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ADAMTS13 ELISA Kit

ADAMTS13 Reaktivität: Human Colorimetric Sandwich ELISA 0.625-40 ng/mL Cell Culture Cells, Cerebrospinal Fluid, Plasma, Saliva, Serum
Produktnummer ABIN5564597
  • Target Alle ADAMTS13 ELISA Kits anzeigen
    ADAMTS13 (ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 13 (ADAMTS13))
    Reaktivität
    • 8
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Human
    Nachweismethode
    Colorimetric
    Methodentyp
    Sandwich ELISA
    Detektionsbereich
    0.625-40 ng/mL
    Untere Nachweisgrenze
    0.625 ng/mL
    Applikation
    ELISA
    Verwendungszweck
    The AssayMax™ Human ADAMTS13 ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of ADAMTS13 in human plasma, serum, saliva, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures ADAMTS13 in 4 hours. A polyclonal antibody specific for ADAMTS13 has been pre-coated onto a 96-well microplate with removable strips. ADAMTS13 in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for ADAMTS13, 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.
    Marke
    AssayMax™
    Proben
    Cell Culture Cells, Cerebrospinal Fluid, Plasma, Saliva, Serum
    Analytische Methode
    Quantitative
    Bestandteile
    Human ADAMTS13 Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human ADAMTS13. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes, which can be cut to fit the format of the individual assay. Human ADAMTS13 Standard: Human ADAMTS13 in a buffered protein base (40 ng, lyophilized). Biotinylated Human ADAMTS13 Antibody (50x): A 50-fold biotinylated polyclonal antibody against ADAMTS13 (140 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).
    Benötigtes Material
    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)
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  • Testdauer
    4 h
    Plattentyp
    Pre-coated
    Protokoll
    • 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 30 minutes.
    • Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.
    Aufbereitung der Reagenzien

    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 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C. Human ADAMTS13 Standard: Reconstitute the 40 ng of Human ADAMTS13 Standard with 1 mL of MIX Diluent to generate a 40 ng/mL standard 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 the standard solution (40 ng/mL) 1:2 with MIX Diluent to produce 20, 10, 5, 2.5, 1.25, and 0.625 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining solution should be frozen at -20 °C and used within 30 days. Standard Point Dilution [ADAMTS13] (ng/mL) P1 Standard (40 ng/mL) 40.00 P2 1 part P1 + 1 part MIX Diluent 20.00 P3 1 part P2 + 1 part MIX Diluent 10.00 P4 1 part P3 + 1 part MIX Diluent 5.000 P5 1 part P4 + 1 part MIX Diluent 2.500 P6 1 part P5 + 1 part MIX Diluent 1.250 P7 1 part P6 + 1 part MIX Diluent 0.625 P8 MIX Diluent 0.000 Biotinylated Human ADAMTS13 Antibody (50x): Spin down the biotinylated antibody briefly and dilute the desired amount of the antibody 1:50 with MIX Diluent. Any remaining solution should be frozen at -20 °C. 5 Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. 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.

    Probennahme
    3 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. Dilute samples 1:200 into MIX Diluent and assay. 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. Dilute samples 1:200 into MIX Diluent and assay. 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 to remove debris. Collect supernatants and assay. Store the remaining samples at -20 °C or below. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using samples tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:10 into MIX Diluent and assay. 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. Dilute samples 1:2 into MIX Diluent and assay. The undiluted samples can be stored at -80 °C for up to 3 months. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines below for further instruction. Guidelines for Dilutions of 1:100 or Greater (for reference only, please follow the insert for specific dilution suggested) 1:100 1:10000 A) 4 μL sample: 396 μL buffer(100x) = 100 fold dilution Assuming the needed volume is less than 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 4 or equal to 400 μL. or equal to 400 μL. 1:1000 1:100000 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.
    Testdurchführung

    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 ADAMTS13 Standard or sample per well. 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 ADAMTS13 Antibody to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate per well 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 per well and incubate for 30 minutes or until 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 l 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. 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

    Ergebnisberechnung
    • 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.
    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Handhabung
    This product is for Research Use Only and is Not For Use In Diagnostic Procedures. Prepare all reagents (working 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
    Lagerung
    4 °C,-20 °C
    Informationen zur Lagerung
    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 desiccants 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.
  • Target Alle ADAMTS13 ELISA Kits anzeigen
    ADAMTS13 (ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 13 (ADAMTS13))
    Andere Bezeichnung
    ADAMTS13 (ADAMTS13 Produkte)
    Synonyme
    ADAMTS13 ELISA Kit, ADAM-TS13 ELISA Kit, ADAMTS-13 ELISA Kit, C9orf8 ELISA Kit, VWFCP ELISA Kit, vWF-CP ELISA Kit, Gm710 ELISA Kit, ADAM metallopeptidase with thrombospondin type 1 motif 13 ELISA Kit, a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 13 ELISA Kit, ADAM metallopeptidase with thrombospondin type 1 motif, 13 ELISA Kit, ADAMTS13 ELISA Kit, Adamts13 ELISA Kit, adamts13 ELISA Kit
    Hintergrund
    ADAMTS13 (a disintegrin-like and metalloproteinase with a thrombospondin type 1 motif 13), also called vonWillebrand factor-cleaving protease (VWFCP), is the 13th member of the ADAMTS family of metalloproteases. It is a multidomain protease synthesized in the liver and secreted into the blood where it cleaves von Willebrand factor (vWF) and thereby limits platelet thrombosis (1, 2). ADAMTS13 encodes a mature 1,353-amino acid protein with a calculated 145 kDa and a glycosylated 190 kDa molecular mass (3).
    Gen-ID
    11093
    UniProt
    Q76LX8
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