Lipopolysaccharides (LPS) Kit ELISA

N° du produit ABIN6574100

Détail du produit Lipopolysaccharides (LPS) Kit ELISA

Antigène: Lipopolysaccharides (LPS)

Reactivité: Différentes espèces

Méthode de détection: Colorimetric

Type de méthode: Competition ELISA

Gamme de detection: 12.35 ng/mL - 1000 ng/mL

Seuil minimal de détection: 12.35 ng/mL

Application: ELISA

Fonction: 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 the kit components. So you can be sure to order a reliable ELISA kit product composed of high quality reagents.

Type d'échantillon: Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate

Analytical Method: Quantitative

Specificité: This assay has high sensitivity and excellent specificity for detection of Lipopolysaccharide (LPS)

Réactivité croisée (Details): No significant cross-reactivity or interference between Lipopolysaccharide (LPS) and analogues was observed.

Sensibilité: 5.41 ng/mL

Ingrédients:

• Pre-coated, ready to use 96-well strip plate, flat buttom
• Plate sealer for 96 wells
• Reference Standard
• Standard Diluent
• Detection Reagent A
• Detection Reagent B
• Assay Diluent A
• Assay Diluent B
• Reagent Diluent (if Detection Reagent is lyophilized)
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

Information d'application

Commentaires:

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.

Volume d'échantillon: 50 μL

Durée du test: 2 h

Plaque: Pre-coated

Protocole:

1. Prepare all reagents, samples and standards,
2. Add 50μL standard or sample to each well.
   Then add 50μL prepared Detection Reagent A immediately.
   Shake and mix. Incubate 1 hour at 37 °C,
3. Aspirate and wash 3 times,
4. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
5. Aspirate and wash 5 times,
6. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
7. Add 50μL Stop Solution. Read at 450 nm immediately.

Préparation des réactifs:

1. 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.
2. 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.
3. Detection Reagent A and Detection Reagent B - If lyophilized reconstitute the Detection Reagent A with 150μL of Reagent Diluent, kept for 10 minutes at room temperature, shake gently (not to foam). Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively.
4. 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).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:

1. Making serial dilution in the wells directly is not permitted.
2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
3. Detection Reagent A and B are sticky solutions, therefore, slowly pipette them to reduce the volume errors.
4. 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.
5. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
6. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
7. Contaminated water or container for reagent preparation will influence the detection result.

Préparation de l'échantillon:

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturationmay occur in these samples, leading to false results. Samples should therefore be stored for a short periodat 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thawcycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged toremove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determinecompatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibilityof causing a deviation due to the introduced chemical substance.The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in therange of the standard curve, the optimal sample dilution for the particular experiment has to be determined.Samples should then be diluted with PBS (pH =7.0-7.2).

Précision du teste: 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%

Restrictions: For Research Use only

Independent Validation (ELISA)

Validation #103828 (ELISA)

by: Nephrology Laboratory, Rush University Medical Center

No.: #103828

Date: 01.05.2019

Antigène: LPS

Numéro du lot: L190202120

Application validée: ELISA

Contrôle positif:

Serum from mice treated with LPS

Contrôle négative:

Naïve serum samples from wt mice and mice knocked-out for gene relevant for our research

Conclusion:

Passed, the LPS ELISA kit ABIN6574100 specifically detects LPS in mice serum.

Validation Images

A. Standard curve and sample measurements using ABIN6574100. B. The standard curves for the assays reported were fitted with a polynomial curve, giving a good quality fit (R2=0.9897).

Protocole

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.

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.

Stockage

Précaution d'utilisation: The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Stock: 4 °C/-20 °C

Stockage commentaire:

1. For unopened kit: All reagents should be stored according to the labels on the vials. The Standard, Detection Reagent A, Detection Reagent B, and 96-well Strip Plate should be stored at -20 °C upon receipt, while the other reagents should be stored at 4 °C.
2. For opened kits: the remaining reagents must be stored according to the above storage conditions. In addition, please return the unused wells to the foil pouch containing the desiccant and seal the foil pouch with the zipper.

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Date de péremption: 6 months

Références pour Lipopolysaccharides (LPS) Kit ELISA (ABIN6574100)

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Détail du antigène

Antigène: Lipopolysaccharides (LPS)

Autre désignation: Lipopolysaccharide (LPS) (Lipopolysaccharides (LPS) Produits)

Classe de substances: Chemical