Human tPA Total ELISA Assay

Additional Information

Assay Background

Tissue-type Plasminogen Actvator (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.

1. Remove microplate from the bag. 
2. Prepare standards as indicated in the provided dilution table. 
   • Note:  The standards should be applied to the plate immediately upon preparation.
3. Add 100 µL of standards and unknowns to the plate.
4. Shake plate at 300 rpm on the 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 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. Add 10 µL of the BSA blocking buffer (see reagent preparation) directly to the lyophilized Primary Antibody. 
7. Add 100 µL of the  BSA/ Primary Antibody to each well.
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. Dilute 1 µL of Secondary Antibody to 10 mL with BSA blocking buffer for a working concentration.
11. Add 100 µL of the working concentration BSA/ Secondary 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. Add 100 µL of TMB substrate to each well and allow to incubate for 15 minutes.
15. Quench reaction with 50 µL per well of 1 N H2SO4­ and read plate at 450 nm.
16. If accounting for substrate background, use 2 to 8 wells as blanks with only substrate in the wells (150 µL/well).  Subtract the average of these absorbance values from the absorbance values of the wells being assayed.