution Buffer (ml)
1.7
3.3
5.0
6.6
8.2
10.0

10. Stop: Add 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.

© 2022 Chondrex, Inc. All Rights Reserved, ELRG-TMB 6.1
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

CALCULATION OF ANTIBODY TITERS
1.

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

2.

Subtract the blank (B) values from the averaged OD values of the standards and test samples.
NOTE: Individual antigens have unique background values. Therefore, blank wells should be used for each different antigen.

3.

Plot the OD values of standards against the units/ml of antibody standard. Using a log/log plot will linearize the data. Figure 1 shows a
representative experiment where the standard range is from 0.25 to 16 units/ml.

4.

The units/ml of antibody in test samples can be calculated using regression analysis.
NOTE: 82 units is approximately 1 µg IgG antibody/ml.

Figure 1 - A Typical Standard Curve for the Rat Anti-Type I and Type II Collagen IgG Antibody ELISA Kit with TMB.
10.00
R² = 0.9969

OD 450

1.00

0.10

0.01
0.1

1
Antibody (units/ml)

10100

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, ELRG-TMB 6.1
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