Chromogenic assay for measuring Factor XIa activity.

INTENDED USE:

BIOPHEN Factor XIa kit is a chromogenic assay for measuring activated Factor XI (FXIa) activity, using a chromogenic method, manual or automated, through Factor IX activation and Factor Xa generation.. APPLICATIONS:

Assay of Factor XIa activity in any therapeutic concentrate or purified milieu, where Factor XIa activity must be measured.

TESTED SPECIMEN:

Purified milieu where Factor XIa needs to be measured or therapeutic concentrates. ASSAY PRINCIPLE:

In presence of Phospholipids (PLPs), calcium and thrombin, activated FXIa present in the tested sample is able to activate Factor IX (in excess and without Factor IXa) into FIXa, that forms an enzymatic complex with its cofactor, Factor VIII:C, also supplied in the assay at a constant concentration and in excess. This complex activates Factor X, present in the assay system, into Factor Xa, which generated amount is directly related to the amount of Factor XIa to be measured,. Generated Factor Xa is then measured by its specific activity on a Factor Xa chromogenic substrate (SXa-11). Factor Xa cleaves the substrate and releases pNA. Finally, there is a direct relationship between the amount of Factor XIa in the assayed sample and the Factor Xa activity generated, measured by the amount of pNA released, and measured at 405nm.

Nota : FVIII:C*: Thrombin activated FVIII:C REAGENTS:

R1A: Reagent 1A: Human Factor X and FVIII:C

Human Factor X, and FVIII:C, lyophilised in presence of a fibrin polymerisation inhibitor and stabilizers.

2 vials (each to be reconstituted with 3.0 mL of distilled water).

R1B: Reagent 1B: Human Factor IX (without Factor IXa)

Human Factor IX, lyophilised in presence of stabilizers.

2 vials (each to be reconstituted with 3.0 mL of distilled water).

R2: Reagent 2: “Activation” Reagent (Thrombin-Calcium-Phospholipids)

Human thrombin, calcium and synthetic phospholipids, lyophilised, in presence of stabilizers.

2 vials (each to be reconstituted with 3.0 mL of distilled water).

R3: Reagent 3: SXa-11 (Sequence: Suc-Ile-Glu-(Pip)Gly-Arg-pNA, HCl)

Chromogenic substrate, specific for Factor Xa (SXa-11), lyophilised.

2 vials of SXa-11 (each to be reconstituted with 3.0 mL of distilled water).

R4: Reagent 4: Specific Tris-BSA Buffer

Specific Tris-BSA Buffer, ready to use. Contains 1% BSA and sodium azide.

2 vials of 25 mL each.

Cal: FXIa calibrator: (established against highly purified human FXIa)

Purified human Factor XIa, lyophilised. When restored with 2.0 ml of distilled water, a solution containing a concentration “C” (expressed in ng/ml) of human FXIa is obtained. This concentration (usually close to 5.0 ng/ml according to the lot), is accurately determined for each lot.

2 vials (each to be reconstituted with 2 mL of distilled water).

The exact concentration of FXIa is indicated on the flyer provided in each kit. The calibration curve covers the range from 0 to about 5.0 ng/ml FXIa.