1Human/Monkey Anti-Type I and Type II Collagen IgG Antibody ELISA Kits with TMB
Catalog # Catalog # 1031T, 1032T, 1033T, 1035T, 2051T, 2052T, 2053T, and 2055T

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to quantify human/monkey anti-collagen antibodies

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

5.25 hours

STANDARD RANGE:

16 units/ml to 0.25 units/ml

NUMBER OF SAMPLES:

Up to 38 (duplicate) samples/standard plate (will vary for custom kits)

SAMPLE TYPES:

Serum and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:100 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C

VALIDATION DATA:N/ANOTES:© 2020 Chondrex, Inc. All Rights Reserved, ELHA-TMB 3.0
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

Human/Monkey Anti-Type I and Type II Collagen IgG Antibody ELISA Kits with TMB
Catalog # Catalog # 1031T, 1032T, 1033T, 1035T, 2051T, 2052T, 2053T, and 2055T

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Human serum, especially from patients with autoimmune diseases, contain high levels of immunoreactive components, which yield highbackground levels in ELISA systems. These non-specific reactions are caused by adhesive immunoglobulins in human serum which sostrongly adhere to plastic surfaces by hydrophilic binding that blocking agents such as bovine serum albumin (BSA) and Tween 20 are notcapable of blocking these non-specific reactions at all. False positive reactions caused by the serum samples themselves are usually
overlooked and are considered real antibody-antigen reactions in many cases, even now. In order to obtain the real values of antigenantibody reactions, it is critical 1) to choose proper blocking agents which block these kinds of non-specific reactions effectively, 2) todetermine the unique non-specific background value of each individual sample using antigen-non-coated wells, and 3) to subtract thatbackground value from the corresponding value determined in antigen-coated wells. In addition, it is important to determine the non-specificreactions caused by the secondary antibody as well. Chondrex, Inc.’s ELISA system incorporates unique blocking agents that inhibit thehydrophobic binding of these serum components onto plastic surfaces and are designed to determine the background values of individual
samples using antigen-non-coated wells.
In general, antibodies to collagen in human serum are highly specific to heterologous collagen such as bovine or chick type I and/or type II
collagen and cross-react to other species and types of collagen including human collagen, regardless of disease. For example, even serum
antibodies from healthy normal controls react to various species and types of collagen. To determine the human serum antibody levels
specific to the desired antigen, Chondrex, Inc. provides various species of type I and type II collagen-coated strips as well as uncoated wells.
This ELISA kit contains enough materials to run two plates on two separate occasions and may be used for monkey serum as well as humanserum.

KIT COMPONENTS
Item

Quantity

AmountStorageStandard IgG Antibody (7010)
Secondary Antibody (Biotin-Conjugated Goat Anti-Human IgG) (7014)
Solution A - Blocking Buffer (9027)
Solution B - Sample/Standard Dilution Buffer (9038)
Solution C - Secondary Antibody Dilution Buffer (9039)
Solution D - Streptavidin Peroxidase Dilution Buffer (9055)

1 vial2 vials
1 bottle
1 bottle
1 bottle
1 bottle

1.1 ml, 16 units/ml
Lyophilized
20 ml
50 ml
20 ml
20 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Streptavidin Peroxidase (9029)
TMB (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
Type I or Type II Collagen-Coated 8-Well Strips

2 vials
2 vials
1 bottle
1 bottle
2 bottles
10 each

50 ml
0.2 ml
20 ml
10 ml
50 ml
8-well strips

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Uncoated 8-Well Strips
Reference Standard Strips (two strips per run)

10 each4 each8-well strips
8-well strips

-20°C
-20°C

© 2020 Chondrex, Inc. All Rights Reserved, ELHA-TMB 3.0
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

3

PLATE COATING AND SETUP
Species
Chick
BovinePorcineHuman

Type I Collagen Color Coding – Catalog #(CI) Gold – 1031T(BI) Dark Blue – 1032T(PI) Brown – 1033T(HI) Silver – 1035T

Type II Collagen Color Coding – Catalog #(CII) Yellow – 2051T(BII) Green – 2052T(PII) Pink – 2053T(HII) Blue – 2055T

UncoatedStandardClear
RedClearRed

Standard ELISA Kit with One Species of Type I or Type II CollagenA standard ELISA kit consisting of five 8-well strips which are uncoatedand serve as a control for background levels of individual samples, five 8well strips coated with one species of type I or type II collagen todetermine specific antibody levels, and two 8-well strips for reference
standards. “B” represents blank wells to determine non-specific reactions
caused by the secondary antibody. Standards and samples are run in
duplicate.
Custom ELISA Kit with Multiple Species of Collagen
A custom kit for assaying antibody levels to various species of type I ortype II collagen in human serum. This custom ELISA plate consists oftwo uncoated 8-well strips, two each of 8-well strips coated with chick(CII), bovine (BII) and human (HII) type II collagen, as well as bovine (BI)type I collagen and two 8-well strips for reference standards. “B”
represents blank wells to determine non-specific reactions caused by
the secondary antibody.

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.

© 2020 Chondrex, Inc. All Rights Reserved, ELHA-TMB 3.0
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

4

ASSAY 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 diluted streptavidin peroxidase 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: The undiluted standard stock solution is 16 units/ml. 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 dilutionsof standard: 4, 2, 1, 0.5 and 0.25 units/ml solutions. The 16 units/ml standard may be stored at –20°C for use in a second assay.
Chondrex, Inc. recommends making fresh serial dilutions for each assay.
250 ml

250 ml

250 ml

250 ml

250 ml

250 ml23456781100 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

13.

Prepare Sample Dilutions: Centrifuge serum samples at 10,000 rpm at room temperature for 3 minutes to remove insoluble materials
and lipids. Dilute samples 1:100 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 necessary, dilute the samples further with Solution B, 1:200 - 1:1000.

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.

© 2020 Chondrex, Inc. All Rights Reserved, ELHG-TMB 4.016928 Woodinville-Redmond Rd 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 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: Dissolve one vial of secondary antibody in 10 ml Secondary Antibody Dilution Buffer (Solution C).
Alternatively, dissolve one vial in 50 µl of Solution C and dilute accordingly. Add 100 µl of secondary antibody solution to each well andincubate at room temperature for 1 hour.
Strip #2
468
1012Secondary 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.

Add Streptavidin Peroxidase: Dilute one vial of streptavidin peroxidase in 10 ml Streptavidin Peroxidase Dilution Buffer (Solution D).
Add 100 µl of streptavidin peroxidase solution to each well and incubate at room temperature for 1 hour.
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. TMB: Dilute one vial of TMB in 10 ml of Chromogen Dilution Buffer just prior to use. Add 100 µl of TMB solution to each well immediatelyafter washing the plate. Incubate for 15 minutes at room temperature.
Strip #2468
1012

TMB (µl)
3466
100132
164
200

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

© 2020 Chondrex, Inc. All Rights Reserved, ELHG-TMB 4.0
16928 Woodinville-Redmond Rd 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

12. Stop: Add 50 µl of 2N sulfuric acid (Stop Solution) to each well.
13. 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.

CALCULATION OF ANTIBODY TITERS
1.

Average the duplicate OD values for the standards, blanks (B) and test samples in uncoated wells and collagen coated wells.

2.

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

3.

Subtract the OD values of samples tested in uncoated wells (background values) from their counterpart OD values in collagen coatedwells from Step 2 to eliminate values associated with non-specific reactions.

4.

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.

5.

The units/ml of antibody in test samples can be calculated using regression analysis.
NOTE: 100 units is approximately 1 µg IgG antibody/ml

Figure 1 - A Typical Standard Curve for the Human/Monkey Anti-Collagen IgG Antibody ELISA Kit with TMB.
10.000R² = 0.9950

OD 450 nm

1.000

0.100

0.010

0.1

110100Human/Monkey Anti-Collagen IgG Antibody (units/ml)

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, ELHG-TMB 4.0
16928 Woodinville-Redmond Rd Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com