DiaPharma Group, Inc.

For in vitro diagnostic use only.

Biopool TintElize® tPA is intended for quantitative determination of human tissue plasminogen activator antigen (tPA) in plasma by enzyme immunoassay. The clinical utility of the assay is to detect disorders of the fibrinolytic system.

Intended Use

TintElize® tPA is intended for quantitative determination of human tissue plasminogen activator antigen (tPA) in plasma by enzyme immunoassay. The clinical utility of the assay is to detect disorders of the fibrinolytic system.

Summary

Tissue-type plasminogen activator (tPA) constitutes an important agent in the fibrinolytic pathway and the levels of tPA are thought to have a major effect on fibrinolytic potential. The physiological role of tPA is to activate plasminogen to plasmin, which in turn degrades fibrin to soluble degradation products. Two forms of the tPA molecule are present in plasma, the single-chain and the two-chain form, and both forms are equally capable of activating plasminogen. Fibrinolysis is regulated by specific molecular interactions between tPA and fibrin as well as between plasmin and the specific plasmin inhibitor, a-2-antiplasmin 1.

Principle

The Biopool TintElize® tPA utilises the double antibody principle. Plasma sample or standard containing tPA is added to a microtest well which is coated with goat anti-tPA IgG and contains soluble non-immune goat IgG. After an incubation sufficient to allow >95% of the tPA to bind to the capture antibodies, HRP-labelled Fab fragments of anti-tPA IgG are added. These are allowed to react with the bound tPA. The wells are emptied and washed to remove unbound conjugate after which peroxidase substrate (OPD/H2O2) is added. The amount of yellow colour developed is directly proportional to the amount of tPA present in the sample. The tPA antigen standard, provided with the TintElize tPA, contains human single-chain tPA and is calibrated against the international standard for tPA, lot 86/670 distributed by NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, England.

Reagent -- Composition, Packaging, Storage and Stability

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

1. Microtest strips, 6 pcs: Framed 16-well strips, precoated with goat anti-tPA IgG and prefilled with non-immune goat IgG and indicator dye. Store tightly sealed in the bag at 2-8°C for one month.
2. Standard 0 ng/ml, 3 vials: Lyophilised human plasma depleted of tPA. Contains merthiolate as preservative. Store on ice and use within 8 hours.
3. Standard 30 ng/ml, 3 vials: Lyophilised human plasma enriched with tPA. Contains merthiolate as preservative. Store on ice and use within 8 hours.
4. Conjugate, 1 vial: Lyophilised HRP-labelled anti-human tPA Fab fragments. Store dark at -20°C for one month. If frozen, thaw at room temperature before re-use. May be thawed and refrozen several times.
5. PET-buffer, 1 vial: PBS-EDTA-Tween 20 buffer substances sufficient to make 1 L solution. Store at 2-8°C and use within one month.
6. Substrate, 1 vial: Lyophilised 1,2 phenylenediamine dihydrochloride (OPD) at 4.17% in buffer salts. Store at -20°C for one month. May be thawed and refrozen.
7. Hydrogen peroxide, 1 vial: 2 ml, 0.15% H2O2 in water. Store in the dark at 2-8°C for one month.

Reagents and equipment required but not provided:

• Pipette 8-channel or repeating for 50-200 ?l
• Pipettes 1-channel covering 20-1000 ?l
• Water purified (glass distilled or water for injection)
• Sulphuric acid concentrated
• Microtest Plate Spectrophotometer operable at 492 nm
• Magnetic stirrer and stir-bar
• Microtest Plate Shaker, orbital movement 3-5 mm, speed 600 rpm.
• Squeeze bottle
• Paper towels or thin sponge
• Small plastic tubes (2-5 ml)

Cleared: FDA, Canadian Licensed