Fibronectin ELISA Kit

Produktnummer ABIN612699

Das Human Fibronectin ELISA Kit (ABIN612699) ist ein Colorimetric ELISA Kit zur Detektion von Human Fibronectin.

Kurzübersicht für Fibronectin ELISA Kit (ABIN612699)

Target: Fibronectin

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Proben: Cell Culture Supernatant

Produktdetails Fibronectin ELISA Kit

Untere Nachweisgrenze: 4 ng/mL

Verwendungszweck: The AssayMax Human Fibronectin ELISA kit is designed for detection of human FN in cell culture media and urine samples

Marke: AssayMax™

Analytische Methode: Quantitative

Bestandteile: FN Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against FN Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. 1 FN Standard: Human FN in a buffered protein base (1 µg, lyophilized) Biotinylated Human FN Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against human FN (80µl). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl) MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml) Wash Buffer Concentrate (10x): A 10-fold concentrated buffered surfactant (2 x 30 ml) Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml) Stop Solution: A 0.5 N hydrochloric acid (12 ml) to stop the chromogen substrate reaction

Benötigtes Material: Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel pipettes) Deionized or distilled reagent grade water

Anwendungsinformationen

Probenmenge: 50 μL

Testdauer: 3.5 h

Plattentyp: Pre-coated

Protokoll: This assay employs a quantitative sandwich enzyme immunoassay technique that measures human FN in 3.5 hours. A polyclonal antibody specific for human FN has been pre-coated onto a 96-well microplate with removable strips. FN in standards and samples is sandwiched by the immobilized polyclonal antibody and biotinylated polyclonal antibody specific for human FN, 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.

Aufbereitung der Reagenzien:

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. Standard Curve: Reconstitute the 1 g of FN Standard with 1 ml of MIx Diluent to generate a 1 g/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 (1 g/ml) 1:4 with MIx Diluent to produce 0.25, 0.063, 0.016 and 0.004 g/ml solutions. MIx Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at

Probennahme: 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. Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes and assay. Dilute samples 1:2 into MIx Diluent. Store undiluted samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.

Testdurchführung:

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, and cover wells and incubate for 2 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 Human FN Antibody to each well and incubate for 60 minutes. Wash five times with 200 µL of Wash Buffer. 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. Add 50 µL of Chromogen Substrate per well and incubate for about 10 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 after the reaction is stopped for about 10 minutes, some black particles may be generated at high concentration point, which will reduce the readings.

Ergebnisberechnung:

Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a Standard Curve, plot 4-parameter graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. T 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.

Testpräzision: Intra-assay and inter-assay coefficients of variation were 5.0% and 7.0% respectively.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: The kit should not be used beyond the expiration date. All human source materials have been tested and found to be negative to HbsAg, HIV-1 and HCV by FDA approved methods.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Store unopened kit at 2-8°C up to expiration date. Opened reagents may be stored for up to 1 month at 2-8°C. Store reconstituted standard and Biotinylated FN 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.

Details zu Fibronectin

Target: Fibronectin

Andere Bezeichnung: Fibronectin (FN)

Hintergrund: Fibronectin (FN) is a major component of the extracellular matrix and blood plasma, and is a specific ligand for several integrin adhesion receptors. FN plays an important role not only in cell adhesion and wound healing , but also in embryogenesis and hematopoiesis. FN is over-expressed in cardiovascular disease states such as atherosclerosis and myocardial infarction. Reduced levels of FN have been reported in patients with Disseminated Intravascular Coagulation (DIC) and low concentrations appear to correlate with a poor prognosis.