Tissue factor Kit ELISA

N° du produit ABIN612783

Détail du produit Tissue factor Kit ELISA

Antigène: Tissue factor (F3) (Coagulation Factor III (thromboplastin, Tissue Factor) (F3))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Seuil minimal de détection: 10 pg/mL

Application: ELISA

Fonction: The AssayMax Human Tissue Factor (TF) ELISA kit is designed for detection of human TF in plasma, serum, tissue, and cell culture lysate

Marque: AssayMax

Type d'échantillon: Plasma

Analytical Method: Quantitative

Specificité: This assay recognizes both natural and recombinant human TF apoprotein and TF/FVII complexes.

Ingrédients: TF Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against TF Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. TF Standard: Human TF in a buffered protein base (1600 pg, lyophilized). Biotinylated TF Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against TF (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). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). 1 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 pipettes) 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 TF in less than 5 hours. A polyclonal antibody specific for TF has been pre-coated onto a 96-well microplate. TF in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for TF, 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. TF Standard: Reconstitute the 1600 pg of TF Standard with 2 ml of MIx Diluent to generate a 800 pg/ml of solution. 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 twofold with equal volume of MIx Diluent to produce 400, 200, 100, 50 and 25 pg/ml. MIx Diluent serves as the zero standard (0 pg/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [TF] (pg/ml) P1 1 part TF Standard 800.00 P2 1 part P1 + 1 part MIx Diluent 400.00 P3 1 part P2 + 1 part MIx Diluent 200.00 P4 1 part P3 + 1 part MIx Diluent 100.00 P5 1 part P4 + 1 part MIx Diluent 50.00 P6 1 part P5 + 1 part MIx Diluent 25.00 P7 MIx Diluent 0.000 Biotinylated TF 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 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. Use undiluted samples or 1:4 diluted samples with MIx Diluent and assay immediately. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Dilute samples 1:4 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 Lysates: The cultured cells are lysed and solubilized with 15 mM octyl--D- glucopyranoside at 370C for 15 minutes. Collect fresh cell lysates. Use undiluted samples or 1:2 diluted samples with MIx Diluent and assay. The undiluted samples can be stored at -20°C or below. Tissue: Extract tissue samples with 50 mM phosphate-buffered saline (pH7.4) containing 1% Triton x-100 and centrifuge at 14000x g for 20 min. Collect the supernatant and measure the protein concentration. Use undiluted samples or 1:2 diluted samples with MIx Diluent and assay. The undiluted samples can be stored at -20°C or below.

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. Hit the plate 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated TF 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 to each 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 about 15 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 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 5.3% and 7.2% 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 zip-seal. May be stored for up to 1 month in a vacuum desiccator.

Détail du antigène

Antigène: Tissue factor (F3) (Coagulation Factor III (thromboplastin, Tissue Factor) (F3))

Autre désignation: Tissue Factor (TF) (F3 Produits)

Synonymes: CD142 Kit ELISA, TF Kit ELISA, TFA Kit ELISA, PRO1557 Kit ELISA, PRO2086 Kit ELISA, TFQTL1 Kit ELISA, f3 Kit ELISA, AA409063 Kit ELISA, Cf-3 Kit ELISA, Cf3 Kit ELISA, tf Kit ELISA, zgc:112151 Kit ELISA, coagulation factor III, tissue factor Kit ELISA, transferrin Kit ELISA, coagulation factor IIIa Kit ELISA, coagulation factor III Kit ELISA, tissue factor Kit ELISA, coagulation factor IIIb Kit ELISA, coagulation factor III (thromboplastin, tissue factor) S homeolog Kit ELISA, F3 Kit ELISA, TF Kit ELISA, f3a Kit ELISA, tf Kit ELISA, f3b Kit ELISA, f3.S Kit ELISA

Sujet: The transmembrane protein Tissue factor (TF) is the physiologic trigger of coagulation in normal hemostasis. TF binds and allosterically activates factor VII. The TF-VIIa complex cleaves factor Ix and x, leading to thrombin generation. Inducible expression of TF in a variety of pathological conditions, including gram-negative sepsis and acute coronary syndromes, is associated with life-threatening thrombosis. In sepsis, TF expression within the vasculature leads to disseminated intravascular coagulation. TF also plays important roles in vasculogenesis, metastasis, and tumor-associated angiogenesis.

Pathways: Positive Regulation of Endopeptidase Activity, Smooth Muscle Cell Migration, Platelet-derived growth Factor Receptor Signaling