DiaPharma Group, Inc.

For in vitro diagnostic use.

Biopool Chromolize PAI-1 is a bio immunoassay (BIA) for the quantitative
determination of active human plasminogen activator inhibitor, type 1 (PAI-
1) in human plasma.

An increased plasma level of PAI-1is an important reason for impaired fibrinolytic function and may be associated with thrombotic diseases. Increased levels of PAI-1 have also been found in conditions such as normal pregnancyand sepsis.

Principle

Chromolizeä PAI-1 is an immunoactivity assay. Active tPA is lyophilized while immobilised on the surface of the microtest well. After reconstitution, plasma samples containing PAI-1 are added and active PAI-1 reacts with the bound tPA. Simultaneously, an HRP-conjugated monoclonal antibody (MA 12A4) to PAI-1 is added. After incubation, unbound sample and conjugated antibody are washed away and the bound PAI-1 quantified with an HRP-sensitive substrate. The amount of colour developed is directly proportional to the concentration of active PAI-1 in the sample.

Definition of PAI Unit

One unit of PAI-1 activity is defined as the amount of PAI-1 that inhibits one international unit of human single chain tPA as calibrated against the International Standard for tPA, lot 86/670 distributed by NIBSC, Holly Hill, London, England. The Chromolizeä PAI-1 assay is standardised against the NIBSC PAI-1 standard 92/654.

Reagents -- Composition, Packaging, Storage and Stability

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

1. Microtest Strips: 12 tPA-coated, 8-well strips containing human tPA immobilised on the strip well surface. Store unused strips in a zip-lock bag at 2-8°C and use within one month
2. PET Buffer: Phosphate, NaCl, EDTA, and Tween 20 buffer substance sufficient for 1 L solution. Store reconstituted PET Buffer at 2-8°C and use within one month.
3. PAI-1 Standard Plasma, 0 IU/ml: 4 vials of 0.250 ml lyophilized plasma with 0 IU/ml PAI-1.Reconstituted PAI-1 standard is stable at 2-8°C for up to 4 hours.
4. PAI-1 Standard Plasma, 50 IU/ml: 4 vials of 0.250 ml lyophilized plasma with 50 IU/ml PAI-1.Store reconstituted conjugate in the dark at -20°C and use within one month. Thaw by hand warmth before reuse. May be thawed and refrozen 4 times. Reconstituted PAI-1 standard is stable at 2-8°C for up to 4 hours.
5. Conjugate, 5 ml: HRP-labelled monoclonal anti-PAI-1 antibody.
6. HRP Substrate Solvent, 20 ml: Phosphate/citrate buffer with hydrogen peroxide.After dissolution: store in the dark at 2-8°C and use within one month.
7. HRP Substrate, 5 mg: 4 tablets containing 5 mg of HRP substrate (1, 2 phenylenediamine dihydrochloride, OPD at 3.33%).After dissolution: store in the dark at 2-8°C and use within one month.
8. ReagentReservoirs: 6 disposable cardboard trays.

Additional Material Required (but not provided):

• 1-channel pipettes covering 0-50 and 200-1000ml.
• 8-channel or repeating pipette for 25-100 ml.
• Purified water (glass distilled or water for injection).
• 1.6 M sulphuric acid.
• Small plastic tubes (2-5 ml) with caps.
• Squeeze bottle.
• Magnetic stirrer and stir bar.
• Paper towels or thin sponge.
• Microtest plate shaker with uniform horizontally circular movement of 3-5 mm.
• Microtest plate spectrophotometer capable of measurements at 492 nm.

Cleared: FDA, Canadian Licensing