Human tPA Total ELISA Assay

$955.00

The Human tPA Total ELISA Assay is for the quantitative determination of total tPA in biological fluids by a ELISA assay (Enzyme-Linked Immunosorbent Assay).

SKU: TPN39-K01 Categories: , ,

Human tPA Total ELISA Assay

The Human tPA Total ELISA Assay is For Research Use Only

Size: 1 x 96 wells
Sensitivity: 0.1 ng/ml
Dynamic Range: 0.1 – 10 ng/ml
Incubation Time: 2 hours
Sample Type: Biological Fluids
Sample Size: 100 µl
Alternative Name: Tissue-type Plasminogen Activator
Product Developed and Manufactured in the USA

Assay Principle

This tPA Total ELISA (Enzyme-Linked Immunosorbent Assay) is for the quantitative analysis of tPA levels in biological fluid.  This test kit operates on the basis of sandwich ELISA where free, latent and complexed tPA is quantified with the use of an HRP labeled secondary antibody.

The various forms of tPA present in the standard or unknown is captured by the tPA capture antibody coated on the well.  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.


Related Products

Human tPA Activity ELISA
Mouse Tissue type Plasminogen Activator (tPA) Total ELISA Assay Kit
Rat Tissue type Plasminogen Activator (tPA) Total ELISA Assay Kit

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.

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Allsbrook, RC;, The Hemostatic Effects of Acute Exposure to Colored Cornstarch Powder During a 5k Run (Dissertation, James Madison University, 2016).

Huschke, AM;, Hemostatic Responses to Exercise in a Polycythemia Vera Patient (Dissertation, James Madison University, 2016).

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