1Mouse Total Immunoglobulin Detection ELISA Kits
IgA, IgG, IgG1, IgG2a, IgG2b, IgG3, and IgMCatalog # 3019, 3023, 3025, 3026, 3027, 3028, and 3024
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify total mouse IgG/IgM/IgA immunoglobulins.

FORMAT:

96-well ELISA plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

100 ng/ml to 1.6 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma (pre-treatment acceptable)

RECOMMENDED SAMPLE DILUTIONS:

Varies (Starting point 1:100 or greater)

CHROMOGEN:

OPD (read at 490 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

3019 (IgA): Intra-Assay (3.2-7.9%)/Inter-Assay (3.3-8.5%)/Spiking Test (102-104%)
3023 (IgG): Intra-Assay (4.0-10.1%)/Inter-Assay (4.2-5.7%)/Spiking Test (93-113%)3025 (IgG1): Intra-Assay (1.6-8.0%)/Inter-Assay (2.8-7.1%)/Spiking Test (93-100%)
3026 (IgG2a): Intra-Assay (8.3-9.0%)/Inter-Assay (4.1-6.9%)/Spiking Test (84-102%)
3027 (IgG2b): Intra-Assay (4.1-7.5%)/Inter-Assay (5.2-9.1%)/Spiking Test (93-100%)
3028 (IgG3): Intra-Assay (0.9-9.7%)/Inter-Assay (0.8-3.6%)/Spiking Test (86-97%)3024 (IgM): Intra-Assay (2.3-6.5%)/Inter-Assay (2.7-5.1%)/Spiking Test (94-112%)

NOTES:

N/A© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.1
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 Total Immunoglobulin Detection ELISA Kits
IgA, IgG, IgG1, IgG2a, IgG2b, IgG3, and IgMCatalog # 3019, 3023, 3025, 3026, 3027, 3028, and 3024
For Research Use Only - Not Human or Therapeutic Use
INTRODUCTION
Animals are consistently exposed to environmental factors that penetrate the body from multiple sites such as the respiratory and digestive
tracts, or through damaged skin. Penetrating antigens can activate the humoral immune response which produces anti-antigen
immunoglobulins to protect the host. Immunoglobulins are classified by their isotypes which differ by biological properties, functional
locations, and ability to deal with different antigens. The main isotypes are summarized below:
1) Immunoglobulin A (IgA) plays a critical role in mucosal immunity and is found in the mucous membranes, especially the respiratory andgastrointestinal tracts, as well as in saliva and tears.
2) Immunoglobulin E (IgE) is involved in allergic reactions and is found in the lungs, skin, and mucous membranes.
3) Immunoglobulin G (IgG) is the most abundant isotype and is found in blood and extracellular fluids. In mice, IgG is categorized into four
subtypes: IgG1, IgG2a, IgG2b, and IgG3.
4) Immunoglobulin M (IgM) is the first isotype produced in a humoral immune response and is found mainly in the blood and lymph fluid.
Total immunoglobulin isotype levels correlate with health or pathological conditions such as hypo- or hyper-gammaglobulinemia, and acuteor chronic infections (1-5). In addition, total immunoglobulin levels are often analyzed relative to antigen-specific antibody levels to evaluate
immune function in patients and animal disease models. Chondrex, Inc. provides mouse total immunoglobulin isotype ELISA kits for IgA,IgE, IgM, and IgG, as well as IgG subtypes: IgG1, IgG2a, IgG2b, and IgG3, along with antigen-specific antibody ELISA kits, such asautoantigens (ssDNA and dsDNA), dietary proteins (collagen, OVA, gliadin), and bacteria (E. coli, LPS, P. gingivalis, and S. aureus). Thesetotal immunoglobulin detection ELISA kits are also suitable for quantifying monoclonal antibody and polyclonal antibody isotypes and/orsubtypes, as well as establishing reliable methods for monitoring antibody production and quality control.

LIST OF MOUSE TOTAL IMMUNOGLOBULIN DETECTION ELISA KITSKit
Total IgE Detection ELISA Kit
Total IgA Detection ELISA Kit
Total IgG Detection ELISA Kit
Total IgM Detection ELISA Kit
Total IgG1 Detection ELISA Kit
Total IgG2a Detection ELISA Kit
Total IgG2b Detection ELISA Kit
Total IgG3 Detection ELISA Kit

Catalog #3005301930233024302530263027
3028© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.1
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 vialLyophilized
1000 ng-20°C

1 vial1 mg/ml, 100 µl-20°C

2 vials

50 µl

-20°C

1 bottle
1 bottle

10 ml
10 ml

-20°C
-20°C

1 bottle
2 vials
1 bottle
1 bottle
1 bottle
1 each50 ml
Lyophilized
20 ml
10 ml
50 ml
8-well Strips x12-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

IgA (30181)
IgG (30111)
IgG1 (30131)
IgG2a (30151)
Standard
IgG2b (30161)
IgG3 (30281)
IgM (30171)
IgA (30192)
IgG (30232)
IgG1 (30232)
Capture Antibody
IgG2a (30232)
IgG2b (30232)
IgG3 (30282)
IgM (30242)
IgA (30193)
IgG (30233)
IgG1 (203011)
Detection Antibody(peroxidase-conjugated IgG2a (203012)
IgG2b (30163)
polyclonal antibodies)
IgG3 (203017)
IgM (30173)
Solution A - Coating Buffer (9052)
Solution B - Blocking Buffer (30235)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30106)
OPD (90021)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
ELISA Plate

PLATE MAPPING
123456789101112A

100

100

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

5050

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C25

25

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D12.512.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E6.36.3S5S5

S13

S13

S21

S21

S29

S29

S37

S37F

3.13.1

S6S6

S14

S14

S22

S22

S30

S30

S38

S38

G1.61.6

S7

S7S15

S15

S23

S23

S31

S31

S39

S39HBB

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.1
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

4ASSAY OUTLINENOTES BEFORE USING ASSAYNOTE 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.

© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.1
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

5ASSAY PROCEDURE
1.

Add Capture Antibody: Centrifuge the Capture Antibody vial at 3000 rpm x 1 minute. Dilute one vial 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.
Strip #24681012Capture Antibody (µl)
1733506682100

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: The recommended standard range is 1.6-100 ng/ml. Dissolve one vial of Mouse Immunoglobulin Standard
in 1 ml of Sample/Standard/Secondary Antibody Dilution Buffer (Solution C) to make a 1000 ng/ml standard stock solution and thendilute the standard stock solution 1:10 with Solution C. For example, mix 50 µl of the standard stock solution (1000 ng/ml) with 450 µlof Solution C to make a 100 ng/ml solution. Then take 250 µl of the diluted standard solution and mix with 250 µl of Solution C to makea 50 ng/ml solution, and then repeat it five more times for 25, 12.5, 6.3, 3.1, and 1.6 ng/ml solutions. The remaining 1000 ng/ml standard
stock solution may be stored at -20°C for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for eachassay.
1000 μl

50 μl

250 μl250 μl250 μl250 μl250 μl250 μl12345678

1000 μl

450 μl250 μl250 μl250 μl250 μl250 μl250 μl250 μl1000 ng/ml

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

Blank5.

Prepare Sample Dilutions: Several factors may influence immunoglobulin levels in serum, such as the adjuvant used for immunizationand the timing of serum collection. In general, it is advisable to make several dilutions of your sample to ensure that the sample OD
values are within the range of the standard curve.

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 µl of standards, Solution C (blank), and samples to wells in duplicate. Incubate at room
temperature for 2 hours

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.

© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.1
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.

Add Detection Antibody: Centrifuge the Detection Antibody vial at 3000 rpm x 1 minute. Dilute one vial of Detection Antibody with 10ml Sample/Standard/Secondary Antibody Dilution Buffer (Solution C). Add 100 µl of detection antibody solution to each well andincubate at room temperature for 1 hour.
Strip #2
4681012Detection Antibody (µl)
81725334250

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 OPD: Dissolve one vial of OPD in 10 ml of Chromogen Dilution Buffer. Add 100 µl of OPD solution to each well immediately after
washing the plate. Incubate for 30 minutes at room temperature.
12. Stop: Add 50 ml of 2N sulfuric acid (Stop Solution) to each well.
13. Read Plate: Read the OD values at 490 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 (B), standards, and test samples.

2.

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

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 the original sample specimens.

Figure 1 - A Typical Standard Curve for the Mouse Total IgG Antibody Detection ELISA Kit
10.00

R² = 0.9865

OD 490 nm

1.00

0.10

0.01
110
Concentration (ng/ml)

100© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.1
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

7ASSAY VALIDATION
Table 1 - Reproducibility Data for the Mouse Total IgA Antibody Detection ELISA Kit (Catalog # 3019)
Test
10 ng/ml
20 ng/ml
Intra-Assay CV (%)
7.9
3.2
Inter-Assay CV (%)
6.7
8.5
Spiking Test*
102%
104%

40 ng/ml
6.3
3.3
103%

Table 2 - Reproducibility Data for the Mouse Total IgG Antibody Detection ELISA Kit (Catalog # 3023)
Test
3.1 ng/ml
12.5 ng/ml
Intra-Assay CV (%)
10.1
8.0
Inter-Assay CV (%)
4.5
4.2
Spiking Test*
93%
104%

50 ng/ml
4.0
5.7113%

Table 3 - Reproducibility Data for the Mouse Total IgG1 Antibody Detection ELISA Kit (Catalog # 3025)
Test
3.1 ng/ml
12.5 ng/ml
Intra-Assay CV (%)
7.6
1.6
Inter-Assay CV (%)
7.1
2.8
Spike Test* (%)
93%
100%

50 ng/ml
8.0
3.6
95%

Table 4 - Reproducibility Data for the Mouse Total IgG2a Antibody Detection ELISA Kit (Catalog # 3026)
Test
3.1 ng/ml
12.5 ng/ml
Intra-Assay CV (%)
8.3
9.0
Inter-Assay CV (%)
6.3
6.9
Spike Test* (%)
102%
84%

50 ng/ml
9.0
4.1
91%

Table 5 - Reproducibility Data for the Mouse Total IgG2b Antibody Detection ELISA Kit (Catalog # 3027)
Test
3.1 ng/ml
12.5 ng/ml
Intra-Assay CV (%)
4.9
7.5
Inter-Assay CV (%)
7.6
9.1
Spike Test* (%)
100%
94%

50 ng/ml
4.1
5.2
93%

Table 6 - Reproducibility Data for the Mouse Total IgG3 Antibody Detection ELISA Kit (Catalog # 3028)
Test
3.1 ng/ml
12.5 ng/ml
Intra-Assay CV (%)
9.75.2
Inter-Assay CV (%)
3.6
0.8
Spike Test* (%)
97%
86%

50 ng/ml
0.9
1.587%

Table 7 - Reproducibility Data for the Mouse Total IgM Antibody Detection ELISA Kit (Catalog # 3024)
Test
3.1 ng/ml
12.5 ng/ml
50 ng/ml
Intra-Assay CV (%)
6.5
2.3
4.2
Inter-Assay CV (%)
5.1
4.8
2.7
Spike Test* (%)
94%
111%
112%
* Known amounts of immunoglobulins were added to samples and then diluted with Sample/Standard/Detection Antibody Dilution
Buffer (Solution C).

© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.1
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

8TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s ELISA FAQ for more information.

REFERENCES
1.

K. J. Hamilton, M. Satoh, J. Swartz, H. B. Richards, W. H. Reeves, Influence of microbial stimulation on hypergammaglobulinemia andautoantibody production in pristane-induced lupus. Clin Immunol Immunopathol 86, 271-279 (1998).

2.

C. B. Reimer et al., Hypergammaglobulinemia associated with human immunodeficiency virus infection. Monogr Allergy 23, 83-96(1988).

3.

S. Senda, E. Cheng, H. Kawanishi, IgG in murine intestinal secretions. Aging effect and possible physiological role. Scand J Immunol
29, 41-47 (1989).

4.

Tana, S. Watarai, E. Isogai, K. Oguma, Induction of intestinal IgA and IgG antibodies preventing adhesion of verotoxin-producing
Escherichia coli to Caco-2 cells by oral immunization with liposomes. Lett Appl Microbiol 36, 135-139 (2003).

5.

M. C. Thurnheer, A. W. Zuercher, J. J. Cebra, N. A. Bos, B1 cells contribute to serum IgM, but not to intestinal IgA, production ingnotobiotic Ig allotype chimeric mice. J Immunol 170, 4564-4571 (2003).

© 2022 Chondrex, Inc. All Rights Reserved, Total ELISAs 4.116928 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