Method for measuring activated factor VII level in a sample

A technology for activating factors and testing samples, applied in biochemical equipment and methods, measuring devices, biological testing, etc., can solve the problems of inaccurate and difficult operation of FVIIa concentration measurement

Inactive Publication Date: 2011-06-01
LFB BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although FVII+FVIIa concentration measurements are accurate, direct FVIIa concentration measurements remain imprecise and difficult to perform

Method used

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  • Method for measuring activated factor VII level in a sample
  • Method for measuring activated factor VII level in a sample
  • Method for measuring activated factor VII level in a sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1: Preparation of plasma lacking FVII

[0121]A polyclonal antibody raised in rabbits against purified human plasma FVII was linked to CNBr-activated Sepharose (Pharmacia), and 2 ml of the obtained gel was placed in a column. The column was equilibrated with 25 ml of equilibration buffer (0.15M NaCl, 10 mM citrate, pH 7.4). Then, 6 mL of human plasma was passed through the column multiple times. Under these conditions, FVII is retained immobilized on the column and the eluate (FVII-deficient plasma) is recovered. The immobilized FVII was eluted with 20 mL of regeneration buffer (50 mM NaCl; 0.1 M glycine, pH 2.4) to regenerate the column, and then the column was reconditioned with 20 mL of equilibration buffer (10 mM citrate; 0.15 M NaCl, pH 7.4). rebalance.

Embodiment 2

[0122] Example 2: Preparation of plasma lacking FVII and FVIII, FIX or FXI

[0123] A polyclonal antibody raised in rabbits against purified human plasma FVII was linked to CNBr-activated Sepharose (Pharmacia), and 2 ml of the obtained gel was placed in a column. The column was equilibrated with 25 ml of equilibration buffer (0.15M NaCl, 10 mM citrate, pH 7.4). Then, 6 mL of commercially available FVIII, FIX or FXI depleted plasma obtained from Diagnostica Stago was passed through the column several times. Under these conditions, FVII is retained immobilized on the column and the eluate (plasma lacking both FVII and FVIII, FIX or FXI) is recovered. The immobilized FVII was eluted with 20 mL of regeneration buffer (50 mM NaCl; 0.1 M glycine, pH 2.4) to regenerate the column, and then the column was reconditioned with 20 mL of equilibration buffer (10 mM citrate; 0.15 M NaCl, pH 7.4). rebalance.

Embodiment 3

[0124] Example 3: Preparation of a standard thrombin generation profile from plasma lacking FVII and FVIII, The concentration of FVII+FVIIa is 50pM

[0125] Obtain a sufficient volume sample of International Standard FVII (SI-FVII) supplied by NIBSC (SI-FVII) and / or FVIIa (SI-FVIIa) also supplied by NIBSC, in order to obtain a standard containing a known amount of activated FVII, which is added to into 80 μL of FVII and FVIII-depleted plasma (FVIII-depleted plasma was obtained from Stargo Diagnostics, or hemophilia A plasma, which was then depleted of FVII as in Example 2), In order to obtain a mixture comprising a fixed activated FVII content between 0% and 100%, a concentration of FVII+FVIIa comprised between 10 pM and 80 pM was included. Add 20 μL of the factor that triggers the thrombin generation reaction (Ca 2+ , phospholipids and TF), at a final concentration of 5 pMTF, 1 μM phospholipids, (Stargo Diagnostics 86195 reagent diluted 1:4) and 16.7 mM Ca 2+ , and 20 μ...

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Abstract

The present invention relates to a method for measuring the activated factor VII level in a sample to be tested, including the steps of: a) mixing said test sample with a plasma free of factor VII (FVII) and free of at least another factor selected from among factor VIII (FVIII), factor IX (FIX), and factor XI (FXI), the test sample + plasma having a final FVII + FVIIa concentration of 10 pM to 80 pM; b) adding initiating components from the thrombin generation reaction; c) obtaining a thrombogram when carrying out a thrombin generation test (TGT) on the mixture from step b); d) comparing at least one of the thrombogram parameters from step c) with a homologous parameter obtained from standard thrombograms established on the basis of standard samples, the activated factor VII level of which is known and varies with each standard sample; e) deducing, from step d), an activated factor VII level measurement in the test sample.

Description

technical field [0001] The present invention relates to a method for measuring the content of activated factor VII (FVIIa) in a sample using a plasma lacking FVII and at least one selected from the group consisting of factor VIII (FVIII), factor IX (FIX) and factor XI ( Other factors of FXI). Background technique [0002] Blood coagulation is a mechanism by which an organism controls bleeding when a blood vessel is injured, and thus avoids bleeding. [0003] Blood coagulation occurs after a series of steps involving the conversion of the different zymogens and procofactors in the blood to their activated forms via proteolytic enzymes. In this sequential step (or series of steps) of coagulation, there are two different pathways, the extrinsic coagulation pathway and the intrinsic coagulation pathway. Both lead to the formation of a complex called prothrombinase, consisting of activated factor X (FXa), activated factor V (FVa), phospholipids and calcium. Prothrombinase acti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/86G01N33/68
CPCG01N2333/96447G01N33/86C12Q1/56G01N33/68
Inventor 莉西安·希尔伯特克劳丁·马聚里耶多米尼克·格瑞尼尔
Owner LFB BIOTECH
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