An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Total and Free Protein S Antigen in citrated human plasma.

Summary and Explanation

Protein S is a vitamin K-dependent protein synthesized in the liver, vascular endothelium, and megakaryocytes, which plays an important physiologic role in the Protein C Anticoagulant System. This anticoagulant system is one of the major regulators of hemostasis by inhibiting clot formation and by promoting fibrinolysis. Protein S functions as a cofactor for activated Protein C on the vascular membrane to facilitate the degradation of clotting factors Va and VIIIa, down-regulating clot formation. In normal plasma approximately 40% of Protein S circulates as a free molecule, while 60% is complexed with C4b, a plasma protein of the classical complement pathway. Only Free Protein S is functionally active and able to bind to activated Protein C, while the complexed form of Protein S is not.

Protein S deficiency, either congenital or acquired, may lead to serious thrombotic events such as thrombophlebitis, deep vein thrombosis, or pulmonary embolism. The prevalence of Protein S deficiency has been estimated to be less than 1 case per 300 in the general population. Two-thirds of patients with a congenital deficiency of Protein S (levels less than 50% of normal) may present with venous thrombosis in young adulthood. In young patients (<35 years) with a history of thrombosis, the prevalence may be as high as 15 to 18%.7 Acquired Protein S deficiency may be seen during pregnancy, oral contraceptive or oral anticoagulant therapy, liver disease, diabetes mellitus, postoperative complications, septicemia and various inflammatory syndromes. A decreased Protein S activity in plasma may be the result of low concentrations or abnormal function of the Protein S molecule.

The laboratory diagnosis of Protein S deficiency may require both quantitative and qualitative (functional) determinations. Quantitative determinations of Protein S Antigen are based on immunologic procedures such as radial immunodiffusion in gel, Laurell rocket immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA).9,10 ELISA procedures are less labor intensive and offer several advantages including more objective, accurate and reproducible results. In addition, ELISA allows automation with commonly available laboratory instrumentation. Measurement of plasma levels of both Total and Free Protein S are useful to determine the type of defect in patients with Protein S deficiency.

Principle of the Test

The Protein S Antigen assay is a sandwich ELISA. A capture antibody specific for human Protein S is coated to 96-microwell polystyrene plates. Diluted patient plasma is incubated in the wells, allowing any available Protein S to bind to the anti-human Protein S antibody on the microwell surface. The plates are washed to remove unbound proteins or other plasma molecules. Bound Protein S is quantitated using horseradish peroxidase (HRP) conjugated anti-human Protein S detection antibody. Following incubation, unbound conjugate is removed by washing. A chromogenic substrate of tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) is added to develop a colored reaction. The intensity of the color is measured in optical density (O.D.) units with a spectrophotometer at 450nm.

Protein S relative percent concentrations in patient plasma are determined against a curve prepared from the reference plasma provided with the kit.

To measure Free Protein S, PEG is added to plasma samples prior to beginning the assay to precipitate the Protein S-C4b binding protein complex. The supernatant fraction containing Free Protein S may be tested along with the untreated plasma sample. Results obtained from diluted plasma samples not pretreated with polyethylene glycol (PEG) represent the Total Protein S concentration for that sample. Both Total (untreated) and Free (PEG-treated) Protein S concentrations are determined following the same assay procedure as described above using separate reference curves.

Reagents
Store at 2 - 8°C. Do Not Freeze. Each REAADS Protein S Antigen 96-microwell Test Kit contains the following reagents:

• 12 x 8 anti-human Protein S antibody coated microwells
• 60mL Sample Diluent (blue-green solution); contains sodium azide.
• 3 vials x 0.5mL lyophilized Reference Plasma, with assay sheet.
• 12mL anti-human Protein S HRP Conjugate (red solution).
• 13mL Substrate (TMB and H2O2).
• 15mL Stopping Solution (0.36 N sulfuric acid).
• 30mL Wash Concentrate (33X PBS with 0.01% Tween 20). Note: turbidity may appear in wash concentrate which will not affect component performance and should disappear when working dilution is prepared.
• 2mL Free Protein S Reagent (PEG).

Materials Required but not Supplied

• Protein S Control Plasma (Total and/or Free). Reconstitute Control Plasma selected for use following manufacturer's instructions, and store as recommended.
• Reagent grade water (1L) to prepare PBS/Tween wash solution, to reconstitute Reference Plasma, and to zero or blank the plate reader during the final assay step.
• Graduated cylinders
• Precision pipettors capable of delivering between 5 and 1000 microliters, with appropriate tips
• Miscellaneous glassware appropriate for small volume handling
• Flask or bottle, 1 liter
• Wash bottles, preferably with the tip partially cut back to provide a wide stream, or an automated or semi-automated washing system
• Disposable gloves, powder-free recommended
• Plate reading spectrophotometer capable of reading absorbance at 450nm (with a 650nm reference if available)
• Multichannel pipettors capable of delivering to 8 wells simultaneously
• Microdilution tubes for patient sample preparation
• Centrifuge