1Mouse Anti-OVA IgE Antibody ELISA Kit
Catalog # 3004
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify mouse non-serum anti-ovalbumin IgE antibodies

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

50 - 0.8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Culture Media & Purified IgE

RECOMMENDED SAMPLE DILUTIONS:

1:10 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (1.6-1.9%) /Inter-Assay (1.7-2.6%) /Spiking Test (89-98%)

NOTES:

This kit cannot be used to assay mouse anti-OVA IgE antibodies in serum© 2022 Chondrex, Inc. All Rights Reserved, 3004 7.0
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-OVA IgE Antibody ELISA Kit
Catalog # 3004
For Research Use Only - Not Human or Therapeutic UseINTRODUCTION
In order to study the pathogenesis of allergic diseases, mice are the most practical experimental animals, due to the variety of inbred strains
and transgenic and gene knockout mice that are available. Serum IgE levels are often raised in allergic diseases and parasitic infections,although serum IgE level alone does not reflect the allergic state and the clinical symptoms of the patient. However, it is apparent that araised serum IgE level aids in the diagnosis of these diseases in humans. As ovalbumin (OVA) is one of the most widely used antigens forstudying allergic diseases in mice (4-7), Chondrex, Inc. provides ELISA kits to determine OVA-specific mouse subtype antibodies and todetermine mouse total serum antibody levels. For more information, please visit www.chondrex.com.
The Mouse Anti-OVA IgE Antibody ELISA Kit (Catalog # 3004) is designed to detect anti-OVA IgE antibodies in limited samples such as
hybridoma cell culture supernatant and solutions containing anti-OVA IgE antibodies without other subclasses of anti-OVA antibodies
present. The secondary antibody used in this kit reacts equally with both IgEa (Balb/c) and IgEb (C57BL/6) allotypes, so it is not necessary
to run two separate assays using two independent IgEa and IgEb standards. It does not cross-react with any other mouse immunoglobulin
subclasses or subtypes (IgA, IgM, IgG, IgG1, IgG2a, IgG2b, IgG2c, or IgG3).
Note: This kit is an indirect ELISA using an OVA-coated plate to detect IgE; this kit cannot assay anti-OVA IgE antibodies in mouse serum.
The competitive binding of other antibody subclasses recognizing the same antigenic epitopes on OVA may reduce anti-OVA IgE binding tothe OVA, as serum IgE levels are usually 1/1000 lower than that of IgG. For assaying anti-OVA IgE antibodies in mouse serum, please use
the Mouse Serum Anti-OVA IgE Antibody Detection ELISA Kit (Catalog # 3010). Please contact us at support@chondrex.com for more
information.

KIT COMPONENTS
Item

Quantity

AmountStorage

Ovalbumin (OVA) (300411)

1 vial100 µl, 1 mg/ml-20°C

Standard Mouse IgE (300412)
Secondary Antibody (300413)
Solution A - Coating Buffer (9052)

1 vial1 vial
1 bottle1000 ng, lyophilized
Lyophilized
10 ml

-20°C
-20°C
-20°C

Solution B - Sample/Standard Dilution Buffer (30055)
Solution C - Secondary Antibody Dilution Buffer (30056)
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

50 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, 3004 7.0
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 OVA into wells

Incubate at 4 degrees Celsius overnight.
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 25 minutes.

Add 50 ml of Stop Solution into wells

Read plates at 450 nm/630 nm

PLATE MAPPING
12

3

4

5678910

1112A

50

50

S1S1S9S9S17S17S25

S25

S33

S33

B

2525

S2S2S10S10

S18

S18

S26

S26S34

S34

C12.5

12.5

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D6.3

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

HB

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2022 Chondrex, Inc. All Rights Reserved, 3004 7.016928 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

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.

ASSAY PROCEDURE
1.

Add OVA Solution: Dilute one vial of Ovalbumin (OVA) with 10 ml of Coating Buffer (Solution A). Alternatively, dilute according to thetable below. Add 100 µl of OVA solution to each well and incubate at 4°C overnight. Any leftover OVA Stock Solution may be stored at-20°C for future assays.
Strip #246810122.

OVA (µl)1733506682100

Solution A (ml)1.73.3
5.0
6.6
8.2
10.0

Prepare Standard Dilutions: The recommended standard range is 0.8-50 ng/ml. Dissolve one vial of Standard in 1 ml ofSample/Standard Dilution Buffer (Solution B) and keep it as a 1000 ng/ml standard stock. Take 50 µl of the standard solution (1000ng/ml) and add it to 950 µl of Solution B to make a 50 ng/ml IgE standard solution. Then serially dilute it with Solution B. For example,
mix 250 µl of the stock solution with an equal volume of Solution B to make a 25 ng/ml stock solution, and then repeat it five more timesfor 12.5, 6.3, 3.1, 1.6, 0.8 ng/ml standard solutions. The remaining 1000 ng/ml standard stock solution can be stored at -20⁰C for use
in a future assay. Chondrex, Inc. recommend making fresh serial dilutions for each assay.

© 2022 Chondrex, Inc. All Rights Reserved, 3004 7.0
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 μl

50 μl

250 μl250 μl250 μl250 μl250 μl250 μl123

456781000 μl

950 μl250 μl250 μl250 μl250 μl250 μl250 μl250 μl1000 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: The sample dilutions (1:10 or more) depend on the types of samples. Chondrex, Inc. recommends assaying
2-3 different dilutions when the anti-OVA IgE concentration in the sample is unknown.

4.

Wash: 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 ml 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 of Secondary Antibody in 50 µl of Solution C and dilute according to the following table. Add 100 µl of
secondary antibody solution to each well and incubate at room temperature for 1 hour.
Strip #2468
1012

Secondary Antibody (µl)
81725
334250

Solution C (ml)
1.7
3.3
5.0
6.6
8.2
10.08.

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)1.73.35.0
6.6
8.2
10.0

© 2022 Chondrex, Inc. All Rights Reserved, 3004 7.0
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

6

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. 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. Add 100 µl of TMB solution to each well immediately after washing the plate and incubate for 25
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

12. Stop: Stop the reaction with 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.

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
an example of a standard curve for anti-OVA IgE antibodies.

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 test samples.

Figure 1 - A Typical Standard Curve for the Anti-OVA IgE Antibody ELISA Kit
10.0

R² = 0.9991

OD 450 nm

1.00.10.0
0.1

110100

Mouse Anti-OVA IgE Antibody Concentration (ng/ml)© 2022 Chondrex, Inc. All Rights Reserved, 3004 7.016928 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

7

VALIDATION DATA
Table 1 - Reproducibility Data for the Mouse Anti-OVA IgE Antibody ELISA Kit
Test
12.5 ng/ml
25 ng/ml
50 ng/ml
Intra-Assay CV (%)
1.9
1.6
1.8
Inter-Assay CV (%)
2.1
2.6
1.7
Spike Test* (%)
98%89%
96%*Known amounts of anti-OVA IgE antibodies were added to samples and then diluted with Sample/Standard Dilution Buffer (Solution B)
to assay anti-OVA IgE antibodies by ELISA.

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

REFERENCES
1. T. Morokawa, et al., Differential susceptibility of C57BL/6 and DBA/2 mice to ovalbumin-induced pulmonary eosinophil regulated by
Th1/Th2 type cytokines. Immunol Letter 70:127-134 (1999).
2. A. Oshiba, et al., Passive transfer of immediate-hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin
IgE and IgG1 in mice. J Clin Invest 97:1398-1408 (1996).
3. E. Hamelmann, et al. Role of IgE in the development of allergic airway inflammation and airway hyperresponsiveness-a marine model.
Allergy 54:297-305 (1999).
4. C. Taube, et al. Mast cells, FcRI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen
exposure in the absence of adjuvant. J Immunol 172:6398-6406 (2004).

© 2022 Chondrex, Inc. All Rights Reserved, 3004 7.0
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