DiaPharma Group, Inc.

For in vitro diagnostic use.

Biopool TintElize® PAI-1 is an enzyme immunoassay (ELISA) for the quantitative determination of human plasminogen activator inhibitor, type 1 (PAI-1) antigen in human plasma.

Summary

Plasminogen activator inhibitor; type 1 (PAI-1) is the primary inhibitor of tissue plasminogen activator (tPA), a key enzyme in fibrinolysis. An increased plasma level of PAI-1 is associated with impaired fibrinolytic function. Elevated levels of PAI-1 have been observed in thrombolytic disease, acute myocardial infarction, deep vein thrombosis, as well as normal pregnancy and sepsis. PAI-1 is produced by vascular endothelial cells, platelets, placenta, and kidney tubule cells. PAI-1 is found in several forms: active, inactive (latent) and complexed to tPA and uPA, all of which are measured in the TintElize PAI-1 assay.

Principle

The Biopool TintElize PAI-1 test is based on a double antibody principle, similar to the ELISA described by Declerck et al. and uses the same coating antibody (MA-7D4B7). The assay uses quenching and normal antibodies to exclude falsely elevated results from the specific response, a limitation of conventional ELISA assay. This method to obtain specific ELISA is "ISAC" (Immunological Specificity and Accuracy Control; patented).

The A-wells (green in colour) contain monoclonal antibodies against PAI-1 immobilized onto the well surface as well as soluble antibodies against PAI-1. The N-wells (blue in colour) contain the same monoclonal antibodies against PAI-1 immobilized onto the well surface and soluble non-immune antibodies. During sample incubation, the PAI-1 antigen in the sample is quantitatively bound to antibodies coating the N-wells, but not the A-wells (prevented from binding by the soluble anti-PAI-1 antibodies). HRP-conjugated anti-PAI-1 antibodies are added and will also bind to the PAI-1 molecule. This forms a sandwich of coating antibody: PAI-1: conjugate antibody. The wells are washed to remove excess conjugate and other unbound material. The HRP substrate (OPD/H2O2) is added and converted to the yellow-coloured product at a rate proportional to the amount of HRP-conjugate bound. The difference in response between the N-well and the A-well is the specific PAI-1 response.

Reagent -- Composition, Packaging, Storage and Stability

The unreconstituted reagents are stable until the expiry date indicated on the label when stored at 2-8 °C.

1. Microtest Strips: 6 ready to use, 16-well, colour-coded strips coated with mouse anti-PAI-1 monoclonal antibodies and pre-filled with control antibodies.Store unused strips in a zip-lock bag at 2-8 °C and use within one month.
2. PET Buffer: PBS-EDTA-Tween 20 buffer concentrate sufficient to make a 1L solution.Store reconstituted PET buffer at 2-8 °C and use within one month.
3. PAI-1 Depleted Plasma, 0.5 ml: Lyophilised plasma depleted of PAI-1 by immunoadsorption on a column containing immobilized antibody to human PAI-1. For use with TintElizeâ PAI-1 Assay only.Store reconstituted reagents capped at 2-8 °C and use within one month.
4. PAI-1 Standard Plasma, 0.5 ml: Lyophilised human plasma containing 40 ng/ml PAI-1. For use with TintElizeâ PAI-1 Assay only.
5. Conjugate, 7 ml: Lyophilised goat anti-human PAI-1 IgG conjugated to horseradish peroxidase.Store reconstituted conjugate at 2-8 °C and use within one month.
6. Substrate, 2 ml: Lyophilised ortho-phenylenediamine (OPD) with buffer salts. Freeze excess reconstituted substrate within 30 minutes, store at -20°C and use within one month. May be thawed and refrozen.
7. Hydrogen Peroxide, 2 ml: 0.15 % H2O2 in water.Store in the dark at 2-8°C and use within one month.
8. Reagent Reservoirs: 6 disposable cardboard trays.

Additional Materials Required (but not provided):

• 8-channel or repeating pipette for 50 - 200 ?l
• Pipettes; 5-1000 ?l, and 10 ml
• Purified water (deionized or distilled)
• 3M sulphuric acid (STOP solution)
• Test tubes: 2-5 ml
• 1000 ml graduated glass cylinder
• Squeeze bottle
• Magnetic stirrer and stir bar
• Paper towels or thin sponge
• Horizontal microtest plate shaker
• Microtest plate spectrophotometer capable of measurements at 492 nm
• 2 levels of quality control material are recommended (see section X).

Cleared: FDA, Canadian Licensed