Mouse Prothrombin/Thrombin Total Antigen ELISA Assay Kit

$750.00

The Eagle Biosciences Prothrombin/Thrombin Total Antigen ELISA Assay Kit is intended for the quantitative determination of total Prothrombin/Thrombin antigen in mouse plasma, serum, cell culture supernatant and tissue extracts. The Eagle Biosciences Prothrombin/Thrombin Total Antigen ELISA Assay Kit is for research use only and should not be used for diagnostic procedures.

Mouse Prothrombin/Thrombin Total Antigen ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 0.319 ng/ml
Dynamic Range: 1-500 ng/mL
Incubation Time: 2 hours
Sample Type: Mouse Plasma, Serum, Tissue Extracts, and Cell Culture Media
Sample Size: 100 µL

Additional Information

Assay Background

Prothrombin (aka Factor II) is a single-chain vitamin K dependent 579 amino acid glycoprotein zymogen. Prothrombin is proteolytically activated to thrombin by the prothrombinase enzyme complex in the coagulation cascade common pathway. The serine protease thrombin converts plasma fibrinogen to insoluble fibrin. Prothrombin levels are decreased by anticoagulant therapy, vitamin K deficiency and severe liver diseases. Elevated plasma prothrombin is associated with a single nucleotide change at position 20210.

Assay Principle

This is an Enzyme Immunoassay for the quantitative determination of total Prothrombin/Thrombin antigen in mouse serum, plasma, cell culture supernatant and tissue extracts. This assay employs the quantitative sandwich enzyme immunoassay technique. An antibody specific for Mouse Prothrombin has been pre-coated onto a microtiter plate. Standards or samples are pipetted into the wells and any Prothrombin/Thrombin antigen present is bound by the immobilized antibody. Biotinylated primary antibody is then added and bound to the captured proteins. After washing away any unbound substances, streptavidinconjugated HRP is added and incubate. After washing steps are carried out, a substrate solution (TMB) is added to the wells and color develops in proportion to the amount of Prothrombin/Thrombin bound in the initial step. The color development is stopped by the addition of acid and the intensity of the color is measured at a wavelength of 450 nm ±2 nm. The concentration of Prothrombin/Thrombin in the sample is then determined by comparing the O.D of samples to the standard curve.

Assay Procedure

1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it.

2. Add 100 µl standards and samples into each well. Incubate for 30 minutes at RT with shaking at 300 rpm.

3. Aspirate each well and wash, repeating the process 4 times for a total 5 washes. Wash by filling each well with wash buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining distilled water by aspirating, decanting or blotting against clean paper towels.

4. Add 100 µl biotinylated anti-mouse Prothrombin/Thrombin antibody into each well. Incubate for 30 minutes at RT with shaking at 300 rpm.

5. Wash as according to step 3.

6. Add 100 µl working Streptavidin-conjugated HRP solution into each well. Incubate for 30 minutes at RT with shaking at 300 rpm.

7. Wash as according to step 3.

8. Add 100 μl of TMB Substrate to each well. Incubate for 2-10 minutes at room temperature in dark.

9. Add 50 μl Stop solution into each well.

10. Read the OD with a microplate reader at 450 nm immediately.

Manual

Product Manual