JPH0694725A - Fibrinogen determination dry reagent - Google Patents

Abstract

PURPOSE:To obtain a dry reagent which can accurately determine fibrinogen quickly by containing protein with thrombin activity, a magnetic particle, and amino acid or its salt or sugars. CONSTITUTION:A dry reagent for determining fibrinogen contains protein with thrombin activity, a magnetic particle, and amino acid or its salt or sugars. For preparing the dry reagent, the protein with thrombin activity is dissolved into any buffer, the magnetic particle and amino acid or its salt or sugars used as an additive are added to a dissolution liquid for creating a final solution, and then a certain amount of the final solution is poured into any reaction slide, frozen, and then frozen and dried. The appropriate amount of thrombin activity contained in the dry reagent for determining fibrinogen is 0.5 to 1.5 NIHU. The appropriate amount of amino acid or its salt is 0.2mg to 0.8mg. The appropriate amount of sugars is 0.2mg to 0.8mg. When no amino acid or its salt or sugars is used in the dry reagent, the solubility of the reagent deteriorates, thus preventing the movement signal of the magnetic particle from being obtained and the determination from being made.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a dry reagent for quantifying fibrinogen, a dry material and a method for producing them.

[0002]

2. Description of the Related Art Quantification of fibrinogen is carried out by activating a partial thromboplastin time (hereinafter abbreviated as APTT), prothrombin time (hereinafter abbreviated as PT), a test for abnormality / normality of blood coagulation ability, or a patient with a large amount of bleeding It is an inspection item that is widely measured as an emergency inspection for

Conventional methods for quantifying fibrinogen are roughly classified into two methods, that is, a method using a solution reagent (hereinafter referred to as a solution reagent) and a method using a dry reagent containing thrombin.

Known methods using a solution reagent include a thrombin time method, a gravimetric method, a salting-out method, a method using an anti-fibrinogen antibody, and an agglutination method using latex particles.

Of these, the salting-out method and the method using an anti-fibrinogen antibody are used in a general blood coagulation test for determining the normality / abnormality of blood coagulation ability because the fibrinogen antigen amount is measured instead of the fibrinogen activity amount. It has not been.

[0006] The gravimetric method is a method in which thrombin is added to plasma and the weight of the formed fibrin clot is directly measured after the reaction. However, it is mostly due to the complexity of the operation and variations in the measured values. not being used.

A quantification method by the agglutination method using latex particles is disclosed in JP-A-57-4551.
The quantification method utilizes the strong affinity of fibrin monomer for latex particles. That is, first, a dilution series of plasma is prepared, a latex particle suspension is added and mixed with the diluted solution, and after culturing, the agglomeration of the particles is examined and the titer is recorded. Next, a latex particle suspension containing thrombin is added to and mixed with the same diluted solution, and after culturing, the agglomeration of the particles is examined and the titer is recorded. The titer of the former corresponds to the concentration of fibrin monomer present in plasma, and the titer of the latter corresponds to the sum of the concentration of fibrinogen and the concentration of fibrin monomer present in plasma. The concentration of fibrinogen is calculated by subtracting the titer of the former. The quantification method is useful for quantifying fibrin monomer and fibrinogen in plasma at the same time, but it is an urgent test because it takes a considerable time from the start of measurement to the calculation of fibrinogen and the preparation of reagents. There is a problem that can not be dealt with.

A commonly used method for the determination of fibrinogen using solution reagents is the thrombin time method found by Clauss (Clauss A, Gerinungsphysiol).
ogishe Schneiomethode Zur Bestimung des Fibrinogen
s, Acta Heamat, 17, 237, 1957). The thrombin time method utilizes that the conversion rate of fibrinogen to fibrin by a fixed amount of thrombin mainly depends on the fibrinogen concentration.

The measuring method of the quantification method is to first dilute plasma in an arbitrary buffer solution, preheat the diluted solution, and then add a reagent solution containing thrombin to measure the coagulation time. The end point of the measurement method is found by an optical measurement for detecting the attenuation of transmitted light or a physical measurement for detecting an increase in viscosity. The coagulation time in the measurement method refers to the time from the addition of the thrombin reagent solution to the end point described above.

This quantification method and the thrombin reagent used in this quantification method are widely accepted in the world.
However, the fibrinogen quantification method using the thrombin reagent requires that the freeze-dried thrombin reagent be reconstituted with distilled water (the reconstituted solution reagent cannot withstand long-term storage), and the plasma diluted solution must be preheated. There is a drawback in that it takes time to perform the measurement because it must be done. Furthermore, since the quantification range for one-time dilution is narrow, in the case of plasma containing fibrinogen at a concentration lower than the quantification range, re-measure by lowering the dilution ratio, and for plasma containing fibrinogen at a concentration higher than the quantification range. In that case, there was a drawback that the dilution ratio had to be increased and remeasurement performed.

On the other hand, the method using a dry reagent containing thrombin has been conceived in recent years.
The method is disclosed in Japanese Patent Publication No. -504076. The dry reagent containing thrombin used in the measuring method is
An arbitrary thrombin reagent solution and a plasminogen reagent solution were mixed, and a solution prepared by adding magnetic particles to the mixed solution was dispensed on a reaction slide in a fixed amount and then freeze-dried. The measurement method using the dry reagent is to place a dry reagent containing thrombin on any reaction holding means,
Next, a certain amount of plasma is added to the dry reagent containing thrombin, and immediately after that, a combination of an oscillating magnetic field and a stationary permanent magnetic field is applied to move the magnetic particles contained in the dry reagent containing thrombin. It is characterized by optically monitoring the motion signals of. The fact that the fall and rise of the kinetic signal corresponded to the rise and fall of the viscosity in the dry reagent containing thrombin suggests the possibility of simultaneously measuring the fibrinogen concentration and the plasminogen activator concentration in plasma. ing. That is, the magnitude of the negative slope of the kinetic signal of magnetic particles that appears immediately after adding plasma is proportional to the fibrinogen concentration in plasma, and the kinetics of the onset of lysis when the kinetic signal of magnetic particles begins to rise again after reaching the plateau. Are inversely proportional to the concentration of plasminogen activator in plasma. In the above publication, there is no specific description about the technical means concerning the method for quantifying fibrinogen using the dry reagent, a specific explanation of the effect, and the correlation with the quantification method using a solution reagent. Not not.

[0012]

If the fibrinogen can be quantified using the dry reagent containing thrombin as described above, it is possible to overcome the current problem that it takes a long time to measure. However, the present inventor
According to the method of Japanese Patent Publication No. -504076, a dry reagent was prepared and measured using the dry reagent and arbitrary plasma,
It was found that the magnitude of the negative slope of the motion signal of the magnetic particles obtained was not very reproducible. Further, when a plurality of plasmas containing known concentrations of fibrinogen were measured by the above method, the magnitude of the negative slope of the motion signal of the magnetic particles obtained did not correspond to the concentration of fibrinogen in plasma. I also knew there was. Therefore, it was found that it is actually difficult to quantify fibrinogen using a dry reagent containing thrombin as disclosed in the method of Japanese Patent Publication No. 3-504076.

[0013] The problem to be solved by the present invention is to develop a new technique for compensating for the above-mentioned drawbacks of the prior art, namely, a method for quantifying fibrinogen using a dry reagent which does not require time before measurement and enables accurate quantification. Is.

[0014]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above technical problems, and found that the solubility of a reagent containing thrombin has a great influence on the fibrinogen quantification performance itself. I understood. That is,
We have found that the uniformity of reagent dissolution leads to reproducibility in fibrinogen quantification, and that the improvement of the solubility of the reagent leads to the expansion of the fibrinogen quantification range. They have found what can be achieved by using an additive, have completed the present invention, and have proposed it here.

That is, the present invention comprises a protein having thrombin activity, magnetic particles, and a fibrinogen quantitative drying reagent characterized by containing an amino acid or a salt or saccharide thereof, and a protein having thrombin activity in a buffer solution. ,
A method for producing a fibrinogen quantitative drying reagent, which comprises freeze-drying a mixed solution containing magnetic particles and an amino acid or a salt or saccharide thereof.

Another aspect of the present invention provides a fibrinogen quantitative dry material containing a protein having a thrombin activity, an amino acid or a salt or saccharide thereof, and a protein, an amino acid having a thrombin activity, a salt or a saccharide thereof in a buffer solution. A method for producing a fibrinogen quantitatively dried material, which comprises freeze-drying a mixed solution containing the same.

The method for preparing the fibrinogen quantitative drying reagent of the present invention will be exemplified. A protein having thrombin activity is dissolved in an arbitrary buffer solution, and then the solution is mixed with magnetic particles and an amino acid or a salt thereof as an additive. A method may be employed in which saccharides are added to give a final solution, a fixed amount of the final solution is dispensed on an arbitrary reaction slide, and the mixture is frozen and then freeze-dried.

The reaction slide used in the above-mentioned preparation method is not particularly limited as long as it can optically monitor the increase in viscosity in the fibrinogen quantitative drying reagent as attenuation of the motion signal of magnetic particles during fibrinogen measurement. Absent. For example, a reaction slide as shown in FIGS. 1 and 2 may be mentioned. FIG. 1 is a view of the reaction slide as viewed from above. A portion surrounded by a dotted line in FIG. 1 is a main portion including a final solution dispensing port for preparing a fibrinogen quantitative drying reagent and a sample addition port.
FIG. 2 shows the structure of the main part in detail. First, the transparent polyester plate B is attached to the white polyester plate C, and then the transparent polyester plate B is attached.
A transparent polyester plate A is further laminated on the above to form the main part. The final solution for fibrinogen quantitative drying reagent is injected from the dispensing port shown in FIG. 1, and the portion D is filled with the final solution. When this type of reaction slide is used, 20 to 30 μl of the final solution for fibrinogen quantitative drying reagent described above is usually dispensed.

Unless otherwise specified, the content of each component in the fibrinogen quantitative drying reagent described below is as shown in FIG.
2 shows the weight and activity per slide when 25 μl was dispensed using the reaction slide shown in FIG. 2 and then freeze-dried.

The protein having thrombin activity used in the present invention refers to a protein capable of converting fibrinogen to fibrin. As this protein, bovine-derived thrombin, human-derived thrombin, snake venom protein having thrombin-like activity, etc. are known, but the origin is not limited in the present invention. The amount of thrombin activity contained in the fibrinogen quantitative drying reagent is not particularly limited and is usually 0.05 NIHU.
You can choose from the above, but 0.5 NIHU ~ 1.5 NIHU
Is preferred. By the way, the above-mentioned protein having thrombin activity is generally sold as a lyophilized product and can be easily obtained. Therefore, it is convenient to use this.

Known magnetic particles can be used in the present invention without any limitation. For example, ferric oxide particles, ferric oxide particles, iron particles, cobalt particles, nickel particles,
Examples of the particles include chromium oxide particles, and fine particles of ferrosoferric oxide are preferably used in terms of the intensity of motion signal of the magnetic particles obtained. The particle size of the magnetic particles is not particularly limited, and it may be selected from those having an average particle size of 0.01 to 10 μm, but those having an average particle size of 0.1 to 3 μm can be recommended.
The amount of the magnetic particles contained in the fibrinogen quantification dry reagent is not particularly limited, but usually 2 × 10 ^{over 6} g to 2 × 10 ^{over 4} g
It may be selected in the range of, 2 × 10 ^over 5 g~1.2 × 10 ^-4
A range of g is preferred.

As the amino acid or salt thereof used in the present invention, any of a neutral amino acid or salt thereof, an acidic amino acid or salt thereof, and a basic amino acid or salt thereof may be used. An acidic amino acid or a salt thereof can be preferably used because the solubility of the amino acid is better and the kinetic signal of the obtained magnetic particles is more reproducible. Examples of typical acidic amino acids or salts thereof include glutamic acid, sodium glutamate, aspartic acid and sodium aspartate. The neutral amino acids or salts thereof include L-glycine, L-glycine hydrochloride, L-alanine and the like, and the basic amino acids or salts thereof include L-lysine, L-lysine hydrochloride, L-arginine and the like. Is mentioned.

The amount of amino acid or salt thereof contained in the fibrinogen quantitative drying reagent of the present invention is 0.02 mg to 1
Although it may be selected in the range of mg, the range of 0.2 mg to 0.8 mg is preferable.

As the saccharide used in the present invention, any saccharide such as a monosaccharide and a polysaccharide may be selected, but a monosaccharide can be preferably used because the motion signal of the magnetic particles obtained is more reproducible. Examples of typical monosaccharides include glucose and fructose. Examples of polysaccharides include sucrose, lactose, dextrin and the like.

The amount of saccharide contained in the fibrinogen assay reagent of the present invention may be selected in the range of 0.02 mg to 1 mg, preferably 0.2 mg to 0.8 mg.

In the dry reagent and dry material of the present invention,
When the above-mentioned amino acid or its salt or saccharide is not used, the solubility of the reagent becomes poor, and as a result, the motion signal of the magnetic particles cannot be obtained and the quantification cannot be performed. Also, when additives other than these are used, the solubility of the reagent may be poor and the motion signal of the magnetic particles may not be obtained in the same manner, and even if the motion signal is obtained, the solubility of the reagent varies. As a result, reproducibility is not seen in the change with time of the motion signal, and thus accurate reproducible quantification cannot be achieved. .

Prior to the freeze-drying, the buffer solution containing the protein having thrombin activity, the magnetic particles, and the amino acid or its salt or saccharide is particularly preferably one having a buffering action between PH 6.0 and PH 8.0. Not limited. For example, 20 mM HEPES buffer (PH7.35))
Alternatively, a 20 mM phosphate buffer solution (PH7.4) and the like are preferable.

The method for drying the buffer solution containing the above-mentioned essential components is preferably the freeze-drying method from the viewpoint of the intensity of the kinetic signal of the magnetic particles obtained and the correlation between the fibrinogen concentration and the coagulation time. That is, in drying by air-drying, since the solubility of the reagent is poor, the motion signal is weak and it is difficult to detect the end point. Even if the end point can be found, the coagulation time obtained from the end point may not correspond to the fibrinogen concentration. .

The freeze-drying method is not particularly limited. To illustrate, the final solution for fibrinogen quantitative drying reagent is shown in FIG.
After being dispensed to the reaction slide from the dispensing port shown in, a general freezing method such as instantaneously freezing the reaction slide with dry ice or liquid nitrogen can be used. Also, the method of drying the frozen final reagent is not particularly limited, but the frozen reaction slide described above is vacuumed at −30 ° C. to room temperature for 7 hours to 1 hour.
A method of linearly increasing the temperature over 3 hours is preferable.

Fibrinogen quantification using the fibrinogen quantification dry reagent of the present invention is carried out by placing the reagent on an arbitrary reaction holding means, then adding a fixed amount of the sample to the reagent, and immediately after that, oscillating magnetic field and stationary permanent magnetic field. Can be performed by moving the magnetic particles contained in the reagent and optically monitoring the motion signal of the magnetic particles.

An example of an apparatus that can be used for fibrinogen quantification using the fibrinogen quantitative drying reagent of the present invention is trade name CG-01 [A & T Sales Co., Ltd.], trade name C
OAG-1 [sold by Wako Pure Chemical Industries, Ltd.] and the like can be mentioned.

Fibrinogen quantification using the fibrinogen quantification dry reagent of the present invention can be carried out based on the change with time of the motion signal intensity of the magnetic particles obtained after adding a sample to the reagent as described above. .

The method for quantifying the fibrinogen concentration of an arbitrary specimen based on the temporal change in the motion signal intensity of magnetic particles is not particularly limited. In addition to the known method that uses the magnitude of the negative slope of the motion signal of the magnetic particles that appears immediately after adding the sample, the end point is any point of the motion signal intensity, Using the time as the coagulation time, a method of utilizing the coagulation time can be used.

The end point is an arbitrary point of the motion signal intensity,
Exemplifying the method of setting the time from the addition of the sample to the end point as the coagulation time, the end point is the point attenuated at a predetermined ratio with respect to the peak value of the motion signal of the obtained magnetic particles,
Examples include a method in which the time from the addition of the sample to the end point is set as the coagulation time. The coagulation time obtained by such a method is correlated with the fibrinogen concentration in the sample.

A method for quantifying fibrinogen in any plasma using the coagulation time is not particularly limited, but a typical example is as follows. First, three types of plasma having known fibrinogen concentrations and different concentrations are arbitrarily selected. After diluting in a dilution buffer and using the diluted solution as a sample and obtaining the coagulation time corresponding to each plasma by the above method, a calibration curve is prepared based on the obtained coagulation time. Next, dilute arbitrary plasma in the same dilution buffer as above with the same dilution ratio, use this diluted solution as a sample, and obtain the coagulation time corresponding to the arbitrary plasma by the above method. The method of finding out the arbitrary plasma fibrinogen concentration using the prepared calibration curve is mentioned. The calibration curve used in the method is preferably a log-log graph in which the X axis is the fibrinogen concentration and the Y axis is the coagulation time.

As another example, a method for preparing a fibrinogen quantitative dry material containing a protein having thrombin activity, an amino acid, or a salt or saccharide thereof will be illustrated.
The protein having thrombin activity is dissolved in any buffer solution,
Next, a method in which a final solution obtained by adding an amino acid or its salt or saccharide to the solution as an additive is dispensed in a predetermined amount in an arbitrary reaction cup, frozen, and then freeze-dried can be employed.

As described above, the fibrinogen quantitative drying material can be a fibrinogen quantitative drying reagent by previously including magnetic particles in a solution state and then drying. However, by combining the quantitative drying material and a steel ball, Fibrinogen can be quantified as follows.

A measuring method using the fibrinogen quantitatively dried material of the present invention will be exemplified. In a reaction cup of KC-10A (manufactured by Amelung Co., Ltd.), a fibrinogen quantitative dry material containing the above-mentioned essential components is prepared. Then, after putting a steel ball (manufactured by Baxter) on the dried material,
Add the sample. The time from the addition of the sample to the movement of the steel ball is measured as the coagulation time. The coagulation time obtained by such a method is correlated with the fibrinogen concentration in the sample. The method for quantifying fibrinogen in arbitrary plasma using the coagulation time is not particularly limited, but first, three types of plasma having known fibrinogen concentrations and different concentrations are diluted with an arbitrary dilution buffer, and the diluted solution is After being used as a sample and obtaining the coagulation time corresponding to each plasma by the above method, a calibration curve is prepared based on the obtained coagulation time. Next, dilute arbitrary plasma in the same dilution buffer as above with the same dilution ratio, use this diluted solution as a sample, and obtain the coagulation time corresponding to the arbitrary plasma by the above method. The method of finding out the arbitrary plasma fibrinogen concentration using the prepared calibration curve is mentioned. The calibration curve used in the method is preferably a log-log graph in which the X axis is the fibrinogen concentration and the Y axis is the coagulation time.

The content of the protein having thrombin activity contained in the above-mentioned fibrinogen quantitatively dried material is not particularly limited and may be usually selected from 0.5 NIHU or more, but 5 NIH.
The range from U to 15 NIHU is preferred.

Similarly, the content of the amino acid or salt thereof, or saccharide contained in the fibrinogen quantitatively dried material may be selected in the range of 0.2 mg to 10 mg.
A range of 0 mg to 8.0 mg is suitable.

In addition, specific examples of the respective constituents, a method for preparing a dry material, and the like are the same as those of the fibrinogen quantitative drying reagent described above.

Also, as a method of drying the dried material, it is preferable to adopt the freeze-drying method for the same reason as described above.

By using this fibrinogen quantitative drying material, it is possible to quantify fibrinogen by using the conventional fibrinogen quantifying apparatus for solution reagents as it is, but without performing a troublesome operation such as preheating. became.

[0044]

EFFECTS OF THE INVENTION By using the fibrinogen quantitative drying reagent of the present invention, there is no need for reagent preparation such as reconstitution of thrombin reagent and sample warming time as compared with the fibrinogen quantitative method by the thrombin time method using a solution reagent. , Fibrinogen can be quantified rapidly only by diluting plasma. Further, since the dry reagent has a wide fibrinogen quantification range, it is substantially unnecessary to perform remeasurement by changing the dilution ratio for plasma having a fibrinogen concentration outside the quantification range as in the case of the solution reagent. It was By the way, the conventional solution reagent can measure only a concentration range of 150 to 800 mg / dl with a 20-fold dilution of a sample, whereas the dry reagent of the present invention can be used to measure a concentration of 50 to 800 mg / dl with a 20-fold dilution of a sample. The range of measurement is possible, and the dry reagent of the present invention has characteristics that it has high sensitivity and can measure even in a low concentration region with the same dilution. .

Furthermore, the correlation between the result of quantifying fibrinogen using the fibrinogen quantitative drying reagent of the present invention and the result of quantifying fibrinogen using the solution reagent is much better than that of the conventional dry reagent containing thrombin, and The reproducibility of the quantification was also good, which enabled more reliable measurement.

As a result of the above, since the fibrinogen quantitative drying reagent of the present invention has been developed, the measuring device can be downsized and the operation is simpler. Therefore, it is possible to perform an emergency test at the bedside without requiring a skilled person. I was able to respond.

Also, when the fibrinogen quantitative dry material of the present invention is used as a reagent of, for example, a fibrinogen quantitative analyzer (trade name KC-10A, manufactured by Amelung Co.) which uses a solution reagent, the coagulation time and plasma obtained can be obtained. A correlation was found with the fibrinogen concentration in the product. As a result, by using this fibrinogen quantitatively dried material, it becomes possible to quantify fibrinogen without using a fibrinogen quantification device for a solution reagent as it is without preparing a troublesome reagent such as preheating.

[0048]

The invention having been generally described, it can be further understood by reference to the following specific examples.
The examples provided herein are for illustration purposes only and are not intended to be limiting unless stated otherwise.

Example 1 and Comparative Example 1 Comparison of changes in kinetic signal of magnetic particles with time according to additive type CG-01 [A & T Sales Co., Ltd.] was used as a device for quantifying fibrinogen using a dry reagent. As the dry reagent, a thrombin reagent containing glucose (hereinafter abbreviated as glucose reagent), a thrombin reagent containing L-sodium glutamate monohydrate (hereinafter abbreviated as glutamate reagent), and bovine serum albumin. Thrombin reagent containing (hereinafter abbreviated as albumin reagent)
And a thrombin reagent containing Tween 80 (hereinafter,
Abbreviated as Tween 80 reagent. ), A thrombin reagent containing PEG6000 (hereinafter abbreviated as PEG6000 reagent), and a thrombin reagent containing glycerol (hereinafter abbreviated as glycerol reagent) as six types of motion signals of magnetic particles of each dry reagent. The changes over time were compared. The glucose reagent was prepared as follows.
Add pure water to the thrombin reagent (made by Dade) and add 100
A U / ml thrombin aqueous solution was prepared. 30 containing the aqueous solution and 1.5% glucose (manufactured by Wako Pure Chemical Industries)
mMHEPES (Dojindo) buffer solution (PH 7.35)
And were mixed 1: 2. Furthermore, the final concentration of the mixed solution is 5 m.
Magnetic particles (made by Rare Metallic Co., Ltd.) to be g / ml
Was added and mixed to give a final solution for glucose reagent. 25 μl of the final solution was dispensed on the reaction slide shown in FIG. The reaction slide was flash frozen in liquid nitrogen and after freezing,
A glucose reagent was prepared by freeze-drying. Freeze-drying was performed in a vacuum state by a method of linearly increasing from -30 ° C to 20 ° C over 8 hours.

Further, the preparation of the glutamic acid reagent was carried out as follows. Pure water was added to the thrombin reagent (made by Dade) to prepare a 100 U / ml thrombin aqueous solution.
30 mM HEPES containing the aqueous solution and 3.0% L-glutamate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.)
A buffer solution (PH 7.35) (manufactured by Dojindo) was mixed at a ratio of 1: 2. Further, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added and mixed to the mixed solution so as to have a final concentration of 5 mg / ml, to obtain a final solution for a glutamic acid reagent. The final solution 2
5 μl was dispensed on the same reaction slide as above. The reaction slide was frozen and freeze-dried under the same conditions as those for the glucose reagent described above.

Next, the albumin reagent was prepared as follows. Pure water was added to the thrombin reagent (made by Dade) to prepare a 100 U / ml thrombin aqueous solution.
The aqueous solution and a 30 mM HEPES (manufactured by Dojindo) buffer (PH 7.35) containing 4.5 mg / ml bovine serum albumin (manufactured by Sigma) were mixed at a ratio of 1: 2. Furthermore, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added to and mixed with the mixed solution so that the final concentration was 5 mg / ml, to give a final solution for albumin reagent. 25 μl of the final solution was dispensed on the same reaction slide as above. The reaction slide was frozen and freeze-dried under the same conditions as those for the glucose reagent described above.

Further, the preparation of Tween 80 reagent was prepared as follows. Pure water was added to the thrombin reagent (made by Dade) to prepare a 100 U / ml thrombin aqueous solution. The aqueous solution and a 30 mM HEPES (manufactured by Dojindo) buffer solution (PH 7.35) containing 0.075% Tween 80 (manufactured by Wako Pure Chemical Industries) were mixed at a ratio of 1: 2. Further, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added and mixed to the mixed solution so as to have a final concentration of 5 mg / ml, and Tween
This was the final solution for the n80 reagent. 25 μl of the final solution was dispensed on the same reaction slide as above. The reaction slide was frozen and freeze-dried under the same conditions as those for the glucose reagent described above.

The PEG6000 reagent was prepared as follows. Pure water was added to the thrombin reagent (made by Dade) to prepare a 100 U / ml thrombin aqueous solution. The aqueous solution and 1.5% PEG6000 (manufactured by Kochwright)
Was mixed with a 30 mM HEPES (manufactured by Dojindo) buffer solution (PH 7.35) containing 1: 2. Furthermore, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added to and mixed with the mixed solution so that the final concentration was 5 mg / ml, to obtain a final solution for PEG6000 reagent. 25 μl of the final solution was dispensed on the same reaction slide as above. The reaction slide was frozen and freeze-dried under the same conditions as those for the glucose reagent described above.

The glycerol reagent was prepared as follows. Pure water was added to the thrombin reagent (made by Dade) to prepare a 100 U / ml thrombin aqueous solution. A 30 mM HEPES (manufactured by Dojindo) buffer solution containing the aqueous solution and 0.75% glycerol (manufactured by Wako Pure Chemical Industries) (P
H7.35) was mixed 1: 2. Further, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added and mixed to the mixed solution so that the final concentration was 5 mg / ml, to give a final solution for glycerol reagent. 25 μl of the final solution was dispensed on the same reaction slide as above. The reaction slide was frozen and freeze-dried under the same conditions as those for the glucose reagent described above. In the comparative method, first, plasma containing 200 mg / dl of fibrinogen is diluted 20 times with Oren buffer (manufactured by Sigma). Next, the fibrinogen quantification device CG-
The above 6 kinds of dry reagents were set in 01, 25 μl of the above-mentioned diluent was added, and then the motion signal of the magnetic particles obtained from each reagent was optically monitored.

FIG. 3 shows the change over time in the motion signal of the magnetic particles after the addition of the diluent. The graph of A is a graph using a glucose reagent, the graph of B is a graph using a glutamic acid reagent, and
Is a graph using the albumin reagent, D is a graph using the Tween 80 reagent, E is a graph using the PEG6000 reagent, and F is the graph. [Fig. 4] is a graph using a glycerol reagent. As is easily understood from FIG. 3, it is the glucose reagent, the glutamic acid reagent, and the glycerol reagent that the change in the motion signal of the magnetic particles with time corresponds to the increase in the viscosity in the reagent. With other reagents, the solubility of the reagents is poor, and the motion signal of the magnetic particles does not correspond to the increase in viscosity in the reagents.

Comparative Example 2 Dry Reagent without Additives CG-01 [A & T Sales Co., Ltd.] was used as a device for quantifying fibrinogen using a dry reagent, and a dry reagent without additives was used. A reagent (hereinafter, abbreviated as a non-added reagent) was prepared as described below, and the temporal change of the motion signal of the magnetic particles was examined.

Pure water was added to the thrombin reagent (made by Dade) to prepare a 100 U / ml thrombin aqueous solution.
The aqueous solution and a 30 mM HEPES (Dojindo) buffer solution (PH 7.35) were mixed at a ratio of 1: 2. Further, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added and mixed to the mixed solution so as to have a final concentration of 5 mg / ml, to obtain a final solution for additive-free reagent. 25 μl of the final solution was dispensed on the reaction slide shown in FIG. The reaction slide was flash frozen in liquid nitrogen, and after freeze-drying, a freeze-dried reagent was prepared. Freeze-drying conditions are vacuum conditions, from -30 ° C to 2
It was carried out by a method of increasing the temperature linearly to 0 ° C. over 8 hours.

The measurement method is as follows. First, plasma containing 200 mg / dl of fibrinogen is diluted 20 times with Oren buffer (manufactured by Sigma). Next, the dry reagent was set in the fibrinogen quantification device CG-01, 25 μl of the above-mentioned diluent was added, and then the motion signal of the magnetic particles obtained from the additive-free reagent was optically monitored.

When the additive-free reagent was used, P of Comparative Example 1 was used.
The graph was similar to that of the EG6000 reagent, and no motion signal of magnetic particles was obtained, and quantification was impossible. Example 2 and Comparative Example 3 Reproducibility of Coagulation Time Four types of glucose reagents of Example 1 and Comparative Example 1, a glutamic acid reagent, a glycerol reagent and a conventional dry reagent disclosed in JP-A-3-504076 are used. CG- is used as a device for quantifying fibrinogen using reagents.
01 [A & T Sales Co., Ltd.] was used to examine the reproducibility of the coagulation time obtained with each of the above reagents.

The test method is as follows. First, plasma containing 200 mg / dl of fibrinogen is diluted 20 times with Oren buffer (manufactured by Sigma). Next, the above reagent was set in the fibrinogen quantification device CG-01, and the above-mentioned diluent 25 was added.
After adding μl, the motion signal of magnetic particles obtained from the reagent was optically monitored. This operation was repeated 5 times for each reagent.

The method for detecting the end point and the method for recognizing the coagulation time were 30% with respect to the peak value of the motion signal in the time-dependent change graph of the motion signal of the magnetic particles shown in FIG.
A method was adopted in which the decayed point was set as the end point, and the time from the addition of the sample to the end point was set as the coagulation time. That is, A in FIG. 4 is the end point, and B in FIG. 4 is the coagulation time.

The results are shown in Table 1. As can be seen from Table 1, only the glucose reagent and the glutamic acid reagent of the present invention show reproducibility in the obtained coagulation time.

Other reagents do not show reproducibility in the temporal change of the motion signal of the magnetic particles obtained because the solubility of the reagents is not uniform. Therefore, it was revealed that the obtained coagulation time was not reproducible.

[0064]

[Table 1]

Example 3 and Comparative Example 4 Comparison of temporal changes in motion signal of magnetic particles due to difference in drying method CG-01 [A & T Sales Co., Ltd.] was used as an apparatus for quantifying fibrinogen using a dry reagent. As the dry reagents, two kinds of fibrinogen quantitative dry reagents containing L-sodium glutamate monohydrate produced by different manufacturing methods as additives were used, and the temporal change of the motion signal of the magnetic particles of the two dry reagents was compared.

Dry reagents of different production methods mean that after freezing,
Lyophilized dry reagent (hereinafter abbreviated as lyophilized reagent)
And air-dried dry reagents (hereinafter abbreviated as air-dried reagents).

The freeze-dried reagent was prepared as follows. Add pure water to the thrombin reagent (made by Dade) and
A thrombin aqueous solution of 00 U / ml was prepared. The aqueous solution and a 30 mM HEPES (manufactured by Dojindo) buffer solution (PH7.35) containing 3.0% sodium L-glutamate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed at a ratio of 1: 2. Further, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added to and mixed with the mixed solution so that the final concentration was 5 mg / ml, to give a final solution for a freeze-dried reagent. 25 μl of the final solution was dispensed on the reaction slide shown in FIG. The reaction slide was flash frozen in liquid nitrogen, and after freeze-drying, a freeze-dried reagent was prepared. Freeze-drying conditions are vacuum state, -3
It was performed by a method of linearly increasing from 0 ° C. to 20 ° C. over 8 hours. On the other hand, the method for preparing the air-dried reagent was as follows. Using the same final solution as above, 25 μl of this final solution was dispensed on the same reaction slide as above. An air-dried reagent was prepared by treating the reaction slide under vacuum at 30 ° C. for 12 hours.

In the comparative method, first, plasma containing 200 mg / dl of fibrinogen is diluted 20 times with Oren buffer (manufactured by Sigma). Next, the fibrinogen quantification device CG-01 was set with the above-mentioned two types of dry reagents, 25 μl of the above-mentioned diluent was added, and then the motion signal of magnetic particles obtained from each reagent was optically monitored. It was

FIG. 5 shows the change over time in the motion signal of the magnetic particles after the addition of the diluent.

The graph of A is a graph using a freeze-dried reagent, and the graph of B is a graph using an air-dried reagent. When the air-dried reagent was used, the motion signal of the magnetic particles obtained was weak, but when the freeze-dried reagent was used, the motion signal of the magnetic particles obtained was sufficiently strong and reproducible. .

Example 4 Correlation between Fibrinogen Concentration and Obtained Coagulation Time The fibrinogen quantitative drying reagent used was the freeze-drying reagent of Example 3, and C was used as an apparatus for quantifying fibrinogen.
The correlation between the obtained coagulation time and the concentration of fibrinogen was examined using G-01 [A & T Sales Co., Ltd.].

The method of detecting the end point and the method of recognizing the solidification time were the same as in Example 2. The method for investigating the correlation between the coagulation time and the fibrinogen concentration was performed as follows. First, human plasma containing 800 mg / dl of fibrinogen and fibrinogen-deficient human plasma (manufactured by Jojiking) were used to prepare a dilution series of human plasma of 50 to 800 mg / dl. Then, each of the plasma of the dilution series was diluted with Oren buffer (manufactured by Sigma)
Diluted 0 times. And a fibrinogen quantification device CG-
Set the above freeze-drying reagent to 01, and use the above-mentioned diluent 25
μl is added, and the coagulation time of each diluted solution is determined by the above method. Finally, the fibrinogen concentration was taken as the X-axis of the log-log graph, and the data was plotted as the coagulation time at which the Y-axis was obtained, and the presence or absence of correlation was examined by whether or not the graph produced had linearity.

FIG. 6 shows a correlation diagram between the fibrinogen concentration and the obtained coagulation time. As can be seen from FIG. 6, a linear relationship is obtained between the fibrinogen concentration and the coagulation time, and it is clear that there is a correlation. Moreover, the result of FIG.
50 to 800 mg / dl required for blood coagulation test
It is shown that the whole region of the fibrinogen concentration range of can be quantified at one time by 20-fold dilution.

Furthermore, whether the above-mentioned plasma was diluted 10-fold to measure the coagulation time or 40-fold to measure the coagulation time, the quantitative range was different from that obtained when diluted 20-fold, but It was confirmed that a linear relationship was obtained between the obtained coagulation time and plasma fibrinogen concentration.

Example 5 Correlation between the solution reagent method and the dry reagent method of the present invention The results of quantifying fibrinogen by a conventional method using a solution reagent using 41 samples of human plasma and fibrinogen using the dry reagent of the present invention The correlation with the quantified results was compared.

Fibrinogen was quantified by the conventional method using a solution reagent, and the reagent was Dataphi Fibrinogen (manufactured by Dade) and the measuring device was KC-1.
It was set to 0 (manufactured by Amelung) and quantified by the method described in the databook of DataFi Fibrinogen.

Quantification of fibrinogen using the dry reagent of the present invention was carried out as follows. The fibrinogen quantitative drying reagent used was the freeze-drying reagent of Example 3, and CG-01 [Co., Ltd.] was used as an apparatus for quantifying fibrinogen.
A & T Sales]. The method of detecting the end point and the method of recognizing the coagulation time were the same as in Example 2. First, human plasma was diluted 20 times with Oren buffer (manufactured by Sigma). Then, the freeze-drying reagent is set in the fibrinogen quantification device CG-01, 25 μl of the above-mentioned diluent is added, and the coagulation time of each diluent is determined by the above method. Finally, a method for finding the fibrinogen concentration in plasma from the obtained coagulation time was used by using the graph shown in FIG. 6 of Example 4 as a calibration curve.

FIG. 7 shows a correlation diagram between the fibrinogen quantitative value determined by the conventional method using a solution reagent and the fibrinogen quantitative value determined by the method of the present invention.

As can be seen from FIG. 7, it is clear that the fibrinogen quantitative value determined by the conventional method using the solution reagent and the fibrinogen quantitative value determined by the method of the present invention have a high correlation.

Example 6 Correlation between coagulation time and fibrinogen concentration by fibrinogen quantitative dry material A fibrinogen quantitative dry material applied to KC-10A (manufactured by Amelung) was prepared as follows.

Pure water was added to the thrombin reagent (made by Dade) to prepare a 100 U / ml thrombin aqueous solution.
The aqueous solution and a 30 mM HEPES buffer solution (manufactured by Dojindo) containing 3.0% L-sodium glutamate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) buffer solution (PH 7.35) were 1: 2.
To obtain a final solution for fibrinogen quantitatively dried material. 300 μl of the final solution was dispensed into a reaction cup (manufactured by Baxter). The reaction cup was flash-frozen with liquid nitrogen, and after freeze-drying, a dried material for measuring fibrinogen was prepared. Freeze-drying conditions are vacuum,
It was carried out by a method of linearly increasing from 30 ° C. to 20 ° C. over 8 hours.

The measuring method is as follows: After setting the fibrinogen quantitatively dried material in KC-10A, put a steel ball (manufactured by Baxter), and then add 300 μl of the sample, and set the time when the steel ball starts to move as the coagulation time. It was adopted.

The method for investigating the correlation between the obtained coagulation time and the fibrinogen concentration was carried out as follows. First,
200, 300, 400, 500 produced in Example 4
Six types of human plasma having a fibrinogen concentration of 600 and 700 mg / dl were each diluted 20 times with an Oren buffer (manufactured by Sigma). Next, using the diluted solution as a sample, the coagulation time of each diluted solution was determined by the above method. Finally, the fibrinogen concentration was taken as the X-axis of the log-log graph, and the data was plotted as the coagulation time at which the Y-axis was obtained, and the presence or absence of correlation was examined by whether or not the graph produced had linearity. FIG. 8 shows a correlation diagram between the fibrinogen concentration and the obtained coagulation time. As can be seen from FIG. 8, a linear relationship is obtained between the fibrinogen concentration and the coagulation time, and it is clear that there is a correlation. Therefore, by using this fibrinogen quantitative drying material of the present invention, it is possible to quantify fibrinogen without using a troublesome preparatory operation such as preheating while using the fibrinogen quantitative determination device for the solution reagent as it is. .

[Brief description of drawings]

FIG. 1 is an example of a representative reaction slide used for fibrinogen quantification by the fibrinogen quantification dry reagent of the present invention.

2 is a partially exploded view of the reaction slide of FIG.

FIG. 3 is a diagram showing changes over time in the intensity of motion signals of magnetic particles after adding a sample to various dry reagents.

FIG. 4 is a diagram showing an endpoint analysis method for obtaining a coagulation time corresponding to the amount of fibrinogen using a fibrinogen quantitative drying reagent.

FIG. 5 is a diagram showing a difference in change over time in the motion signal intensity of magnetic particles of a freeze-dried reagent and an air-dried reagent.

FIG. 6 is a diagram showing the relationship between coagulation time and plasma fibrinogen concentration obtained using the endpoint analysis method of FIG.

FIG. 7 is a correlation diagram of the fibrinogen quantitative value in the conventional solution reagent method and the fibrinogen quantitative value when the fibrinogen quantitative dry reagent of the present invention is used.

FIG. 8 is a diagram showing the relationship between the coagulation time and the plasma fibrinogen concentration obtained using the fibrinogen quantitatively dried material.

[Explanation of symbols]

 A transparent resin plate B transparent resin plate C white resin plate D reagent filling section

Claims (4)

[Claims] 1. A fibrinogen quantitative drying reagent comprising a protein having thrombin activity, magnetic particles, and an amino acid or a salt or saccharide thereof. 2. The fibrinogen quantitative drying reagent according to claim 1, wherein a mixed solution containing a protein having thrombin activity, magnetic particles, and an amino acid or a salt or saccharide thereof in a buffer solution is freeze-dried. Production method. 3. A fibrinogen quantitative dry material comprising a protein having thrombin activity, an amino acid, or a salt or saccharide thereof. 4. The method for producing a fibrinogen quantitatively dried material according to claim 3, wherein a mixed solution containing a protein having a thrombin activity, an amino acid or a salt or saccharide thereof in a buffer solution is freeze-dried.