Assay for tissue factor in a sample

• US 7,604,932 B2
• Filed: 11/06/2003
• Issued: 10/20/2009
• Est. Priority Date: 11/13/2002
• Status: Expired due to Fees

• Current Assignee: Haematologic Technologies Incorporated
• Original Assignee: Haematologic Technologies Incorporated
• Inventors: Jenny, Richard J., Butenas, Saulius
• Primary Examiner(s): Saucier; Sandra
• Assistant Examiner(s): HANLEY, SUSAN MARIE
• Application Number:
  US10/534,187
• Publication Number:
  US 20060148022A1
• Time in Patent Office: 2,175 Days
• Field of Search: None
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 2333/96447 Factor VII (3.4.21.21)

Abstract

The invention provides assays for detecting and quantitating tissue factor and factor VIIa in simple and complex biological systems. The assays are performed by detecting and/or measuring the tissue factor cofactor activity and factor VIIa enzymatic activity using aminonapthalene-based fluorogenic substrates.

Claims

1. A method for determining the concentration of tissue factor (TF) in a sample suspected to contain TF, comprising:

(a) combining the sample and a molar excess of factor VIIa (fVIIa) compared to the moles of TF in the sample to produce a TF/fVIIa enzyme complex;

(b) detecting the enzymatic activity of the complex using a fluorogenic or chromogenic substrate;

(c) generating numerical values correlated with the enzymatic activity of the sample; and

(d) comparing the numerical values with a standard curve of TF-dependent enzymatic activity, wherein the standard curve is generated by quantifying TF-dependent enzymatic activity of the TF/fVIIa complex in samples with known concentrations of TF.

2. The method of claim 1, wherein the substrate is a compound of the formula:

3. The method of claim 1, where the substrate is a chromogenic substrate.

4. The method of claim 3, wherein the chromogenic substrate is a para-nitroaniline based substrate.

5. The method of claim 1, wherein the TF is native human tissue factor.

6. The method of claim 1, wherein the sample is obtained from is brain tissue, placenta, endothelial cells, tissue extract, plasma, cell extract, synthetic or naturally derived thromboplastin, or recombinant human tissue factor.

7. The method of claim 1, wherein the fVIIa is native human factor VIIa or recombinant factor VIIa.

8. The method of claim 1, wherein the TF and the fVIIa are not of human origin.

9. The method of claim 1, wherein the concentration of TF is from 0.1 pM to 1 mM.

10. The method of claim 1, wherein the reaction mixture contains divalent metal ion or a metal ion chelator.

11. The method of claim 10, wherein the divalent metal ion is calcium ion, magnesium ion or manganese ion.

12. The method of claim 10, wherein the metal ion chelator is ethylenediamimetetraacetic acid (EDTA) or ethylene glycol-bis(2-aminoethylether)-N,N,N′

,N′

-tetraacetic acid (EGTA).