Prekallikrein (total) ELISA Assay Kit

$1,070.00

The Eagle Biosciences Prekallikrein ELISA Assay Kit is intended for the quantification of Prekallikrein (total) in Human plasma samples. The Eagle Biosciences Prekallikrein ELISA Assay Kit is for research use only and should not be used for diagnostic procedures.

Prekallikrein (total) ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Dynamic Range: 20-10000 ng/ml
Incubation Time: 2 hours
Sample Type: Plasma
Sample Size: 100 µL

Additional Information

Assay Background

Prekallikrein is the glycosylated single chain zymogen precursor of the plasma serine protease kallikrein. Plasma prekallikrein circulates with kininogen and is activated by Factor XIIa in the intrinsic coagulation pathway. Kallikrein activates plasminogen in fibrinolysis and cleaves kininogen in the bradykinin system of vasodilation. Prekallikrein deficiency is rare and causes increased activated partial thromboplastin time. Elevated plasma prekallikrein is associated with diabetes and cardiovascular disease.

Assay Principle

The Eagle Biosciences Prekallikrein ELISA Assay Kit employs the sandwich enzyme immunoassay technique for the detection of Human Prekallikrein (total) in human plasma samples. Human Prekallikrein will bind to the capture antibody coated on the microtiter plate. After appropriate washing steps, anti-Human Prekallikrein primary antibody binds to the captured protein. Following a washing to remove unbound substances, secondary antibody conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After washing away any unbound antibody-enzyme reagent, a substrate solution (TMB) is added to the wells and color develops in proportion to the amount of Prekallikrein 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 450nm ±2nm. The concentration of Prekallikrein 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 of standards, samples and zero controls into appropriate wells. Shake plate on a microplate shaker at 300 rpm for 30 minutes at RT.

3. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1X wash buffer (300 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 buffer by aspirating, decanting or blotting against clean paper towels.

4. Add 100 µl of working Primary Antibody into each well. Shake plate on a microplate shaker at 300 rpm for 30 minutes at RT.

5. Wash as according to step 3.

6. Add 100 µl of working HRP-conjugated secondary Antibody into each well. Shake plate on a microplate shaker at 300 rpm for 30 minutes at RT.

7. Wash as according to step 3.

8. Add 100 μl of TMB substrate to each well. Incubate for 2-10 minutes at RT in dark. Substrate will change from colorless to different strengths of blue.

9. Add 50 μl of 1N H2SO4 or HCl to each well. The color of the solution should change from blue to yellow. Mix thoroughly by gently shaking the plate.

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

Manual

Product Manual