Mouse tPA Active ELISA Assay

$995.00

The Mouse tPA Active ELISA Assay is for the quantitative determination of active tPA in mouse biological fluids by a ELISA assay (Enzyme-Linked Immunosorbent Assay).

Mouse tPA Active ELISA Assay

The Mouse tPA Active ELISA Assay is For Research Use Only

Size: 1 x 96 wells
Sensitivity: 0.1 ng/ml
Dynamic Range: 0.1 – 50 ng/ml
Incubation Time: 2.5 hours
Sample Type: Biological Fluids
Sample Size: 100 µl

Product Developed and Manufactured in the USA


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.


SAMPLE COLLECTION, STORAGE, AND PREPARATION
Samples should be collected using 0.1 M trisodium citrate or acidified citratein a 1:10 ratio of collection media to blood. Immediately upon collection of blood, the samples should be centrifuged at 3000 x g for 15 minutes. This should ensure the removal of platelets as they can release PAI-1, that in turn complexes with tPA. The plasma should be transferred to a clean plastic tube to be kept on ice prior to analysis or stored frozen for up to one month. Samples are stable for approximately 24 hours when stored at 4°C or one month if stored at -20°C and thawed three times without loss of tPA activity. Note: Detergents such as Triton X cause interference with the assay. If using detergent extracted samples, it is necessary to dialyze the samples overnight to remove the detergent.


Related Products

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

Additional Information

Assay Principle


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 100 µl of reconstituted Biotinylated PAI-1 to all of the wells. Shake the plate at 300 rpm for 30 minutes at room temperature (RT).
  2. Wash the plate 3 times according to the following wash procedure:
    1. Remove the contents of each well by inversion of the plate.
    2. Tap out the remaining contents of the plate onto a lint free paper towel.
    3. Add 300 mL of 1x Wash Buffer.
    4. Let stand for 2-3 minutes.
    5. Repeat procedure two more times, then proceed to step “f”.
    6. Remove the contents of each well by inversion of plate into an appropriate disposal device.
    7. Tap out the remaining contents of the plate onto a lint free paper towel, then proceed to step 3.
  3. Add 100 µl of the Standards and unknowns to the wells in duplicate.  Shake the plate at 300 rpm for 30 minutes at RT.
  4. Wash the plate three times 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 as in step 2.
  7. Add 100 ml of the Secondary Antibody to each well. Shake the plate at 300rpm for 30 minutes at RT.
  8. Wash the plate three times as in step 2.
  9. Add 100 µl of TMB Substrate to each well. Shake the plate at 300 rpm for 10-20 minutes at RT.
  10. Stop the reaction by adding 50 µl of 1N H2SO4 to each well and read the plate at 450 nm.

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