Lipopolysaccharides (LPS) ELISA Kit ELISA Kit
- Lipopolysaccharides (LPS)
- Detection Method
- Method Type
- Competition ELISA
- Detection Range
- 12.35 ng/mL - 1000 ng/mL
- Minimum Detection Limit
- 12.35 ng/mL
The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of lipopolysaccharide in serum, plasma, tissue homogenates, cell lysates, cell culture supernates.
We offer validation data (WB) for each of the kit components. So you can be sure to order a reliable ELISA kit product composed of high quality reagents.
- Sample Type
- Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
- Analytical Method
- This assay has high sensitivity and excellent specificity for detection of Lipopolysaccharide (LPS)
- Cross-Reactivity (Details)
- No significant cross-reactivity or interference between Lipopolysaccharide (LPS) and analogues was observed.
- 5.15 ng/mL
- Pre-coated, ready to use 96-well strip plate
- Plate sealer for 96 wells
- Standard Diluent
- Detection Reagent A
- Detection Reagent B
- Assay Diluent A
- Assay Diluent B
- TMB Substrate
- Stop Solution
- Wash Buffer (30 x concentrate)
- Instruction manual
- Material not included
- Microplate reader with 450 ± 10nm filter.
- Precision single or multi-channel pipettes and disposable tips.
- Eppendorf Tubes for diluting samples.
- Deionized or distilled water.
- Absorbent paper for blotting the microtiter plate.
- Container for Wash Solution
- 0.01Mol/L (or 1x) Phosphate Buffered Saline(PBS), pH 7.0-7.2.
- Discover our best selling ELISA Kit
- Application Notes
- Limited by the current condition and scientific technology, we cannot completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
- The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
- Kits from different batches may be a little different in detection range, sensitivity and color developing time.
- Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
- Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.
- There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
- Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
- Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
- Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
- Kits from different manufacturers for the same item might produce different results, since we have not compared our products with other manufacturers.
Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.
Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.
Information on antibodies:
The provided antibodies and their host vary in different kits.
- Sample Volume
- 50 μL
- Assay Time
- 2 h
- Prepare all reagents, samples and standards,
- Add 50μL standard or sample to each well.
And then add 50μL prepared Detection Reagent A immediately.
Shake and mix. Incubate 1 hour at 37 °C,
- Aspirate and wash 3 times,
- Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
- Aspirate and wash 5 times,
- Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
- Add 50μL Stop Solution. Read at 450 nm immediately.
- Reagent Preparation
- Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
- Standard - Reconstitute the Standard with 0.5 mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 1,000 ng/mL. Please prepare 5 tubes containing 0.6 mL Standard Diluent and produce a triple dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted standard such as 1,000 ng/mL, 333.33 ng/mL, 111.11 ng/mL, 37.04 ng/mL, 12.35 ng/mL, and the last EP tubes with Standard Diluent is the blank as 0 ng/mL.
- Detection Reagent A - Reconstitute the Detection Reagent A with 150μL of Reagent Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). Dilute to the working concentration with Assay Diluent A (1:100).
- Detection Reagent B - Briefly spin or centrifuge the stock Detection B before use. Dilute to the working concentration with Assay Diluent B (1:100).
- Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
- TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
- Making serial dilution in the wells directly is not permitted.
- Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
- Detection Reagent A and B are sticky solutions, therefore, slowly pipette them to reduce the volume errors.
- Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for one pipetting.
- The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
- If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
- Contaminated water or container for reagent preparation will influence the detection result.
- Assay Procedure
- Determine wells for diluted standard, blank and sample. Prepare wells for standard points and blank. Add 50μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells, respectively. And then add 50μL of Detection Reagent A to each well immediately. Shake the plate gently (using a microplate shaker is recommended). Cover with a Plate sealer. Incubate for 1 hour at 37 °C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
- Aspirate the solution and wash with 350μL of 1X Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
- Add 100μL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37 °C after covering it with the Plate sealer.
- Repeat the aspiration/wash process for total 5 times as conducted in step 2.
- Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at 37 °C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution.
- Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
- Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately.
- Assay preparation: Keep appropriate numbers of wells for each experiment and remove extra wells from microplate. Rest wells should be resealed and stored at -20 °C.
- Samples or reagents addition:Please use the freshly prepared Standard. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of standards, samples, and reagents. Also, use separated reservoirs for each reagent.
- Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be controlled.
- Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and false elevated absorbance reading.
- Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.
- TMB Substrate is easily contaminated. Please protect it from light.
- The environment humidity which is less than 60 % might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.
- Calculation of Results
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between target concentration in the sample and the assay signal intensity. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log or semi-log graph paper, with the log of target concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the log of concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. The O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects). Typical standard curve below is provided for reference only.
- Assay Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level of target were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level of target were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV < 10%
Inter-Assay: CV < 12%
- For Research Use only
Validation #103828 (ELISA)Validation ImagesFull Methods
- Nephrology Laboratory, Rush University Medical Center
- Lot Number
- Method validated
- Positive Control
Serum from mice treated with LPS
- Negative Control
Naïve serum samples from wt mice and mice knocked-out for gene relevant for our research
Passed, the LPS ELISA kit ABIN6574100 specifically detects LPS in mice serum.
- Primary Antibody
- Secondary Antibody
- Full Protocol
- Treat wt and ko mouse with LPS by intraperitoneal injection.
- Collect the urine and serum 1h, 3h, and 6h after LPS injection.
- Prepare all reagents and standards according to the manufacturer’s protocol.
- Dilute samples 10x.
- Add 50μl standard or sample to each well.
- Add 50μl prepared Detection Reagent A immediately.
- Shake and mix.
- Cover plate with plate sealer and incubate for 1h at 37 °C.
- Aspirate and wash 3x with 350µl 1x Wash Solution.
- Add 100μl prepared Detection Reagent B.
- Cover plate with plate sealer and Incubate for 30min at 37°C.
- Aspirate and wash 5x with 350µl 1x Wash Solution.
- Add 90μL Substrate Solution.
- Cover plate with plate sealer and incubate 10min at 37°C.
- Add 50μL Stop Solution and read plate at 450nm immediately.
- Experimental Notes
LPS in urine is undetectable even though the spike controls work well in urine. Possibly, the 1:10 dilution of the urine samples caused the LPS concentration to fall below the kit's detection limit. However, it was not possible to run undiluted urine samples because the mice got sick and had less urine after LPS treatment.
When measuring LPS in urine samples the LPS ELISA kit ABIN6574100 proved to be superior to the Endotoxin Assay Kit ABIN491527. Unlike ABIN6574100, ABIN491527 did not work with the spike control in urine.
- Precaution of Use
- The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
- Handling Advice
The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.
- 4 °C,-20 °C
- Storage Comment
- For unused kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon receipt while the others should be at 4 °C.
- For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch.
It is highly recommended to use the remaining reagents within 1 month provided this is prior to the expiration date of the kit. For the expiration date of the kit, please refer to the label on the kit box. All components are stable up to the expiration date.
- Expiry Date
- 6 months
Kiang, Smith, Cannon, Anderson, Ho, Zhai, Cui, Xiao: "Ghrelin, a novel therapy, corrects cytokine and NF-κB-AKT-MAPK network and mitigates intestinal injury induced by combined radiation and skin-wound trauma." in: Cell & bioscience, Vol. 10, pp. 63, 2020 (PubMed).
Sánchez-Tapia, Aguilar-López, Pérez-Cruz, Pichardo-Ontiveros, Wang, Donovan, Tovar, Torres: "Nopal (Opuntia ficus indica) protects from metabolic endotoxemia by modifying gut microbiota in obese rats fed high fat/sucrose diet." in: Scientific reports, Vol. 7, Issue 1, pp. 4716, 2019 (PubMed).
Chen, Shen, Wang, Ding, Zhao, Wang, Fu, Wang: "Megasphaera elsdenii Lactate Degradation Pattern Shifts in Rumen Acidosis Models." in: Frontiers in microbiology, Vol. 10, pp. 162, 2019 (PubMed).
Bluemel, Wang, Martino, Lee, Wang, Williams, Horvath, Stadlbauer, Zengler, Schnabl: "The Role of Intestinal C-type Regenerating Islet Derived-3 Lectins for Nonalcoholic Steatohepatitis." in: Hepatology communications, Vol. 2, Issue 4, pp. 393-406, 2018 (PubMed).
Feng, Wang, Zhang, Gao, Tao, Ge, Zhu, Bi: "Modulation of gut microbiota contributes to curcumin-mediated attenuation of hepatic steatosis in rats." in: Biochimica et biophysica acta, Vol. 1861, Issue 7, pp. 1801-1812, 2017 (PubMed).
Rosas-Villegas, Sánchez-Tapia, Avila-Nava, Ramírez, Tovar, Torres: "Differential Effect of Sucrose and Fructose in Combination with a High Fat Diet on Intestinal Microbiota and Kidney Oxidative Stress." in: Nutrients, Vol. 9, Issue 4, 2017 (PubMed).
Hartmann, Seebauer, Mazagova, Horvath, Wang, Llorente, Varki, Brandl, Ho, Schnabl: "Deficiency of intestinal mucin-2 protects mice from diet-induced fatty liver disease and obesity." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 310, Issue 5, pp. G310-22, 2016 (PubMed).
Wosiewicz, Żorniak, Hartleb, Barański, Hartleb, Onyszczuk, Pilch-Kowalczyk, Kyrcz-Krzemień: "Portal vein thrombosis in cirrhosis is not associated with intestinal barrier disruption or increased platelet aggregability." in: Clinics and research in hepatology and gastroenterology, 2016 (PubMed).
Gao, Wen, Tong, Wang, Yang, Tang, Yan, Tai, Ye, Liu, Huang, Tang, Yang, Tang: "Inhibition of cyclooxygenase-2 alleviates liver cirrhosis via improvement of the dysfunctional gut-liver axis in rats." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 310, Issue 11, pp. G962-72, 2016 (PubMed).
Zhao, Zhang, Chai, Li, Cui, Wang, Meng, Liu, Wang, Li, Bai, Xiao: "Oxymatrine attenuates CCl4-induced hepatic fibrosis via modulation of TLR4-dependent inflammatory and TGF-β1 signaling pathways." in: International immunopharmacology, Vol. 36, pp. 249-55, 2016 (PubMed).
Avila-Nava, Noriega, Tovar, Granados, Perez-Cruz, Pedraza-Chaverri, Torres: "Food combination based on a pre-hispanic Mexican diet decreases metabolic and cognitive abnormalities and gut microbiota dysbiosis caused by a sucrose-enriched high-fat diet in rats." in: Molecular nutrition & food research, Vol. 61, Issue 1, 2016 (PubMed).
Keshavarzi, Khaksari: "The effects of female sexual steroids on gastric function and barrier resistance of gastrointestinal tract following traumatic brain injury." in: Journal of pharmacy & bioallied sciences, Vol. 7, Issue 1, pp. 75-80, 2015 (PubMed).
Karl, Fu, Wang, Zhao, Shen, Zhang, Wolfe, Saltzman, Zhao, Booth: "Fecal menaquinone profiles of overweight adults are associated with gut microbiota composition during a gut microbiota-targeted dietary intervention." in: The American journal of clinical nutrition, Vol. 102, Issue 1, pp. 84-93, 2015 (PubMed).
Han, Suk, Kim, Kim, Baik, Kim, Cheon, Choi, Ham, Shin, Kim: "Effects of probiotics (cultured Lactobacillus subtilis/Streptococcus faecium) in the treatment of alcoholic hepatitis: randomized-controlled multicenter study." in: European journal of gastroenterology & hepatology, Vol. 27, Issue 11, pp. 1300-6, 2015 (PubMed).
Barros, Borges, Ferreira, Carmo, Rosado, Fouque, Mafra: "Is there interaction between gut microbial profile and cardiovascular risk in chronic kidney disease patients?" in: Future microbiology, Vol. 10, Issue 4, pp. 517-26, 2015 (PubMed).
Plaza-Diaz, Gomez-Llorente, Abadia-Molina, Saez-Lara, Campaña-Martin, Muñoz-Quezada, Romero, Gil, Fontana: "Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on hepatic steatosis in Zucker rats." in: PLoS ONE, Vol. 9, Issue 5, pp. e98401, 2014 (PubMed).
Chen, Zhang, Dang, Liu, Tian, Zhao, Chen, Zhang, Chen: "Antidiabetic effect of Lactobacillus casei CCFM0412 on mice with type 2 diabetes induced by a high-fat diet and streptozotocin." in: Nutrition (Burbank, Los Angeles County, Calif.), Vol. 30, Issue 9, pp. 1061-8, 2014 (PubMed).
He, Wang, Wu, Gong, Hong, Xia, Zhang, Bao, Gao: "Immunological activity difference between native calreticulin monomers and oligomers." in: PLoS ONE, Vol. 9, Issue 8, pp. e105502, 2014 (PubMed).
Gram, Sveinsdottir, Ruscher, Hansson, Cinthio, Akerström, Ley: "Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation." in: Journal of neuroinflammation, Vol. 10, pp. 100, 2013 (PubMed).
Wang, Wang, Moreno-Vinasco, Lang, Siegler, Mathew, Usatyuk, Samet, Geyh, Breysse, Natarajan, Garcia: "Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation." in: Particle and fibre toxicology, Vol. 9, pp. 35, 2012 (PubMed).
- Kiang, Smith, Cannon, Anderson, Ho, Zhai, Cui, Xiao: "Ghrelin, a novel therapy, corrects cytokine and NF-κB-AKT-MAPK network and mitigates intestinal injury induced by combined radiation and skin-wound trauma." in: Cell & bioscience, Vol. 10, pp. 63, 2020 (PubMed).
- Lipopolysaccharides (LPS)
- Alternative Name
- Lipopolysaccharide (LPS) ()
- Target Type