1Staphylococcal Enterotoxin A and B Detection Kits
Catalog # 6029 and 6030

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify SEA and SEB in samples

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISA

TIME:

1.5 hours

STANDARD RANGE:

10 ng/ml to 0.16 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Liquid samples

RECOMMENDED SAMPLE DILUTIONS:

1:1 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°CVALIDATION DATA:

6029: Intra-Assay (1.3-3.7%)/Inter-Assay (4.1-7.8%)/Spiking Test (92-103%)
6030: Intra-Assay (1.2-2%)/Inter-Assay (1.1-8.6%)/Spiking Test (98-100%)

NOTES:© 2020 Chondrex, Inc. All Rights Reserved, 6029, 6030 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

Staphylococcal Enterotoxin A and B Detection Kits
Catalog # 6029 and 6030

For Research Use Only - Not Human or Therapeutic UseINTRODUCTION
The contamination of pathogenic microorganisms and their toxins in food and water is a serious issue for human health and safety (1,2). For instance, enterotoxins (SEs) produced by Staphylococcus aureus (S. aureus) are heat-stable, meaning pathological activity
remains even after exposure to sterilization techniques and digestive proteases. Among the SEs, staphylococcal enterotoxin A (SEA)and B (SEB) are confirmed toxins which cause enteritis and food poisoning. Symptoms include nausea, vomiting, diarrhea, which insevere cases, may lead to fatalities in children and the elderly (3-5). These SEs, known as superantigens, non-specifically activate Tcells, leading to proliferation which ultimately results in T-cell elimination. This activation directly and indirectly induces a massive releaseof inflammatory cytokines (6).
In addition to acute poisoning, researchers reported that these toxins may play roles in the pathogenesis of autoimmune diseases. Morespecifically, intestinal dysbiosis (enteromicrobial imbalance) was found in patients with rheumatoid arthritis (RA) and which may
overwhelm the host immune defense functions by chronic exposure to excess amounts of these pathogens (7, 8). In animal models, SEs
synergistically play a role in the pathogenesis of autoimmune-related diseases (9), such as atopic dermatitis (8, 10), food allergies (11,12), colitis (13, 14), arthritis (11, 15-17), and systemic lupus erythematosus (11, 18).
Several methods exist for detecting pathogenic microorganisms and their toxins, such as polymerase chain reaction PCR), mass
spectrometry, biosensor-based techniques, reversed passive latex agglutination, and enzyme-linked immunosorbent assay (ELISA) (5,19). Of these techniques, PCR is ideal to detect the presence of microorganisms through their DNA or RNA. On the other hand, ELISA
is widely used for detecting toxins in samples because of its high assay sensitivity and simplicity. For example, ELISAs were used toassay SEB in food samples as well as synovial fluids, sera, and urine from RA patients (6, 19).
Chondrex, Inc. provides ELISA kits for detecting SEA and SEB (Catalog# 6029, 6030) in food, feces, intestinal fluids, and other liquidsamples. Chondrex, Inc. also provides mouse anti-SEA and SEB IgG antibody and IgG subtype antibody ELISA kits and a S. aureus IgG
antibody ELISA kit. For further requests and consultation, please contact us at support@chondrex.com or visit www.chondrex.com.

KIT COMPONENTS
Item
SEA Standard (60291)
SEB Standard (60301)
SEA Detection Antibody (60293)
SEB Detection Antibody (60303)
Solution B - Sample/Standard/Detection Antibody Dilution Buffer (67015)
TMB (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
Capture Antibody Coated 96-Well ELISA Plate (SEA: Yellow, SEB: Green)

Quantity

AmountStorage

1 vial10 ng, lyophilized-20°C

1 vial

50 µl-20°C

1 bottle
2 vials
1 bottle
1 bottle
1 bottle
1 each50 ml
0.2 ml
20 ml
10 ml
50 ml
8-well strips x 12-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

© 2020 Chondrex, Inc. All Rights Reserved, 6029, 6030 2.016928 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

3

ASSAY OUTLINE
Add 50 ml of diluted standards and samples.

Step 1
Add 50 ml of Detection Antibody.
Mix plate with a plate mixer or pipet* individual wells.
Incubate at room temperature for 1 hour.
Wash plates.

Add 100 ml of TMB.

Step 2

Incubate plate at room temperature for 25 minutes.

Step 3

Add 50 ml of Stop Solution.

Step 4

Read plate at 450 nm/630 nm.

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

PLATE LAYOUT123456789101112A

1010

S1S1S9

S9

S17

S17

S25

S25

S33

S33

B

55

S2S2S10

S10S18

S18

S26

S26

S34

S34C

2.52.5

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

1.25

1.25

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

0.63

0.63

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

0.31

0.31

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

0.16

0.16

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

HB

B

S8

S8

S16

S16

S24

S24

S32S32S40

S40Standards

Standards© 2020 Chondrex, Inc. All Rights Reserved, 6029, 6030 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.

Prepare Standard Dilutions: The recommended standard range is 0.16-10 ng/ml. Dissolve one vial of standard in 1 ml ofSample/Standard/Detection Antibody Dilution Buffer (Solution B) for the 10 ng/ml standard. Then serially dilute it with Solution B. For
example, mix 200 ml of the standard (10 ng/ml) with an equal volume of Solution B to make a 5 ng/ml solution, and then repeat it five
more times for 2.5, 1.25, 0.63, 0.31, and 0.16 ng/ml solutions. The remaining 10 ng/ml standard stock may be stored at -20°C for use
in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 ml

200 ml200 ml200 ml200 ml200 ml200 ml23456781000 ml

200 ml200 ml200 ml200 ml200 ml200 ml200 ml10 ng/ml

5 ng/ml

2.5 ng/ml

1.25 ng/ml

0.63 ng/ml

0.31 ng/ml

0.16 ng/ml

Blank

1

2.

Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids. Dilutethe supernatants with an equal volume of Solution B. For example, take 100 µl of supernatant, and mix with 100 µl of Solution B. If
the SEA or SEB level is higher than 10 ng/ml, re-assay the sample at a higher dilution.

3.

Prepare Detection Antibody: Dilute one vial in 5 ml of Sample/Standard/Detection Antibody Dilution Buffer (Solution B).

© 2020 Chondrex, Inc. All Rights Reserved, 6029, 6030 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

5

Strip #2
468
1012Detection Antibody (µl)
8172533
4250

Solution B (ml)
0.8
1.7
2.5
3.3
4.2
5.0

4.

Add Standards and Samples: Add Standards, Samples, and Detection Antibody: vortex individual standard, sample, and detection
antibody tubes well. Add 50 µl of Solution B (blank), standards, and samples to designated wells. Add 50 µl of diluted detection
antibody solution to all wells. Mix all wells by pipetting or with a plate shaker. Cover the plate with a plate sealer and incubate at room
temperature for 1 hour.

5.

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.

6.

Add TMB Solution: Use new tubes when preparing TMB solution. Just prior to use, dilute one vial of TMB solution with 10 ml of
Chromogen Dilution Buffer. Add 100 µl of TMB solution to each well immediately after washing the plate and incubate for 25 minutesat room temperature.
Strip #
2468
1012TMB (µl)
3466100132
164
200

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

7.

Stop: Stop the reaction with 50 ml of 2N Sulfuric Acid (Stop Solution) to each well.

8.

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.

CALCULATING RESULTS
1.

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

2.

Subtract the averaged blank OD values from the averaged OD values of the standards and test samples.

3.

Plot the OD values of the standards against the ng/ml of antibody standard. Using a log/log plot will linearize the data. Figure 1 shows
representative experiments where the standard range is 0.16 -10 ng/ml.

4.

The ng/ml of antibody in test samples can be calculated using regression analysis. Multiply it by the sample dilution factor to obtain theantibody concentration (ng/ml) in original sample specimens.

© 2020 Chondrex, Inc. All Rights Reserved, 6029, 6030 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

6Figure 1- Typical Standard Curves for the SEA and SEB Detection ELISA Kits

10.010.0
R² = 0.9971

R² = 0.9989OD 450 nm

1.0

OD 450 nm

1.00.10.1

0.00.00.1

1100.1

1

SEA Concentration (ng/ml)

10SEB Concentration (ng/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the SEA Detection ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

0.32 ng/ml
3.7
7.8
100%

1.25 ng/ml
1.3
7.2
103%

5 ng/ml
2.1
4.1
92%

1.25 ng/ml
2.0
3.6
98%

5 ng/ml
1.2
8.6
100%

Table 2 - Reproducibility Data for the SEB Detection ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

0.32 ng/ml
1.3
1.199%* Known amounts of SEA or SEB were added to samples and then diluted with Sample/Standard/Detection Antibody Dilution Buffer(Solution B).

1201201001008080

Recovery (%)

Recovery (%)

Figure 2 - Recovery Tests Using Liquid Samples (Left: SEA, Right: SEB). Samples needed to be adjusted to pH 7.5 before running
the assay; best results are obtained if diluted with Solution B.

60
Milk

40

60

40

Milk

Soy milk
20

Soy milk
20

Orange Juice

Orange Juice

Orange Juice, pH adjusted

Orange Juice, pH adjusted
001234Dilution

56781234Dilution

5678© 2020 Chondrex, Inc. All Rights Reserved, 6029, 6030 2.016928 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

7TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s ELISA FAQ for more information.

REFERENCES
1.

I. Sospedra, C. Soler, J. Mañes, J. Soriano, Rapid Whole Protein Quantitation of Staphylococcal Enterotoxins A and B by Liquid
chromatography/mass Spectrometry. J Chromatogr A 1238, 54-9 (2012).

2.

P. Mead, L. Slutsker, V. Dietz, L. McCaig, J. Bresee, et al., Food-related Illness and Death in the United States. Emerg Infect Dis 5,607-25 (1999).

3.

J. Hennekinne, A. Ostyn, F. Guillier, S. Herbin, A. Prufer, S. Dragacci, et al., How Should Staphylococcal Food Poisoning Outbreaks
Be Characterized? Toxins (Basel) 2, 2106-16 (2010).

4.

E. Cook, X. Wang, N. Robiou, B. Fries, Measurement of Staphylococcal Enterotoxin B in Serum and Culture Supernatant With a Capture
Enzyme-Linked Immunosorbent Assay. Clin Vaccine Immunol 14, 1094-101 (2007).

5.

O. Pepe, G. Blaiotta, F. Bucci, M. Anastasio, M. Aponte, F. Villani, et al., Staphylococcus Aureus and Staphylococcal Enterotoxin A in
Breaded Chicken Products: Detection and Behavior During the Cooking Process. Appl Environ Microbiol 72, 7057-62 (2006).

6.

L. Vanier, G. Prud'homme, Cyclosporin A Markedly Enhances Superantigen-Induced Peripheral T Cell Deletion and Inhibits AnergyInduction. J Exp Med 176, 37-46 (1992).

7.

K. Terato, C. Do, H. Shionoya, Slipping Through the Cracks: Linking Low Immune Function and Intestinal Bacterial Imbalance to theEtiology of Rheumatoid Arthritis. Autoimmune Dis 2015, 636207 (2015).

8.

N. Kamada, S. Seo, G. Chen, G. Núñez, Role of the Gut Microbiota in Immunity and Inflammatory Disease. Nat Rev Immunol 13, 32135 (2013).

9.

J. Li, J. Yang, Y. Lu, S. Wu, M. Wang, J. Zhu, et al., Possible Role of Staphylococcal Enterotoxin B in the Pathogenesis of Autoimmune
Diseases. Viral Immunol 28, 354-9 (2015).

10. S. Hong, M. Kim, E. Lee, J. Kim, Y. Kim, et al., Extracellular Vesicles Derived From Staphylococcus Aureus Induce Atopic DermatitisLike Skin Inflammation. Allergy 66, 351-9 (2011).
11. E. Baharav, H. Gur, P. Fishman, L. Ziporen, M. Blank, et al., Superantigens and Experimental SLE Induced by Idiotypic Dysregulation.
Clin Exp Rheumatol 14, 359-66 (Jul-).
12. K. Ganeshan, C. Neilsen, A. Hadsaitong, R. Schleimer, X. Luo, P. Bryce, et al., Impairing Oral Tolerance Promotes Allergy andAnaphylaxis: A New Murine Food Allergy Model. J Allergy Clin Immunol 123, 231-238 (2009).
13. P. Yang, C. Wang, Z. An, A Murine Model of Ulcerative Colitis: Induced With Sinusitis-Derived Superantigen and Food Allergen. BMC
Gastroenterol 5, 6 (2005).
14. J. Lu, A. Wang, S. Ansari, R. Hershberg, D. McKay, Colonic Bacterial Superantigens Evoke an Inflammatory Response and Exaggerate
Disease in Mice Recovering From Colitis. Gastroenterology 125, 1785-95 (2003).
15. P. Wooley, B. Cingel, Staphylococcal Enterotoxin B Increases the Severity of Type II Collagen Induced Arthritis in Mice. Ann Rheum
Dis 54, 298-304 (1995).
16. H. Nagai, Y. Takaoka, H. Kamada, H. Mori, The Model of Arthritis Induced by Superantigen in Mice. Life Sci 55, PL233-7 (1994).
17. H. Nagai, Y. Takaoka, K. Kuwabara, H. Kamada, K. Kitagaki, The Effect of SM-8849 on Experimental Arthritis in Mice. Pharmacology
52, 377-86 (1996).
18. D. Scott, W. Kisch, A. Steinberg, Studies of T Cell Deletion and T Cell Anergy Following in Vivo Administration of SEB to Normal andLupus-Prone Mice. J Immunol 150, 664-72 (1993).
19. H. Kuang, W. Wang, L. Xu, W. Ma, L. Liu, et al., Monoclonal Antibody-Based Sandwich ELISA for the Detection of Staphylococcal
Enterotoxin A. Int J Environ Res Public Health 10, 1598-608 (2013).

© 2020 Chondrex, Inc. All Rights Reserved, 6029, 6030 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