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Hemopexin Kit ELISA

HPX Reactivité: Humain Colorimetric Sandwich ELISA Cell Culture Supernatant, Plasma
N° du produit ABIN612796
  • Antigène Voir toutes Hemopexin (HPX) Kits ELISA
    Hemopexin (HPX)
    Reactivité
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    Humain
    Méthode de détection
    Colorimetric
    Type de méthode
    Sandwich ELISA
    Seuil minimal de détection
    4 ng/mL
    Application
    ELISA
    Fonction
    The AssayMax Human Hemopexin ELISA kit is designed for detection of human and murine Hemopexin in plasma, serum and cell culture supernatants
    Marque
    AssayMax
    Type d'échantillon
    Plasma, Cell Culture Supernatant
    Analytical Method
    Quantitative
    Ingrédients
    Hemopexin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human hemopexin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes, which can be cut to fit the format of the individual assay. Hemopexin Standard: Human Hemopexin in a buffered protein base (800 ng, lyophilized). Biotinylated Hemopexin Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against hemopexin (90µl). 1 MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
    Matériel non inclus
    Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water.
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  • Volume d'échantillon
    50 μL
    Durée du test
    3.5 h
    Plaque
    Pre-coated
    Protocole
    This assay employs a quantitative sandwich enzyme immunoassay technique that measures hemopexin in 3.5 hours. A polyclonal antibody specific for hemopexin has been pre-coated onto a microplate. Hemopexin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for hemopexin, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
    Préparation des réactifs

    Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Hemopexin Standard: Reconstitute the 800 ng of human hemopexin Standard with 2 ml of MIx Diluent to generate a stock solution of 400 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the Standard solution (400 ng/ml) 1:2 with MIx Diluent to produce 200, 100, 50, 25, 12.5 and 6.25 ng/ml. Sample Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Hemopexin] (ng/ml) P1 1 part Standard (400 ng/ml) 400.00 P2 1 part P1 + 1 part MIx Diluent 200.00 P3 1 part P2 + 1 part MIx Diluent 100.00 P4 1 part P3 + 1 part MIx Diluent 50.00 P5 1 part P4 + 1 part MIx Diluent 25.00 P6 1 part P5 + 1 part MIx Diluent 12.50 P7 1 part P6 + 1 part MIx Diluent 6.25 P8 MIx Diluent 0.00 Biotinylated Hemopexin Antibody (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C.

    Prélèvement de l'échantillon
    Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Dilute samples 1:10 into MIx Diluent. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:10 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
    Procédure de l'essai

    Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated Hemopexin Antibody to each well and incubate for one hour. Wash five times with 200 µL of Wash Buffer. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer. Add 50 µL of Chromogen Substrate per well and incubate for about 10 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. 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 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

    Précision du teste
    Intra-assay and inter-assay coefficients of variation were 4.8 % and 7.5% respectively.
    Restrictions
    For Research Use only
  • Conseil sur la manipulation
    The kit should not be used beyond the expiration date.
    Stock
    4 °C/-20 °C
    Stockage commentaire
    Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
  • Pohl, Danz, Gross, John, Urban, Patzer, Habbig, Feldkötter, Witzke, Walther, Rhode: "Diagnosis of Alport syndrome-search for proteomic biomarkers in body fluids." dans: Pediatric nephrology (Berlin, Germany), (2013) (PubMed).

  • Antigène Voir toutes Hemopexin (HPX) Kits ELISA
    Hemopexin (HPX)
    Autre désignation
    Hemopexin (HPX Produits)
    Synonymes
    HPX Kit ELISA, HX Kit ELISA, Hpxn Kit ELISA, hx Kit ELISA, wu:fi17f11 Kit ELISA, wu:fi19a10 Kit ELISA, hemopexin Kit ELISA, HPX Kit ELISA, Mmar10_1124 Kit ELISA, Hpx Kit ELISA, hpx Kit ELISA
    Sujet
    Hemopexin is a heme-binding plasma glycoprotein which, after haptoglobin, forms the second line of defense against hemoglobin-mediated oxidative damage during intravascular hemolysis. A decrease in plasma hemopexin concentration reflects a recent release of heme compounds in the extracellular compartment. Heme-hemopexin complexes are delivered to hepatocytes by receptor-mediated endocytosis after which hemopexin is recycled to the circulation. Studies indicated that increased hemopexin level associate with minimal change disease (MCD) , sporadic Alzheimer's disease (AD) , heavy-drinking chronic alcoholics , hemolytic anemias, chronic neuromuscular diseases and acute intermittent porphyria.
    Pathways
    Transition Metal Ion Homeostasis, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response
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