For in vitro diagnostic use

A chromogenic kit for the quantitative determination of protein C activity in human citrated plasma. No influence of heparin levels up to 3 IU/ml. Simple and rapid performance gives optimal user convenience. Excellent accuracy and precision as well as reagent stability. Validated and documented protocols for a wide range of instruments. Suitable for both large and small laboratories.

Background and summary

Protein C is a vitamin K dependent plasma protein which plays an important role in the anticoagulant regulatory mechanisms. It circulates as a zymogen and is converted to an active serine protease, activated Protein C (APC), by the action of thrombin in presence of thrombomodulin. APC regulates the coagulation system by proteolytic cleavage and inactivation of activated factors V and VIII. Hereditary and/or acquired Protein C deficiency has been shown to be a risk factor for development of venous thrombosis.

Measurement principle

Protein C in plasma is activated by a specific enzyme from Southern Copperhead Snake venom. The amount of activated protein C is determined by the rate of hydrolysis of the chromogenic substrate S-2366. The pNA release measured at 405 nm is proportional to the Protein C level in the range from 0-120% of normal plasma.

Reagents -- Packaging, Storage and Stability

1. S-2366 6 mg 2 vials. Lyophilized chromogenic substrate pyroGlu-Pro-Arg-pNA·HCl. Stability after reconstitution: 3 months at 2-8°C in the original vial.

2. Protein C activator 1.2 U 2 vials. Lyophilized venom enzyme from Southern Copperhead Snake (Agkistrodon Contortrix Contortrix), with bovine serum albumin (stabilizer) and Ciprofloxacin® (preservative). Stability after reconstitution: 3 months at 2-8°C in the original vial.

The sealed reagents are stable at 2-8°C until the expiry date printed on the label.

WARNING: Do not use reagents beyond the expiry date printed on the package label. Discard if the substrate solution appears yellow.

Reagent preparation

For microplate and test tube techniques reconstitute each reagent with 7.2 mL of water (see REAGENTS 3.) Replace the stopper and swirl gently. Make sure of the complete reconstitution of the product. Keep reagent at 15-25°C for 10-30 min and invert before use.

NOTE: Other reagent reconstitution volumes may apply for automated methods. Reagents are not interchangeable between lots.

Reagents required but not provided:

• 3. Deionized water, filtered through 0.22 ?m or NCCLS type II water6.
• 4. Human normal plasma with a Protein C activity of 100%. Different standard dilutions are obtained by diluting normal plasma with water (see REAGENTS 3). A lyophilized preparation of human normal plasma, calibrated against International standard for Protein C is recommended.
• 5. Acetic acid 20% or citric acid 2%.

Materials required but not provided:

• Spectrophotometer 405 nm (405 nm and 490 nm for microplate procedure)
• Incubator 37°C ± 0.2°C
• Microplates* or semi-micro cuvettes
• Centrifuge, 2000 x g
• Plastic test tubes
• Stopwatch
• Vortex mixer
• Calibrated pipettes
• Linear graph paper

*NOTE: Do not use microplates intended for coating!

Number of determinations:
Test tube: 72
Microplate: 288
Automated: up to 180