1

Mouse anti-Lipopolysaccharide from Porphyromonas gingivalis Antibody ELISA Kits
Catalog # 6222, 6223, and 6224
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify mouse anti-PG LPS IgG/IgM/IgG3 antibodies
6222: Mouse Anti-PG LPS IgG Antibody ELISA Kit
6223: Mouse Anti-PG LPS IgM Antibody ELISA Kit
6224: Mouse Anti-PG LPS IgG3 Antibody ELISA Kit

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

6222/6224: 500 ng/ml to 8 ng/ml
6223: 100 ng/ml 1.6 ng/ml

NUMBER OF SAMPLES:

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

SAMPLE TYPES:

Serum and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:200 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C

VALIDATION DATA:

6222: Intra-Assay (5.6-9.5%)/Inter-Assay (1.8-5.2%)/Spiking Test (98-105%)
6223: Intra-Assay (2.7-4.1%)/Inter-Assay (2.9-9.5%)/Spiking Test (90-97%)6224: Intra-Assay (3.5-7%)/Inter-Assay (5.7-9.4%)/Spiking Test (98-108%)

NOTES:

N/A© 2020 Chondrex, Inc. All Rights Reserved, M PG LPS 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-Lipopolysaccharide from Porphyromonas gingivalis Antibody ELISA Kits
Catalog # 6222, 6223, and 6224
For Research Use Only - Not Human or Therapeutic Use
INTRODUCTION
Recent studies indicate that environmental factors, especially intestinal microbiota and their toxins, may play roles in the development of
autoimmune diseases such as rheumatoid arthritis (RA) (1-7), inflammatory bowel diseases (IBDs) (8, 9), systemic lupus erythematosus(SLE) (10), and other chronic disorders (11-13). In fact, germ-free mice fail to develop ankylosing enthesopathy, a naturally occurring joint
disease in mice with many parallels to human ankylosing spondylitis (15). Moreover, several studies indicate that increased intestinal mucosal
permeability due to stress, surgery, and minor gastrointestinal disorders such as irregularity, may contribute to excess translocation of gut
bacteria and their toxins into the body (16-18). These events suggest the pathological importance of intestinal bacteria with regard toautoimmune disorders.
Among the many environmental factors, gram-negative bacteria and their toxins, such as Porphyromonas gingivalis (P. gingivalis),
Escherichia coli (E. coli), and lipopolysaccharide (LPS), induce inflammation by increasing levels of inflammatory cytokines, and may be
involved in various types of chronic autoimmune disorders. In fact, chronic P. gingivalis oral infection prior to arthritis induction increases thelikelihood of Th17 cell responses, ultimately accelerating collagen-induced arthritis (CIA) development in mice (19). Interestingly, orally
administered P. gingivalis also perturbs gut microbiomes in CIA mice, affecting the gut immune system and the gut microbiota composition(20). Although intraperitoneal E. coli-LPS combined with sub-arthritogenic levels of anti-type II collagen antibodies induces severe arthritis
in mice, mice who were instead given LPS from P. gingivalis (PG-LPS) failed to develop arthritis (21). Thus, the contribution of these factors
in autoimmune disorders requires more studies.
In order to advance these microbiome-related research fields, Chondrex, Inc. provides mouse anti- P. gingivalis (33277) and P. gingivalis LPS (33277) antibody ELISA kits to elucidate the host immune response to potential environmental pathogens. For a complete list of available
mouse anti-bacteria and bacterial toxins antibody ELISA kits, such as E. coli (O111:B4), E. coli -LPS (O111:B4), and staphylococcal
enterotoxins, please visit www.chondrex.com or contact us at support@chondrex.com.

KIT COMPONENTS
Item
Standard
IgG (62221)
IgM (62231)
IgG3 (62241)
Secondary Antibody (peroxidase-conjugated polyclonal antibodies)
IgG (62223)
IgM (62233)
IgG3 (62243)
Solution A - Blocking Buffer (61026)
Solution B - Sample/Standard Dilution Buffer (61027)
Solution C - Detection Antibody Dilution Buffer (61025)
TMB (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
PG-LPS Coated 96-Well ELISA Plate (Pink)

Quantity

AmountStorage

1 vialLyophilized
IgG/IgG3: 500 ng
IgM: 100 ng-20°C

2 vials

50 µl

-20°C

1 bottle
1 bottle
1 bottle
2 vials

10 ml
50 ml
20 ml
0.2 ml

-20°C
-20°C
-20°C
-20°C

1 bottle
1 bottle
1 bottle
1 each

20 ml
10 ml
50 ml
8-well Strips x12

-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 PG LPS 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
E. coli LPS (O111:B4)
E. coli
P. gingivalis LPS (PG-LPS)
P. gingivalis (PG)

Color Coding
Red
Yellow
Pink
OrangeASSAY OUTLINE© 2020 Chondrex, Inc. All Rights Reserved, M PG LPS 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

PLATE MAPPING
Map the plate based on the number of samples and sample dilution. For example, if sample dilution is higher than 1:200, it is not necessary
to run antigen un-coated wells, but if sample dilution is less than 1:200, it may be necessary to run antigen uncoated wells to determine thebackground noise reaction OD values of individual samples. An antigen uncoated plate (Catalog # 9026) for lower sample dilutions is notincluded. Please contact support@chondrex.com to place an order.
Standard Layout of Antigen Coated Plate

Standard Layout of Antigen Coated and Uncoated Plate

1

2

3

4

5

6

7

8

9

10

11

12

A

500

500

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

250

250

S2

S2

S10

S10

S18

S18

S26

S26

S34

C

125

125

S3

S3

S11

S11

S19

S19

S27

S27

D

63

63

S4

S4

S12

S12

S20

S20

S28

E

31

31

S5

S5

S13

S13

S21

S21

F

16

16

S6

S6

S14

S14

S22

G

8

8

S7

S7

S15

S15

H

B

B

S8

S8

S16

S16

Standards

1

2

3

4

5

6

7

8

9

10

11

12

A

500

500

S1

S1

SB1

SB1

S9

S9

SB9

SB9

S17

S17

S34

B

250

250

S2

S2

SB2

SB2

S10

S10

SB10

SB10

S18

S18

S35

S35

C

125

125

S3

S3

SB3

SB3

S11

S11

SB11

SB11

S19

S19

S28

S36

S36

D

63

63

S4

S4

SB4

SB4

S12

S12

SB12

SB12

S20

S20

S29

S29

S37

S37

E

31

31

S5

S5

SB5

SB5

S13

S13

SB13

SB13

SB17

SB17

S22

S30

S30

S38

S38

F

16

16

S6

S6

SB6

SB6

S14

S14

SB14

SB14

SB18

SB18

S23

S23

S31

S31

S39

S39

G

8

8

S7

S7

SB7

SB7

S15

S15

SB15

SB15

SB19

SB19

S24

S24

S32

S32

S40

S40

H

B

B

S8

S8

SB8

SB8

S16

S16

SB16

SB16

SB20

SB20

Standards

Samples

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 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.

© 2020 Chondrex, Inc. All Rights Reserved, M PG LPS 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

5

ASSAY 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 1:200 or less is assayed, antigen non-coated strips should be used. Solution A must be added to
the 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). Incubate for 1
hour at room temperature.

2.

Prepare Standard Dilutions: Dissolve one vial of standard with 1 ml of Sample/Standard Dilution Buffer (Solution B) to make thehighest standard concentration - labeled “1” below. Prepare serial dilutions by mixing 250 µl of the 1X standard with 250 µl of Solution
B - labeled “2”. Then repeat this procedure to make five additional serial dilutions of standard. The 1X standard may be stored at -20°C
for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 ml
250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

2

3

4

5

6

7

8

1000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 mlIgG/IgG3

500 ng/ml

250 ng/ml

125 ng/ml

63 ng/ml

31 ng/ml

16 ng/ml

8 ng/ml

IgM

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

1

3.

Blank

Prepare 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 250 µl of the sample stock
solution and mix with 250 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 blotting 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. Incubate atroom 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).

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 Dilute one vial of secondary antibody solution with 10 ml of Secondary Antibody Dilution Buffer (Solution C).
Add 100 µl of secondary antibody solution to each well and incubate at room temperature for 1 hour.

© 2020 Chondrex, Inc. All Rights Reserved, M PG LPS 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

6

Strip #
2
4
6
8
10
12

Secondary Antibody (µl)
9
17
25
33
41
50

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, dilute one vial of TMB solution in 10 ml Chromogen
Dilution Buffer. The prepared TMB cannot be stored for the next assay. 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

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.

1.

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 8 - 500 ng/ml.

2.

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 sample specimens.

© 2020 Chondrex, Inc. All Rights Reserved, M PG LPS 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

7

Figure 1 - A Typical Standard Curve for the Mouse Anti-PG-LPS IgG Antibody ELISA Kit

VALIDATION DATA
Table 1 - Reproducibility data for Mouse Anti-PG LPS IgG Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

16 ng/ml
5.6
4.6
105%

63 ng/ml
8.6
1.8
100%

250 ng/ml
9.5
5.2
98%

12.5 ng/ml
4.1
3.8
95%

50 ng/ml
3.0
9.5
97%

63 ng/ml
7.0
5.7
103%

250 ng/ml
3.9
9.4
108%

Table 2 - Reproducibility data for Mouse Anti-PG IgM Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

3.1 ng/ml
2.7
2.9
90%

Table 3 - Reproducibility data for Mouse Anti-PG IgG3 Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

16 ng/ml
3.5
9.2
98%

* Known amounts of anti-PG LPS antibodies were added to standards and then diluted with Sample/Standard Dilution Buffer to assay antiPG antibodies

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 PG LPS 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

8

REFERENCES
1.

S. Aoki, K. Yoshikawa, T. Yokoyama, T. Nonogaki, S. Iwasaki, et al., Role of Enteric Bacteria in the Pathogenesis of Rheumatoid
Arthritis: Evidence for Antibodies to Enterobacterial Common Antigens in Rheumatoid Sera and Synovial Fluids. Ann Rheum Dis 55,
363-9 (1996).

2.

I. van der Heijden, B. Wilbrink, I. Tchetverikov, I. Schrijver, L. Schouls, et al., Presence of Bacterial DNA and Bacterial Peptidoglycans
in Joints of Patients With Rheumatoid Arthritis and Other Arthritides. Arthritis Rheum 43, 593-8 (2000).

3.

K. Terato, X. Ye, H. Miyahara, M. Cremer, M. Griffiths, Induction by Chronic Autoimmune Arthritis in DBA/1 Mice by Oral Administrationof Type II Collagen and Escherichia Coli Lipopolysaccharide. Br J Rheumatol 35, 828-38 (1996).

4.

R. Peltonen, M. Nenonen, T. Helve, O. Hänninen, P. Toivanen, E. Eerola, et al., Faecal Microbial Flora and Disease Activity inRheumatoid Arthritis During a Vegan Diet. Br J Rheumatol 36, 64-8 (1997).

5.

P. Toivanen, Normal Intestinal Microbiota in the Aetiopathogenesis of Rheumatoid Arthritis. Ann Rheum Dis 62, 807-11 (2003).

6.

J. Vaahtovuo, E. Munukka, M. Korkeamäki, R. Luukkainen, P. Toivanen, Fecal Microbiota in Early Rheumatoid Arthritis. J Rheumatol
35, 1500-5 (2008).

7.

C. Edwards, Commensal Gut Bacteria and the Etiopathogenesis of Rheumatoid Arthritis. J Rheumatol 35, 1477-14797 (2008).

8.

D. Shi, J. Das, G. Das, Inflammatory Bowel Disease Requires the Interplay Between Innate and Adaptive Immune Signals. Cell Res
16, 70-4 (2006).

9.

S. Nell, S. Suerbaum, C. Josenhans, The Impact of the Microbiota on the Pathogenesis of IBD: Lessons From Mouse Infection Models.
Nat Rev Microbiol 8, 564-77 (2010).

10. T. Cavallo, N. Granholm, Bacterial Lipopolysaccharide Induces Long-Lasting IgA Deficiency Concurrently With Features of Polyclonal
B Cell Activation in Normal and in Lupus-Prone Mice. Clin Exp Immunol 84, 134-8 (1991).
11. W. Penhale, P. Young, The Influence of the Normal Microbial Flora on the Susceptibility of Rats to Experimental Autoimmune Thyroiditis.
Clin Exp Immunol 72, 288-92 (1988).
12. M. Murakami, T. Tsubata, R. Shinkura, S. Nisitani, M. Okamoto, et al., Oral Administration of Lipopolysaccharides Activates B-1 Cells
in the Peritoneal Cavity and Lamina Propria of the Gut and Induces Autoimmune Symptoms in an Autoantibody Transgenic Mouse. J
Exp Med 180, 111-21 (1994).
13. M. Nymark, P. Pussinen, A. Tuomainen, C. Forsblom, P. Groop, et al., Serum Lipopolysaccharide Activity Is Associated With theProgression of Kidney Disease in Finnish Patients With Type 1 Diabetes. Diabetes Care 32, 1689-93 (2009).
14. J. Taurog, J. Richardson, J. Croft, W. Simmons, M. Zhou, et al., The Germfree State Prevents Development of Gut and Joint
Inflammatory Disease in HLA-B27 Transgenic Rats. J Exp Med 180, 2359-64 (1994).
15. Z. Reháková, J. Capková, R. Stĕpánková, J. Sinkora, A. Louzecká, et al., Germ-free Mice Do Not Develop Ankylosing Enthesopathy,
a Spontaneous Joint Disease. Hum Immunol 61, 555-8 (2000).
16. P. Anderlik, I. Szeri, Z. Bános, Z. Barna, Bacterial Translocation After Cold Stress in Young and Old Mice. Acta Microbiol Hung 37, 28994 (1990).
17. A. Velin, A. Ericson, Y. Braaf, C. Wallon, J. Söderholm, Increased Antigen and Bacterial Uptake in Follicle Associated Epithelium
Induced by Chronic Psychological Stress in Rats. Gut 53, 494-500 (2004).
18. I. Khalif, E. Quigley, E. Konovitch, I. Maximova, Alterations in the Colonic Flora and Intestinal Permeability and Evidence of Immune
Activation in Chronic Constipation. Dig Liver Dis 37, 838-49 (2005).
19. J. Marchesan, E. Gerow, R. Schaff, A. Taut, S. Shin, et al., Porphyromonas Gingivalis Oral Infection Exacerbates the Development
and Severity of Collagen-Induced Arthritis. Arthritis Res Ther 15, R186 (2013).
20. K. Sato, N. Takahashi, T. Kato, Y. Matsuda, M. Yokoji, et al., Aggravation of Collagen-Induced Arthritis by Orally Administered
Porphyromonas Gingivalis Through Modulation of the Gut Microbiota and Gut Immune System. Sci Rep 7, 6955 (2017).
21. K. Terato, D. Harper, M. Griffiths, D. Hasty, X. Ye, et al., Collagen-induced Arthritis in Mice: Synergistic Effect of E. Coli
Lipopolysaccharide Bypasses Epitope Specificity in the Induction of Arthritis With Monoclonal Antibodies to Type II Collagen.
Autoimmunity 22, 137-47 (1995).

© 2020 Chondrex, Inc. All Rights Reserved, M PG LPS 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