Electrochemical determination of factor XA inhibitors

• US 9,594,042 B2
• Filed: 12/20/2013
• Issued: 03/14/2017
• Est. Priority Date: 10/31/2006
• Status: Active Grant

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Doerge, Liesel, Galm, Margarete, Horn, Carina
• Primary Examiner(s): Martin, Matthew
• Assistant Examiner(s): ROSENWALD, STEVEN ERIC
• Application Number:
  US14/136,769
• Publication Number:
  US 20140106383A1
• Time in Patent Office: 1,180 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 27/26 by investigating electrochemical variables; by using electrolysis or electrophoresis
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

Methods and devices for determining factor Xa inhibitors, in particular heparins and fractionated or low-molecular-weight heparins, as well as direct factor Xa inhibitors in blood samples. The methods include contacting a blood sample with a detection reagent that contains at least one thrombin substrate having a peptide residue that can be cleaved by thrombin and is amidically bound via the carboxyl end to an electrogenic substance, and with a known amount of factor X reagent and an activator reagent which induces the conversion of factor X into factor Xa. Subsequently, in a second step, the amount or activity of the electrogenic substance that is cleaved from the thrombin substrate by the factor Xa-mediated thrombin activation and/or the time course thereof is determined as the measurement signal using electrochemical methods. In a third step, the amount of the factor Xa inhibitor in the sample of the blood to be analyzed or a measured quantity that correlates therewith, in particular a clotting time that correlates therewith, is determined on the basis of this measurement signal.

Claims

1. A method for determining the amount an amount of a factor Xa inhibitor in a blood sample comprising:

(a) contacting a blood sample with a dried reagent comprising a known amount of factor X, an activator reagent that induces the conversion of factor X into factor Xa, and a detection reagent comprising a peptidic thrombin substrate that can be cleaved by thrombin and comprising a carboxyl end that is amidically bound to an electrogenic substance, wherein the reagent comprising factor X is added to the sample in a common step with the addition of the activator reagent to the sample;

(b) measuring the amount or activity of the electrogenic substance that is cleaved from the thrombin substrate by measuring an electrochemical signal associated with the amount or activity of the electrogenic substance;

(c) relating the amount or activity of the electrogenic substance to the amount of Xa inhibitor in the sample, thereby determining the amount of the factor Xa inhibitor in the sample.

2. The method of claim 1, wherein the reagent comprising factor X and the activator reagent are present on the element as dry chemistry reagents in spatially separated compartments.

3. The method of claim 1, wherein the activator reagent is arranged at least partially in front of the reagent comprising factor X in a flow direction of the sample.

4. The method of claim 1, wherein the reagent comprising factor X and the activator reagent are present on the element as at least partially separated dry chemistry reagents.

5. The method of claim 1, wherein the reagent comprising factor X and the activator reagent are present on the element as dry chemistry reagents in a common compartment.

6. The method of claim 5, wherein the reagent comprising factor X and the activator reagent are in a single dry chemistry reagent line or spot.

7. The method of claim 1, wherein the detection reagent is added to the sample spatially separated from or at a separate time from the addition of the activator reagent to the sample.

8. The method according to claim 1, wherein the detection reagent is added to the sample after the activator reagent has been added to the sample.

9. The method according to claim 1, wherein the factor Xa inhibitor is selected from the group consisting of unfractionated heparin, fractionated heparin, low-molecular-weight heparin, an indirect selective factor Xa inhibitor, and a direct factor Xa inhibitor.

10. A test element for determining an amount of a factor Xa inhibitor, the element comprising an inert carrier comprising at least two electrodes, a reagent comprising a known amount of factor X, an activator reagent for converting factor X into factor Xa, and a detection reagent that contains at least one peptidic thrombin substrate that can be cleaved by thrombin and comprising a carboxyl end that is amidically bound to an electrogenic substance, wherein the reagent comprising factor X and the activator reagent are present on the element as dry chemistry reagents in a common compartment.

11. The test element of claim 10, wherein the reagent comprising factor X and the activator reagent are in a single dry chemistry reagent line or spot.

12. The test element of claim 10, wherein the Xa inhibitor is selected from the group consisting of unfractionated heparin, fractionated heparin, low-molecular-weight heparin, indirect selective factor Xa inhibitors, and direct factor Xa inhibitors.

13. An analytical system comprising at least one device for measuring current or voltage, and a test element according to claim 10.

14. A test element for determining an amount of a factor Xa inhibitor, the element comprising an inert carrier comprising at least two electrodes and a dried reagent line comprising a reagent comprising a known amount of factor X and an activator reagent for converting factor X into factor Xa, the element further comprising a detection reagent that contains at least one peptidic thrombin substrate that can be cleaved by thrombin and comprising a carboxyl end that is amidically bound to an electrogenic substance.

15. The test element of claim 14, wherein the Xa inhibitor is selected from the group consisting of unfractionated heparin, fractionated heparin, low-molecular-weight heparin, indirect selective factor Xa inhibitors, and direct factor Xa inhibitors.

16. An electrochemical test element analytical system comprising at least one device for measuring current or voltage, and a test element according to claim 14.