EPX Kit ELISA

N° du produit ABIN5564595

Détail du produit EPX Kit ELISA

Antigène: EPX (Eosinophil Peroxidase (EPX))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 1.953-125 ng/mL

Seuil minimal de détection: 1.953 ng/mL

Application: ELISA

Fonction: The AssayMax™ Human EPO ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of EPO in human plasma, serum, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human EPO in approximately 4 hours. A polyclonal antibody specific for human EPO has been pre-coated onto a 96-well microplate with removable strips. EPO in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human EPO, which is recognized by a streptavidin- peroxidase (SP) conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Marque: AssayMax™

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

Analytical Method: Quantitative

Ingrédients: Human EPO Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human EPO. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human EPO Standard: Human EPO in a buffered protein base (175 ng, lyophilized). Biotinylated Human EPO Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human EPO (120 l). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution to stop the chromogen substrate reaction (12 ml).

Matériel non inclus: Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)

Information d'application

Durée du test: 4 h

Plaque: Pre-coated

Protocole:

• Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
• Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 20 minutes.
• Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Préparation des réactifs:

Freshly dilute all reagents and bring all reagents to room temperature before use. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. 4 Human EPO Standard: Reconstitute the Human EPO Standard (175 ng) with 1.4 mL of EIA Diluent to generate a 125 ng/mL standard stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (125 ng/mL) 2-fold with equal volume of EIA Diluent to produce 62.5, 31.25, 15.625, 7.813, 3.906, and 1.953 ng/mL solutions. EIA Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be stored at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. Standard Point Dilution [EPO] (ng/mL) P1 1 part Standard (125 ng/mL) 125 P2 1 part P1 + 1 part EIA Diluent 62.5 P3 1 part P2 + 1 part EIA Diluent 31.25 P4 1 part P3 + 1 part EIA Diluent 15.625 P5 1 part P4 + 1 part EIA Diluent 7.813 P6 1 part P5 + 1 part EIA Diluent 3.906 P7 1 part P6 + 1 part EIA Diluent 1.953 P8 EIA Diluent 0.0 Biotinylated Human EPO Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with EIA Diluent to produce a 1x solution. The undiluted antibody should be stored at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with EIA Diluent to produce a 1x solution. The undiluted conjugate should be stored at -20 °C.

Prélèvement de l'échantillon: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes and collect plasma. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes and remove serum. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes at 4 °C to remove debris and collect supernatants. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles.

Procédure de l'essai:

Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human EPO Standard or sample to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have 5 formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human EPO Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 1 hour. Wash the microplate as described above. Add 50 l of SP Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 20 minutes or until the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

Calcul des résultats:

• Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
• To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
• Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. 2 The kit should not be used beyond the expiration date.

Stock: 4 °C,-20 °C

Stockage commentaire: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Détail du antigène

Antigène: EPX (Eosinophil Peroxidase (EPX))

Autre désignation: Eosinophil Peroxidase (EPO) (EPX Produits)

Synonymes: EPX Kit ELISA, pmr-1 Kit ELISA, EPO Kit ELISA, EPP Kit ELISA, EPX-PEN Kit ELISA, eosinophil peroxidase Kit ELISA, eosinophil peroxidase L homeolog Kit ELISA, LOC788751 Kit ELISA, EPX Kit ELISA, epx.L Kit ELISA, Epx Kit ELISA

Sujet: Eosinophil peroxidase (EPO, EPX), an abundant protein in the matrix of the eosinophil granule, is a member of the peroxidase family. The preproprotein is proteolytically processed into covalently attached heavy (57 kDa) and light (11 kDa) chains to form the mature enzyme (1). EPO catalyzes the formation of hypohalous acids from hydrogen peroxide and halide ions in solution. It functions as a potent oxidant and plays important roles in human defense against microorganisms in eosinophils. The enzyme is released at sites of parasitic infection or allergen stimulation to mediate bacterial fragmentation and lysis (2-3).

ID gène: 8288

UniProt: P11678