1Mouse Anti-dsDNA IgG Antibody ELISA Kit
Mouse Anti-ssDNA IgG Antibody ELISA Kit
Catalog # 3031 and 3041
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify mouse IgG antibodies against single-stranded DNA (ssDNA) anddouble-stranded DNA (dsDNA).
3031: Mouse Anti-dsDNA IgG Antibody ELISA Kit
3041: Mouse Anti-ssDNA IgG Antibody ELISA Kit

FORMAT:

96-well ELISA plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

50 ng/ml to 0.8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma (pre-treatment acceptable)

RECOMMENDED SAMPLE DILUTIONS:

1:100 or greater

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

3031: Intra-Assay (6.3-9.4%)/Inter-Assay (3.9-9.9%)/Spiking Test (93.4-106%)
3041: Intra-Assay (7.5-9.8%)/Inter-Assay (3.8-8.1%)/Spiking Test (94-101.4%)© 2020 Chondrex, Inc. All Rights Reserved, 3031, 3041 4.0
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-dsDNA IgG Antibody ELISA Kit
Mouse Anti-ssDNA IgG Antibody ELISA Kit
Catalog # 3031 and 3041
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
High levels of serum autoantibodies against deoxyribonucleic acid (DNA) are observed in most patients with systemic lupus erythematosus(SLE) (1, 2), therefore the presence of anti-DNA antibodies in serum is considered a valuable marker for the diagnosis of SLE. Especiallyconsidering that the serum anti-DNA antibodies form anti-DNA/DNA immune complexes which play important roles in theimmunopathogenesis of renal injury also known as lupus nephritis (3). With regards to the specificity of anti-DNA antibodies, anti-single
stranded DNA (ssDNA) lgG antibodies are elicited in the early stages of SLE, whereas anti-double stranded DNA (dsDNA) lgG antibody
levels correlate with the severity of SLE. On the other hand, anti-dsDNA lgM antibodies are not specific to SLE, but correlate with theprognosis of lupus nephritis in patients with SLE (4, 5). Therefore, evaluating immunoglobulin levels of different isotypes against individual
DNA types may indicate the stages and prognosis of SLE.
Mouse models, which provide relevant information to the human condition, elucidate the cellular and genetic requirements for inducing SLE.
For example, in spontaneous murine NZB/W F1 lupus models, anti-dsDNA antibody isotype class switching from lgM to lgG indicates renal
failure which is a similar trend in human SLE (6). Nonetheless, in artificial pristane-induced Balb/c lupus models, anti-ssDNA lgM antibodies
solely induce SLE (7-9). In order to advance studies in these fields, Chondrex, Inc. provides mouse anti-DNA antibody ELISA kits to elucidate
the diverse roles of anti-DNA antibodies in these mouse SLE models. For a complete list of available mouse anti-DNA antibody ELISA kits,please visit www.chondrex.com for more information.

KIT COMPONENTS
Item

Quantity

AmountStorageStandard Mouse anti-DNA IgG monoclonal Antibody (30311)
dsDNA (30312) or ssDNA (30412)
Secondary Antibody (Peroxidase-Conjugated Goat Anti-Mouse IgG
Polyclonal Antibody) (7012)
Solution A - Coating Buffer (9052)
Solution B - Blocking Buffer (30313)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer(30314)
TMB Solution (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
ELISA Plate

1 vial1 vial

50 ng/vial, lyophilized
0.5 mg/ml, 100 µl

-20°C
-20°C

2 vials

50 µl

-20°C

1 bottle
1 bottle

10 ml
10 ml

-20°C
-20°C

1 bottle

50 ml

-20°C

2 vials
1 bottle
1 bottle1 bottle
1 each0.2 ml
20 ml
10 ml
50 ml
96-well (8-well strips x 12)

-20°C
-20°C
-20°C
-20°C
-20°C

© 2020 Chondrex, Inc. All Rights Reserved, 3031, 3041 4.0
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 µl of diluted DNA to all wells
Incubate at 4 degrees Celsius overnight.
Wash plate.
Add 100 µl of Solution B to all wells
Incubate at room temperature for 1 hour.
Wash plate.
Add 100 µl of diluted Standards and Samples
Incubate at room temperature for 2 hours.
Wash plate.
Add 100 µl of diluted Secondary Antibody solution
Incubate at room temperature for 1 hour.
Wash plate.

Add 100 µl of TMB solution

Incubate at room temperature for 25 minutes.
Add 50 µl of Stop Solution

Read plates at 450 nm/630 nm

PLATE MAPPING
123

45

6789101112A

50

50

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B25

25

S2S2

S10

S10

S18

S18

S26

S26

S34

S34

C

12.5

12.5

S3

S3

S11S11S19S19

S27

S27

S35

S35

D6.36.3

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

3.13.1

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

1.61.6

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G0.8

0.8

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© 2020 Chondrex, Inc. All Rights Reserved, 3031, 3041 4.0
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 fordisposal.

ASSAY PROCEDURE
1.

Add DNA Solution: Dilute one vial of DNA with 10 ml of Solution A. Alternatively, dilute according to the table below. Add 100 µl ofDNA solution to each well and incubate at 4°C overnight. Any leftover DNA Stock Solution may be stored at -20°C for future assays.
Strip #24681012DNA (µl)
17335066
82
100Solution A (ml)
1.7
3.3
5.0
6.6
8.2
10.0

2.

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 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 0.8-50 ng/ml. Dissolve one vial of Standard (50 ng/vial) in 1 ml ofSample/Standard/Secondary Antibody Dilution Buffer (Solution C) and keep it as a standard stock. Then serially dilute it with Solution
C. For example, mix 250 µl of the 50 ng/ml solution with an equal volume of Solution C to make a 25 ng/ml solution, and then repeat it
five more times for 12.5, 6.3, 3.1, 1.6, and 0.8 ng/ml standard solutions.

© 2020 Chondrex, Inc. All Rights Reserved, 3031, 3041 4.0
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 ml

250 ml23456781000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 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

1

5.

Prepare Sample Dilutions: Mouse serum dilution varies (1:100 or more) depending on the animal model and timing of serum collection.
In general, no IgG antibodies against DNA 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 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.

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 #24681012

2nd 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 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
4681012

TMB (µl)
3466
100132
164
200

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

© 2020 Chondrex, Inc. All Rights Reserved, 3031, 3041 4.0
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

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 RESULTS1.

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. Figures 1a and1b show examples of standard curves for anti-DNA 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 theantibody concentration (ng/ml) in the original test samples.

Figure 1a - A Typical Standard Curve for the Anti-dsDNA IgG Antibody ELISA Kit
10.00
R² = 0.996

OD 450 nm

1.00

0.100.01
110100dsDNA concentration (ng/ml)

Figure 1b - A Typical Standard Curve for the Anti-ssDNA IgG Antibody ELISA Kit
10.00
R² = 0.9987

OD 450 nm

1.00

0.100.01
110100ssDNA concentration (ng/ml)© 2020 Chondrex, Inc. All Rights Reserved, 3031, 3041 4.016928 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-dsDNA IgG Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

1.6 ng/ml
9.4
9.9
104.0 %

6.3 ng/ml
8.6
9.5
106.0 %

25 ng/ml
6.3
3.9
93.4 %

Table 2 - - Reproducibility Data for the Mouse Anti-ssDNA IgG Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
25 ng/ml
Intra-Assay CV (%)
9.89.5
7.5Inter-Assay CV (%)
3.8
4.2
8.1
Spike Test* (%)
101.4 %
97.6 %
94.0 %* Standard was added to known amounts of anti-dsDNA or anti-ssDNA antibodies and then diluted with Sample/Standard/Secondary
Antibody Dilution Buffer to assay anti-dsDNA or anti-ssDNA IgG antibodies by ELISA.

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

REFERENCES
1.

D. Koffler, V. Agnello, R. Winchester, H. G. Kunkel, The occurrence of single-stranded DNA in the serum of patients with systemic lupus
erythematosus and other diseases. J Clin Invest 52, 198-204 (1973).

2.

H. Sarvas, M. Gripenberg, M. Leirisalo-Repo, Anti-DNA antibodies: the choice of assays for routine diagnostic work. Acta Pathol
Microbiol Immunol Scand C 93, 13-18 (1985).

3.

D. Adu, J. Dobson, D. G. Williams, DNA-anti-DNA circulating complexes in the nephritis of systemic lupus erythematosus. Clin ExpImmunol 43, 605-614 (1981).

4.

H. Krippner, S. Merle, K. Jörgens, K. Pirlet, Antibodies to dsDNA and ssDNA in the immunoglobulin classes IgG and IgM: prognostic
value in the course of SLE. Z Rheumatol 43, 265-271 (1984).

5.

M. Okamura et al., Significance of enzyme linked immunosorbent assay (ELISA) for antibodies to double stranded and single stranded
DNA in patients with lupus nephritis: correlation with severity of renal histology. Ann Rheum Dis 52, 14-20 (1993).

6.

N. Talal, Disordered immunologic regulation and autoimmunity. Birth Defects Orig Artic Ser 14, 197-207 (1978).

7.

D. Perry, A. Sang, Y. Yin, Y. Y. Zheng, L. Morel, Murine models of systemic lupus erythematosus. J Biomed Biotechnol 2011, 271694(2011).

8.

D. M. Tillman, N. T. Jou, R. J. Hill, T. N. Marion, Both IgM and IgG anti-DNA antibodies are the products of clonally selective B cellstimulation in (NZB x NZW)F1 mice. J Exp Med 176, 761-779 (1992).

9.

M. Satoh, A. Kumar, Y. S. Kanwar, W. H. Reeves, Anti-nuclear antibody production and immune-complex glomerulonephritis inBALB/c mice treated with pristane. Proc Natl Acad Sci U S A 92, 10934-10938 (1995).

© 2020 Chondrex, Inc. All Rights Reserved, 3031, 3041 4.0
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