DiaPharma Group, Inc.

For in vitro diagnostic use only.

Biopool Chromolize tPA is a bio-functional immunosorbent assay (BIA) intended for the quantitative determination of human tissue plasminogen activator activity in plasma. The clinical utility of the assay is to detect disorders of the fibrinolytic system.

Intended Use

Biopool's Chromolize tPA is a bio-functional immunosorbent assay (BIA) intended for the quantitative determination of human tissue plasminogen activator activity in plasma. The clinical utility of the assay is to detect disorders of the fibrinolytic system.

Summary

Tissue-type plasminogen activator (tPA) constitutes an important protein in the fibrinolytic pathway, and its activity is thought to play a major role in the fibrinolytic system. The physiological role of tPA is to activate plasminogen to plasmin, which degrades fibrin to soluble fibrin degradation products (FDP). The tPA molecule is present in either the single-chain or two-chain form with similar plasminogen activating potentials. Fibrinolysis is regulated by specific interactions between tPA and fibrin, as well as between plasmin and the specific plasmin inhibitor, a2-antiplasmin.

In the assay of tPA, the fast-acting specific inhibitor for single-chain tPA, plasminogen activator inhibitor type 1 (PAI-1), is usually present in large excess over tPA and must be inhibited from quenching tPA activity. This is accomplished using Stabilyte blood collection tubes, which provide mild acidification and stabilisation of the sample.

Principle

The Biopool Chromolize tPA has been briefly described. The sample tPA is captured by antibodies on the microtest wells. The SP-322 monoclonal antibody used allows excellent tPA recovery at pH 5.9 without inhibiting tPA activity. After the test plasma is discarded, the wells are washed with mild detergent. The tPA substrate consisting of plasminogen, a plasmin-sensitive chromogenic substrate, and tPA activity promoters, all in HEPES buffer pH 8.5, is added and the microtest wells are incubated at ambient temperature (18-25°C) for 90 minutes with agitation. Samples are read at 405 nm. The amount of color developed is proportional to the amount of tPA activity in the sample. The tPA activity standard provided contains human singlechain tPA and is calibrated against the international standard for tPA, lot 86/670 distributed by NIBSC, South Mimms, Potters Bar, Hartfordshire, UK.

Reagents -- 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. 12 Framed Microtest Strips: Ready-to-use, lyophilised, 8-well strips coated with monoclonal antibody SP-322 directed against tPA. Store unused strips in a zip-lock bag at 2-8°C and use within 4 weeks.
2. PET Buffer: Phosphate, NaCl, EDTA, and Tween 20 buffer concentrate sufficient for 1 L solution. Store dissolved reagent at 2-8°C and use within 4 weeks.
3. tPA Activity Standard, 3 x 0.5 ml: Lyophilised human plasma containing 2.0 IU/ml human melanoma tPA. Store reconstituted reagent on ice and use within 8 hours.
4. Substrate Reagent, 6 ml: Lyophilised H-D-But-CHT-Lys-pNA and poly-D-lysine. Store reconstituted reagent in the dark at -20°C for 4 weeks. May be thawed and refrozen twice.
5. Plasminogen Reagent, 6 ml: Lyophilised plasminogen and FDP. Store reconstituted reagent in the dark at -20°C for 4 weeks. May be thawed and refrozen twice.
6. Citrate Buffer, 8 ml: Ready-to-use citrate buffer, pH 5.9.
7. HEPES Buffer, 13 ml: Ready-to-use buffer containing HEPES, EDTA, Tween 20, and 0.02% sodium azide, pH 8.5.

Additional Material Required (but not provided):

• Reagent grade water
• Squeeze bottle
• Adjustable pipettes for 20-500 ml
• Repeating pipette for 50 and 100 ml
• Paper towels or thin sponge
• Small plastic tubes (2-5 ml)
• Magnetic stirrer and stir-bar
• Glacial acetic acid
• Microtest plate shaker with a uniform horizontally circular movement of 3-5 mm (e.g., IKA-Schuttler, Janke & Kunkel Gmbh &Co.KG).
• Microtest plate spectrophotometer capable of measurements at 405 nm and 492 nm.

Cleared: FDA, Canadian Licensed