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.