1HMGB1 Detection ELISA kit
Catalog # 6010

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify HMGB1FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

2 hours*

STANDARD RANGE:

50 ng/ml to 0.8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Cell culture, Serum, and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:1 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C

VALIDATION DATA:

Human Serum: Intra-Assay (2.7-3.6%)/Inter-Assay (1.3-5.1%)/Recovery (101+/-25.1%)
Mouse Serum: Intra-Assay (1.4-8.1%)/Inter-Assay (0.9-4.5%)/Recovery (104+/8.3%)

NOTES:*This kit has an overnight incubation step© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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

HMGB1 Detection ELISA kit
Catalog # 6010

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
HMGB1 (high mobility group box 1) (1) was recently rediscovered as a late lethal mediator of endotoxin (2) and is currently considered a
pro-inflammatory cytokine that plays crucial roles in a variety of acute and chronic inflammatory diseases. HMGB1 contains 216 amino acids(3) and maintains 99% of its sequence identity among mice (4), rats (5), bovines (6), and humans (7). HMGB1 consists of three structural
domains (8), termed “A box (9-85)”, “B box (88-162)”, and a negatively charged carboxyl terminus (186-216). Moreover, it has been previously
shown that the B box recapitulates the pro-inflammatory activity whereas the A box acts as an antagonist of HMGB1 (9,10).
Several lines of evidence highlight the significance of HMGB1 in the immune inflammatory response. For example, it has been shown that
HMGB1 is actively released by a variety of cells such as macrophages when stimulated by lipopolysaccharides (LPS), TNF-α, and IL-1 (2),and is passively released by injured or necrotic cells associated with collapsing cell structures. In fact, patients who died from septic shock
had higher serum HMGB1 levels than surviving sepsis patients (11). Similarly, high serum HMGB1 levels are observed in sepsis animalmodels and in collagen-induced arthritis animal models (12). With regard to the function of the protein itself, HMGB1 has also been shownto stimulate the release of TNF-α and IL-1 (13,14), as well as bind LPS and synergistically increase peripheral blood mononuclear cell IL-6
production (15). Together, these observations demonstrate that HMGB1 plays important roles in the inflammatory cascade.
Chondrex, Inc. provides an HMGB1 Detection ELISA kit (Cat # 6010) to determine HMGB1 levels in cell culture media and sera. This kit
contains enough reagents to measure 40 samples in duplicate together with standards.

KIT COMPONENTS
Item

Quantity

AmountStorage

HMGB1 Standard (60101)
Capture Antibody (Anti-HMGB1 Monoclonal Antibody) (60102)
Detection Antibody (Anti-HMGB1 Monoclonal Antibody) (60103)
Solution A - Coating Buffer (9052)

2 vials
1 vial1 vial
1 bottle

50 µl/vial
100 µl/vial
Lyophilized
10 ml

-20°C
-20°C
-20°C
-20°C

Solution B - Sample/Standard Dilution Buffer (601010)
Solution C - Detection Antibody Dilution Buffer (60106)
Solution D - Streptavidin Peroxidase Dilution Buffer (9055)
Streptavidin Peroxidase (9029)
TMB Solution (contains DMSO) (90023)
Chromogen Dilution Buffer (90022)

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

20 ml
10 ml
20 ml
50 µl
0.2 ml
20 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
ELISA Plate

1 bottle
1 bottle
1 each

10 ml
50 ml
96-well (8-well strips x 12)

-20°C
-20°C
-20°C© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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

ASSAY OUTLINE
Add 100 ml of diluted Capture Antibody Solution to wells.
Incubate plate at 4 C overnight.
Wash plates.
Add 50 ml of diluted standards and samples to wells.

Add 50 ml of diluted Detection Antibody Solution to wells.
Mix plate with a plate mixer or *pipet individual wells.
Incubate at 37 C for 1 hour.
Transfer plate to 4 C overnight.
Wash plates.
Add 100 ml of diluted Streptavidin Peroxidase Solution to wells.
Incubate plate at room temperature for 30 minutes.
Wash plates.

Add 100 ml of diluted TMB solution to wells.

Incubate plate at room temperature for 30 minutes.
Add 50 ml of Stop Solution to wells.

Read plate at 450 nm/630 nm.

* Use one tip per sample or standards.
Do not crosscontaminate samples or standards by re-using pipet tips.
A
multi-channel pipet is recommended.

PLATE LAYOUT
123456789101112A

50

50

S1S1S9S9S17

S17

S25

S25

S33

S33

B

2525

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C

12.5

12.5

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

6.3

6.3

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

3.1

3.1

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

1.6

1.6

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

0.8

0.8

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

H

B

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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 fordisposal.
NOTE 8: This kit can be used to determine HMGB1 levels in sera and cell culture media samples. However, special concern should beconsidered for assaying HMGB1 in human serum because autoantibodies to HMGB1 are determined in 9-89% of sera from patients withautoimmune and inflammatory diseases (16-19). These reports indicate that human serum polyclonal antibodies to HMGB1 might mask theepitopes recognized by the capture and detection antibodies used in this kit, resulting in interference against the assay.

ASSAY PROCEDURE
1.

Add Capture Antibody: Dilute 1 vial (100 µl) of Capture Antibody with 10 ml of Coating Buffer (Solution A). Add 100 µl of capture
antibody solution to each well and incubate at 4°C overnight. If planning to use less, the remaining stock solution can be stored in its
original vial at -20°C.
Strip #246810122.

Capture Antibody (µl)
1733506682100

Solution A (ml)
1.7
3.3
5.0
6.6
8.2
10.0

Prepare Standard Dilutions: The recommended standard range is 0.8-50 ng/ml. Dilute one vial of HMGB1 Standard with 950 µl ofSample/Standard Dilution Buffer (Solution B) - 100 ng/ml. Prepare serial dilutions of the standard by mixing 200 µl of the 100 ng/ml
standard with 200 µl of Solution B - 50 ng/ml. Then repeat this procedure to make six more serial dilutions of standard - 25, 12.5, 6.25,
3.1, 1.6, and 0.8 ng/ml solutions. Partially used 100 ng/ml standard stock cannot be saved for future assays. Discard unused, diluted
standard solution. Chondrex, Inc. recommends making fresh standard and serial dilutions for each assay.

© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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

5950 μl

200 μl200 μl200 μl200 μl200 μl200 μl200 μl123456781000 μl

200 μl200 μl200 μl200 μl200 μl200 μl200 μl200 μl

100 ng/ml

50 ng/ml

25 ng/ml

12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

0.8 ng/ml

Blank

3.

Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids and use
the supernatant as samples. Dilute samples at least 1:1 with an equal volume of Solution B. For example, take 100 µl of a serum, andmix with 100 µl of Solution B.

4.

Prepare Detection Antibody: Reconstitute one vial of Detection Antibody with 50 µl of distilled water to make a stock solution. Dilutethe 50 µl of detection antibody stock solution in 5 ml of Detection Antibody Dilution Buffer (Solution C). If planning to use less, theremaining stock solution can be stored in its original vial at -20°C.
Strip #24681012Detection Antibody (µl)
81725334250Solution C (ml)
0.8
1.7
2.5
3.3
4.2
5.0

5.

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.

6.

Add Standards, Samples, and Detection Antibody: Vortex standards, samples, and detection antibody tubes well. Add 50 µl ofSolution B (blank), standards, and samples to appropriate wells. Add 50 µl of diluted detection antibody solution to all wells. Mix all
wells by pipetting or use a plate shaker. Cover the plate with a plate sealer and incubate at 37°C for 1 hour, then transfer plate to 4°C
overnight.

7.

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.

8.

Add Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Peroxidase Dilution Buffer (SolutionD). If planning to use less, the remaining stock solution can be stored in its original vial at -20°C. Add 100 µl of streptavidin peroxidase
solution to each well and incubate at room temperature for 30 minutes.
Strip #24681012

Streptavidin Peroxidase (µl)
91725334150

Solution D (ml)1.83.4
5.0
6.6
8.2
10.0

© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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

69.

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.

10. Add TMB: Use new tubes when preparing TMB. Dilute one vial of TMB with 10 ml of Chromogen Dilution Buffer just prior to use. Add
100 µl of TMB solution to all wells immediately after washing the plate. Incubate for 30 minutes at room temperature. If planning to use
less, the remaining stock solution can be stored in its original vial at -20°C.
Strip #246
8
1012TMB (µl)
3466100132164200Chromogen Dilution Buffer (ml)
1.7
3.3
5.0
6.68.2
10.0

11. Stop: Add 50 µl of 2N sulfuric acid (Stop Solution) to each well.
12. Read Plate: Read the OD values at 450 nm (a 630 nm filter can be used as a reference) immediately. If the OD values of samples are
greater than the OD values of the highest standard, re-assay the samples at a higher dilution.

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank, standards, and samples.

2.

Subtract the averaged blank (B) OD value from the averaged standard and sample OD values.

3.

Plot the OD values of standards against the concentration of HMGB1 (ng/ml) using a log scale. Figure 1 shows a typical standard curve
where the HMGB1 range is from 0.8-50 ng/ml.

4.

The concentration of HMGB1 (ng/ml) in samples can be calculated using regression analysis. Multiply the results by the dilution factors(usually 2 without extra dilution). For additional assistance, please download a sample calculation worksheet from www.chondrex.com.

Figure 2 - A Typical Standard Curve for the HMGB1 Detection ELISA Kit© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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

7

VALIDATION DATA
Table 1 - Reproducibility Data for the HMGB1 Detection ELISA Kit
Human Serum
Test At
Intra-Assay CV (%)
Inter-Assay CV (%)

2 ng/ml
2.7
1.3

7.5 ng/ml
3.4
5.1

30 ng/ml
3.6
3.1

Mouse Serum
Test At
Intra-Assay CV (%)
Inter-Assay CV (%)

2 ng/ml
1.4
3.7

7.5 ng/ml
3.2
4.5

30 ng/ml
8.1
0.9RecoverySpecies
Human (5 sera)
Mouse (5 sera)

Averaged Recovery Results
101 ± 25.1%
104 ± 8.3%

Specificity: Average cross reactivity with bovine HMGB2 is 12.4%.

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

REFERENCES
1.

L. Einck, M. Bustin, The Intracellular Distribution and Function of the High Mobility Group Chromosomal Proteins. Exp Cell Res 156,
295-310 (1985).

2.

H. Wang, O. Bloom, M. Zhang, J. Vishnubhakat, M. Ombrellino, et al., HMG-1 as a Late Mediator of Endotoxin Lethality in Mice. Science285, 248-51 (1999).

3.

J. Parkkinen, E. Raulo, J. Merenmies, R. Nolo, E. Kajander, et al., Amphoterin, the 30-kDa Protein in a Family of HMG1-type
Polypeptides. Enhanced Expression in Transformed Cells, Leading Edge Localization, and Interactions With Plasminogen Activation. JBiol Chem 268, 19726-38 (1993).

4.

S. Ferrari, L. Ronfani, S. Calogero, M. Bianchi, The Mouse Gene Coding for High Mobility Group 1 Protein (HMG1). J Biol Chem 269,28803-8 (1994).

5.

G. Paonessa, R. Frank, R. Cortese, Nucleotide Sequence of Rat Liver HMG1 cDNA. Nucleic Acids Res 15, 9077 (1987).

6.

D. Kaplan, C. Duncan, Full Length cDNA Sequence for Bovine High Mobility Group 1 (HMG1) Protein. Nucleic Acids Res 16, 10375(1988).

© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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

87.

L. Wen, J. Huang, B. Johnson, G. Reeck, A Human Placental cDNA Clone That Encodes Nonhistone Chromosomal Protein HMG-1.
Nucleic Acids Res 17, 1197-214 (1989).

8.

J. Li, H. Wang, J. Mason, J. Levine, M. Yu, et al., Recombinant HMGB1 With Cytokine-Stimulating Activity. J Immunol Methods 289,
211-23 (2004).

9.

M. Bustin, Regulation of DNA-dependent Activities by the Functional Motifs of the High-Mobility-Group Chromosomal Proteins. Mol Cell
Biol 19, 5237-46 (1999).

10. H. Yang, M. Ochani, J. Li, X. Qiang, M. Tanovic, H. Harris, et al. Reversing established sepsis with antagonists of endogenous highmobility group box 1. Proc Natl Acad Sci U S A. 101:296-301 (2004).
11. U. Andersson, H. Wang, K. Palmblad, A. Aveberger, O. Bloom, H. Erlandsson-Harris, et al. High mobility group 1 protein (Hmg-1)
stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med. 192:565-570 (2000).
12. R. Kokkola, J. Li, E. Sundberg, A. Aveberger, K. Palmblad, et al., Successful Treatment of Collagen-Induced Arthritis in Mice and Rats
by Targeting Extracellular High Mobility Group Box Chromosomal Protein 1 Activity. Arthritis Rheum 48, 2052-8 (2003).
13. H. Wang, J. Vishnubhakat, O. Bloom, M. Zhang, M. Ombrellino, et al., Proinflammatory Cytokines (Tumor Necrosis Factor andInterleukin 1) Stimulate Release of High Mobility Group protein-1 by Pituicytes. Surgery 126, 389-92 (1999).
14. E. Abraham, J. Arcaroli, A. Carmody, H. Wang, K. Tracey, HMG-1 as a Mediator of Acute Lung Inflammation. J Immunol 165, 2950-4(2000).
15. H. Hreggvidsdottir, T. Ostberg, H. Wähämaa, H. Schierbeck, A. Aveberger, et al., The Alarmin HMGB1 Acts in Synergy With
Endogenous and Exogenous Danger Signals to Promote Inflammation. J Leukoc Biol 86, 655-62 (2009).
16. B. Wittemann, G. Neuer, H. Michels, H. Truckenbrodt, F. Bautz, Autoantibodies to Nonhistone Chromosomal Proteins HMG-1 andHMG-2 in Sera of Patients With Juvenile Rheumatoid Arthritis. Arthritis Rheum 33, 1378-83 (1990).
17. H. Uesugi, S. Ozaki, J. Sobajima, F. Osakada, H. Shirakawa, et al., Prevalence and Characterization of Novel pANCA, Antibodies tothe High Mobility Group Non-Histone Chromosomal Proteins HMG1 and HMG2, in Systemic Rheumatic Diseases. J Rheumatol 25,703-9 (1998).
18. J. Sobajima, S. Ozaki, H. Uesugi, F. Osakada, M. Inoue, et al., High Mobility Group (HMG) Non-Histone Chromosomal Proteins HMG1and HMG2 Are Significant Target Antigens of Perinuclear Anti-Neutrophil Cytoplasmic Antibodies in Autoimmune Hepatitis. Gut 44,867-73 (1999).
19. J. Sobajima, S. Ozaki, F. Osakada, H. Uesugi, H. Shirakawa, et al., Novel Autoantigens of Perinuclear Anti-Neutrophil CytoplasmicAntibodies (P-ANCA) in Ulcerative Colitis: Non-Histone Chromosomal Proteins, HMG1 and HMG2. Clin Exp Immunol 107, 135-40(1997).

© 2022 Chondrex, Inc. All Rights Reserved, 6010 8.0
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