IL-10 Kit ELISA

N° du produit ABIN612718

Kit ELISA IL-10 Humain, Colorimetric test pour la quantification de Humain IL-10.

Aperçu rapide pour IL-10 Kit ELISA (ABIN612718)

Antigène: IL-10 (IL10) (Interleukin 10 (IL10))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Type d'échantillon: Plasma, Cell Culture Supernatant

Détail du produit IL-10 Kit ELISA

Seuil minimal de détection: 100 pg/mL

Fonction: The AssayMax Human IL-10 ELISA kit is designed for detection of IL-10 in human plasma, tissue extracts or cell culture supernatants

Marque: AssayMax™

Analytical Method: Quantitative

Specificité: This assay recognizes both natural and recombinant human IL-10.

Ingrédients: IL-10 Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a murine monoclonal antibody against IL-10. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. IL-10 Standard: Human IL-10 in a buffered protein base (24 ng, lyophilized). Biotinylated IL-10 Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against IL-10 (80µl). 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). 1 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.

Information d'application

Volume d'échantillon: 50 μL

Durée du test: < 5 h

Plaque: Pre-coated

Protocole: This assay employs a quantitative sandwich enzyme immunoassay technique that measures IL-10 in less than 5 hours. A murine monoclonal antibody specific for human IL-10 has been pre-coated onto a microplate. IL-10 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human IL-10, 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 Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 24 ng of human IL-10 Standard with 3 ml of MIx Diluent to generate a standard solution of 8 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 IL-10 standard solution twofold with equal volume of MIx Diluent to produce 4, 2, 1, 0.5, 0.25, and 0.125 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [IL-10] (ng/ml) P1 Standard (8 ng/ml) 8.000 P2 1 part P1 + 1 part MIx Diluent 4.000 P3 1 part P2 + 1 part MIx Diluent 2.000 P4 1 part P3 + 1 part MIx Diluent 1.000 P5 1 part P4 + 1 part MIx Diluent 0.500 P6 1 part P5 + 1 part MIx Diluent 0.250 P7 1 part P6 + 1 part MIx Diluent 0.125 P8 MIx Diluent 0.000 Biotinylated IL-10 Antibody (100x): Spin down the antibody 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: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Store serum at -20°C or below. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. Tissue: Extract tissue samples with 0.1M Tris-buffered saline (pH7.4) containing 0.5% Triton x-100 and centrifuge at 14000 x g for 30 min. Collect the supernatant and measure the protein concentration. Dilute the tissue extract 1:2 into MIx Diluent and assay. Freeze the remaining extract at -20°C or below. 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 IL-10 Antibody to each well and incubate for two hours. Wash five times with 200 µL of Wash Buffer as above. 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 as above. Add 50 µL of Chromogen Substrate per well and incubate for approximately 15 minutes or till the optimal blue color density develops. Gently tap the 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.

Calcul des résultats:

Calculate the mean value of the 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 of the linear portion of the curve. Determine the unknown sample concentration from the Standard Curve and multiply the dilution factor. 3 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 5.1% and 7.4% respectively.

Restrictions: For Research Use only

Stockage

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 with zip-seal. May be stored for up to 1 month in a vacuum desiccator.

Détail du antigène

Antigène: IL-10 (IL10) (Interleukin 10 (IL10))

Autre désignation: Interleukin-10 (IL-10)

Sujet: Interleukin-10 (IL-10) is a regulatory cytokine, and its principal role in vivo is to limit inflammatory response. IL-10 has been shown to influence both the susceptibility and course of various diseases. High IL-10 expression whereas monocytes from cardiac-disease, patients may be committed to induction of inflammatory responses related to high TNF-alpha expression. Interleukin 10 (IL-10) is a key cytokine produced by a multitude of immune effector cells and possesses distinct regulatory effects on immune functioning in the skin. The accelerated alveolar bone loss observed in IL-10 (-/-) mice is a late-onset condition and that lack of IL-10 may have an effect on bone homeostasis.

Pathways: Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Production of Molecular Mediator of Immune Response, Maintenance of Protein Location, Cancer Immune Checkpoints