JPS58220697A - Activity measurement method of antithrombin-3 and kit using it - Google Patents

Abstract

PURPOSE:To raise reproducibility in the activity measurement for antithrombin- III, by measuring the inhibitory action of antithrombin-III on thrombin by the anticoagulating action measurement using fibrinogen in the presence of albumin. CONSTITUTION:In measuring the inhibitory action of antithrombin-III on thrombin by the anticoagulating action measurement using fibrinogen, albumin is added to the measurement system. Thrombin, fibrinogen, and antithrombin IIIreference standard to be used are preferably derived from man or horse. Albumin is preferably derived from man, cow, horse, goat, sheep, or chicken. In the above-mentioned measurement, each reagent is conveniently processed into a kit. The kit to be used comprises at least four constitutional element of a reagent consisting of thrombin, a reagent consisting of fibrinogen, an antithrombin III reference standard, and a diluent solvent.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring the activity of antithrombin-111 and a kit for measuring the activity. As the name antithrombin-1uld-t suggests, it exhibits an inhibitory effect on thrombin and also on other coagulation and fibrinolytic factors. For example, it also has an inhibitory effect on activated factor (I), factor Pathological condition of so-called generalized coagulation syndrome □... 1° total amount. Therefore, measuring the in vivo activity of antithrombin-III is important for the prevention and diagnosis of various diseases.

Two methods are known for measuring antithrombin-1: an immunological method based on its antigenicity, and a method for measuring anticoagulant activity based on its biological activity. Among the latter methods, the method involving thrombin, fibrinokene, and kumquat is recommended as a measurement system close to in vivo.

However, a drawback of this measurement system is its low reproducibility. Particularly when measuring the activity of low titer antithrombin-nt, it is difficult to judge the end point of clotting time.

The inventors conducted research to solve this drawback and completed the present invention based on the discovery that reproducibility can be dramatically improved by adding albumin to the measurement system.

That is, the present invention provides a method for measuring the activity of antithrombin-III in which when the inhibitory effect of antithrombin-III on thrombin is measured by the anticoagulant effect measurement method using fibrinoken, the addition of albumin to the measurement system is a sign. The present invention relates to a method and a kit for measuring antithrombin-1,11 activity used to carry out the determination method.

In relation to the present invention, the anticoagulant effect measurement method refers to the measurement of residual thrombin activity by reacting antithrombin-■ with an excessive amount of thrombin, and then using the fact that fibrinokene coagulates into fibrin due to the action of thrombin. death,
This measures the total activity of antithrombin-1.

In this case, the amount of residual thrombin can be measured, for example, by the time it takes for fibrinokene to coagulate into fibrin.

In the measurement method of the present invention, it is convenient to prepare each reagent in a kit, so the present invention will be fully explained below using the kit as an example.

The kit according to the present invention includes a reagent consisting of thrombin, a reagent consisting of fibrinokene, and antithrombin-111.
The kit consists of at least four components: a standard product and a solvent for dilution, and in some cases, a reagent consisting of albumin may be included as one of the kit components.

The origin of the thoron, fibrinoken, and antithrombin-111 standard products used in the present invention is not limited to bamboo, but is preferably derived from humans, cows, and horses (especially humans). things are used. These can be produced by conventional means, and in the kit of the present invention, it is preferable to prepare them as a completely dried product (in particular, a lyophilized product) and to dissolve them in a diluting solvent before use. .

There are no particular restrictions on the origin of albumin, but it is preferably derived from 1. Those derived from goats, sheep, and chickens (especially humans) are used.

The albumin I/I may be added in advance to a thrombin reagent, a fibrinogen reagent, a dilution solvent, or an antithrombin-111m quasi-product, or may be added to the measurement system during measurement. good.

The most preferred embodiment is that it is added commercially to thrombin. When adding it to a measurement system, it is recommended to add a reagent other than albumin, which is included as one of the reagents in the kit. In that case, albumin is kept as a dry product (in particular, a lyophilized product) and dissolved in a dilution solvent before use.

The concentration of albumin in the measurement system is 0.01-0.5w
/v% concentration is preferable.

In addition, when albumin is added to thrombin in advance, a/' humin f is added to the thrombin aqueous solution.
It is preferable to add it to a concentration of 0.2 to 5 w/v% and freeze-dry it.

As the diluting solvent, preferably a buffer having a pH of 7 to 8 is used, for example an imidazole buffer, especially an imidazole buffer containing sodium chloride.

The reagent consisting of thrombin and the reagent consisting of fibrinoken related to the present invention may contain desired additives in addition to albumin. For example, when producing a lyophilized product of fibrinoken, it is preferable to add sodium citrate, glucose, etc. as a stabilizer.
The additive may coexist as it is in the reagent consisting of fibrinoken.

+i′ Antithrombin-LIT using the kit of the present invention
Measurement of active cases is done in the following way. That is, a thrombin solution dissolved in a total thrombin dilution solvent was dispensed, and equal amounts of antithrombin standard diluted to various concentrations with a dilution solvent were added to the solution.
The reaction was carried out at 5 to 30°C (preferably 28°C) for about 10 minutes, and a specific amount of this reaction solution was blown into an equal volume of a solution diluted with a solvent for diluting the entire fibrinoken to a concentration of, for example, 1%. Determine the coagulation time of fibrinokene at various concentrations of the standard product and calculate the calibration mt-
create. Next, the fipurinogon coagulation time of the specimen is determined by the same procedure, and this is compared with the previously prepared calibration curve to measure the total antithrompin-1I [activity] in the specimen.

Comparative Experimental Example A comparative experiment was conducted to confirm the effect of adding albumin. The anticoagulant effect was measured by adding an equal volume of standard antithrombin-■ diluted solution to thrombin and incubating it for 10 minutes at 28°C.
After reacting for a minute, add 0.2 ml of this reaction mixture.
f Blow into a 1% fibrinoken solution at 0.2 d, measure the entire coagulation time of fibrinoken, and create a calibration curve for the coagulation time. For preparing the thrombin solution and fibrinoken solution and diluting the sample, add O, 15M NaCl and 0.02
M imidazole buffer 1 pH 7, 3y was used. These 3
Human or bovine albumin' (rO, 01-0
．． The concentration was adjusted to 5 w/v and the clotting time was measured according to the method for preparing the leaf detection line described above. Antithrompin-1 - [A solution of active O units/W/, i.e. negative control and Antithrompin-■ 2 units/IWe diluted 20 times with the specified solvent O, 1 unit/rsl solution of 2 Each sample was measured for 10 days, and all tests were conducted to determine whether the clotting time could be measured with good reproducibility. All measurement systems listed in Table 1 were prepared, and the results shown in Tables 2 to 4 were obtained.

Table 1 Note) Measurement system 1 Human albumin 0.001-0.5 w/v% in a solution of human thrombia 10 units/d.
When adding % concentration.

Human fibrinokene 1% concentration and alkaline solvent for sample dilution. No humins added.

Measurement system 2 Human albumin 0.00 in the sample dilution solvent
1 to 0.5 w/v% concentration plus Kabakura.

Human thrombin solution and The fibrinokene solution contains a No ruhumin was added.

Measurement system 3: When human Φ albumin was added to a human fibrinogen solution at a concentration of 0.001 to 5 w/v.

Human thrombin solution and test Albumin is used as a solvent for body dilution. Not added.

Measurement system 4: When albumin is not added to all solutions.

In addition, AT-Ill in Tables 2 to 4 means antithrompine.

(Left below) , '1' Table 2 Reproducibility of clotting time measurement when albumin is added to nitthrombin solution Table 3 Measurement system 2: When albumin is added to the sample dilution solvent Table 4 Above As shown in the experimental results, it was found that adding albumin to the measurement system improved the reproducibility of cyanantithrombin-111 measurement.

Albumin can also be added to fibrinoken, but
Antithrombin-1 ■ Sample! :) Reproducibility is somewhat higher if it is added during the reaction with rhombin, that is, at the beginning of the measurement. For that purpose, it is better to add it into thrombin or a diluting solvent. However, since a relatively large amount of dilution solvent is used in this measurement system, a large amount of albumin is also used.
From an economical point of view, it is desirable to add it to the thrombin solution. Further, it is more preferable to add albumin to the thrombin solution in advance, since this also provides a stabilizing effect when the thrombin solution is freeze-dried.

The present invention will be explained in more detail below with reference to Examples, but the present invention is not limited thereto.

Example 1 Human albumin fl w/v % concentration was added to an aqueous solution containing 50 units/d of human thrombin, and 1 rad aliquot was freeze-dried. After lyophilization, the thrombin titer was 9.7 units/unit when redissolved in 5 Mt imidazole buffer.
At, almost no inactivation of thrombin was observed during freeze-drying.As a coagulating protein of human fibrinogen, sodium citrate was added to an aqueous solution of 1 w/v.
O, 58 w/v % concentration and glucose f 1.
In addition to the concentration of 6 w/v%, 5111/ml was aliquoted and lyophilized. After freeze-drying, when a 5Hg imidazole buffer solution was added, the mixture was rapidly dissolved and no insoluble substances were observed. Plasma obtained by blood collection with citric acid from healthy subjects with a diameter of 100 Å or more was treated at -56°C for 4 minutes to remove all insoluble matter (mainly fibrinochene), aliquoted into 1.3 d portions, and lyophilized (antithrombin and citric acid). -III standard product). No difference in titer was observed before and after freeze-drying, and both were 1.2 monoWwi.
Met.

The imidazole buffer solution was prepared by adding imidazole (final concentration 0.02 M) and sodium azide (final concentration 0.1 w/v) to NaCt (final concentration 0.15 M), and adjusting the pH to 7.3 using specified salt. i! 11! Nodrombin, fibrinokene, and antithrompin were prepared and redissolved in unknown samples using 5 J, 5 m+/, and 41 I/' (z) of imidazole buffer, respectively. Antitron and N-ill
r & sex was measured. As an unknown sample, the 1st V fraction obtained by Mr. Chon's alcohol fractionation method was dissolved in the above-mentioned imidazole buffer, and citrate blood was collected from a healthy subject 5.
Name (A, BXC, D, and E) plasma and patient plasma with decreased antitron and III were used. These samples were measured five times, and antithrompin-■
The antithrombin-1 titer in each sample was determined from a calibration curve prepared using standard products.

As shown in Table 5, O was measured with good reproducibility (the margins below) Table 5 Antithrombin-111 activity measurement results in unknown samples

Claims (1)

[Scope of Claims] (1) Antithrombin-1 is characterized in that albumin is entirely present in the measurement system when the inhibitory effect of antithrombin-1 on thrombin is measured by an anticoagulant effect measuring method using fibrinogen. III activity measurement method. (2) The concentration of albumin present in the measurement system is 0°O1
A method according to claim 1, wherein the concentration is ~0.5%. (3) The method according to claim (1), characterized in that albumin is added to thrombin in advance. (4) The method according to claim (1), characterized in that albumin is added in advance to the dilution solvent. (5) The method according to claim (1), wherein the albumin is derived from human, bovine, rabbit, horse, goat, sheep, or chicken. (6) Thrombin and fibrinogen are each derived from The method according to claim (1), which is characterized in that it is derived from human, bovine or horse origin. (7) Reagent consisting of thrombin, reagent consisting of fipurinogun, antithrombin - Standard product and for dilution A kit for measuring antithrombin-I activity in which a solvent is all essential components, and albumin is present in at least one of the above kit components, or a kit consisting of all the reagents consisting of albumin as a kit component. (8) Reagent consisting of thrombin, reagent consisting of fibrinogen, antithrombin -■ Standard product and reagent consisting of albumin are each lyophilized product?f
- The kit described in item (7) of the scope of the patent. (9) The lyophilized thrombin product is produced by adding albumin to an aqueous solution of thrombin to a concentration of 0.2 to 5 w/v% and lyophilizing the product. ) Kit described in section.