Chromogenic method for quantitative determination of antithrombin (AT) activity in human plasma


Antithrombin (AT) is the major physiological coagulation inhibitor. It inhibits coagulation serine esterases, especially Thrombin, Factor Xa and Factor IXa, regulates coagulation pathway and prevents from thrombosis. When complexed to heparin, AT becomes a potent and fast acting inhibitor of coagulation serine esterases. This anti-IIa assay thus measures the heparin dependent anti-IIa activity of AT1,2,3,4.


The BIOPHEN™ AT (Anti-IIa) method is an assay based on the inhibition of a constant and in excess amount of Thrombin (IIa), by the tested AT in presence of an excess of heparin, and hydrolysis of a Thrombin specific chromogenic substrate, by Thrombin in excess. pNA is then released from the substrate. The amount of pNA released (measured by absorbance at 405nm) is then a relation of the residual Thrombin quantity. There is an inverse relationship to the concentration of AT present in the tested sample.

Variant Method (Progressive Antithrombin Activity)

For the identification of type II abnormality, HBS (Heparin Binding Site), a variant method can be performed using a specific “Tris buffer without heparin” (AR104A) instead of “R3 buffer”. A specific calibration curve must be done with Plasma Calibrator and the patient AT activity (HBS) is directly read on the curve. The specific protocol is available upon request. In presence of the HBS variant, the patient has a normal AT activity with this method.

Ordering information

Product Name Reference Package
BIOPHENTM AT Anti-IIa (CE-IVD) 221122  2 x 2.5 ml
BIOPHENTM  Plasma Calibrator (CE-IVD) 222101 12 x 1 ml
BIOPHENTM  Normal Controle Plasma (CE-IVD) 223201 12 x 1 ml
BIOPHENTM  Abnormal Controle Plasma (CE-IVD) 223301 12 x 1 ml


  1. Leslie B. et al. Investigation of the anticoagulant mechanism of a covalent antithrombin-heparin complex. The Journal of Biological Chemistry. 1999.
  2. Tsiang M et al. Functional requirements for inhibition of Thrombin by Antithrombin III in the presence and absence of heparin. The journal of Biological Chemistry.1997.
  3. Tollefsen D.M. Laboratory Diagnosis of Antithrombin and Heparin Cofactor II deficiency.
    Seminars in Thromb haemost. 1990.
  4. Mortensen J.Z. Inherited ATIII deficiency. Fast and slow inactivation of thrombin and Factor Xa
    Thromb. Res. 1984.