REAADS Monoclonal Free Protein S uses a monoclonal antibody specific for free Protein S to measurement free Protein S levels in citrated human plasma. No pretreatment of samples with polyethylene glycol (PEG) is required. Results are reported in percent (%) of normal, relative to a reference plasma that has been standardized against the Secondary Standard for Coagulation / International Society on Thrombosis and Hemostasis  (SSC/ISTH) preparation, which is calibrated to World Health Organization (WHO) standards.

  • Utilizes a monoclonal antibody specific for Free Protein S
  • Convenient ELISA procedure
  • Objective, accurate and reproducible
  • Reagent complete kit
  • Total incubate time: 60 minutes at room temperature

REAADS Anti-Beta2 Glycoprotein I Anti-Prothrombin Anti-Cardiolipin Anti-Phosphatidylserine

Reagents

  • 12 x 8 Mouse Monoclonal antibody to human Free Protein S coated microwells.
  • 60 ml Sample Diluent (blue-green solution); contains sodium azide.
  • 3 x 0.5 ml Lyophilized Reference Plasma, with assay sheet.
  • 12 ml Rabbit Anti-human Protein S HRP Conjugate (red solution).
  • 13 ml Substrate (TMB and H2O2).
  • 15 ml Stopping Solution (0.36 N sulfuric acid).
  • 30 ml 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.

Store at 2 – 8°C. Do Not Freeze.

Materials Required but not Supplied

  • Free Protein S Control Plasma. Reconstitute Control Plasma selected for use following manufacturer’s instructions, and store as recommended.
  • Reagent grade water (1 L) 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 450 nm (with a 650 nm reference, if available)
  • Multichannel pipettors capable of delivering to 8 wells simultaneously
  • Microdilution tubes for patient sample preparation
  • Centrifuge

Principle

REAADS Monoclonal Free Protein S ELISA uses a monoclonal antibody specific for free Protein S to measurement free Protein S levels in citrated human plasma. No pretreatment of samples with polyethylene glycol (PEG) is required. Results are reported in percent (%) of normal, relative to a reference plasma that has been standardized against the Secondary Standard for Coagulation / International Society on Thrombosis and Hemostasis  (SSC/ISTH) preparation, which is calibrated to World Health Organization (WHO) standards.

Procedure

Diluted citrated patient plasma is incubated in microwells coated with a monoclonal capture antibody specific for free Protein S, allowing patient free Protein S to bind to the surface. After washing to remove unbound plasma proteins, HRP-conjugated polyclonal anti-human Protein S detection antibody is added, which attaches to the surface bound free Protein S antigen during a second incubation. The wells are washed, and a chromogenic substrate is added, resulting in a soluble colored product that is measured in a spectrophotometer at 450nm after the addition of stop solution. The concentration of free Protein S in the test sample is determined from a standard curve prepared from the reference plasma provided in the kit. Total assay incubation time is 60 minutes at room temperature.

Clinical Performance

The clinical performance was determined by testing plasma samples from 35 healthy individuals and 20 patients with known Protein S deficiency with REAADS Monoclonal Free Protein S assay and REAADS Protein S Antigen test kit (PEG method). As shown in the table, a good correlation was seen between the two methods for the combined test population (r = 0.980), with a P value of 0.739 by single factor Anova.

Technical Performance

Intra-assay precision, expressed in %CV, was 4.7% when samples ranging in value from 6 – 150% were tested in duplicate with REAADS Monoclonal Free Protein S assay. Inter-assay precision was shown to be 5.2%. Accuracy was demonstrated by testing the recovery of plasma samples spiked with known levels of free Protein S; the mean % recovery was 101.2% across three production lots. Linearity was determined by linear regression of the log-log curve expressed as the coefficient of determination (r2) = 0.994.

REAADS Monoclonal Free Protein S Assay is a rapid, accurate and precise method for the determination of free Protein S levels in human plasma, offering improved specificity, convenience and significant time savings over traditional method that require PEG precipitation.

REAADS Monoclonal Free Protein SREAADS Protein S Assay (PEG Method)
NormalsMean105%100%
Range65 – 144%61 – 130%
DeficientsMean20%22%
Range8 – 40%12 – 34%
Correlation (r) = 0.980; P value = 0.739

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%. 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). ELISA procedures are less labor intensive and offer several advantages including more objective, accurate, and reproducible results. In addition, the ELISA format allows automation with commonly available laboratory instrumentation.

Measurement of plasma levels of both Total and Free Protein S are useful in determining the type of defect in patients with Protein S deficiency. Historically, ELISA procedures measuring Protein S used a polyclonal antibody specific to both the free and bound forms of Protein S. The addition of polyethylene glycol (PEG) to precipitate the bound Protein S in the patient sample allowed determination of levels of free Protein S. While the PEG precipitation procedure allows the measurement of Free Protein S, it is non-specific, time consuming, and difficult to perform accurately. This assay utilizes a monoclonal antibody specific for Free Protein S in an ELISA format to measure Free Protein S directly, without PEG precipitation.

What is the interaction between Protein S and C4b-binding protein?
The affinity of protein S to C4BP is several hundred-fold increased by calcium ions. C4BP is an acute-phase reactant, and its concentration in plasma may increase as much as four-fold. This does not result in free PS levels approaching 0 however, because C4BP exists in 2 forms: under normal circumstances, about 10 – 15% of the C4BP circulating in plasma lacks the b-chain and hence does not bind PS. During an acute phase reaction, the C4BP which lacks the b-chain is increased and therefore no significant decrease of free PS occurs.
How is functional activity of protein S tested?
Many labs perform functional clotting assays to test free PS activity. These will detect PS deficiency, but will not discriminate between Types I, II, and III. Clotting tests utilize an excess of protein S deficient plasma, thereby keeping the amount of prothrombin essentially constant. APTT and PT methods commonly used. In a FXa based method, coagulation is triggered by FXa in the presence of calcium ions and phospholipids. This method is not currently used in routine settings, however.
Are elevated levels of Protein S clinically significant?
Though not as well studied as PS deficiency, there has been shown an associated between free and total PS with cholesterol and triglycerides. Though more studies are needed, preliminary findings suggest that elevated free PS may be associated with an increased risk of ischemic heart disease.
What are the different types of protein S deficiency?
The classification of PS deficiency is as follows:

Type 1: Low Protein S Ag Total, Low Protein S Ag Free, Low Protein S Activity
Type II: Normal PS Ag Total, Normal PS Ag Free, Low PS Activity
Type III: Normal PS Ag Total, Low PS Ag Free, Low PS Activity

Type II deficiency seems to be quite rare. Acquired PS is also possible, such as with liver disease, DIC, IBD, and APS. Some research suggests that free PS rather than total PS should be measured in the diagnosis of PS deficiency.

What is the clinical significance of Protein S?
Although the exact role of PS in vivo in the protein C anticoagulant pathway has not been clarified, there is no doubt that PS is an important anticoagulant protein and that PS deficiency is primarily associated with venous thromboembolism. The reported prevalence of PS deficiency in thrombosis patients varies between 1.5-7%, the difference being due to methods used and to the selection of patients. Clinical symptoms in patients affected with PS deficiency are very similar to those with protein C deficiency (DVT). Superficial thrombosis seems to be more common than for antithrombin deficient patients.
What is the interaction between Protein S and APC?
Only the free, native form of PS binds to APC and functions as a cofactor. PS has the highest affinity for negatively charged phospholipids of all the vitamin K-dependent proteins and has been shown to increase by approximately 10-fold, the affinity of APC for membranes or vesicles containing such phospholipids. This may be of physiological importance since APC degrades preferentially membrane-bound FVa and FVIIIa, but not the circulating, inactivated co-factors. In addition to increasing the affinity of APC to membranes, PS also enhances the cleavage of FVa by APC and works in concert with FV to increase the ability of APC to inactivate FVIIIa.
How do Protein S and APC interact?
Only the free, native form of PS binds to APC and functions as a cofactor. PS has the highest affinity for negatively charged phospholipids of all the vitamin K-dependent proteins and has been shown to increase by approximately 10-fold, the affinity of APC for membranes or vesicles containing such phospholipids. This may be of physiological importance since APC degrades preferentially membrane-bound FVa and FVIIIa, but not the circulating, inactivated co-factors. In addition to increasing the affinity of APC to membranes, PS also enhances the cleavage of FVa by APC and works in concert with FV to increase the ability of APC to inactivate FVIIIa.
Protein concentrations in plasma
ComponentMolecular
Weight kDa
Plasma
Concentration
mg/l
Plasma
Concentration
μmol/l
Fibrinogen33030009
Prothrombin721502
Factor V330200.05
Factor VII500.50.01
Factor VIII3300.10.0003
Factor IX5650.09
Factor X5980.13
Factor XI16050.03
Factor XII80300.4
Factor XIII320100.03
Protein C6240.06
Protein S7010 (free)0.14
Protein Z6220.03
Prekallikrein86500.6
HMW kininogen120700.6
Fibronectin4503000.7
Plasminogen922002
t-PA600.0050.0001
Urokinase530.0040.0001
Antithrombin581452.5
Heparin Cofactor II66801.2
Plasmin Inhibitor63601
Protein C Inhibitor5740.07
α2-Macroglobulin72520003