Rat tPA Active ELISA Assay

Additional Information

Assay Background

Tissue-type Plasminogen Activator (tPA) is a member of the serine proteinase family.  tPA functions to lyse fibrin clots into soluble plasmin fragments.  tPA is active in two forms, single-chain and two-chain.  The two-chain tPA is created via interaction with the plasmin product cleaving the single chain.  This two-chain form is regarded as the more active form.  Both single-chain and two-chain tPA are complexable with PAI-1.  PAI-1 acts as an inhibitor for tPA by binding to the tPA and thus stifling its ability to lyse fibrin.  tPA can serve as an indicator of both myocardial infarction for patients with impaired fibrinolytic systems as well as a marker for type-II diabetes.

Assay Procedure

1. Remove microplate from the bag.
2. Make a working solution of Biotinylated PAI-1 by reconstituting according to the amount indicated on the vial using 3% BSA Blocking Buffer.
3. Add 100 µL of the Biotinylated PAI-1 Solution to both the standard and test wells.
4. Shake the plate at 300 rpm on a plate shaker for 30 minutes.
5. Wash wells according to the following wash procedure:
   • Remove contents of the plate by inversion into an appropriate disposal device.
   • Tap out remaining contents of the plate onto a lint free paper towel.
   • Add 300 µL of Wash Buffer.
   • Let stand for 2-3 minutes.
   • Repeat procedure 2 more times and then proceed to next step.
   • Remove contents of the plate by inversion into an appropriate disposal device.
   • Tap out the remaining contents of the plate onto a lint free paper towel then proceed to step 6.
6. Prepare the Standards as indicated in the provided dilution table.
   • Note:  The standards should be applied to the plate immediately upon preparation.
7. Add 100 µL of Standards or unknowns to the plate.  See Scheme I for suggested template design.
8. Shake plate at 300 rpm on the plate shaker for 30 minutes.
9. Wash wells according to step 5 located above in this section.
10. Make a working solution of Primary Antibody by reconstituting according to the amount indicated on the vial using 3% BSA Blocking Buffer.
11. Add 100 mL of the Primary Antibody Solution to each well.
12. Shake plate at 300 rpm on the plate shaker for 30 minutes.
13. Wash wells according to step 5 located above in this section.
14. Make a working solution of Secondary Antibody by reconstituting according to the amount indicated on the vial using 3% BSA Blocking Buffer.
15. Add 100 µL of the Secondary Antibody Solution to each well.
16. Shake plate at 300 rpm on the plate shaker for 30 minutes.
17. Wash wells according to step 5 located above in this section.
18. Add 100 µL of TMB Substrate to each well and incubate for 10 minutes.
19. Stop the reaction with 50 µL per well of 1 N H2SO4­ and read plate at 450 nm.

If accounting for substrate background, use 2 to 8 wells as blanks with only substrate in the wells (100 mL/well).  Subtract the average of these absorbance values from the absorbance values of the wells being assayed.