Prothrombin ELISA Kit

Produktnummer ABIN456298

Produktdetails Prothrombin ELISA Kit

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

Bindungsspezifität: Fragment 1, Fragment 2

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 0.78-50 ng/mL

Untere Nachweisgrenze: 0.78 ng/mL

Applikation: ELISA

Verwendungszweck: This immunoassay kit allows for the specific measurement of human prothrombin fragment 1+2, F1+2 concentrations in cell culture supernates, serum and plasma.

Proben: Cell Culture Supernatant, Serum, Plasma

Analytische Methode: Quantitative

Spezifität: This assay recognizes recombinant and natural human F1+2.

Kreuzreaktivität (Details): No significant cross-reactivity or interference was observed.

Sensitivität: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Produktmerkmale: Homo sapiens,Human,Prothrombin,Coagulation factor II,F2,3.4.21.5

Bestandteile: Reagent (Quantity): Assay plate (1), 2 Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Anwendungsinformationen

Probenmenge: 100 μL

Plattentyp: Pre-coated

Protokoll: This assay employs the quantitative sandwich enzyme immunoassay technique. A antibody specific for F1+2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any F1+2 present is bound by the immobilized antibody. An enzyme-linked antibody specific for F1+2 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of F1+2 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Aufbereitung der Reagenzien:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 2,000 pmol/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (2,000 pmol/mL). The Sample Diluent serves as the zero standard (0 pmol/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using 3 Assay Diluent A and B (1:100), respectively.

Probennahme: Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.

Testdurchführung:

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser 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.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:
1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 4
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

Ergebnisberechnung:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the F1+2 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Details zu Prothrombin

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

Andere Bezeichnung: F2 (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 a carbohydrate-protein compound in plasma essential to coagulation. In response to bleeding, a complex series of clotting-factor interactions leads to its conversion by thromboplastin to thrombin, which transforms fibrinogen in plasma into fibrin. Fibrin and platelets combine to form a clot. Hemophilia is caused by a hereditary lack of one of the clotting factors. Vitamin K is needed to synthesize prothrombin, so conditions that impair the vitamin's absorption result in prothrombin deficiency and a tendency to prolonged bleeding. Prothrombin and prothrombin fragment F1+2 (F1+2) were demonstrated in the tumor stroma on cancer cells and on small blood vessels in areas of neoangiogenesis at the host-tumor interface (gastric and pancreatic cancer tissues). F1+2 is an indicator of local activation of blood coagulation in cancer tissue. Thrombin itself is impossible to quantitate and so the use of surrogate markers is necessary. The measurement of F1+2 would be an excellent marker of thrombin generation. This is helped by the fact that F1+2 is not generated in vivo by any other mechanism. Fragment 1+2 has a half life of about 1 hour and is cleared from the bloodstream by the liver.

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