DiaPharma Group, Inc.

For in vitro diagnostic use

Intended Use

COALIZA® Protein S - Free is intended for the quantitative determination of free protein S antigen in human citrated plasma by ELISA (enzyme-linked ligand sorbent assay)immunoassay.

Summary

Protein S is a vitamin K-dependent, potent natural anticoagulant plasma glycoprotein. Protein S has a molecular weight of approximately 70 kDa and circulates in plasma at a concentration of 25 µg/mL with a half-life of approximately two days.^1-4 About 40% of Protein S in plasma is in a free form, whereas 60% is complexed with C4b-binding protein (C4BP). Only the free Protein S (FPS) functions as a cofactor to APC in the APC-dependent degradation of FVa and FVIIIa.^5-6 Protein S deficiency is associated with an increased risk of thrombosis.

The Coaliza Protein S Free kit method is based on a procedure described by Dahlbäck and colleagues.¹² The microplate wells are precoated with C4BP, which has a very high affinity for binding free Protein S (FPS) antigen in plasma. A monoclonal antibody (HPS 54) conjugated with the enzyme horseradish peroxidase (HRP) is added together with the plasma sample. After the sample and conjugate incubation, unbound material is washed away and bound protein S, in complex with C4BP is detected with the addition of a substratechromogen.

The amount of color in the wells, when expressed in a double logarithmic scale, is directly proportional to the amount of free Protein S antigen in the plasma sample.

Reference: Tusar Kanti Giri, Andreas Hillarp, Yiva Härdig, Bengt Zöller, Björn Dahlbäck. A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen. Thormb Haemost 79, 767-72 (1998).

Measurement Principle

Kit Components -- Composition, Packaging, Storage and Stability
All kit reagents are stable until the expiration dates shown on the labels when stored at 2-8°C.

1. Microtitre Plate 12 x 8. Wells coated with human purified C4BP. To avoid condensation in the wells, the bag containing the microtitre plate should be brought to room temperature before it is opened. Store unused strips at 2-8°C in the resealable bag with the silica gel sachet provided in the kit.
2. Conjugate Concentrate 1 x 0.3 mL. A monoclonal antibody anti-Protein S (HPS 54) conjugated with peroxidase, containing preservatives and protein stabilisers. Diluted conjugate is stable for 14 days when stored at 2-8°C.
3. Diluent (Conjugate and Sample) 2 x 60 mL. Tris buffer containing additives and preservatives.
4. Wash Buffer 2 x 50 mL. Phosphate buffer (10x concentrate) containing Tween 20 (1%) and 2-Chloroacetamide. Diluted wash buffer is stable for 14 days when stored at 2-8°C.
5. Substrate Buffer 2 x 14 mL. Citrate-acetate buffer containing hydrogen peroxide.
6. Chromogen TMB 1 x 1.5 mL. 3,3',5,5' -Tetramethylbenzidine (TMB). Store the chromogen protected from light. As the Substrate-Chromogen TMB solution is not stable once prepared, instructions for use should be closely followed.
7. Calibration Plasma 2 x 1 mL (lyophilised). Human plasma containing a known concentration (IU/mL) of Free Protein S as specified in the enclosed data sheet. The concentration of the Free protein S in the calibration plasma has been referenced against the 1st International Standard 93/950. After reconstitution, aliquots can be stored for 4 months at -20°C. Do not refreeze. Thoroughly mix all dilutions of the plasma.
8. Normal Control Plasma 1 x 1 mL (lyophilised). Human plasma containing a known concentration range (IU/mL) of Free Protein S as specified in the enclosed data sheet. After reconstitution, aliquots can be stored for 4 months at -20°C. Do not refreeze. Thoroughly mix all dilutions of the plasma.
9. Stopping Solution 1 x 12 mL. Sulfuric acid (1N H2SO4)
10. Adhesive Plate Covers x 6. Adhesive seals to cover the microplate strips during incubations.
11. Resealable Bag x 1. For storage of unused microplate strips.

Material required but not provided:

• Sterile distilled or deionized water or NCCLS Type II water15
• Multichannel pipettes capable of delivering 100 µL and 200 µL
• Micropipettes for pipetting volumes of 10 µL, 500 µL and 1000 µL
• Glass or plastic graduated cylinders for preparation of wash buffer
• Manual, semi-automated or automated wash system
• Microplate reader at 450 nm (preferably with a 620 nm / 630 nm reference filter)
• Microtubes/containers for preparation of the calibrators, controls, sample dilutions, conjugate and substrate working solutions
• Vortex mixer