1Human/Monkey Anti-Type V Collagen IgG Antibody ELISA Kits with TMB
Catalog # 2082T, 2085T, and 20851T

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, ELHVG-TMB 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

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.
Type V collagen is a minor collagen component that forms interstitial collagen fibrils with type I collagen and plays a critical role in theregulation of fibrillogenesis. Type V collagen from most tissues consists of two α1(V) chains and one α2(V) chain, [α1(V)]2α2(V), whereas
the placenta contains two subtypes of type V collagen, [α1(V)]2α2(V) and α1(V)α2(V)α3(V). Immune reactions to type V collagen may playa role in graft rejection following lung transplant. For studying the possible involvement of type V collagen immunity in organ allograft rejection
in both in vitro and in vivo experiments, Chondrex Inc. provides type V collagen-coated ELISA plates (wells) in addition to uncoated wells foruse as blanks. This ELISA kit contains enough materials to run two plates on two separate occasions and may be used for monkey serum
as well as human serum.

KIT COMPONENTS
Item

Quantity

AmountStorageStandard IgG Antibody (7010)
Secondary Antibody (Biotin-Conjugated Goat Anti-Human IgG) (7014)

1 vial2 vials

1.1 ml, 16 units/ml
Lyophilized-20°C-20°C

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)
Streptavidin Peroxidase (9029)
TMB (90023)

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

20 ml
50 ml
20 ml
20 ml
50 ml
0.2 ml

-20°C-20°C
-20°C
-20°C
-20°C
-20°C

Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
Type V Collagen-Coated 8-Well Strips
Uncoated 8-Well Strips
Reference Standard Strips (two strips per run)

1 bottle
1 bottle
2 bottles
10 each10 each4 each

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

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

© 2020 Chondrex, Inc. All Rights Reserved, ELHVG-TMB 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3

PLATE COATING AND SETUPSpecies
Bovine Amnion
Human Amnion
Human Placenta
Uncoated

Type V Collagen Color Coding – Catalog #(BV) Green – 2082T(HV) Blue – 2085T(HVP) Silver – 20851T
ClearStandardRedStandard ELISA Kit with One Species of Type I or Type IICollagen

HVA standard ELISA kit consisting of five 8-well strips which areuncoated and serve as a control for background levels of
individual samples, five 8-well strips coated with one species oftype V collagen to determine specific antibody levels, and two 8well strips for reference standards. “B” represents blank wells todetermine non-specific reactions caused by the secondary
antibody. Standards and samples are run in duplicate.

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© 2020 Chondrex, Inc. All Rights Reserved, ELHVG-TMB 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

4

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 for
disposal.

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

1

3.

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.

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.

© 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

56.

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
1012

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
the plate 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 #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. 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 immediately
after washing the plate. Incubate for 15 minutes at room temperature.
Strip #24681012TMB (µl)
3466
100
132
164
200

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

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.

© 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

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.
10.0
R² = 0.998

OD 450 nm

1.00.10.00.1

110100

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

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s ELISA FAQ for more information.
REFERENCES
1.

K. M. Mak, C. Y. M. Png, D. J. Lee, Type V Collagen in Health, Disease, and Fibrosis. Anat Rec. 299, 613–629 (2016).

2.

D. E. Birk, Type V collagen: heterotypic type I/V collagen interactions in the regulation of fibril assembly. Micron. 32, 223–237 (2001).

3.

J. A. Madri, H. G. Foellmer, H. Furthmayr, Type V collagens of the human placenta: trimer alpha-chain composition, ultrastructuralmorphology and peptide analysis. Collagen and Related Research Clinical and Experimental. 2, 19–29 (1982).

4.

C. Niyibizi, P. P. Fietzek, M. van der Rest, Human placenta type V collagens. Evidence for the existence of an alpha 1(V) alpha 2(V)alpha 3(V) collagen molecule. J. Biol. Chem. 259, 14170–14174 (1984).

5.

K. Yasufuku et al., Oral Tolerance Induction by Type V Collagen Downregulates Lung Allograft Rejection. Am J Respir Cell Mol Biol.
25, 26–34 (2001).

© 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