Prothrombin ELISA Kit

Produktnummer ABIN1440257

Produktdetails Prothrombin ELISA Kit

Target: Prothrombin (F2) (Coagulation Factor II (thrombin) (F2))

Reaktivität: Maus

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Untere Nachweisgrenze: ~ 0.7 ng/mL

Applikation: ELISA

Verwendungszweck: The AssayMax Mouse Prothrombin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of mouse Prothrombin in plasma, serum, urine, and cell culture supernatants. This assay employs a quantitative sandwich enzyme immunoassay technique that measures mouse Prothrombin in less than 4 hours. A polyclonal antibody specific for mouse Prothrombin has been pre-coated onto a 96-well microplate with removable strips. Prothrombin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for mouse prothrombin, 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: Serum, Urine, Plasma, Cell Culture Supernatant

Analytische Methode: Quantitative

Kreuzreaktivität (Details): Cross-Reactivity: Rat 5%
This kit cross-reacts with thrombin.

Produktmerkmale: Standard Added Value: 2 - 20 ng/mL

Bestandteile: Mouse Prothrombin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against mouse Prothrombin. AssayMax Mouse Prothrombin ELISA Kit Catalog No. EMP3022-1 This protocol serves as an example for the above. Do not use this protocol in conjunction with any purchased kit. 2
Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay.
Mouse Prothrombin Standard: Mouse Prothrombin in a buffered protein base (50 ng, lyophilized).
Biotinylated Mouse Prothrombin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against Prothrombin (140 µL).
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 µL).
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 450 nm.
Pipettes (1-20 µL, 20-200 µL, 200-1000 µL and multiple channel).
Deionized or distilled reagent grade water.

Anwendungsinformationen

Testdauer: < 4 h

Plattentyp: Pre-coated

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 1:10 with reagent grade water. Store for up to 1 month at 2-8°C.
Standard Curve: Reconstitute the 50 ng of mouse Prothrombin Standard with 1 mL of MIX Diluent to generate a stock solution of 50 ng/mL. 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 (50 ng/mL) 1:2 with MIX Diluent to produce 25, 12.5, 6.25, 3.13, 1.56, and 0.78 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining solution should be frozen at -20°C.
Biotinylated H. Prothrombin Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:50 with MIX Diluent. Any remaining solution should be frozen at -20°C.
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.

Aufbereitung der Proben:

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. Dilute samples 1:8000 into MIX Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. (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. Dilute samples 1:8000 into MIX Diluent. 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 samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Urine: Collect urine using sample tube. Centrifuge samples at 600 x g for 10 minutes and assay. Store 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 two hours. Start the timer after the last sample addition.
Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid.
Add 50 µL of Biotinylated Prothrombin Antibody to each well and incubate for one hour.
Wash the microplate as described 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 the microplate as described above.
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. 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.

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 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.

Testpräzision: Intra-assay and inter-assay coefficients of variation were 5.1% and 7.1% respectively.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Prepare all reagents (working diluent buffer, wash buffer, standards, 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 protocol. 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 kit should not be used beyond the expiration date.
The Stop Solution is an acidic solution.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Store components of the kit at 2-8°C or -20°C upon arrival 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
Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Diluent (1x) may be stored for up to 1 month at 2-8°C.
Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Details zu Prothrombin

Target: Prothrombin (F2) (Coagulation Factor II (thrombin) (F2))

Andere Bezeichnung: Prothrombin (F2 Produkte)

Synonyme: PT ELISA Kit, RPRGL2 ELISA Kit, THPH1 ELISA Kit, Cf-2 ELISA Kit, Cf2 ELISA Kit, FII ELISA Kit, thrombin ELISA Kit, zgc:66319 ELISA Kit, wu:fb57c10 ELISA Kit, wu:fd42e09 ELISA Kit, wu:fd59d01 ELISA Kit, prothrombin ELISA Kit, F2 ELISA Kit, coagulation factor II, thrombin ELISA Kit, coagulation factor II ELISA Kit, coagulation factor II (thrombin) ELISA Kit, F2 ELISA Kit, f2 ELISA Kit

Hintergrund: Prothrombin is also known as Factor II. The conversion of Factor X to Xa changes prothrombin into its active form, thrombin, which then accelerates the formation of fibrin. The level of the plasma prothrombin in the circulating blood decreases during its passage through the pulmonary capillaries. The bleeding tendency in acute chloroform intoxication is due to deficiency in both plasma fibrinogen and plasma prothrombin. On the other hand, in severe Alzheimer's disease, prothrombin was localized within the wall and neuropil surrounding microvessels. It has been reported that plasma prothormbin level increases in sepsis patients, and in chronic gastrointestinal disorders.

Pathways: Komplementsystem, Peptide Hormone Metabolism, Regulation of G-Protein Coupled Receptor Protein Signaling