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

Reagents

  • 12 x 8 anti-human Protein C antibody coated microwells.
  • 60 ml Sample Diluent (blue-green solution); contains sodium azide.
  • 3 vials x 0.5 ml lyophilized Reference Plasma, with assay sheet
  • 12 ml anti-human Protein C HRP Conjugate (blue solution).
  • 13 ml Substrate (TMB and H2O2).
  • 15 ml Stopping Solution (0.36 N sulfuric acid).
  • 30 ml Wash Concentrate (33X phosphate buffered saline 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

  • Protein C Control Plasma. Reconstitute Control Plasma selected for use following manufacturer’s instructions, and store as recommended.
  • Reagent grade water (1L) to prepare PBS/Tween 20 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

Principle

The Protein C Antigen assay is a sandwich ELISA. A capture antibody specific for human Protein C is coated to 96-microwell polystyrene plates. Diluted patient plasma is incubated in the wells, allowing any available Protein C to bind to the anti-human Protein C antibody on the microwell surface. The plates are washed to remove unbound proteins and other plasma molecules. Bound Protein C is quantitated using horseradish peroxidase (HRP) conjugated anti-human Protein C 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 C Antigen relative percent concentrations in patient plasma are determined against a curve prepared from the reference plasma provided with the kit.

 

Procedure

Diluted citrated patient plasma and controls are incubated in microwells coated with capture antibody specific for human Protein C. During an incubation period, patient Protein C is allowed to bind to the surface.

Following a wash to remove any unbound plasma, horseradish peroxidase (HRP) conjugated anti-human Protein C detection antibody is added to the wells. After washing to remove unbound conjugate, a chromogenic substrate is added, resulting in a soluble colored product that is measured in a spectrophotometer at 450nm following the addition of a stop solution.

Patient Protein C levels are determined from a sixpoint curve prepared from the reference plasma provided in the kit. Total incubation time is 60 minutes.

1. Remove any microwell strips that will not be used from the frame and store them in the bag provided.
2. Assay each reference plasma dilution in duplicate. Duplicate determinations are also recommended for patient and control samples. One well should be run as a reagent blank; sample diluent without serum is added to the well as explained in step 7 of this section. This well is treated the same as a control or patient sample in subsequent assay steps. A water blank well should be included with each plate; it is to remain empty until 200 µl of reagent grade water is added at the completion of the assay, immediately prior to reading the plate. The water blank well is to be used to zero the plate reader.
3. Pre-dilute all plasmas (1:2 dilution in Sample Diluent) as follows: Reference plasma: add 100 µl reference plasma to 100 µl Sample Diluent Control and patient samples: add 20 µl plasma to 20 µl Sample Diluent Mix well. These pre-dilutions are utilized in preparing the working dilutions in steps 4 and 5.
4. Using the 1:2 reference plasma dilution from step 3, prepare six working reference dilutions as described below.

 

Volume Reference Plasma (1:2)Volume Sample Diluent*Reference Level
30 μl+500 μl=150
20 μl+500 μl=100
15 μl+500 μl=75
10 μl+500 μl=50
10 μl+1000 μl=25
10 μl+2000 μl=12.5
* Reference level value to be used for constructing reference curve only

5. Prepare working dilutions of control and patient samples by adding 20 µl of prediluted plasma (1:2 dilution from step 3) to 500 µl Sample Diluent. (Note: these dilutions correspond to the 100% reference plasma dilution.)
6. Mix thoroughly, and add 100 µl of the working dilutions (reference plasmas, controls and patient samples) to the appropriate microwells.
7. Add 100 µl of Sample Diluent to the reagent blank well. Leave the water blank well empty.
8. Incubate 40 minutes at room temperature. After the incubation is complete, carefully invert the microwells and dump the sample fluid. Do not allow samples to contaminate other microwells.
9. Wash 4 times with working wash solution (PBS/Tween 20). Each well should be filled with wash solution per wash. Wash solution in the empty well intended to serve as a water blank will not interfere with the procedure. Invert microwells between each wash to empty fluid. Use a snapping motion of the wrist to shake the liquid from the wells. The frame must be squeezed at the center on the top and bottom to retain microwell modules during washing. Blot on absorbent paper to remove residual wash fluid. Do not allow wells to dry out between steps.
10. Add 100 µl Conjugate (blue) to each well (except the water blank well).
11. Incubate for 10 minutes at room temperature. After the incubation is complete, carefully invert the microwells and dump the conjugate solution.
12. Wash 4 times with working wash solution (PBS/Tween 20) as in step 9. Wash solution in the water blank well does not interfere with the procedure. Use a snapping motion to drain the liquid, and blot on absorbent paper after the final wash. Do not allow the wells to dry out.
13. Add 100 µl Substrate to each well (except for the water blank well) and incubate for 10 minutes at room temperature. Add the substrate to the wells at a steady rate. Blue color will develop in wells with positive samples.
14. Add 100 µl Stopping Solution (0.36 N sulfuric acid) to each well (except for the water blank well) to stop the enzyme reaction. Be sure to add Stopping Solution to the wells in the same order and at the same rate as the Substrate Solution was added. Blue Substrate will turn yellow and colorless substrate will remain colorless. Do not add Stopping Solution to the water blank well. Instead, add 200 µl of reagent grade water to the water blank well. Blank or zero the plate reader against the water blank well. Read the O.D. of each well at  450nm, against a 650nm reference filter (if available). For best results, the O.D. values should be measured within 30 minutes after the addition of Stopping Solution.

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 C ELISA with a well established, commercially available Protein C Antigen Rocket EID method. As shown in the table, the results correlated well, and were shown to be statistically similar by single factor Anova.

Technical Performance

Intra-assay precision of REAADS Protein C ELISA is 7.0% while intra-assay precision is 7.5% Linearity, expressed as the coefficient of determination (r2) is 0.992 with a mean accuracy of 99.4%REAADS Protein C ELISA is a rapid, convenient, highly accurate and precise method for the quantitative determination of Protein C levels in human plasma.

 

Protein C is a vitamin K-dependent protein synthesized primarily by hepatocytes in the liver and plays an important physiologic role in the Protein C Anticoagulant System. Protein C, thrombin from blood clots, and endothelial cells, through complex interactions with other factors of the coagulation cascade, contribute to the maintenance of normal hemostatic mechanisms by down-regulating clot formation and by promoting fibrinolysis. The Protein C Anticoagulant System is activated by the binding of thrombin to thrombomodulin, a transmembrane protein receptor on endothelial cells. The thrombin-thrombomodulin binding on endothelial cell membranes activates circulating Protein C. Activated Protein C binds to Protein S on the membrane of endothelial cells or platelets. In this Protein C-Protein S complex, activated Protein C is now capable of inactivating coagulation factors Va and VIIIa, down-regulating clot formation. Activated Protein C also enhances the function of tissue plasminogen activator (TPA) by dissociating this molecule from its inhibitor, plasminogen activator inhibitor-1 (PAI-1), thereby facilitating clot dissolution or fibrinolysis.

Protein C deficiency, either congenital or acquired, may lead to serious thrombotic events such as thrombophlebitis, deep vein thrombosis, or pulmonary embolism. Patients with a congenital heterozygous deficiency may present with venous thrombosis in young adulthood, while patients with the rare homozygous deficiency present with massive thrombosis (purpura fulminans) during the neonatal period. The prevalence of Protein C deficiency in the general population has been estimated at 1 in 300. In younger patients (<40-45 years) with recurrent venous thrombosis, the frequency of Protein C deficiencies may be as high as 10 to 15%. Acquired Protein C deficiency may be seen in liver disease, extensive thrombotic episodes, surgery, oral anticoagulant therapy, antiphospholipid syndrome, etc. A decreased Protein C activity in plasma may be the result of low concentrations and function (type I) or only low function (type II).

The laboratory diagnosis of Protein C deficiency may require both quantitative and qualitative (functional) determinations. Quantitative determinations of Protein C 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, ELISA allows automation with commonly available laboratory instruments.