1

Mouse Anti-OVA Antibody Subtype/Subclass ELISA Kits
Catalog # 3011, 3013, 3015, 3016, 3017, 3018, and 3029
For Research Use Only - Not Human or Therapeutic Use

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

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

3011 (IgG): 12.5 - 0.2 ng/ml

3013 (IgG1): 12.5 - 0.2 ng/ml

3015 (IgG2a) : 50 - 0.8 ng/ml
3016 (IgG2b) : 50 - 0.8 ng/ml
3017 (IgM): 100 - 1.6 ng/ml

3018 (IgA): 12.5 - 0.2 ng/ml

3029 (IgG2c) : 50 - 0.8 ng/ml
NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:100 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

3011: Intra-Assay (2.6-9.5%)/Inter-Assay (0.4-3.7%)/Spiking Test (93-107%)
3013: Intra-Assay (4.7-9.1%)/Inter-Assay (3.6-9.5 %)/Spiking Test (96-114%)
3015: Intra-Assay (5.9-8.9%)/Inter-Assay (6.3-9.4%)/Spiking Test (98-104%)
3016: Intra-Assay (3.9-9.9%)/Inter-Assay (6.3-9.4%)/Spiking Test (94-106%)
3017: Intra-Assay (2.9-4.6%)/Inter-Assay (6.6-9.1%)/Spiking Test (93-116%)
3018: Intra-Assay (0.2-7.8%)/Inter-Assay (1.2-7.9%)/Spiking Test (93-98%)
3029: Intra-Assay (5.5-9.6%)/Inter-Assay (2.9-7%)/Spiking Test (100-109%)

NOTES:

N/A© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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 Antibody Subtype/Subclass ELISA Kits
Catalog # 3011, 3013, 3015, 3016, 3017, 3018, and 3029
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Ovalbumin (OVA) is a widely used antigen for inducing allergic reactions in experimental animals (1-4). To study the contribution of antibodies
to allergic reactions, Chondrex, Inc. provides a variety of subtype and subclass anti-OVA mouse antibody ELISA kits. Investigators studying
the pathogenesis of allergic diseases in mice should consider several issues such as mucosal immunity, antibody responses to different
subtypes (IgA, IgE, IgG, and IgM) and subclasses (IgG1, IgG2a, IgG2b, and IgG2c), and even the types of adjuvants used for immunization.
The following may be informative for studies on allergic reactions in experimental animals.
1) Mucosal immunity: The mucosal immune system is the first line of defense against potential pathogenic and non-pathogenic
environmental factors such as bacteria, viruses, and dietary proteins. Importantly, poor mucosal immune function may lead to abnormal
absorption of mimic antigens such as food components and bacterial cell walls, which elicit antibodies that may cross-react withautologous components, also known as “autoantibodies”. IgG, the dominant immunoglobulin subtype in serum, protects the host from
pathogens and unwanted antigens that have penetrated the body. On the other hand, IgA, a known mucosal immunoglobulin, prevents
the penetration of pathogens and other unwanted antigenic substances through mucosal membranes. Therefore, to study mucosal andsystemic immune responses to allergens in mouse models, OVA is a valuable and convenient antigen (5).
2) Allergy: In general, an allergic reaction is mediated by IgE-antigen complexes. More specifically, IgE molecules cross-linked by apolyvalent antigen on the surfaces of mast cells trigger their degranulation which initiates the ensuing allergic cascade. Although the roleof IgG antibodies in allergic reactions is not yet clear, two opposing roles are postulated: 1) IgG antibodies which share epitopes with IgE
antibodies may competitively bind the epitopes on the allergen and modulate the allergic reaction, or 2) IgG antibodies may enhance theallergic reaction by providing aggregated allergens to IgE on mast cells. In addition, the roles of antibody subtypes and subclasses may
differ depending on the allergic reaction, as IgG1 and IgE are regulated by Th2 cells, whereas IgG2a, IgG2b and IgG2c are dependenton Th1 cells. Thus, to investigate the immune responses involved in allergic reactions in OVA-induced allergic mouse models (1-4), antiOVA IgE, IgG, and IgA antibody ELISA kits are valuable tools.
3) Adjuvants: The type of adjuvant used can elicit specific antibody subtypes. For example, aluminum adjuvant is widely used to elicit IgE
antibodies (7), whereas Cholera toxins are effective at eliciting IgA antibodies (6). Moreover, Complete Freund’s Adjuvant (CFA) is widelyused for stimulating IgG and IgM antibody production (8).

LIST OF MOUSE ANTI-OVA ANTIBODY SUBTYPE/SUBCLASS ELISA KITS
Kit
Mouse Anti-OVA IgG Antibody ELISA Kit
Mouse Anti-OVA IgG1 Antibody ELISA Kit
Mouse Anti-OVA IgG2a Antibody ELISA Kit
Mouse Anti-OVA IgG2b Antibody ELISA Kit
Mouse Anti-OVA IgM Antibody ELISA Kit
Mouse Anti-OVA IgA Antibody ELISA Kit
Mouse Anti-OVA IgG2c Antibody ELISA Kit

Catalog #
3011
3013
3015
3016
3017
3018
3029

© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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

KIT COMPONENTS
Item

Quantity

AmountStorage

1 vial1000 ng, Lyophilized-20°C

IgG (30111)
IgG1 (30131)
IgG2a (30151)
Standard
IgG2b (30161)
IgM (30171)
IgA (30181)
IgG2c (30291)
IgG (30113)
IgG1 (30133)
Secondary Antibody
IgG2a (30153)(peroxidase-conjugated IgG2b (30163)
polyclonal antibodies)
IgM (30173)
IgA (30183)
IgG2c (30293)
Ovalbumin (OVA) (300411)
Solution A - Coating Buffer (9052)
Solution B - Blocking Buffer (30105)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30106)
TMB Solution (90023)

2 vials

50 µl

-20°C

1 vial
1 bottle
1 bottle
1 bottle
2 vials

100 µl
10 ml
10 ml
50 ml
0.2 ml

-20°C
-20°C
-20°C
-20°C
-20°C

Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
ELISA Plate

1 bottle
1 bottle
1 bottle
1 each

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

-20°C
-20°C
-20°C
-20°C

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 the
protocol 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.
NOTE 8: Depending on the isotypes, subtypes, and targeting epitopes of antibodies, the binding affinity of individual antibodies varies
significantly. Therefore, the total IgG antibody concentration calculated as the sum of individual IgG subtypes might not perfectly match thetotal IgG concentration as determined by the Mouse Anti-OVA IgG Antibody ELISA Kit (Cat # 3011).

© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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 OUTLINE
Add 100 ml of diluted OVA solution into wells

Incubate at 4 degrees Celsius overnight.
Wash plate.
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 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
Example of the Mouse Anti-OVA IgG Antibody ELISA Kit
1

2

3

4

5

6

7

8

9

10

11

12

A

12.5

12.5

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

6.3

6.3

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C

3.1

3.1

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

1.6

1.6

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

0.8

0.8

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

0.4

0.4

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

0.2

0.2

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© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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

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.
Strip #
2
4
6
8
10
12

OVA (µl)
17
33
50
66
82
100

Solution A (ml)
1.7
3.3
5.0
6.6
8.2
10.0

2.

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.

3.

Add Blocking Buffer: Add 100 µl of the Blocking Buffer (Solution B) to each well and incubate at room temperature for 1 hour.

4.

Prepare Standard Dilutions: Please see the figure below for each assay’s recommended standard range. Dissolve one vial of Standard
in 1 ml of Sample/Standard/Secondary Antibody Dilution Buffer (Solution C) and keep it as a standard stock. Dilute the standard stock
accordingly to get the first stock solution. Then serially dilute it with Solution C. For example, mix 250 µl of the first stock solution withan equal volume of Solution C to make the second stock solution, and then repeat it five more times. The original standard stock solution
may be stored at -20⁰C for use in a future assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 μl*

250 μl1

250 μl250 μl250 μl250 μl250 μl

2

3

4

5

6

7

8

250 μl250 μl250 μl250 μl250 μl250 μl250 μl12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

0.8 ng/ml

0.4 ng/ml

0.2 ng/ml

Blank

IgG2a/IgG2b/IgG2c: 25 μl + 475 μl

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

IgM: 50 μl + 450 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

Blank1000 μl1000 ng/ml*
IgG/IgG1/IgA: 25 μl + 1975 μl

5.

Prepare Sample Dilutions: The dilution of serum from mouse immunized with OVA varies (1:100 or more) depending on the
immunization schedule and timing of serum collection. In general, no antibodies against OVA are observed in normal serum at a 1:100
dilution.

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 Standards and Samples: Add 100 ml of standards, Solution C (blank), and samples to wells in duplicate. Incubate at room
temperature for 2 hours.

© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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

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 Secondary Antibody: Dilute one vial of Secondary Antibody in 10 ml Sample/Standard/Secondary Antibody Dilution Buffer(Solution C). Add 100 µl of secondary antibody solution to each well and incubate at room temperature for 1 hour.
Strip #
2
4
6
8
10
12

2nd Antibody (µl)
8
17
25
33
42
50

Solution C (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. 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 #
2
4
6
8
10
12

TMB (µl)
34
66
100
132
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 IgG 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 the
antibody concentration (ng/ml) in original test samples.

© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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

Figure 1 - Typical Standard Curves for the Anti-OVA Antibody ELISA Kits
IgG

IgG1

10.00

10.00

R² = 0.9998

OD 450 nm

IgG2a

10.00

R² = 0.9912

R² = 0.993

1.00

1.00

0.10

OD 450 nm

OD 450 nm

1.00

0.10

0.01

0.10

0.01
0

1

10

100

0.01
0.1

1

10

100

0.1

IgG2b

IgM

10.00

R² = 0.999

R² = 0.9894

OD 450 nm

1.00

OD 450 nm

OD 450 nm

1.00

0.10

0.10

0.01

0.10

0.01
1

10

100

1

10

100

Anti-OVA IgM Concentration (ng/ml)

Anti-OVA IgG2b Concentration (ng/ml)

100

10.00

R² = 0.9946
1.00

10

IgA

10.00

0.1

1

Anti-OVA IgG2a Concentration (ng/ml)

Anti-OVA IgG1 Concentration (ng/ml)

Anti-OVA IgG Concentration (ng/ml)

0.01
0.1

1

10

100

Anti-OVA IgA Concentration (ng/ml)

IgG2c
10.00

R² = 0.9938

OD 450

1.00

0.10

0.01
0.1

1

10

100

Anti-OVA IgG2c Concentration (ng/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the Mouse Anti-OVA IgG Antibody ELISA Kit
Test
2.1 ng/ml
4 ng/ml
Intra-Assay CV (%)
9.5
5.2
Inter-Assay CV (%)
3.7
0.4
Test
0.4 ng/ml
1.6 ng/ml
Spike Test* (%)
107%
95%

8.8 ng/ml
2.6
3.3
6.3 ng/ml
93%© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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

8

Table 2 - Reproducibility Data for the Mouse Anti-OVA IgG1 Antibody ELISA Kit
Test
0.4 ng/ml
1.6 ng/ml
Intra-Assay CV (%)
9.1
6.3
Inter-Assay CV (%)
3.6
9.4
Spike Test* (%)
114%
105%

6.3 ng/ml
4.7
9.5
96%

Table 3 - Reproducibility Data for the Mouse Anti-OVA IgG2a Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
Intra-Assay CV (%)
5.9
8.3
Inter-Assay CV (%)
9.4
8.6
Spike Test* (%)
102%
104%

25 ng/ml
8.9
6.3
98%

Table 4 - Reproducibility Data for the Mouse Anti-OVA IgG2b Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
Intra-Assay CV (%)
9.9
9.5
Inter-Assay CV (%)
9.4
8.6
Spike Test* (%)
104%
106%

25 ng/ml
3.9
6.3
94%

Table 5 - Reproducibility Data for the Mouse Anti-OVA IgM Antibody ELISA Kit
Test
3.1 ng/ml
12.5 ng/ml
Intra-Assay CV (%)
2.9
4.6
Inter-Assay CV (%)
6.6
9.1
Spike Test* (%)
112%
116%

50 ng/ml
4.4
9.0
93%

Table 6 - Reproducibility Data for the Mouse Anti-OVA IgA Antibody ELISA Kit
Test
2 ng/ml
Intra-Assay CV (%)
7.8
Inter-Assay CV (%)
7.9
Spike Test* (%)
97%

8 ng/ml
2.1
5.2
93%

4 ng/ml
0.2
1.2
98%

Table 7 - Reproducibility Data for the Mouse Anti-OVA IgG2c Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
25 ng/ml
Intra-Assay CV (%)
5.8
9.6
5.5
Inter-Assay CV (%)
7.0
4.0
2.9
Spike Test* (%)
101%
109%
100%
*Known amounts of anti-OVA antibodies were added to samples and then diluted with Sample/Standard/Secondary Antibody Dilution
Buffer to assay anti-OVA antibodies by ELISA.

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

© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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

9

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).
5. Y. Hagiwara, et al. Protective Mucosal Immunity in Aging Associated with Functional CD4+ T Cells in Nasopharyngeal-AssociatedLymphoreticular Tissue. J Immunol 170:1754-1762 (2003).
6. A. K. Gloudemans, et al. The mucosal adjuvant cholera toxin B instructs non-mucosal dendritic cells to promote IgA production via retinoic
acid and TGF-β. PLoS One. 8(3): e59822 (2013).
7. N. Mizutani, et al. Establishment and characterization of a murine model for allergic asthma using allergen-specific IgE monoclonal
antibody to study pathological roles of IgE. Immunol Lett 141(2):235-45 (2012).
8. J. H. Kim, et al. Effects of diphenyl dimethyl dicarboxylate on oral tolerance to ovalbumin in mice. J Toxicol Sci 20(4):375-82 (1995).

© 2024 Chondrex, Inc. All Rights Reserved, OVA Isotypes 3.0
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