1Mouse Anti-Staphylococcal Enterotoxin B (SEB) Antibody ELISA Kits
Catalog # 6214-6217
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify mouse anti-SEB IgG/IgG1/IgG2a/IgG2b antibodies
6214: Mouse Anti-SEB IgG Antibody ELISA Kit
6215: Mouse Anti-SEB IgG1 Antibody ELISA Kit
6216: Mouse Anti-SEB IgG2a Antibody ELISA Kit
6217: Mouse Anti-SEB IgG2b Antibody ELISA Kit

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

100 ng/ml to 1.6 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) diluted samples/plate and up to 20 (duplicate) low dilution samples/plate

SAMPLE TYPES:

Serum and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:200 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C

VALIDATION DATA:

6214: Intra-Assay (2.3-10.7%)/Inter-Assay (1.5-4.6%)/Spiking Test (83-89%)6215: Intra-Assay (8.5-10%)/Inter-Assay (1.5-8.8%)/Spiking Test (87-91%)
6216: Intra-Assay (7.5-8.9%)/Inter-Assay (3-9.8%)/Spiking Test (91-96%)
6217: Intra-Assay (2.6-8.6 %)/Inter-Assay (4.9-8.1%)/Spiking Test (89-100%)

NOTES:N/A© 2020 Chondrex, Inc. All Rights Reserved, M SEB ELISA 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

Mouse Anti-Staphylococcal Enterotoxin B (SEB) Antibody ELISA Kits
Catalog # 6214-6217
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Staphylococcus aureus (S. aureus) is a gram-positive coccal bacterium frequently found in the human respiratory tract and on the skin. S.
aureus infections contribute to a broad spectrum of illnesses, including endocarditis, septic arthritis, toxic-shock syndrome, scalded-skinsyndrome, and food poisoning (1-4). Recently, antibiotic-resistant strains (e.g. methicillin-resistant S. aureus) are being isolated more
frequently which presents a challenge to clinical medicine.
This bacterium produces a variety of toxic exoproteins that can trigger massive pro-inflammatory cytokine release, leading to severe
inflammation and tissue damage (5-7). Among these toxins, staphylococcal enterotoxins A and B (SEA and SEB) are known superantigens
that bridge MHC-class II molecules and T-cell receptors, resulting in a potent stimulation of the host immune system, which is believed tocontribute to the pathogenicity of autoimmune diseases (8). For instance, a study found that the presence of anti-SEA and anti-SEB IgE
antibodies correlated with the severity of skin lesions in children with atopic dermatitis (9). SEA and SEB have also been used in vaccine
development to protect against staphylococcal infections (5,6). Anti-SEA and/or SEB IgG antibodies may work as vaccine-induced, allergenspecific IgG antibodies to occupy the binding sites intended for IgE antibodies.
To study the pathological roles of SEA and SEB in mice, Chondrex, Inc. provides mouse anti-SEA IgG antibody and IgG subtype antibody
ELISA kits (Cat # 6218 - 6221) and mouse anti-SEB IgG antibody and IgG subtype antibody ELISA kits (Cat # 6218 - 6221). In addition,Chondrex, Inc. also provides a variety of antibody ELISA kits against various bacteria and toxins. Please visit www.chondrex.com for more
information. For further requests and consultation, please contact Chondrex, Inc. at support@chondrex.com.

KIT COMPONENTS
Item
IgG (62141)
IgG1(62151)
IgG2a (62161)
IgG2b (62171)
IgG (62143)
Secondary Antibody (PeroxidaseIgG1(62153)
Conjugated Goat Anti-Mouse
IgG2a (62163)
Polyclonal Antibodies)
IgG2b (62173)
Solution A - Blocking Buffer (61026)
Solution B - Sample/Standard Dilution Buffer (61027)
Solution C - Secondary Antibody Dilution Buffer (61025)
Standard
Antibody

TMB Solution containing DMSO (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
SEB-coated 8-Well Strips (Green)

Quantity

AmountStorage

1 vial100 ng, lyophilized-20°C

2 vials

50 µl/vial-20°C

1 bottle
1 bottle
1 bottle

10 ml
50 ml
20 ml

-20°C
-20°C
-20°C

2 vials
1 bottle
1 bottle
1 bottle
12 each

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

-20°C
-20°C
-20°C
-20°C
-20°C

An antigen uncoated plate (Cat # 9026) for lower sample dilutions is not included. Please contact support@chondrex.com to place anorder.

© 2020 Chondrex, Inc. All Rights Reserved, M SEB ELISA 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

3

IDENTIFICATION OF ANTIGEN-COATED STRIPS
Antigens
S. aureus
SEA
SEB

Color Coding
Blue
Yellow
Green

IgG
6213
6218
6214

IgG162196215

IgG2a6220
6216

IgG2b6221
6217

PLATE MAPPINGStandard Layout of Antigen Coated Plate
1234567Standard Layout of Antigen Coated and Uncoated Plate
891011123456789101112S33100100

S1S1SB1SB1S9S9SB9SB9S17

S17S34

S34

B

5050

S2S2SB2SB2S10S10SB10SB10S18

S18

S27

S35

S35C2525

S3S3SB3SB3S11S11

SB11

SB11

S19

S19

S28

S28

S36

S36

D

12.5

12.5

S4S4SB4SB4

S12

S12

SB12

SB12S20

S20

S21

S29

S29

S37

S37

E6.3

6.3

S5S5

SB5SB5S13

S13

SB13

SB13

SB17

SB17S22

S22

S30

S30

S38

S38F

3.13.1

S6

S6SB6

SB6

S14

S14SB14SB14SB18

SB18

S15

S23

S23

S31

S31

S39

S39

G1.61.6S7S7SB7SB7S15

S15

SB15

SB15

SB19

SB19

S16

S24

S24

S32

S32

S40

S40

H

B

B

S8

S8

SB8

SB8

S16

S16

SB16

SB16

SB20

SB20

100100

S1S1

S9

S9

S17

S17

S25

S25

S33

B

5050

S2

S2

S10

S10

S18

S18

S26

S26

C2525

S3

S3

S11

S11

S19

S19

S27

D

12.5

12.5

S4S4

S12

S12

S20

S20E

6.3

6.3

S5

S5

S13

S13

S21F3.1

3.1

S6S6

S14S14G

1.6

1.6

S7S7S15HBB

S8S8S16Standards

12AA

SamplesStandards

Samples

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, M SEB ELISA 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

ASSAY OUTLINEASSAY PROCEDURE
1.

Add Blocking Buffer: Add 100 µl of the Blocking Buffer (Solution A) to each well and incubate for 1 hour at room temperature
NOTE: If a sample with a dilution of 1:100 or less is assayed, antigen non-coated strips should be used. Solution A must be added tothe non-coated wells without prior washing because any contaminants in the vessel containing the washing buffer will bind to the strips.
For example, add 100 µl of Solution A to the antigen-coated strips (S1) and the corresponding uncoated strips (SB1) as mapped out inFigure 1b. Incubate for 1 hour at room temperature.

2.

Prepare Standard Dilutions: The recommended standard range is 1.6-100 ng/ml. Dissolve one vial of standard (100 ng/vial) in 1 ml
Standard/Sample Dilution Buffer (Solution B) to make a 100 ng/ml stock standard solution. Then, serially dilute it with Solution B. Forexample, mix 250 ml of the 100 ng/ml solution with an equal volume of Solution B to make a 50 ng/ml solution, and then repeat it five
more times for 25, 12.5, 6.3, 3.1, and 1.6 ng/ml standard solutions. The leftover standard stock may be kept at -20°C for future use.

© 2020 Chondrex, Inc. All Rights Reserved, M SEB ELISA 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

51000 ml
250 ml

250 ml

250 ml

250 ml

250 ml23456781000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml100 ng/ml

50 ng/ml

25 ng/ml

12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

Blank

1

3.

250 mlPrepare Sample Dilutions: Add 10 µl of mouse serum sample to 990 µl of Solution B (1:100) and keep it as a stock solution for future
assays. Then, further dilute the sample with Solution B depending on the antibody levels. For example, take 200 µl of the sample stock
solution and mix with 200 µl of Solution B to make a 1:200 dilution. If it is necessary to assay antibodies at less than 1:200 due to low
antibody levels, antigen uncoated control strips will be necessary. Please contact support@chondrex.com for more information.
NOTE: Chondrex, Inc. recommends running a preliminary assay using various dilutions of sera (1:200, 1:1,000, 1:5,000) in order todetermine the optimal dilution of your samples, especially before assaying a large number of 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 blot 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 Solution B (blank), standards, and samples to designated wells in duplicate according tothe layouts in Figure 1a or 1b. Incubate at room temperature for 2 hours.
NOTE: If a sample with a dilution of 1:100 or less is assayed, add 100 µl of the diluted samples to the antigen-coated strips (S1) andthe corresponding uncoated strips (SB1) as mapped out in Figure 1b.

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 blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.

7.

Add Secondary Antibody: Prepare the secondary antibody solution with Secondary Antibody Dilution Buffer (Solution C) as shown inthe following table. Add 100 µl of secondary antibody solution to each well and incubate at room temperature for 1 hour.
Strip #24681012

Secondary Antibody (µl)
91725334150

Solution C (ml)
1.8
3.4
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 TMB Solution: Use new tubes when preparing TMB solution. Just prior to use, prepare TMB solution with Chromogen Dilution
Buffer as shown in the following table. The prepared TMB cannot be stored for the next assay. Add 100 µl of TMB solution to each wellimmediately after washing the plate and incubate for 25 minutes at room temperature.

© 2020 Chondrex, Inc. All Rights Reserved, M SEB ELISA 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

6Strip #246810
12

TMB (µl)
3466
100
132
164200Chromogen Dilution Buffer (ml)
1.7
3.3
5.0
6.6
8.2
10.0

10. Stop: Stop the reaction with 50 µl of 2N Sulfuric Acid (Stop Solution) to each well.
11. Read Plate: Read the OD values at 450 nm. A 630 nm filter can be used as a reference. If the OD values of samples are greater thanthe 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 test samples.

2.

Subtract the “blank” (B) values from the averaged OD values in step 1.
NOTE: If antigen-uncoated wells were used, subtract the OD values of samples tested in uncoated wells (background values) from theircounterpart OD values in antigen-coated wells from step 2 to eliminate values associated with non-specific reactions.

3.

Plot the OD values of standards against the concentration of standard antibody (ng/ml). Using a log/log plot will linearize the data. Figure
1 shows a representative experiment where the standard range is 1.6 - 100 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.

Figure 1 - A Typical Standard Curve for the Mouse Anti-SEB IgG Antibody ELISA Kit
10.0

OD 450 nm

R² = 0.9992

1.00.1

1

10100

Mouse Anti-SEB Antibody Concentration (ng/ml)© 2020 Chondrex, Inc. All Rights Reserved, M SEB ELISA 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

7VALIDATION DATA
Table 1 - Reproducibility data for Mouse Anti-SEB IgG Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spiking Test*

3.1 ng/ml
2.3
4.688%

12.5 ng/ml
7.8
1.5
83%

50 ng/ml
10.7
4.589%

12.5 ng/ml
8.5
7.788%

50 ng/ml
9.2
8.891%

12.5 ng/ml
7.5
3.091%

50 ng/ml
7.6
7.096%

12.5 ng/ml
8.68.189%

50 ng/ml
7.3
5.495%

Table 2 - Reproducibility data for Mouse Anti-SEB IgG1 Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spiking Test*

3.1 ng/ml
10.0
1.587%

Table 3 - Reproducibility data for Mouse Anti-SEB IgG2a Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spiking Test*

3.1 ng/ml
8.9
9.896%

Table 4 - Reproducibility data for Mouse Anti-SEB IgG2b Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spiking Test*

3.1 ng/ml
2.64.9100%* Known amounts of anti-SEB antibodies were added to standards and then diluted with Sample/Standard Dilution Buffer to assay anti-SEBantibodies

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, M SEB ELISA 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

8REFERENCES
1.

P. Sharma, N. Wang, A. Chervin, C. Quinn, J. Stone, D. Kranz, et al., A Multiplex Assay for Detection of Staphylococcal andStreptococcal Exotoxins. PLoS One 10, e0135986 (2015).

2.

M. Shirtliff, J. Mader, Acute Septic Arthritis. Clin Microbiol Rev 15, 527-44 (2002).

3.

B. Baker, The Role of Microorganisms in Atopic Dermatitis. Clin Exp Immunol 144, 1-9 (2006).

4.

G. Archer, M. Climo, Staphylococcus Aureus Bacteremia--Consider the Source. N Engl J Med 344, 55-6 (2001).

5.

G. Lowell, R. Kaminski, S. Grate, R. Hunt, C. Charney, et al., Intranasal and Intramuscular Proteosome-Staphylococcal Enterotoxin B(SEB) Toxoid Vaccines: Immunogenicity and Efficacy Against Lethal SEB Intoxication in Mice. Infect Immun 64, 1706-13 (1996).

6.

R. LeClaire, R. Hunt, S. Bavari, Protection Against Bacterial Superantigen Staphylococcal Enterotoxin B by Passive Vaccination. InfectImmun 70, 2278-81 (2002).

7.

L. Faulkner, A. Cooper, C. Fantino, D. Altmann, S. Sriskandan, The Mechanism of Superantigen-Mediated Toxic Shock: Not a Simple
Th1 Cytokine Storm. J Immunol 175, 6870-7 (2005).

8.

H. Acha-Orbea, Bacterial and Viral Superantigens: Roles in Autoimmunity? Ann Rheum Dis 52 Suppl 1, S6-16 (1993).

9.

F. Ide, T. Matsubara, M. Kaneko, T. Ichiyama, T. Mukouyama, S. Furukawa, et al., Staphylococcal Enterotoxin-Specific IgE Antibodies
in Atopic Dermatitis. Pediatr Int 46, 337-41 (2004).

© 2020 Chondrex, Inc. All Rights Reserved, M SEB ELISA 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