Mouse tPA Total ELISA Assay

Additional Information

Assay Principle

In the Mouse Tissue-type Plasminogen Activator (tPA) Total Antigen ELISA Assay kit, mouse tPA will bind to the capture antibody coated on the microtiter plate. Free and complexed enzyme will react with the capture antibody on the plate. A standard calibration curve is prepared using dilutions of tPA along with the samples to be measured. After appropriate washing steps, monoclonal anti-mouse tPA primary antibody binds to the captured enzyme. Excess antibody is washed away and bound monoclonal antibody is then reacted with the secondary antibody conjugated to horseradish peroxidase. TMB substrate is used for color development at 450 nm.

1. Add 100 µl of the Standards and unknowns to the wells in duplicate. Shake the plate at 300 rpm for 30 minutes at room temperature (RT). See Scheme 1 below for a sample plate layout.
2. Wash the plate three times with 300 µL of Wash Buffer.  Remove excess Wash Buffer by gently tapping the plate on a paper towel.
3. Add 100 ml of the Primary Antibody to each well. Shake the plate at 300 rpm for 30 minutes at RT.
4. Wash the plate three times with 300 µL of Wash Buffer as in step 2.
5. Add 100 ml of the Primary Antibody to each well. Shake the plate at 300 rpm for 30 minutes at RT.
6. Wash the plate three times with 300 µL of Wash Buffer as in step 2.
7. Add 100 µl of TMB Substrate to each well. Shake the plate at 300 rpm for 2-10 minutes at RT.
8. Stop the reaction by adding 50 µl of 1N H2SO4 and read the plate at 450 nm.