Assay overview
The Protein C pathway is a complex system where activators, inhibitors and accelerators like the Protein C (PC) cofactor Protein S (PS) play a major rol. One goes not whiteout the other.
Nodia has a broad range of Protein C, Protein S, and Factor V-Leiden assays for both clinical and research applications. Find out more about this tests on this Protein C system specific page.
Protein C assays
Biochemistry
Protein C is a vitamin K dependent human protein of the coagulation system, synthesized in the liver, of about 62 KDa. PC is usually present in plasma as a proenzyme, at a concentration of about 4-5 µg/ml. When activated by thrombin (and thrombomodulin), in presence of calcium and phospholipids, activated Protein C, in presence of Protein S, inhibits and regulates coagulation through specific cleavages of Factors Va and VIIIa, suppressing their procoagulant cofactor activity.
Classification
Protein C deficiencies can be congenital or acquired for example during disease, therapy or in DIC. A diagnosis is then preferable. When doing laboratory assessment a distinction can be made between two types of PC malfunctions. There is Type I, a quantitative defect and there is Type II, a qualitative defect. The later is the more rare type and will only be detected when preforming a Clotting assay. Though, for routine assessment a Chromogenic assay is preferred.
Introduction
The HEMOCLOT™ Protein C reagent is an aPTT like clotting assay, performed in presence of a Protein C activator (Protac®, extracted from snake venom Agkistrodon Contortrix) for the quantitative determination of Protein C in human citrated plasma.
Introduction
The BIOPHEN™ Protein C (LRT) kit is a chromogenic assay for the in vitro quantitative determination of Protein C activity in human citrated plasma.
Reaction principle
Intended Use
Diagnosis of congenital or acquired Protein C deficiencies in human plasma samples and Protein C concentrates.
Major Features
- Activity assay
- Very good on board stability (can be frozen)
- Applications available for all analysers
Reaction principle
Intended Use
Diagnosis of congenital or acquired Protein C deficiencies in human plasma samples and Protein C concentrates.
Major Features
- Ready to use due to Liquid reagent technology
- long onboard stability
- CE applications for the most common instruments available
Protein S assays
Biochemistry
Protein S (PS) is a vitamin K dependent glycoprotein with a molecular weight of 80,000 daltons, which is synthesized in the liver. The balance between the free form and the C4bBP bound form of PS plays an important role because only the Free Protein S is believed to serve as a cofactor for Protein C.
In presence of its cofactor Protein S, phospholipids and Calcium, Activated Protein C (APC) inactivates Factor Va and Factor VIIIa.
Pathology
Under physiological conditions, Thrombin binds to Thrombomodulin exposed on endothelial cells and which is abundant in capillaries, and looses its procoagulant activity, whilst it acquires an anticoagulant function capable of activating PC to APC. Clotting pathways are then stopped by inactivation of Factors Va and VIIIa. This PC/PS inhibition pathway has a decreased activity if the free form of Protein S is deficient or abnormal. Congenital or acquired Protein S deficiencies are a risk factor for venous thrombosis.
PS deficiency is the underlying cause in a small proportion of cases with disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT) and pulmonary embolism (PE). Rare deficiencies due to an acquired or transitory auto-antibody to Protein S have been reported..
classification
Inherited protein S deficiencies are classified into three types:
- Type I: decreased total and free PS levels (quantitative defects).
- Type II: decreased PS activity but normal antigenic levels (rare qualitative defects).
- Type III: normal levels of total PS but decreased free PS (quantitative defect).
Type I and III deficiencies account for 95% of cases of PS deficiency.
Introduction
Introduction
The HEMOCLOT™ Protein S method is an APTT like clotting assay, triggered by Factor IXa in presence of phospholipids, Calcium, and of a constant in excess amount of Activated Protein C (APC). First, the diluted assayed plasma is mixed with Protein S deficient plasma (R1). Then, the Activator Reagent (R2)is added in a constant and optimised concentration. Finally clotting is initiated and the clotting time is recorded. Protein S being the limiting factor, there is a direct relationship between the Protein S concentration and the corresponding clotting time.
The LIAPHEN™ Free Protein S assay is an immunoturbidimetric method, based on antigen-antibody reaction: Free PS antigen of the sample reacts with Latex particles sensitized with two mouse monoclonal anti-Free PS antibodies, leading to latex particles agglutination. This agglutination can be directly detected by a change of absorbance. The absorbance change is directly proportional to the amount of Free PS:Ag in the sample
Reaction principle
Intended Use
Major Features
- Clotting Based Activity assay
- Very high on board stability (can be frozen)
- Excellent correlation with Free PS
- Applications available for all analysers
Reaction principle
Intended Use
Major Features
- Ready-to-use Liquid Reagent.
- Specific and reliable assay, without interferences of anticoagulants and sample content.
- Extended measurement range (6-300% Free PS).
- Validated application guide for the major coagulation analyzers
- Long shelf life at 2-8°C in original packaging
- Stable after opening:
- 10 days onboard,
Activated Protein C resistance - Factor V-Leiden
Factor V is a key coagulation factor, acting as a FXa co-factor in the activation of Prothrombin to Thrombin. FV is present in its inactive form in the blood circulation, and will be activated via proteolitic cleavage by Thrombin. Activated FV will then expresses its full procoagulante cofactor activity. A build-in feedback loop is nesscessary to prevent FVa from extensive clotting. This is where the APC-PS complex comes in. Via cleavage at various sites, including the R506Q one it will inactivate FVa and restore normal coagulation.
In 1993 the group of Björn Dahlbäck linked the decreased inhibition by activated Protein C (APC) of Factor V with a higher occurrence of thrombosis. Later, it was demonstrated that this defect is due to a point mutation (R0506) in the FV-gene, now called Factor V-Leiden (FV-L), which prolongs the activated FV survival. In the laboratory, screening assays where introduced. Most of the existing screening assays are aPTT based. The ratio of the tests performed with or with-out APC is calculated. For these tests, the cut off value is instrument and reagent dependent. To get rid of these variabilities, Hyphen BioMed developed the Hemoclot™ Quanti-V Leiden.
Prevalence
The incidence of Factor VL (R506Q mutation) is variable according to the geographical area. It is about 5-6% in the U.S. and Canada. It is higher than 15% in Scandinavian countries and of about 5% in Mediterranean countries. This mutation is absent in the Chinese or Japanese populations.
Introduction
The HEMOCLOT™ Quanti-V Leiden assay is a total quantitative clotting method for Factor V-Leiden (FV-L) measurement in citrated plasma, based on FV-L’s resistance to Activated Protein C (APC). Normal FV is not measured with the assay, as it is totally inhibited by APC, whilst FV-L keeps its clotting activity.
- Patients with low concentrations of total FV clotting activity (<25%) must be identified, and the diagnosis confirmed by comparing FV-L and total FV clotting activity (normals < 0.1)
Reaction principle
Controls and Calibrators
A set of specific APC-resistance calibrators and controls is not provided with the kit but can be purchased separately.
Intended Use
Major Features
- Easy to perform.
- Only one test required which makes the result easy to interpret and the test cost effective.
- Excellent discrimination (for both clotting times and FV-L concentrations) between normals, heterozygous and homozygous patients (for the R506Q mutation).
- Fully automatable on laboratory instruments.
- No interference of Heparin or Dicoumarol therapy.
Protein C antigen
By definition, the 100 % Protein C concentration corresponds to the concentration in a normal human citrated plasma pool, obtained by pooling plasmas from healthy males or females aged from 18 to 55 years, and out of any medication or disease. The Protein C concentration in adults is usually between 70 and 140%.
The Protein C concentration is decreased in neonates. It is then independent from age or gender.
Sandwich ELISA assay for measuring human Protein C (PC) antigen in plasma, or in any biological fluid where Protein C can be present.
DIY
Antibodies for immunoblotting, immunostaining of cells and several types of immunoassays where a higher signal-to-noise is required:
- Anti-human Protein C, Mouse, Monoclonal IgG
- Anti-human Protein C, Sheep, HRP conjugated IgG
- Anti-human Protein C, Sheep, Affinity Purified IgG
- Anti-human Protein C, Sheep, Purified IgG
- Anti-human Protein C, Goat, HRP conjugated IgG
- Anti-human Protein C, Goat, Affinity Purified IgG
- Anti-human PCI-Protein C Inhibitor, Goat, HRP conjugated IgG
- Anti-human PCI-Protein C Inhibitor, Goat, Affinity purified IgG
Matched pair of antibodies:
Protein S antigen
In general
The Protein S concentration in normal human plasma is of about 25 µg/ml. About 40% (i.e. 10 µg/ml) is in the Free form and 60% (i.e. 15 µg/ml) circulates in blood as a non-covalent complex with C4b-BP. It is believed that only the Free form has an anticoagulant activity as cofactor of Activated Protein C. Because of this the balance between the free form and the C4bBP bound form of protein S plays an important role. The Total Protein S concentration in normal human plasma is usually in the range 70–150%. The concentration is higher in males than in females. It tends to increase with age, and with blood lipid concentration.
The ZYMUTEST™ Total Protein S kit is a one step, two-site immuno-assay, for measuring human Total Protein S in plasma, or in any fluid where Total Protein S can be present.
When present, the Protein S (free or complexed with C4b-BP) binds onto the monoclonal antibody coated solid phase through one epitope, and fixes the second monoclonal antibody coupled to HRP by another epitope.
DIY
Antibodies for immunoblotting, immunostaining of cells and several types of immunoassays where a higher signal-to-noise is required:
- Anti-human Protein S, Goat, HRP conjugated IgG
- Anti-human Protein S, Goat, Purified IgG
- Anti-human Protein S, Sheep, HRP conjugated IgG
- Anti-human Protein S, Sheep, Affinity Purified IgG
- Anti-human Protein S, Sheep, Purified IgG
Matched pair of antibodies:
Controls and Calibrators
A set of specific Protein S plasma controls for Elisa can be purchased separately.
Anti-Protein S antigen
The ZYMUTEST™ Anti-Protein S IgM kit is a standardised and optimised enzyme immunoassay designed for measuring autoantibodies to Protein S of the IgM isotype, in human plasma or in any biological fluid where autoantibodies to Protein S must be measured.
This kit is for research use only and should not be used for patient diagnosis or treatment.
Protein C and Protein S Inhibitor and Deficient plasma
Deficient plasma products
The Protein C Deficient Plasma and Protein S Deficient Plasma can be used for the quantitative determination of Protein C and Protein S activity respectively in human citrated plasma using a clotting method via a manual or automated method.
In case Frozen Deficient plasma is preferred, you can chose for 5-Diagnostics Human Protein C Deficient Plasma, Frozen and Human Protein S Deficient Plasma, Frozen.
Inhibitor plasma products
Protein C inhibitor Plasma is produced from normal human plasmas from which specific factors have been removed by selective affinity immuno-adsorption and an antibody inhibitory to the specific factor is added to provide neutralizing activity.