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

  • Total and Free protein S
  • Convenient ELISA procedure
  • Reagent complete kit – no extra expense for additional reagents – cost effective
  • Six point reference curve
  • Accurate measurement of a specific protein
  • No reconstitution required, reducing risk of dilution errors
  • Kit reagents optimized for more accurate results
  • Plate and components are stable after opening through shelf-life – cost effective
  • Calibrated for Protein C, S and vWF assays to ISTH standards and optimized for each lot for more accurate results
  • Assay formats available for both manual and select automated platforms
  • Total incubation time: 60 minutes

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).

Store at 2 – 8°C. Do Not Freeze.

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

Principle

The REAADS Protein S Antigen test kit is a double antibody capture assay for measuring total and free Protein S levels in human plasma, expressed in relative percent (%) of normal. The assay is intended to be used as an aid in the diagnosis of Protein S deficiency in patients with thrombotic disorders. The REAADS Protein S Antigen test kit will accurately detect antigen levels as low as 5% of normal.

Procedure

Diluted citrated patient plasma is incubated in microwells coated with capture antibody specific for human Protein S, allowing patient Protein S to bind to the surface. The human Protein S detection antibody is added. After incubation, the wells are washed, substrate is added and color development is measured in a spectrophotometer at 450nm following the addition of a stop solution. Patient Protein S levels are determined from a six-point curve prepared from the reference plasma provided in the kit. Free Protein S levels can be measured simultaneously by testing samples that have been pretreated with PEG, following the same assay procedure. Total incubation time is 60 minutes.

Clinical Performance

Plasma samples from healthy blood donors and from patients with a history of thrombosis were tested to define and compare the clinical performance of REAADS Protein S ELISA with a well established, commercially available Protein S Antigen Rocket EID method. As shown in the table, the results correlated well, and were shown to be statistically similar by single factor ANOVA.

REAADSRocket EID
Total Protein SHealthyMean105%98%
Range64 – 150%65 – 143%
PatientsMean59%58%
Range23 – 140%23 – 148%
Free Protein SHealthyMean97%102%
Range61 – 162%62 – 160%
PatientsMean47%52%
Range12 – 115%20 – 127%
Correlation (r) = 0.934; P value = 0.346

Technical Performance

Intra-assay precision is 10.1% for REAADS Total Protein S, and 6.6% for the Free Protein S assay. Inter-assay precision is 11.0% for Total, and 10.5% for Free Protein S. Linearity, expressed as the coefficient of determination (r2) is 0.985 for Total, and 0.992 for Free Protein S. Mean accuracy is 101% for Total, and 98% for Free Protein S assays. REAADS Protein S ELISA is a rapid, convenient, highly accurate and precise method for the quantitative determination of Protein S levels in human plasma.

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.