1LPS from E. coli (O111:B4) Detection ELISA Kit
Catalog # 6039
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify E. coli (O111:B4) LPS levels in samples

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

5.5 hours

STANDARD RANGE:

4 ng/ml to 0.06 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:1 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C

VALIDATION DATA:

Intra-assay (2.6-6.6%)/Inter-assay (4.6-7.7%)/Spiking Test (106-110%)NOTES:© 2021 Chondrex, Inc. All Rights Reserved, 6039 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

2LPS from E. coli (O111:B4) Detection ELISA Kit
Catalog # 6039
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Lipopolysaccharide (LPS) is a major component of the outer-membrane in gram-negative bacteria. While the main role of LPS is to maintainthe integrity of the bacteria envelope, LPS also binds to the CD14/TLR4/MD2 receptor complex on many cell types (1,2), such as B cells,
macrophages, and dendritic cells, resulting in pro-inflammatory cytokine release from these cells (3). This activation of the innate immune
response causes inflammation which may play important roles in obesity and chronic inflammatory diseases such as Crohn’s Disease,
Chronic Fatigue Syndrome, Ulcerative Colitis, and Rheumatoid Arthritis. Translocation of bacteria toxins from intestinal flora may also trigger
the pathogenesis of autoimmune diseases (4,5). To examine the possible link between pathogenic toxins from intestinal bacteria andautoimmune diseases, Chondrex Inc. provides an LPS (E. coli O111:B4) detection kit as well as ELISA kits for detecting staphylococcal
enterotoxins A and B (Catalog # 6029 & 6030). For further requests and consultation, please visit www.chondrex.com or contact Chondrex,Inc. Customer Support at support@chondrex.com.
NOTE: Because E. coli (O111:B4) may not be a natural resident in SPF mouse guts, inoculation of the E. coli or its LPS (i.e. immunizationor oral feeding) may be required for animal studies. Chondrex, Inc. recommend testing LPS levels in naive animals to determine baseline
LPS levels that can result from different vendors, animal strains, and housing conditions.

KIT COMPONENTS
Item

Quantity

AmountStorage

LPS from E. Coli (O111:B4) Standard (60391)
Detection Antibody (60393)

1 vial1 vial4 ng, lyophilized
50 µl-20°C-20°C

Solution B - Sample/Standard/Detection Antibody Dilution Buffer (67015)
Solution D – Streptavidin Peroxidase Dilution Buffer (9055)
Streptavidin Peroxidase (9029)
TMB (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)

1 Bottle
1 Bottle
2 vials
2 vials
1 Bottle
1 Bottle

50 ml
20 ml
50 µl
0.2 ml
20 ml
10 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Wash Buffer, 20X (9005)
Capture Antibody Coated 96-Well ELISA Plate (Purple)

1 Bottle
1 each50 ml
8-well Strips x12-20°C-20°C© 2021 Chondrex, Inc. All Rights Reserved, 6039 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

3ASSAY OUTLINE
Add 100 ml of diluted standards and samples into wells

Incubate at room temperature for 2 hours.
Wash plate.
Add 100 ml of diluted Detection Antibody

Incubate at room temperature for 2 hours.
Wash plate.
Add 100 ml of diluted Streptavidin Peroxidase

Incubate at room temperature for 1 hour.
Wash plate.
Add 100 ml of TMB solution

Incubate at room temperature for 25 minutes.

Add 50 ml of Stop Solution

Read plates at 450 nm/630 nm

PLATE LAYOUT
123456789101112A

44

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

22

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C

11S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

0.50.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

0.25

0.25

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

0.120.12

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G0.06

0.06

S7S7S15S15S23

S23

S31S31S39S39HBB

S8S8S16

S16S24

S24S32S32

S40S40StandardsSamples© 2021 Chondrex, Inc. All Rights Reserved, 6039 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

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 for
disposal.

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 0.06 - 4 ng/ml. Dissolve one vial of LPS standard in 1 ml ofSample/Standard/Detection Antibody Dilution Buffer (Solution B) for the 4 ng/ml standard. Then serially dilute it with Solution B. Forexample, mix 250 µl of the standard (4 ng/ml) with an equal volume of Solution B to make a 2 ng/ml solution, and then repeat it five
more times for 1, 0.5, 0.25, 0.12, and 0.06 ng/ml solutions. The remaining 4 ng/ml standard stock may be stored at –20°C for use in asecond assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 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 ml4 ng/ml

2 ng/ml

1 ng/ml

0.5 ng/ml

0.25 ng/ml

0.12 ng/ml

0.06 ng/ml

Blank

1

2.

Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids. Dilute thesupernatants with an equal volume of Solution B. For example, take 100 µl of supernatant, and mix with 100 µl of Solution B. If the LPS
level is higher than 4 ng/ml, re-assay the sample at a higher dilution.
NOTE 1: Buffer used to prepare the original samples may be contaminated with LPS. Chondrex, Inc. recommends assaying the buffer
to avoid overestimated assay results.
NOTE 2: Dilute samples at least 1:1 with Solution B depending on the estimated LPS level in the samples. Two to three different sample
dilutions are recommended if the LPS levels in the samples are unknown.

3.

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.

© 2021 Chondrex, Inc. All Rights Reserved, 6039 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

54.

Dilute Wash Buffer: Dilute 50 ml of Wash Buffer, 20X 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 Detection Antibody Solution: Dilute one vial of Detection Antibody in 10 ml of Sample/Standard/Detection Antibody Dilution
Buffer (Solution B). Add 100 µl of detection antibody solution to each well and incubate at room temperature for 2 hours.
Strip #2
46
81012Detection Antibody (µl)
8.5
1725
33
4150Solution B (ml)
1.7
3.3
5.0
6.6
8.2
10.0

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 Streptavidin Peroxidase Solution: Dilute one vial of streptavidin peroxidase solution in 10 ml of 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 #2
4681012Streptavidin Peroxidase (µl)
8.51725334150

Solution D (ml)
1.7
3.3
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. Dilute one vial of TMB in 10 ml of Chromogen Dilution Buffer. Add100 µl of TMB solution to each well immediately after washing the plate and incubate for 25 minutes at room temperature.
Strip #24681012TMB (µl)
3466100132
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. 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.

© 2021 Chondrex, Inc. All Rights Reserved, 6039 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

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 showsa representative experiment where the standard range is 0.06 - 4 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 original sample specimens.

Figure 1 - A Typical Standard Curve for the LPS Detection ELISA Kit

10.0
R² = 0.9961

OD 450 nm

1.0

0.1

0.0

0.01

0.1

110LPS Concentration (ng/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the LPS Detection ELISA Kit
Test
0.13 ng/ml
0.5 ng/ml
2 ng/ml
Intra-Assay CV (%)
6.6
5.1
2.6
Inter-Assay CV (%)
5.2
7.7
4.6
Spike Test* (%)
110%
109%
106%
* Known amounts of LPS were added to samples and then diluted with Sample/Standard/Detection Antibody Dilution Buffer (Solution B).

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

© 2021 Chondrex, Inc. All Rights Reserved, 6039 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

REFERENCES
1.

Poltorak, X. He, I. Smirnova, M. Liu, H. Van, et al., Defective LPS Signaling in C3H/HeJ and C57BL/10ScCr Mice: Mutations in Tlr4
Gene. Science 282, 2085-8 (1998).

2.

R Shimazu, S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, et. al, MD-2, a molecule that confers lipopolysaccharide
responsiveness on Toll-like receptor 4. J Exp Med 189,1777-82 (1999).

3.

Ulmer, H. Flad, T. Rietschel, T. Mattern, Induction of Proliferation and Cytokine Production in Human T Lymphocytes by
Lipopolysaccharide (LPS). Toxicology 152, 37-45 (2000).

4.

K. Terato, T. Waritani, R. Fukai, H. Shionoya, H. Itoh, K. Katayama, et al., Contribution of Bacterial Pathogens to Evoking Serological
Disease Markers and Aggravating Disease Activity in Rheumatoid Arthritis. PLoS One 13, e0190588 (2018).

5.

K. Terato, C. Do, H. Shionoya, Slipping Through the Cracks: Linking Low Immune Function and Intestinal Bacterial Imbalance to theEtiology of Rheumatoid Arthritis. Autoimmune Dis 2015, 636207 (2015).

© 2021 Chondrex, Inc. All Rights Reserved, 6039 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