Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Patent
1995-02-17
1997-07-08
Kight, John
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
435 4, 435810, 435975, 435 795, 436 63, 436 69, 436 74, C12Q 156, C12Q 100, G01N 3353, G01N 3348
Patent
active
056460074
DESCRIPTION:
BRIEF SUMMARY
INDUSTRIAL FIELD OF APPLICATION
The present invention relates to a method for the determination of antithrombin III (hereinafter abbreviated as ATIII) activity in body fluids. More specifically, it relates to an improved method for the determination of ATIII activity in body fluids comprising reacting a sample with thrombin in the presence of heparin and a salt and then measuring a color developed from a chromogenic substrate to determine the remaining thrombin activity. The present invention further relates to a reagent kit to be used in the method for the determination of ATIII activity.
PRIOR ART AND PROBLEMS
ATIII is a serine protease inhibitor present in blood in a large amount (20 to 30 mg/dl) and is known as a blood coagulation inhibitor. Blood coagulation takes place by an amplification reaction due to cascade-like function of many proteases. In the final stage of blood coagulation, thrombin thus formed converts fibrinogen into fibrin and, in addition, thrombin activates blood coagulation factor XIII. The activated blood coagulation factor XIII forms crosslinkages between fibrins to form thrombi. A most important regulator of this formation of thrombi is ATIII and ATIII binds to thrombin and proteases which participate in blood coagulation such as blood coagulation factor X and the like to inhibit them. When ATIII in blood is lowered, thrombi are liable to be formed. Therefore, lowering of ATIII in blood causes trouble. It has been known that lowering of ATIII in blood occurs by, for example, low nutriture, severe hepatic diseases, coagulation sthenia such as diffuse intravascular coagulation (DIC), acute thrombosis and the like, nephrotic syndrome, and congenital ATIII deficiency or abnormality. Then, ATIII is an important indication in clinical diagnosis.
As methods for the determination of ATIII, immunological methods for the determination of immunizing doses, methods using fibrinogen or chromogenic substrates, and methods using ATIII deficient plasma containing coagulation factors participating in extrinsic reactions have already been known. We have invented a method using ATIII deficient plasma containing coagulation factors participating in extrinsic reactions (PCT/JP89/00173 (Nov. 2, 1989); and Thrombosis Research, Vol. 57, 729-736 (1990)). In this method, ATIII can be simply, easily and accurately determined by measuring a blood coagulation time with minimum influence of heparin cofactor II (hereinafter abbreviated as HCII). However, since a blood coagulation time is measured in this method, it is difficult to apply the method to general automatic analyzers.
At present, methods using chromogenic substrates are widely used because they are readily applicable to automatic analyzers.
In a method using a chromogenic substrate, an excess amount of thrombin is added to a sample to react ATIII in the sample with thrombin in the presence of heparin and a salt and then a chromogenic substrate is added to the reaction mixture to measure a color developed to determine the remaining thrombin activity, that is, to indirectly determine ATIII in the sample. This determination of ATIII is described by, for example, Rinsho Byori, Special Vol. 70, 173-177 (1987); U. Abildgaard, et al., Thrombosis Research, Vol. 11, 549-553 (1977).
In this method for the determination of ATIII with a chromogenic substrate, there are two major problems. One problem is dilution of a sample. In general, it is necessary to dilute a sample so as to avoid influence of its inherent color and turbidity upon measurement. In addition, since a sample contains a large amount of ATIII, when the sample is used without any dilution, a very large amount of thrombin must be added because the amount of thrombin should exceed the amount of ATIII. In such a case, since 0% of ATIII activity corresponds to the activity of thrombin added for preparing a calibration curve, a color released from a chromogenic substrate reaches a measurable limit of absorbance within a very short period of time. For applying to various automatic analyzers, it is u
REFERENCES:
patent: 4473639 (1984-09-01), Sommer et al.
patent: 4736018 (1988-04-01), Reutelingsperger
patent: 4918001 (1990-04-01), Kolde
patent: 4948724 (1990-08-01), Yin
patent: 5093237 (1992-03-01), Enomoto
patent: 5221614 (1993-06-01), Enomoto
patent: 5248596 (1993-09-01), Esmon et al.
patent: 5320945 (1994-06-01), Dessauer et al.
Abildgaard et al., "Antithrombin (Heparin Cofactor) Assay with New Chromogenic Substrates (s-2238 and Chromozyn TH)", Thrombosis Research, vol. 11, 1977, pp. 549-553.
M. Ukita, "ATIII, Heparin", Clinichopathologic, Rinsyo Byori, Special, vol. 70, 1987, pp. 173-180, with full English transistion.
M. Enomoto et al., "Antithrombin-III Assay without Influence of Heparin Cofactor II", Thrombosis Research, vol. 57, 1990, pp. 729-736.
K. Takahashi et al., Rinsho Byori, Special vol. 70, 1987, pp. 181-186.
D. Tollefsen et al., "Activation of Heparin Cofactor II by Dermatan Sulfate", Jour. Biological Chemistry, vol. 258, No. 11, 1983, pp. 6713-6716.
D. Tollefsen et al., "Heparin Cofactor II", Jour. Biological Chemistry, vol. 257, No. 5, 1982, pp. 2162-2169.
F. Church et al., "Antithrombin Activity of Fucoidan", Jour. Biological Chemistry, vol. 264, No. 6, 1989, pp. 3618-3632.
J. Conard et al., "Bovine or Human Thrombin in Amidolytic AT III Assays", Thrombosis Research, vol. 41, 1986, pp. 873-878.
Enomoto Masayasu
Nishimura Haruhiko
Kight John
Leary Louise
Nippon Shoji Kaisha Ltd.
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