Plasminogen Activator Inhibitor-1 (PAI-1) is a glycoprotein and member of the

serine proteinase inhibitor (serpin) superfamily. PAI-1 is the primary inhibitor

of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen

activator (uPA). This inhibition exhibits antiproteolytic properties that can

lead to myocardial infarction, thromboembolic disease with elevated levels of

PAI-1. Additionally, PAI-1 is thought to play a role in the function of tissue

remodeling and tumor metastasis.

This is an ELISA (Enzyme-Linked Immunosorbent Assay) for the quantitative analysis of

PAI-1 levels in biological fluid. This test kit operates on the basis of sandwich ELISA

where active PAI-1 complexes with uPA and is quantified with the use of an HRP labeled

secondary antibody.

The functional or active PAI-1 will bind to the uPA coated on the well of the microtiter plate.

The latent and complexed forms of PAI-1 will not bind and are discarded at a later washing

step. Next, a PAI-1 primary antibody is added to the wells, binding to the captured PAI-1

on the microtiter plate. HRP conjugated secondary antibody is then added for detection of

the active PAI-1. Quantitative test results are obtained by the measure and comparison of

the sample and standard absorbance readings.

1. Human PAI-1 Activity Standard (0 U/mL): 1 vial of 2.0 mL lyophilized plasma

with 0 U/mL PAI-1.

2. Human PAI-1 Activity Standard (~200 U/mL): 1 vial of 2.0 mL lyophilized

plasma with lot specific PAI-1 activity. Please refer to the enclosed dilution table for

exact activity information.

3. Substrate: 10 mL. Stabilized 3,3', 5,5' Tetramethylbenzidine (TMB) plus Hydrogen

4. Anti-Human PAI-1 Primary Antibody: 1 vial of lyophilized monoclonal antihuman

PAI-1 antibody.

5. HRP Secondary Antibody: 1 vial of HRP conjugated secondary antibody.

6. Wash Buffer (10x): 30 mL of 10 x wash bufer. Dilute prior to use.

7. EIA Buffer: 10 mL of EIA buffer for diluting both sample and standard. Supplied

ready to use.

8. Primary Antibody Buffer: 10 mL of diluent specifically formulated for use with the

primary antibody.

9. Secondary Antibody Buffer: 10 mL of diluent specifically formulated for use with

the secondary antibody.

10. Coated Plate: A 96 well microplate with uPA precoated on each well. The plate is

ready for use as is. DO NOT WASH!

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2. DI water.

3. Microplate reader with 450 nm filter.

4. Microplate shaker with uniform horizontal circular movement up to 300 rpm.

5. Beakers, flasks, cylinders, etc. required for preparation of reagents.

7. Plastic film or plate cover to cover plate during incubation.

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1. PAI-1 standards are of human origin and have been screened and found to be negative

for HBAG, anti-HIV 1+2, anti-HBC and anti-HCV. It is recommended that handling of

this component be treated as a Biosafety Level 2 - a potentially infectious human serum or

blood specimen – and follow the guidelines as established by the Center for Disease

Control/National Institutes of Health manual "Biosafety in Microbiological and Biomedical

Laboratories".

1. DO NOT use components beyond expiration date.

2. DO NOT mix any reagents or components of this kit with any reagents or

components of any other kit. This kit is designed to work properly as provided.

3. DO NOT pipette reagents by mouth.

4. Always pour substrate out of the bottle into a clean test tube - DO NOT pipette out of

the bottle (if your tip is unclean you could contaminate your substrate).

5. All specimens should be considered potentially infectious. Exercise proper handling

precautions.

6. DO NOT smoke, eat or drink in areas where specimens or reagents are being handled.

7. Use aseptic technique when opening and removing reagents from vials and bottles.

8. Keep plate covered except when adding reagents, washing or reading.

9. Kit components should be stored as instructed when not in use.

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1. Always use new pipette tips for the buffer, conjugate, standards, samples etc.

2. Before pipetting a reagent, rinse the pipette tip three times with that reagent (i.e. fill the

tip with the desired amount of reagent and dispense back into the same vial - repeat 2

times). Now the tip is properly rinsed and ready to dispense the reagent into your well

or test tube.

3. When pipetting into the wells, DO NOT allow the pipette tip to touch the inside of the

well, or any of the reagents already in the well - this can cause cross contamination.

4. Standards and samples should be assayed in duplicate.

5. To quantitate, always run a standard curve when testing samples.

6. Gently mix specimens and reagents before use. Avoid vigorous agitation.

7. Before taking an absorbance reading, wipe the outside bottom of the wells with a lintfree

wiper to remove dust and fingerprints.

8. Desiccant bag must remain in foil pouch with unused strips. Keep pouch sealed when

not in use to maintain a dry environment. Seal with a heat sealer. If a heat sealer is not

available, thoroughly close the open end with tape. Try to remove excess air before

sealing.

(DiaPharma) collection media. Collection should be in accordance with the collection vials

manufacturers instructions or in a 1:10 ratio of collection media to blood.

Immediately, upon collection of blood, the samples should be centrifuged at 3000 x g. This

should ensure the removal of platelets as they can release PAI-1 that in turn complexes with

uPA. The plasma can be transferred to a clean plastic tube and stored frozen for up to one

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.

1. Remove microplate from the bag.

2. Reconstitute both standards with 2 mL of DI water and prepare each as indicated in the

provided dilution table insert. Note: The standards should be applied to the plate

immediately upon preparation.

suggested template design.

5. Shake the plate at 300 rpm on a plate shaker for 30 minutes.

6. Wash wells according to the following wash procedure:

a. Remove contents of the plate by inversion into an appropriate disposal device.

b. Tap out remaining contents of the plate onto a lint free paper towel.

d. Let stand for 2-3 minutes.

e. Repeat procedure 2 more times then proceed to step "f".

Remove contents of the plate by inversion into an appropriate disposal device.

g. Tap out remaining contents of the plate onto a lint free paper towel –proceed to

the next step.

Note: The decanted wells should be void of visible moisture before proceeding. If

moisture is visible then follow step b. until satisfactory results are obtained.

7. Add 10 mL of the primary antibody buffer directly to the primary antibody vial and

slightly agitate until completely dissolved.

unknown test wells.

9. Shake plate at 300 rpm on the plate shaker for 30 minutes.

10. Wash wells according to step 6 located above in this section.

buffer and slightly agitate until completely dissolved.

12. Shake plate at 300 rpm on the plate shaker for 30 minutes.

13. Wash wells according to step 6 located above in this section.

20. If accounting for substrate background, use 2 to 8 wells as blanks with only the

from the absorbance values of the wells being assayed. NOTE: Some

microplate readers can be programmed to do these subtractions automatically when

reading the plate. Consult your instrument manual.

1. Subtract the average O.D. value of the blank wells (BLK) from all other pairs of wells.

2. Average the O.D. values for each pair of duplicate wells.

3. Plot a standard curve using the average O.D. value for each standard value versus the

concentration of standard.

4. Determine the concentration of each unknown by interpolation from the standard

curve.

Conversion Factor: 1 PAI-1 unit = 1.34 ng.

Assay Range: 0.0 - ~200 U/mL (please refer to lot specific standard)

Samples with uPA levels higher than the lot specific PAI-1 activity (~200 U/mL) should be

diluted in similar media devoid of active uPA.

1. Declerck, P. J., et al. J Intern Med (1994) 236:425-432

2. Winman, B., et al. Scand J Clin Lab Invest Suppl (1999) 230:23-31

3. Winman, B, et al. The fast inhibitor of tissue plasminogen activator during

pregnancy. Thromb Haemostas (1984) 5-1:R9-R17

4. Colucci, M. Beneration in plasma of a fast acting inhibitor plasminogen activator in

response to endotoxin stimulation. J Clin Invest (1985) 75: 818-824

5. Kruithof, EK Plasminogen activator inhibitor-1: development of a

radioimmunoassay and observations on its plasma concentration during venous

occlusion and after platelet aggregation. Blood (1987) 70:1645-1653

6. Ranby, M Activity of plasminogen activator inhibitor type 1 in a population of

Northern Sweden. Fibrinolysis (1990) 4:54-55.