HMGB1 Kit ELISA

N° du produit ABIN578817

Détail du produit HMGB1 Kit ELISA

Antigène: HMGB1 (High Mobility Group Box 1 (HMGB1))

Reactivité: Rat

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 31.2-2000 pg/mL

Seuil minimal de détection: 31.2 pg/mL

Application: ELISA

Fonction: This immunoassay kit allows for the in vitro quantitative determination of rat high mobility group box-1 protein, HMGB1 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Type d'échantillon: Cell Culture Supernatant, Plasma, Serum

Analytical Method: Quantitative

Specificité: This assay recognizes recombinant and natural rat HMGB1.

Réactivité croisée (Details): No significant cross-reactivity or interference was observed.

Attributs du produit: Rattus norvegicus,Rat,High mobility group protein B1,Amphoterin,Heparin-binding protein p30,High mobility group protein 1,HMG-1,Hmgb1,Hmg-1,Hmg1

Ingrédients: Reagent (Quantity):

• Assay plate (1),
• Standard (2),
• Sample Diluent (1×20 mL),
• Assay Diluent A (1×10 mL),
• Assay Diluent B (1×10 mL),
• Detection Reagent A (1×120 μL),
• Detection Reagent B (1×120 μL),
• Wash Buffer(25 x concentrate) (1×30 mL),
• Substrate (1×10 mL),
• 2 Stop Solution (1×10 mL),
• Plate sealer for 96 wells (5),
• Instruction (1)

Matériel non inclus: Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water.

Information d'application

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole: The microtiter plate provided in this kit has been pre-coated with an antibody specific to HMGB1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for HMGB1 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain HMGB1, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of HMGB1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Préparation des réactifs:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard. The Sample Diluent serves as the zero standard (0 ng/ml).

Prélèvement de l'échantillon: Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.

Procédure de l'essai:

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 °C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 °C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
1. Add 100 μL of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 °C .
2. Remove the liquid of each well, don ’ t wash.
3. Add 100 μL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 °C . Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 μL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 °C .
6. Repeat the aspiration/wash as in step 4.
7. Add 90 μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37 °C . Protect from light.
8. Add 50 μL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
Important Note:
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
3. 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 have been added to the well strips, DO NOT let the 5 strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.

Calcul des résultats:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the SAA concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Stock: 4 °C/-20 °C

Stockage commentaire: The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C.

Références pour HMGB1 Kit ELISA (ABIN578817)

Wang, Wang, Li, Han, Gong: "Ethyl pyruvate prevents inflammatory factors release and decreases intestinal permeability in rats with D-galactosamine-induced acute liver failure." dans: Hepatobiliary & pancreatic diseases international : HBPD INT, Vol. 12, Issue 2, pp. 180-8, (2013) (PubMed).

Wang, Wang, Chen, Fan, Li, He, Gong: "Ethyl pyruvate protects against experimental acute-on-chronic liver failure in rats." dans: World journal of gastroenterology, Vol. 18, Issue 40, pp. 5709-18, (2013) (PubMed).

Hou, Qin, Zheng, Lu, Wang, Peng, Yu, Xin, Ji, Xiong: "Severity of sepsis is correlated with the elevation of serum high-mobility group box 1 in rats." dans: Chinese medical journal, Vol. 122, Issue 4, pp. 449-54, (2009) (PubMed).

Détail du antigène

Antigène: HMGB1 (High Mobility Group Box 1 (HMGB1))

Autre désignation: Hmgb1 (HMGB1 Produits)

Synonymes: HMG1 Kit ELISA, HMG3 Kit ELISA, SBP-1 Kit ELISA, DEF Kit ELISA, HMG-1 Kit ELISA, Hmg1 Kit ELISA, amphoterin Kit ELISA, p30 Kit ELISA, hmgb1 Kit ELISA, ik:tdsubc_1a5 Kit ELISA, wu:fb23c02 Kit ELISA, xx:tdsubc_1a5 Kit ELISA, zgc:56110 Kit ELISA, zgc:77104 Kit ELISA, hmg-1 Kit ELISA, hmg3 Kit ELISA, sbp-1 Kit ELISA, hmg1 Kit ELISA, HMGB1 Kit ELISA, Ac2-008 Kit ELISA, high mobility group box 1 Kit ELISA, high-mobility group box 1 Kit ELISA, high mobility group box 1a Kit ELISA, high mobility group box 1 L homeolog Kit ELISA, high mobility group protein B1 Kit ELISA, HMGB1 Kit ELISA, Hmgb1 Kit ELISA, hmgb1 Kit ELISA, hmgb1a Kit ELISA, hmgb1.L Kit ELISA, LOC100359149 Kit ELISA

Sujet: HMGB1 was first purified from nuclei 30 years ago, and named high mobility group for its rapid mobility on electrophoresis gels. Nuclear HMGB1 has been implicated in diverse cellular functions, including determination of nucleosomal structure and stability, and binding of transcription factors to cognate DNA sequences. In addition to the nucleus and cytoplasm, HMGB1 has also been localized to the cell membrane of neurites and tumor cells, where it colocalizes and interacts with tissue plasminogen activator and the receptor for advanced glycation end products (RAGE). Engagement of RAGE with HMGB1 activates mitogen-activated protein kinase (MAPK) pathways, and the blockade of HMGB1-RAGE interaction suppresses the activation of MAPKs and the growth and metastases of tumors in mice .

ID gène: 3175

Pathways: Signalisation p53, Regulation of Muscle Cell Differentiation, Skeletal Muscle Fiber Development, Positive Regulation of Endopeptidase Activity, Regulation of Carbohydrate Metabolic Process, Toll-Like Receptors Cascades, Smooth Muscle Cell Migration, Inflammasome