Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-10-11
1999-04-06
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 7021, 436518, 436519, 5303871, 53038825, G01N 3353, G01N 33543, C12P 2106, C07K 1600
Patent
active
058916474
DESCRIPTION:
BRIEF SUMMARY
This application is filed as a 371 of the international patent application PCT/FR95/00456 filed 10 Apr. 1995.
The present invention relates to an immunological method for the assay of antithrombin III (AT III) activated by one of the sulphated glycosaminoglycans which are known to act on the process of blood coagulation at AT III level. In the presence of heparin, AT III exerts its anticoagulant activity by inhibiting various serum proteases involved in the reactions leading to the formation of insoluble fibrin, especially thrombin and factor Xa.
Various biochemical analogue glycosaminoglycans are known which, on binding to AT III, interrupt the succession of enzyme reactions ending in the clot; natural porcine or bovine heparins and low molecular weight heparins, several types of which are on the market, may be mentioned in particular. Some sulphated oligosaccharides having the same properties are also known, including those described in Patent Applications EP-A-84,999, EP-A-133,599 and EP-A-356,275, in particular the pentasaccharide recognized as constituting the sequence of the heparin chains to which AT III binds.
These compounds may be used in man as anticoagulants and antithrombotics in prophylactic or therapeutic treatments; however, monitoring of the state of the coagulation system during these treatments is essential given their potential side-effects, and in particular their risks of haemorrhage which vary from individual to individual.
Monitoring is currently based on biological methods which consist in determining in vitro the inhibitory action at factor Xa level of the sulphated glycosaminoglycans present in the patient's blood, either by a specific coagulation test for this factor, or by measuring its residual activity on a chromogenic substrate. For a description of these tests, Duclos--Editions Masson (1984).
In man, the efficacious blood concentrations of antithrombotic glycosaminoglycans are low, less than 10 .mu.M, and in the absence of specific antibodies, no direct assay of these products which can be used routinely in biological analytical laboratories has been developed.
However, it should be noted that the results obtained with the above two methods are sometimes divergent and poorly correlated with the antithrombotic activity observed in vivo and with the extent of the risks of haemorrhage, as mentioned by A. Leizorovicz, L. Bara, M. M. Samama and M. C. Haugh dans Haemostasis (1993), 23, (sup. 1), pp. 89-98. In addition, they do not always reflect the clinical situation and, in particular, they underestimate high levels of heparin and of similar glycosaminoglycans.
It was hence desirable to have at one's disposal a simple and reliable method enabling the extent of the action of one of these sulphated glycosaminoglycans on plasma AT III in the coagulation cascade to be assessed. It is known that this action, during which heparin or its analogues bind transiently to AT III, manifests itself in particular by a conformational modification of the protein which is the source of the increase in the rate of inhibition of factor Xa.
A determination of the proportion, probably low, of AT III in this activated state, that is to say in a form modified by the binding of heparin or of glycosaminoglycans, which precedes the covalent binding of AT III to the other proteabes of coagulation, should enable the intensity of the inhibitory action of the sulphated glycosaminoglycan present in the plasma to be assessed better than by the previous biological methods.
Monoclonal antibodies are known to be very suitable for the identification and determination of specific spatial conformations.
Application JP 6094713 describes a method for the assay of AT III activity by affinity chromatography.
According to this method, the determination of heparin-AT III complexes employs only a single type of labelled antibody specific for these complexes. Moreover, Application DE-2,708,985 describes a method for the evaluation of plasma heparin levels, according to which the amount of AT III complexed with heparin is me
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S. Knoller et al., European Journal of Biochemistry, Feb. 1989, vol. 180, No. 2, pp. 319-326, Berlin de.
Search report (France) for corresponding application.
Translation (English) of international preliminary examination report.
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Lormeau Jean-Claude
Milleblandine Renee Georgette
Paolucci Francis Egiste Joseph
Pau Bernard
Devi S.
Housel James C.
Pasteur Sanofi Diagnostics
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