DiaPharma Group, Inc.

For research use only. Not for use in diagnostic procedures.

Competitive Enzyme Immuno Assay (CELIA) of Fibrino Pepdide A (FPA), for measuring FPA on bentonite adsorbed human plasma or any biological sample where FPA must be measured. If tested sample contains fibrinogen, this latter must be removed (i.e. bentonite adsorption), in order to avoid any cross-reactivity. The assay contains all the necessary reagents for the assay, and particularly the bentonite suspension and the anticoagulant solution. All the reagents are ready to use and the microELISA plate is already precoated with synthetic human FPA and stabilized.

Structure & Mechanism of Action

FPA is a 16 amino acid peptide, with a molecular weight of 1536, released from the amino (NH2) terminal end of fibrinogen Aa chains upon the action of thrombin during blood coagulation. Two molecules of FPA are released from one molecule of fibrinogen. The total FPA releasable from fibrinogen is then of 0.9% of the fibrinogen concentration. FPA has a very short half-life in body (< 3 min.). The FPA concentration in normal human plasma is usually below 3 ng/ml.

The end result of the clotting pathway is the production of thrombin for the conversion of fibrinogen to fibrin. Fibrinogen is a dimer soluble in plasma. Exposure of fibrinogen to thrombin results in rapid proteolysis of fibrinogen and the release of fibrinopeptide A. The loss of small peptide A is not sufficient to render the resulting fibrin molecule insoluble, a proces that is required for clot formation, but it tends to form complexes with adjacent fibrin and fibrinogen molecules. A second peptide, fibrinopeptide B, is then cleaved by thrombin, and the fibrin monomers formed by this second proteolytic cleavage polymerize spontaneously to form an insoluble gel.

Significance

In vivo, FPA is used as a marker to determine the rate of conversion of fibrinogen to fibrin by thrombin. An increased FPA level (> 3 ng/ml), indicates the existence of an excess of thrombin activity. FPA is elevated in many clinical situations associated with blood activation, such as DIC, evolutive thrombosis, and malignancies. It is therefore a marker of hypercoagulable states induced in these pathological conditions. The FPA assay can be used, for research purposes, for evidencing the existence of an excess of thrombin turnover, or the presence of thrombin generation during storage of blood or plasma fraction.

Assay Principle

Cross-reactive fibrinogen must be removed by bentonite adsorption. This is done by adding the solution to the anticoagulated plasma sample, centrifuging, collecting the supernatant and repeating the procedure. The plasma is now fibrinogen free and ready for testing. The assay is performed like a standard ELISA. The standard or sample is added to the coated well, pre-incubated with a constant and limited amount of affinity purified rabbit antibodies specific for human FPA. The unreacted anti-FPA antibodies are then measured using a microplate coated with synthetic FPA and stabilised. Free antibodies bind to immobilised FPA. Following a washing step, the immunoconjugate, a goat polyclonal antibody specific for rabbit IgGs and coupled to HRP, is introduced into microwells and binds to immobilised anti-FPA. Following a new washing step, the peroxidase substrate, TMB, in presence of H2O2, is introduced and a colour develops. There is an indirect relationship between the colour developed and the concentration of FPA in the tested sample.

Calibration Curve

Assay Features

· Bentonite treatment of plasma ensures no cross-reactivity of fibrinogen
· Sensitive Assay: Measuring range is 0.5 - 50 ng/ml
· Calibrators and controls included
· Specific for human FPA, both synthetic or released from fibrinogen

Performance Characteristics

· Test sample: Bentonite adsorbed plasma collected on the special FPA anticoagulant, assayed undiluted (or diluted for higher FPA concentrations)
· Dynamic range: 0.5 to 50 ng/ml
· Detection threshold: 0.5 ng/ml
· Total assay time: 2 hours
· Calibrator: FPA released from purified human fibrinogen at an exactly determined concentration
· Specificity: Human FPA
· Quality control: test with synthetic FPA and FPA released from fibrinogen
· Miscellaneous: disposable kit

Kit Composition

· One vial of 50 ml of bentonite suspension, ready to use.
· One vial of 5 ml of 2% Tween 20, ready to use.
· 6 x 16 well Micro ELISA plate, coated with synthetic human FPA, then stabilized
· One vial containing 50 ml of sample diluent, ready to use
· Three vials of FPA calibrator, lyophilized
· Three vials of affinity purified rabbit antibodies specific for human FPA
· Three vials of anti-rabbit IgG-HRP immunoconjugate, a goat polyclonal antibody coupled to HRP, lyophilized
· One vial of 25 ml of conjugate diluent, ready to use
· One vial of 50 ml of 20 fold concentrated wash solution
· Peroxidase substrate (OPD/Urea peroxide tablets or ready to use TMB solution)
· One vial of 20 ml of special anticoagulant solution for the assay of FPA

Number of determinations:12 x 8 strip-wells

Kit Components: FPA Anticoagulant Solution, 20 ml, Catalog number: AR013A
Contains TriSodium citrate, Heparin, Aprotinin and Hirudin.