Human tPA Activity ELISA

Additional Information

Assay Principle

This Human tPA Activity ELISA Assay kit (Enzyme-Linked Immunosorbent Assay) is for the quantitative analysis of active tPA levels in biological fluid.  This test kit operates on the basis of sandwich ELISA where free, active, tPA enzyme complexes with PAI-1 and is quantified with the use of an HRP labeled secondary antibody.  First the biotinylated PAI-1 binds to the avidin coated wells.  Next, active tPA present in the standard or unknown, complexes with PAI-1.  Inactive or complexed tPA is removed in a subsequent wash step.  A primary antibody specific for tPA is then added to each well followed by the HRP conjugated secondary antibody.  The bound conjugated secondary antibody is detected by the addition of substrate, which generates an optimal color after 10 minutes. Quantitative test results may be obtained by the measure and comparison of the sample and standard absorbance readings when read with a microplate reader at 450 nm.

1. Add the indicated amount of BSA blocking buffer solution directly to the Biotinylated PAI-1 vial and slightly agitate until completely dissolved.
2. Add 100 µL of the BSA/Biotinylated PAI-1 mixture to the both standard and test wells.
3. Shake the plate at 300 rpm on a plate shaker for 30 minutes.
4. Wash wells according to the following wash procedure:
   1. Remove contents of the plate by inversion into an appropriate disposal device.
   2. Tap out remaining contents of the plate onto a lint free paper towel.
   3. Add 300 mL of wash buffer.
   4. Let stand for 2-3 minutes.
   5. Repeat procedure 2 more times then proceed to next step.
   6. Remove contents of the plate by inversion into an appropriate disposal device.
   7. Tap out the remaining contents of the plate onto a lint free paper towel then proceed to step 6.
5. Prepare standards as indicated in the provided dilution table.
   • Note:  The standards should be applied to the plate immediately upon preparation.
6. Add 40 µL of EIA buffer to the wells of the plate followed by 60 µL of standards or unknowns.
7. Shake plate at 300 rpm on the plate shaker for 30 minutes.
8. Wash wells according to step 5 located above in this section.
9. Add the indicated volume of the 3% BSA blocking buffer directly to the Anti-Human tPA primary antibody and slightly agitate until completely dissolved.
10. Add 100 µL of the reconstituted Anti-Human tPA Primary Antibody to each well.
11. Shake plate at 300 rpm on the plate shaker for 30 minutes.
12. Wash wells according to step 5 located above in this section.
13. Make a stock concentration of secondary antibody by adding the indicated amount of secondary antibody stock solution to 10 µL of 3% BSA blocking buffer for a working concentration.
14. Add 100 µL of the working concentration BSA/ secondary antibody solution to each well.
15. Shake plate at 300 rpm on the plate shaker for 30 minutes.
16. Wash wells according to step 5 located above in this section.
17. Add 100 µL of TMB substrate to each well and allow to incubate for 10 minutes.
18. Quench reaction with 50 µL per well of 1 N H2SO4­ and read plate at 450 nm.
19. If accounting for substrate background, use 2 to 8 wells as blanks with only substrate in the wells (150 mL/well).  Subtract the average of these absorbance values from the absorbance values of the wells being assayed.