Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Patent
1989-11-02
1992-03-03
Lacey, David L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
436 69, 436175, 530381, 530393, C12Q 100
Patent
active
050932373
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method for determination of biological activity of antithrombin III (hereinafter abbreviated as AT III) in plasma and a reagent therefor.
BACKGROUND OF THE INVENTION
AT III is a serine protease inhibitor present in blood in a large amount (from 20 to 30 mg/dl) and is known as a coagulation inhibitor. As described hereinafter, the blood coagulation reaction includes an intrinsic coagulation reaction pathway and an extrinsic coagulation reaction pathway. Various coagulation factors take part in respective pathways and AT III inhibits most of the activated coagulation factors. Particularly, as the action of AT III, the inhibition of thrombin, which is activated factor II (IIa), and activated factor X (Xa) is considered to be of importance. And, since the reaction of coagulation factors is inhibited more effectively at an early stage in the blood coagulation system, AT III is considered to play a particularly important role in the inhibition of factor Xa which takes part in a stage earlier than factor IIa.
Clinically, attention is directed to AT III in AT III production depressed conditions such as hepatocirrhosis and undernutrition, AT III consuming conditions such as disseminated intravascular coagulation (DIC) and other progressive coagulation conditions, conditions wherein AT III is lost into urine such as nephrosis syndrome, congenital AT III deficiency disease and the like. Therefore, for diagnosis of these conditions, it has been requested to establish a precise, rapid and easy method the for determination of biological activity of AT III.
Hitherto, as a method for determination of biological activity of AT III, there have been known a method using a synthetic substrate and a method utilizing coagulation activity. Among them, the method using synthetic substrate is carried out by adding a synthetic substrate and an excess amount of factor IIa to a specimen plasma and measuring anti-IIa activity of AT III in the specimen. This method is excellent because the determination can be carried out within a short period of time. However, the method has many problems. For example, the method has different specificity depending upon the kind of synthetic substrates and a synthetic substrate is very expensive. Further, factor IIa to be used is less stable and, since IIa factor is apt to be adsorbed by glass, a plastic cell should be used in the measurement. Furthermore, since the method requires measuring apparatuses such as a spectrophotometer and a fluorophotometer, economical burden becomes large.
On the other hand, in the coagulation method, a concentration of AT III is determined by subjecting a specimen plasma to heat treatment to remove fibrinogen, adding an excess amount of factor IIa and measuring a coagulation time due to conversion of fibrinogen to fibrin to obtain the anti-IIa activity of AT III in the specimen. However, these operations of this method are complicated and the determination takes a long period of time, for example, not less than one hour.
OBJECTS OF THE INVENTION
Under these circumstances, the present inventor has studied intensively in order to establish a precise, rapid and easy method for determination of AT III without the above problems. As a result, the present inventor has found that this objective can be attained by a novel method for determination of AT III wherein a coagulation method is employed by utilizing the extrinsic coagulation reaction pathway of the blood coagulation reaction and using AT III deficient plasma wherein AT III has been removed.
Measurement of coagulation factor activity using a factor deficient plasma wherein a desired factor has been removed is utilized in the determination of various blood coagulation factors [for example, Kensa to Gijutsu, Vol. 13, No. 7, pages 611 to 616 (July, 1985)]. In addition, the use of protein S deficient plasma is proposed for determination of biological activity of protein S which is one of blood coagulation inhibitors (Japanese Patent Laid Open Publication No.
REFERENCES:
patent: 4409327 (1983-10-01), Bartl et al.
patent: 4948724 (1990-08-01), Yin
Edwards Newton
Lacey David L.
Nippon Shoji Kabushiki Kaisha
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