Tel:
+49 (0)241 95 163 153
Fax:
+49 (0)241 95 163 155
E-Mail:
orders@anticorps-enligne.fr

Haptoglobin Kit ELISA

HP Reactivité: Humain Colorimetric Sandwich ELISA 3.125-200 ng/mL Cell Culture Cells, Cerebrospinal Fluid, Milk, Saliva, Urine
N° du produit ABIN612708
  • Antigène Voir toutes Haptoglobin (HP) Kits ELISA
    Haptoglobin (HP)
    Reactivité
    • 6
    • 5
    • 4
    • 4
    • 4
    • 3
    • 3
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    Humain
    Méthode de détection
    Colorimetric
    Type de méthode
    Sandwich ELISA
    Gamme de detection
    3.125-200 ng/mL
    Seuil minimal de détection
    3.125 ng/mL
    Application
    ELISA
    Fonction
    The AssayMax Human Haptoglobin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human haptoglobin in human urine, saliva, milk, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human haptoglobin in approximately 4 hours. A polyclonal antibody specific for human haptoglobin has been pre-coated onto a 96-well microplate with removable strips. Haptoglobin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human haptoglobin, which is recognized by a streptavidin-peroxidase 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, Cerebrospinal Fluid, Milk, Saliva, Urine
    Analytical Method
    Quantitative
    Specificité
    10% FBS in culture media will not affect the assay.
    Ingrédients
    Human Haptoglobin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human haptoglobin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Haptoglobin Standard: Human haptoglobin in a buffered protein base (1800 ng, lyophilized). Biotinylated Human Haptoglobin Antibody (40x): A 40-fold concentrated biotinylated polyclonal antibody against human haptoglobin (150 l). 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, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). 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 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water. Incubator (37 °C)
    Featured
    Discover our best selling HP Kit ELISA
    Top Product
    Discover our top product HP Kit ELISA
  • Volume d'échantillon
    50 μL
    Durée du test
    2 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 10 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. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C. Human Haptoglobin Standard: Reconstitute the Human Haptoglobin Standard (1800 ng) with 4.5 mL of MIX Diluent to generate a 400 ng/mL standard stock solution. Allow the standard 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 (400 ng/mL) 2-fold with MIX Diluent to produce 200, 100, 50, 25, 12.5, 6.25, and 3.125 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be frozen at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. Standard Point Dilution [Haptoglobin] (ng/mL) P1 1 part Standard (400 ng/mL) + 1 part MIX Diluent 200 P2 1 part P1 + 1 part MIX Diluent 100 P3 1 part P2 + 1 part MIX Diluent 50 P4 1 part P3 + 1 part MIX Diluent 25 P5 1 part P4 + 1 part MIX Diluent 12.5 P6 1 part P5 + 1 part MIX Diluent 6.25 P7 1 part P6 + 1 part MIX Diluent 3.125 P8 MIX Diluent 0.0 Biotinylated Human Haptoglobin Antibody (40x): Spin down the antibody briefly and dilute the desired amount of the antibody 40-fold with MIX Diluent. 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. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent. The undiluted conjugate should be stored at -20 °C. 5

    Prélèvement de l'échantillon
    Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. A 4-fold sample dilution is suggested into MIX Diluent, 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. Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 100-fold sample dilution is suggested into MIX Diluent, 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. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 400-fold sample dilution is suggested into MIX Diluent, 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. CSF: Collect cerebrospinal fluid (CSF) using sample pot. Centrifuge samples at 3000 x g for 10 minutes. A 100-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -80 °C 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. Refer to Sample Dilution Guidelines for further instruction. Guidelines for Dilutions of 100-fold or Greater (for reference only, please follow the insert for specific dilution suggested) 100x 10000x A) 4 μL sample: 396 μL buffer (100x) = 100-fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000-fold dilution Assuming the needed volume is less than or equal to 400 μL. 1000x 100000x A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000-fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000-fold dilution Assuming the needed volume is less than or equal to 240 μL. 4
    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 Haptoglobin Standard or sample 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 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 Haptoglobin 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 Streptavidin-Peroxidase 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 10 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. 6

    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.
    Précision du teste
    Intra-assay and inter-assay coefficients of variation were 5.0% and 7.1 % respectively.
    Restrictions
    For Research Use only
  • 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. The kit should not be used beyond the expiration date. 2
    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.
  • Kamada, Akita, Takeda, Yamada, Fujii, Sawai, Doi, Asazawa, Nakayama, Mizutani, Fujii, Yakushijin, Miyazaki, Ezaki, Hiramatsu, Yoshida, Kiso, Imai, Kawada, Takehara, Miyoshi: "Serum Fucosylated Haptoglobin as a Novel Diagnostic Biomarker for Predicting Hepatocyte Ballooning and Nonalcoholic Steatohepatitis." dans: PLoS ONE, Vol. 8, Issue 6, pp. e66328, (2013) (PubMed).

    Galea, Cruickshank, Teeling, Boche, Garland, Perry, Galea: "The intrathecal CD163-haptoglobin-hemoglobin scavenging system in subarachnoid hemorrhage." dans: Journal of neurochemistry, Vol. 121, Issue 5, pp. 785-92, (2012) (PubMed).

  • Antigène Voir toutes Haptoglobin (HP) Kits ELISA
    Haptoglobin (HP)
    Autre désignation
    Haptoglobin (HP Produits)
    Synonymes
    HP Kit ELISA, wu:fb64e01 Kit ELISA, BP Kit ELISA, HP2ALPHA2 Kit ELISA, HPA1S Kit ELISA, HP-1 Kit ELISA, HPR Kit ELISA, Zonulin Kit ELISA, haptoglobin Kit ELISA, haptoglobin-like Kit ELISA, HP Kit ELISA, hp Kit ELISA, Hp Kit ELISA, LOC479668 Kit ELISA, LOC101102413 Kit ELISA
    Sujet
    Haptoglobin (HP, zonulin) is a plasma protein with hemoglobin-binding capacity and a plasma glycoprotein that forms a stable complex with hemoglobin to aid the recycling of heme iron (1).
    ID gène
    3240
    UniProt
    P00738
    Pathways
    Transition Metal Ion Homeostasis
Vous êtes ici:
Support technique