DiaPharma Group, Inc.

Principle

Spectrolyse®/pL PAI is a two-stage, indirect enzymatic assay intended for the quantitative determination of Plasminogen Activator Inhibitor (PAI-1) activity in plasma. PAI-1 levels in plasma samples are determined using a method first described by Chmielewska et al. (8) and Eriksson et al. (11).

In stage one, a fixed amount of tPA is added to the plasma sample and allowed to react with the PAI-1 present. Then the sample is acidified to destroy alpha-2-antiplasmin and other potential plasmin inhibitors which would otherwise interfere with the tPA assay. Subsequently, the sample is diluted.

In stage two, the residual tPA activity is measured by adding the sample to a mixture of Glu-plasminogen, poly-D-lysine and chromogenic substrate at neutral pH. The residual tPA activity in the sample will catalyse the conversion of plasminogen to plasmin, which in turn will hydrolyse the chromogenic substrate. The amount of color developed is proportional to the amount of tPA activity in the sample. Poly-D-lysine is present as a stimulator of the tPA catalysed conversion of plasminogen to plasmin.

The PAI content of the sample is then identified as the difference between the amount of tPA added and the amount of tPA recovered.

Background

An increased plasma level of PAI is an important reason for impaired fibrinolytic function and may be associated with thrombotic diseases. In addition, increased levels have been found in conditions such as normal pregnancy and sepsis.

Definition of PAI Unit

One unit of PAI activity is defined as the amount of PAI that inhibits one international unit of human single chain tPA as calibrated against the International Standard for tPA lot 86/670 distributed by NIBSAC, Holly Hill, London, England.

Reagents -- Composition, Packaging, Storage and Stability

Store at 2 - 8°C in sealed protective foils and use before expiry date.

1. PAR/pL: 1 vial 0.5 mg lyophilised human Glu-plasminogen, D-But-CHT-Lys-pNA and poly-D-lysine.
2. tPA Standard: 1 vial 10 ?g lyophilised human melanoma singlechain tPA.
3. tPA/PAI depleted plasma: 1 vial 1.0 ml lyophilised human plasma.
4. Imidazole 10X: 1 vial 8 ml, 10 X concentrated assay buffer, pH 7.2. Contains Sodium Azide and imidazole.
5. Acetate Buffer: 1 vial of 8 ml 1.0 mol/L sodium acetate buffer, pH 3.9. Contains Potassium hydroxide 0.69% and acetic acid.
6. STOP/pL Reagent: 1 vial 8 ml, 0.8 M potassium acetate, 3.2 M guanidine HCl, pH 3.7, ready-to-use stop solution. Contains 0.51% Potassium hydroxide.

Reagents and equipment required but not provided

• 3 ml plastic test tubes
• 37± 0.1 °C water bath
• Variable volume pipettors 20-1000 ?l
• Spectrophotometer operable at 405 nm and 492 nm
• Purified water (distilled or deionized)
• Ice

Determinations:

• 60 tests in micro test plates
• 25 tests in test tubes

Cleared: FDA, Canadian Licensed