ASSAY FOR MEASURING FACTOR VIIa-ANTITHROMBIN COMPLEXES

Inactive Publication Date: 2009-07-09
OKLAHOMA MEDICAL RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One cannot measure thrombin activity levels in plasma directly because the active enzyme has an extremely short plasma half-life (owing to plasma's high content of protease inhibitors).
In the case of factors IX and X, as with thrombin, the active proteases (factors IXa and Xa) have very short half-lives in plasma, so it is not possible to measure their levels directly.
However, when factor VIIa binds to tissue factor, it becomes susceptible to inhibition both by antithrombin and TFPI.

Method used

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  • ASSAY FOR MEASURING FACTOR VIIa-ANTITHROMBIN COMPLEXES
  • ASSAY FOR MEASURING FACTOR VIIa-ANTITHROMBIN COMPLEXES
  • ASSAY FOR MEASURING FACTOR VIIa-ANTITHROMBIN COMPLEXES

Examples

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example 1

ELISA Assay for Measuring the Level of Factor VIIa-AT Complexes in Plasma Using p-NPP Substrate (With Use of Blocking Agent)

[0039]The ELISA assay was used to measure the concentration of factor VIIa-antithrombin (factor VIIa-AT) complexes in human plasma and other samples.

Preparation of Factor VIIa-AT Complexes for Standardizing the Factor VIIa-AT ELISA

[0040]A 1.1 ml solution of 100 nM factor VIIa [Factor VIIa (recombinant); catalog #407recB; American Diagnostica, Inc., Greenwich, Conn.] and 150 nM sTF [recombinant soluble tissue factor protein produced by methods known in the art] was prepared in HBSA / calcium [HBSA / calcium: 30 mM Hepes buffer, pH 7.4, 100 Mm NaCl, 5 mM CaCl2, 0.1% w / v bovine serum albumin, and 0.1% w / v sodium azide and stored at 4° C.].

[0041]A 0.1 ml aliquot was removed and held on ice to form factor VIIa-sTF complexes.

[0042]To the remaining 1.0 ml reaction mixture, heparin (catalog #H3393; Sigma Chemical Co. St. Louis, Mo.) was added to a final concentration of 1 ...

example 2

ELISA for Factor VIIa-Antithrombin (VIIa-AT) Complexes (Without Blocking Agent)

[0053]Materials. The materials used in this example were as follows:[0054]Anti-Factor VII Monoclonal Antibody—a calcium independent murine monoclonal antibody raised against human factor VII in the Morrissey lab, the hybridoma cells deposited as ATCC No. PTA-3497.[0055]Anti-Human Antithrombin III Rabbit polyclonal antibody)—DAKO catalog number A0296.[0056]Bovine Serum Albumin—Calbiochem catalog number 12659 (“Albumin, bovine serum, fraction V, low heavy metals”)[0057]Donkey Anti-rabbit IgG (H+ L), (min.times.BvChGtGuHaHsHnMsRtSh Sr Prot), Immunopure.RTM., Alkaline Phosphatase Conjugated—Pierce catalog number 31345.[0058]ELISA plates—Flat bottom Costar EIA / RIA Plates from Corning Inc. (Corning / Costar number 9018).[0059]Factor VII-deficient plasma—Congenital factor VII-deficient plasma (citrated) from George King BioMedical, Inc. (www.kingbiomed.com)[0060]pNPP (p-nitrophenyl phosphate)—Sigma catalog number ...

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Abstract

A method for determining the concentration of factor VIIa-antithrombin complexes is disclosed which has application to estimating the level of intravascular exposure of tissue factor, assessing patient risk for hypercoagulation or other coagulopathies, and monitoring patients for factor VIIa-antithrombin complexes over time which can reveal changes in risk for hypercoagulation or other coagulopathies and / or effectiveness of anticoagulant therapy. Antibodies suitable for use in an in vitro assay for determining the concentration of factor VIIa-antithrombin complexes and methods for making the same are also disclosed.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of prior copending U.S. patent application Ser. No. 10 / 481,345 filed 27 Feb. 2004, which is a § 371 national stage application of PCT / US02 / 21081 filed 2 Jul. 2002, which is a non provisional of 60 / 302,867 filed 3 Jul. 2001.TECHNICAL FIELD OF INVENTION[0002]This invention relates to in vitro assays for blood coagulation disorders.BACKGROUND OF THE INVENTION[0003]A number of clinical assays have been designed to measure the degree of turnover of the blood clotting system. These assays are routinely used to assess the severity of coagulopathies such as disseminated intravascular coagulation (DIC). Many of these assays are also being investigated for their utility in predicting hypercoagulable states that may predispose a person to developing deep vein thrombosis (DVT), heart attack, ischemic stroke, or other thrombotic and thromboembolic disorders.[0004]Examples of such assays are measurements of the plasma l...

Claims

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Application Information

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IPC IPC(8): G01N33/53C07K16/36C07K16/40C12N5/10C12N15/02C12P21/08C12Q1/56G01N33/543G01N33/577G01N33/86
CPCC07K16/40C12Q1/56G01N33/54306Y10S435/971G01N2800/52Y10T436/106664Y10S435/973G01N33/86
Inventor MORRISSEY, JAMES H.
Owner OKLAHOMA MEDICAL RES FOUND
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