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CD40 Ligand Kit ELISA

CD40LG Reactivité: Humain Colorimetric Sandwich ELISA 6-6000 pg/mL Cell Culture Supernatant, Plasma, Serum
N° du produit ABIN1979623
  • Antigène Voir toutes CD40 Ligand (CD40LG) Kits ELISA
    CD40 Ligand (CD40LG)
    Reactivité
    • 9
    • 9
    • 5
    • 5
    • 4
    • 3
    • 3
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    Humain
    Méthode de détection
    Colorimetric
    Type de méthode
    Sandwich ELISA
    Gamme de detection
    6-6000 pg/mL
    Seuil minimal de détection
    6 pg/mL
    Application
    ELISA
    Fonction
    Human CD40 Ligand (TNFSF5) ELISA Kit for cell culture supernatants, plasma, and serum samples.
    Type d'échantillon
    Plasma, Cell Culture Supernatant, Serum
    Analytical Method
    Quantitative
    Specificité
    This ELISA kit shows no cross-reactivity with any of the cytokines tested: Human Angiogenin, BDNF, BLC, ENA-78, FGF-4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin, MCP-1, MCP-2, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.
    Sensibilité
    < 6 pg/mL
    Attributs du produit
    • Strip plates and additional reagents allow for use in multiple experiments
    • Quantitative protein detection
    • Establishes normal range
    • The best products for confirmation of antibody array data
    Ingrédients
    • Pre-Coated 96-well Strip Microplate
    • Wash Buffer
    • Stop Solution
    • Assay Diluent(s)
    • Lyophilized Standard
    • Biotinylated Detection Antibody
    • Streptavidin-Conjugated HRP
    • TMB One-Step Substrate
    Matériel non inclus
    • Distilled or deionized water
    • Precision pipettes to deliver 2 μL to 1 μL volumes
    • Adjustable 1-25 μL pipettes for reagent preparation
    • 100 μL and 1 liter graduated cylinders
    • Tubes to prepare standard and sample dilutions
    • Absorbent paper
    • Microplate reader capable of measuring absorbance at 450nm
    • Log-log graph paper or computer and software for ELISA data analysis
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  • Indications d'application
    Recommended Dilution for serum and plasma samples3 fold
    Volume d'échantillon
    100 μL
    Plaque
    Pre-coated
    Protocole
    1. Prepare all reagents, samples and standards as instructed in the manual.
    2. Add 100 μL of standard or sample to each well.
    3. Incubate 2.5 h at RT or O/N at 4 °C.
    4. Add 100 μL of prepared biotin antibody to each well.
    5. Incubate 1 h at RT.
    6. Add 100 μL of prepared Streptavidin solution to each well.
    7. Incubate 45 min at RT.
    8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
    9. Incubate 30 min at RT.
    10. Add 50 μL of Stop Solution to each well.
    11. Read at 450 nm immediately.
    Préparation des réactifs
    1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
      2. Sample dilution: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1 x Assay Diluent B (Item E) should be used for dilution of culture supernantants and urine. Suggested dilution for normal serum/plasma: 3 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
      3. Assay Diluent B should be diluted 5-folds with deionized or distilled water.
      4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium and urine) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 80 µL CD40L standard from the vial of Item C, into a tube with 586.7 µL Assay Diluent A or 1x Assay Diluent B to prepare a 6000 pg/mL stock standard solution. Pipette 400 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 200 µL 200myl 200 µL 200 µL 200 µL 200 µL 80 µL standard +586.7 µL 6000 2000 666.7 222.2 74.07 24.6 8.23 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
      5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
      6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-folds with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
      7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 600-folds with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 20 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a final 600 fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well.
    Procédure de l'essai
    1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
      2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
      3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette 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.
      4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
      5. Discard the solution. Repeat the wash as in step
      6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
      7. Discard the solution. Repeat the wash as in step
      8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
      9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.
    Calcul des résultats

    Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
    Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent A Human CD40 Ligand concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10 Assay Diluent B Human CD40 Ligand concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10
    Sensitivity: The minimum detectable dose of CD40L is typically less than 6 pg/mL.
    Recovery: Recovery was determined by spiking various levels of human CD40L into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 93.57 83-103 Plasma 94.63 84-105 Cell culture media 96.35 86-105
    Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 88 88 90 Range ( %) 84-103 85-105 85-106 1:4 Average % of Expected 90 93 95 Range ( %) 85-106 86-105 85-105
    Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

    Précision du teste
    Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %
    Restrictions
    For Research Use only
  • Conseil sur la manipulation
    Avoid repeated freeze-thaw cycles.
    Stock
    -20 °C
    Stockage commentaire
    The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.
    Date de péremption
    6 months
  • Xu, Xu, Wei, Tan, Zhao, Zhao, Wu: "Self-complementary adeno-associated virus 5-mediated gene transduction of a novel CD40L mutant confers direct antitumor effects in lung carcinoma." dans: Molecular medicine reports, Vol. 11, Issue 1, pp. 482-8, (2014) (PubMed).

    Diaconu, Cerullo, Hirvinen, Escutenaire, Ugolini, Pesonen, Bramante, Parviainen, Kanerva, Loskog, Eliopoulos, Pesonen, Hemminki: "Immune response is an important aspect of the antitumor effect produced by a CD40L-encoding oncolytic adenovirus." dans: Cancer research, Vol. 72, Issue 9, pp. 2327-38, (2012) (PubMed).

    Alderson, Armitage, Tough, Strockbine, Fanslow, Spriggs: "CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40." dans: The Journal of experimental medicine, Vol. 178, Issue 2, pp. 669-74, (1993) (PubMed).

    Splawski, Fu, Lipsky: "Immunoregulatory role of CD40 in human B cell differentiation." dans: Journal of immunology (Baltimore, Md. : 1950), Vol. 150, Issue 4, pp. 1276-85, (1993) (PubMed).

  • Antigène Voir toutes CD40 Ligand (CD40LG) Kits ELISA
    CD40 Ligand (CD40LG)
    Autre désignation
    CD40L (CD40LG Produits)
    Synonymes
    CD154 Kit ELISA, cd40l Kit ELISA, CD40L Kit ELISA, HIGM1 Kit ELISA, IGM Kit ELISA, IMD3 Kit ELISA, T-BAM Kit ELISA, TNFSF5 Kit ELISA, TRAP Kit ELISA, gp39 Kit ELISA, hCD40L Kit ELISA, CD40-L Kit ELISA, Cd40l Kit ELISA, Ly-62 Kit ELISA, Ly62 Kit ELISA, Tnfsf5 Kit ELISA, CD40 ligand Kit ELISA, TNF superfamily member 5 Kit ELISA, CD40LG Kit ELISA, cd40l Kit ELISA, Cd40lg Kit ELISA
    Sujet
    CD40L is expressed predominantly on activated CD4+T lymphocytes, and also found in other types of cells, like NK cells, mast cells, basophils and eosinophils. CD40 is expressed on all B-lymphocytes during various stages of development, activated T-cells and monocytes, follicular dendritic cells, thymic epithelial cells, and various carcinoma cell lines. CD40L mediates a range of activities on B cells including induction of activation-associated surface antigen, entry into cell cycle, isotype switching and Ig secretion and memory generation. CD40 ligation has been shown to induce cell death by apotosis in transformed cells of mesenchymal and epithelial origin. The Human CD40L ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human CD40L in serum, plasma (EDTA or heparin), cell culture supernatants and urine. This assay employs an antibody specific for human CD40L coated on a 96-well plate. Standards and samples are pipetted into the wells and CD40L present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human CD40L antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of CD40L bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.
    ID gène
    959
    UniProt
    P29965
    Pathways
    Signalisation NF-kappaB, Production of Molecular Mediator of Immune Response, Cancer Immune Checkpoints
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