JP2980468B2 - Fibrinogen determination method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for quantifying fibrinogen using a fibrinogen quantitative drying reagent.

[0002]

2. Description of the Related Art Quantification of fibrinogen is widely used as a test for examining abnormal or normal blood coagulation ability together with activated partial thromboplastin time and prothrombin time, or as an urgent test for patients with a large amount of bleeding. It is.

[0003] Conventional fibrinogen determination methods include:
The method is roughly classified into two methods, a method using a thrombin reagent solution and a method using a dry reagent containing thrombin.

A commonly used method for determining fibrinogen using a thrombin reagent solution is Claus
thrombin time method (Claus
sA, Gerinungsphysiology
SchneiomethodZur Bestim
ung des Fibrinogens, Acta
Heamat, 17, 237, 1957). The thrombin time method utilizes the fact that the rate of conversion of fibrinogen into fibrin by a fixed amount of thrombin mainly depends on the fibrinogen concentration.

[0005] In the measurement method of the quantitative method, first, plasma is diluted with an arbitrary buffer, the diluted solution is preliminarily heated, and a thrombin-containing reagent solution is added to measure the coagulation time. The end point of the measuring method is a method that is found by optical measurement for detecting attenuation of transmitted light or physical measurement for detecting increase in viscosity. The clotting time in the measurement method refers to the time from the addition of the thrombin reagent solution to the end point described above.

The quantification method and the thrombin reagent used in the quantification method are widely accepted in the world.
However, the method of quantifying fibrinogen using a thrombin reagent requires that the freeze-dried thrombin reagent must be reconstituted with distilled water or the like each time it is used (the reconstituted solution does not withstand long-term storage), and the plasma diluent is reserved. There is a disadvantage that it takes time to perform the measurement due to the necessity of heating and the like. Furthermore, since the quantification range in one dilution of the sample is narrow, in the case of plasma containing a lower concentration of fibrinogen than the quantification range, re-measure with a lower dilution factor,
In the case of plasma containing a higher concentration of fibrinogen than the quantification range, there is a disadvantage that the dilution ratio must be increased and the measurement must be performed again.

On the other hand, a method for quantifying fibrinogen using a thrombin-containing dry reagent has recently been considered and is disclosed in Japanese Patent Publication No. 3-504076. The dry reagent containing thrombin used in the measurement method is obtained by mixing an arbitrary thrombin reagent solution and a plasminogen reagent solution, further dispensing a fixed amount of a solution obtained by adding magnetic particles to the mixed solution, and then dispensing the solution to a reaction slide. Lyophilized.

In the measurement method using the dry reagent, a dry reagent containing thrombin is placed on an arbitrary reaction holding means,
Next, a certain amount of plasma is added to the thrombin-containing dry reagent, and immediately thereafter, a combination of an oscillating magnetic field and a static permanent magnetic field is applied, and the magnetic particles contained in the thrombin-containing dry reagent are moved, and the magnetic particles are added. It is characterized by optically monitoring the movement signal. The fact that the decrease and increase of the kinetic signal correspond to the increase and decrease of the viscosity in the thrombin-containing dry reagent 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 the magnetic particles appearing immediately after the addition of plasma is proportional to the concentration of fibrinogen in the plasma, and the lysis start time at which the kinetic signal of the magnetic particles starts to rise again after reaching the plateau. States that it is inversely proportional to the concentration of plasminogen activator in plasma. In the above-mentioned publication, there are no technical means relating to the quantification of fibrinogen using the dry reagent, a specific description of the effect thereof, and specific description of the consistency with a conventional quantification method using a solution reagent, etc. Not done.

[0009]

If fibrinogen can be quantified using a dry reagent containing thrombin as described above, it is possible to overcome the current problem that it takes time to measure fibrinogen. However, the present inventor has reported that Japanese Patent Publication No.
A dry reagent was prepared according to Japanese Patent No. 4076 and measured using the dry reagent and arbitrary plasma. As a result, the magnitude of the negative gradient of the kinetic signal of the magnetic particles obtained was not so reproducible. I knew it wasn't. In addition, when a plurality of plasmas containing a known concentration of fibrinogen were measured by the above method, the magnitude of the negative slope of the obtained kinetic signal of the magnetic particles did not correspond to the fibrinogen concentration in the plasma. I knew there was.

Therefore, it has been found that it is actually difficult to quantify fibrinogen by using a thrombin-containing dry reagent and a measuring method disclosed in Japanese Patent Publication No. 3-504076.

The present inventor has found that when a dry reagent contains an amino acid or a salt or saccharide thereof in addition to a protein and magnetic particles having thrombin activity, a dry reagent having good solubility in plasma can be obtained. Was. With the use of the reagent, the change over time of the obtained kinetic signal of the magnetic particles can be obtained with good reproducibility, so that even when the method of quantifying fibrinogen from the magnitude of the negative gradient of the magnetic particles described above is used, it is constant. In the concentration range of, quantification was possible to some extent. However, unless the measurement range for measuring the magnitude of the negative slope is strictly defined, the magnitude of the negative slope may not correspond to the fibrinogen concentration in plasma, and even if the measurement range is defined, the quantitative It turns out that the possible range is narrow. Furthermore, the magnitude of the negative slope obtained is significantly affected by the above measurement range, so if a small change in the manufacturing conditions leads to a small delay in the solubility of the reagent, the magnitude of the negative slope obtained will It was also found that reproducibility was often not obtained.

Therefore, the method of quantifying fibrinogen from the magnitude of the negative slope reported in Japanese Patent Publication No. 3-504076 is not an effective means of quantification in practice.

The problem to be solved by the present invention is to develop a new technique for compensating for the above-mentioned disadvantages of the prior art, that is, a method for quantifying fibrinogen with a wide fibrinogen quantitative range and high reproducibility.

[0014]

Means for Solving the Problems In order to solve the above-mentioned technical problems, the present inventors have developed a reagent for quantitatively determining thrombin-containing fibrinogen and a blood capable of optically monitoring the kinetic signal of magnetic particles contained in the reagent. As a result of extensive research using a coagulation measuring device, assuming that a certain range of the kinetic signal of the magnetic particles, that is, a certain range of the change in viscosity increase, is the end point, the elapsed time from the addition of the sample to the end point is the time in the sample. Has a very good correlation with the fibrinogen concentration of Furthermore, they found that when the elapsed time was used for quantification of fibrinogen, the quantification range was expanded from the current state, and the present invention was completed and proposed here.

That is, the present invention relates to a fibrinogen quantitative drying reagent containing a protein having thrombin activity, magnetic particles, an amino acid or a salt thereof or a saccharide, and adding and mixing the specimen. The change in the viscosity of the dried reagent is monitored, the elapsed time from the addition of the sample to the increase in the viscosity to a specific viscosity is measured, and the measured elapsed time is an index representing the amount of fibrinogen in the sample. A method for determining the amount of fibrinogen in a sample to be evaluated as, wherein the specific viscosity is obtained by multiplying a minimum value of the viscosity of the fibrinogen quantitative dry reagent after addition of the sample by a specific magnification in a range of 1.05 to 2 times. This is a method for determining the amount of fibrinogen in a sample, characterized by having a low viscosity.

The fibrinogen quantitative drying reagent used in the present invention comprises a protein having thrombin activity, magnetic particles, and an amino acid or a salt or saccharide thereof. By containing an amino acid, a salt thereof, or a saccharide as an essential component, the solubility of the reagent when a sample is added is improved, and the reproducibility is improved.

As an example of a method for preparing the above-mentioned fibrinogen quantitative drying reagent, a protein having thrombin activity is dissolved in an arbitrary buffer solution, and then the magnetic particles and an amino acid or a salt or saccharide thereof as an additive are added to the solution. Is added to obtain a final solution, a predetermined amount of the final solution is dispensed to an arbitrary reaction slide, 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 kinetic signal of the magnetic particles at the time of fibrinogen measurement. Absent. By way of example, a reaction slide as shown in FIGS. 1 and 2 may be mentioned. FIG. 1 is a diagram of a reaction slide viewed from above. A portion surrounded by a dotted line in FIG. 1 is a main portion including a dispensing port of a final solution for preparing a fibrinogen quantitative drying reagent and a sample addition port.
FIG. 2 shows the structure of the main part in detail. First, a transparent polyester plate B is pasted to the white polyester plate C, and then the pasted transparent polyester plate B is pasted.
The main part is constituted by pasting a transparent polyester plate A on the above. The final solution for the fibrinogen quantitative dry reagent is injected from the dispensing port shown in FIG. 1, and part 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 is usually dispensed.

Unless otherwise specified, the contents of the respective components in the fibrinogen quantitative drying reagent described below are as shown in FIG.
And using the reaction slide shown in FIG.
The weight and activity per slide when 5 μl was dispensed and then lyophilized are shown.

The protein having thrombin activity used in the fibrinogen quantitative drying reagent refers to a protein capable of converting fibrinogen to fibrin. As this protein, bovine-derived thrombin, human-derived thrombin, a snake venom protein having thrombin-like activity and the like are known, but in the present invention,
Its origin is not limited. The amount of thrombin activity contained in the fibrinogen quantitative drying reagent is not particularly limited, and may be usually selected to be 0.05 NIHU or more.
A range from U to 1.5 NIHU is preferred. By the way, since the protein having the thrombin activity is generally sold as a lyophilized product and can be easily obtained, it is convenient to use this.

Known magnetic particles can be used for the dry reagent without any limitation. For example, triiron tetroxide particles, iron sesquioxide particles, iron particles, cobalt particles, nickel particles, chromium oxide particles and the like are mentioned. It is preferably used. The particle size of the magnetic particles is not particularly limited, and may be usually 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 × 1
May be selected in the range of 0 ^{over 4} g but, 2 × 10 ^over 5 g~1.2 ×
Range of 10 ^{@ 4} g are preferred.

As the amino acid or its salt used in the drying reagent, any of a neutral amino acid or its salt, an acidic amino acid or its salt, or a basic amino acid or its salt may be used. An acidic amino acid or a salt thereof can be preferably used in that solubility is better when a sample is added, and in that the obtained magnetic particles have a more reproducible kinetic signal. Representative examples of acidic amino acids or salts thereof include glutamic acid, sodium glutamate, aspartic acid, sodium aspartate and the like. Further, as a neutral amino acid or a salt thereof, L-glycine, L-glycine hydrochloride,
Examples of L-alanine and the like and basic amino acids and salts thereof include L-lysine, L-lysine hydrochloride, L-arginine and the like. The amount of the amino acid or a salt thereof contained in the dry reagent may be selected in the range of 0.02 mg to 1 mg, but is preferably in the range of 0.2 mg to 0.8 mg.

Similarly, as the saccharide used for the dry reagent, any saccharide such as a monosaccharide and a polysaccharide may be selected, but a monosaccharide is preferably used in that the obtained magnetic particles have a more reproducible kinetic signal. Can be used. To illustrate a typical monosaccharide,
Glucose, fructose and the like can be mentioned. Examples of the polysaccharide include sucrose, lactose, dextrin and the like. The amount of saccharides contained in the dry reagent is 0.02 mg-
You may choose in the range of 1mg, 0.2mg ~ 0.8mg
Is suitable.

When the amino acid or its salt or saccharide is not used in the dry reagent, the solubility of the reagent is deteriorated, and as a result, the kinetic signal of the magnetic particles is not obtained, and the quantification tends to be difficult. In addition, when additives other than these are used, the solubility of the reagent is similarly poor and the kinetic signal of the magnetic particles may not be obtained, and even if the kinetic signal is obtained, the solubility of the reagent varies. As a result, reproducibility is not observed in the temporal change of the motor signal, and as a result, accurate quantification cannot be performed with good reproducibility.

Prior to freeze-drying, a buffer containing and adjusting a protein having thrombin activity, magnetic particles, an amino acid or a salt or saccharide thereof has a pH of 6.0 to PH8.
There is no particular limitation as long as there is a buffering action between 0. As an example, a 20 mM HEPES buffer (PH7.
35) or 20 mM phosphate buffer (PH 7.4) is preferred.

The method for drying the buffer solution containing the above essential components, ie, the final solution for the fibrinogen quantitative drying reagent, is not particularly limited, but may include the strength of the kinetic signal of the obtained magnetic particles, the correlation between the fibrinogen concentration and the coagulation time, and the like. The freeze-drying method is preferred from the viewpoint.

The freeze-drying method is not particularly limited. For example, the final solution for the fibrinogen quantitative drying reagent is shown in FIG.
After dispensing the reaction slide from the dispensing port shown in (1), a general freezing method such as instantaneously freezing the reaction slide with dry ice or liquid nitrogen can be used. The method of drying the frozen final reagent is not particularly limited, but a method is preferable in which the temperature of the above-mentioned frozen reaction slide is linearly increased from −30 ° C. to room temperature in 7 to 13 hours under vacuum.

In the present invention, after a sample is added to and mixed with the fibrinogen quantitative drying reagent, a change in viscosity of the fibrinogen quantitative drying reagent is detected and monitored. The change in viscosity at this time can be grasped as a change in the motion signal of the magnetic particles when a combination of an oscillating magnetic field and a static magnetic field is applied to the reagent, as described later. And
As shown in FIG. 3, the change in viscosity at that time is generally
Immediately after the addition of the sample, it decreases with the dissolution of the reagent, and after reaching the minimum (minimum), increases with the progress of the coagulation reaction.
In addition, the intensity of the kinetic signal of the magnetic particles in FIG. 3 is inversely related to the viscosity of the reagent (that is, the intensity of the kinetic signal is low when the viscosity of the reagent is high, and the intensity is high when the viscosity is high. The intensity of the movement signal increases). In the present invention, when the viscosity of the reagent after addition of the sample reaches a minimum (minimum) and then rises again, the viscosity is reduced from the range of 1.05 to 2 times the minimum value after the addition of the sample. The elapsed time until a viscosity multiplied by a predetermined specific magnification (hereinafter, also referred to as a specific viscosity) is measured, and the elapsed time thus measured is evaluated as an index representing the amount of fibrinogen in the sample. That is, one of the features of the present invention is the elapsed time measured as described above, as described at the beginning of "Means for Solving the Problems", and as will be apparent from the examples described later. Has found that there is a very good correlation with the fibrinogen concentration in the sample, using this correlation, specifically by converting from the "elapsed time" using a calibration curve etc. created using a standard sample The point is to determine the fibrinogen concentration (amount) in the sample. At this time, when determining the “elapsed time”, the ratio of the “specific viscosity”, which is the viscosity of the reagent at the elapse of the “elapsed time”, to the “minimum viscosity” (hereinafter, also referred to as a set magnification) is 1.05. It is indispensable to set so as to be twice. When the set magnification is less than 1.05 times, the sensitivity is reduced, and when the set magnification exceeds 2 times, the quantitative range is narrowed. In particular, from the viewpoint of reproducibility, the set magnification is preferably set to 1.4 to 1.7 times.

The change in the viscosity of the dried reagent of the present invention can be monitored by using the magnetic particles contained in the reagent.
The method for monitoring the change in viscosity is not particularly limited. To specifically illustrate the monitoring method, a fibrinogen quantitative drying reagent is placed on an arbitrary reaction holding means, then a certain amount of a sample is added to the reagent, and immediately thereafter, a combination of an oscillating magnetic field and a stationary permanent magnetic field is applied, and A method of moving the magnetic particles contained in the magnetic particles and optically monitoring the movement signal of the magnetic particles.

As an example of an apparatus that can be used, a trade name CG
01 [A & T Sales], trade name COAG-1 [Wako Pure Chemical Industries, Ltd. sales] and the like.

When the above-mentioned apparatus is used, a change with time of the kinetic signal of the magnetic particles which is inversely corresponding to the change in the viscosity of the dried reagent is obtained. To explain the present invention in this case, the minimum value of the viscosity is the point at which all the components in the dry reagent are dissolved, that is, the peak value of the kinetic signal of the magnetic particles obtained immediately after the addition of the sample. Here, assuming that the peak value of the motion signal is X and the signal value at the time when the viscosity rises and reaches a certain viscosity is Y, the viscosity rise at this point is X /
It becomes Y times. In other words, for the peak value of the movement signal,
The point in time when the signal intensity decreases to (X−Y) × 100 / X (%) corresponds to the point in time when the increase in viscosity becomes X / Y times. That is, the point at which the viscosity becomes 1.05 times the minimum value is
This corresponds to a point attenuated by about 5% with respect to the peak value of the movement signal of the magnetic particles (see FIG. 3).

The “elapsed time” in the present invention refers to the time from the addition of the sample to the quantitative dry reagent to the end point. The elapsed time is correlated with the fibrinogen concentration in the sample. As shown in FIG. 3, the viscosity increases after the end point (the intensity of the motion signal of the magnetic particles decreases).
As can be seen from the above, the quantitative dry reagent to which the sample is added is not always completely coagulated at the above-mentioned end point. However, the “elapsed time” has a very good correlation with the fibrinogen concentration in the sample, and similarly to the “clotting time” in the method using the conventional thrombin reagent solution, the fibrinogen concentration in the sample is It can be treated as an index representing (amount).
Therefore, in the present specification, the “elapsed time” is hereinafter referred to as a coagulation time.

The method for quantifying fibrinogen in plasma using the clotting time is not particularly limited. As a representative example, first, three types of plasma having known and different concentrations of fibrinogen are diluted in an arbitrary dilution buffer, and the diluted solution is used as a sample. After obtaining the corresponding clotting time, a calibration curve is generated based on it. Next, any plasma is diluted in the same dilution buffer as described above at the same dilution ratio, and the diluted solution is used as a sample, and after the clotting time corresponding to each plasma is obtained by the above method, There is a method of finding fibrinogen concentration in any plasma using the prepared calibration curve. The calibration curve used in the method is preferably a logarithmic graph in which the X-axis is the fibrinogen concentration and the Y-axis is the clotting time.

[0034]

The quantification of the fibrinogen concentration in the specimen according to the method of the present invention is different from the method of quantifying fibrinogen using the negative slope of the movement signal of magnetic particles described in Japanese Patent Publication No. 3-504076. The quantification range is wide. Therefore, the operation of re-measuring plasma having a fibrinogen concentration out of the quantification range by changing the dilution factor in the fibrinogen quantification method using the negative gradient of the movement signal of the magnetic particles became practically unnecessary.

Also, the results of quantification of fibrinogen using the method of the present invention and the results of quantification of fibrinogen using conventional solution reagents agree very well. In addition, reproducibility of quantification was good, and more reliable measurement was possible.

[0036]

The invention has been described generally, but can be better understood by reference to the following specific examples.
The examples shown herein are for illustrative purposes only and are not intended to be limiting unless otherwise specified.

Example 1 Correlation between fibrinogen concentration and coagulation time The fibrinogen quantitative drying reagent used was prepared by the following method.

Pure water was added to a thrombin reagent (manufactured by Dade) to prepare a 100 U / ml thrombin aqueous solution.
30 mM HEPES containing the aqueous solution and 3.0% sodium L-glutamate monohydrate (manufactured by Wako Pure Chemical Industries)
(Made by Dojindo Chemical Co., Ltd.) and a buffer solution (PH 7.35) were mixed at a ratio of 1: 2. Further, magnetic particles (manufactured by Rare Metallic Co., Ltd.) were added to the mixed solution so as to have a final concentration of 5 mg / ml to obtain a final solution for a lyophilized reagent. 25 μ of the final solution
1 was dispensed to the reaction slide shown in FIGS. The reaction slide was flash-frozen in liquid nitrogen, freeze-dried, and then freeze-dried to prepare a lyophilized reagent. The freeze-drying was performed by a method of linearly increasing the temperature from -30 ° C to 30 ° C over 13 hours in a vacuum state.

As a device for quantifying fibrinogen,
CG01 [A & T Sales] was used.

The correlation between the obtained clotting time and the concentration of fibrinogen was examined using the above-mentioned fibrinogen quantitative drying reagent and the blood coagulation measuring device CG01.

The method of detecting the end point and the method of recognizing the coagulation time are as follows. The end point is a point attenuated by 30% with respect to the peak value of the motion signal of the magnetic particles shown in FIG. A method of setting the time as the coagulation time was employed.
That is, A in FIG. 3 is the end point, and B in FIG. 3 is the coagulation time. The above-mentioned end point corresponds to a viscosity increase of 100/70 (about 1.43) times.

The method for examining the correlation between the coagulation time and the fibrinogen concentration was carried out as follows. First, 900m
Using human plasma containing g / dl fibrinogen and fibrinogen deficient human plasma (George King), dilution series of human plasma from 30 to 900 mg / dl were made. Next, each of the plasmas in the dilution series was diluted 20-fold with Orlen buffer (manufactured by Sigma). Then, the lyophilized reagent is set in the blood coagulation measuring device CG01, 25 μl of the diluent described above is added, and the coagulation time of each diluent is determined by the above method. Finally, the X-axis of the log-log graph is used as the fibrinogen concentration, and the data is plotted as the coagulation time at which the Y-axis is obtained. The presence or absence of the correlation is examined based on whether or not the prepared graph shows linearity. The quantification range was determined from which concentration deviated from the straight line.

FIG. 4 shows a correlation diagram between the fibrinogen concentration and the obtained coagulation time. FIG. 4 clearly shows that a linear relationship was obtained between the fibrinogen concentration and the coagulation time, and that there was an extremely good correlation. In addition, the results of FIG.
It shows that the entire region of the fibrinogen concentration range of dl can be quantified at one time.

Comparative Example 1 Correlation between the conventional negative gradient method and the concentration of fibrinogen The fibrinogen quantitative drying reagent used was the lyophilized reagent of Example 1, and C was used as an apparatus for quantifying fibrinogen.
Using G01 [A & T Sales], the correlation between the obtained negative slope and the fibrinogen concentration was examined.

The measurement of the negative inclination was performed as follows. FIG.
As shown in FIG. 5, first, the peak value of the movement signal is recorded (A in FIG. 5), and then the movement signal value 5 seconds after the movement signal reaches the peak value (A in FIG. 5). B) was recorded and finally 1 was calculated using both motor signal values.
The amount of change in the motor signal per second was calculated as a negative slope. That is, the measurement range of the negative slope in the present comparative example is 5 seconds after the peak of the motion signal of the magnetic particles, and if the negative slope is expressed using the symbol in FIG.
B) / 5 [count / sec].

The method for examining the correlation between the negative slope and the fibrinogen concentration was carried out as follows. First, 800m
A dilution series of human plasma up to 50-800 mg / dl was made using human plasma containing g / dl fibrinogen and fibrinogen deficient human plasma (George King). Next, each of the plasmas in the dilution series was diluted 20-fold with Orlen buffer (manufactured by Sigma). Then, the lyophilized reagent was set in the blood coagulation measuring device CG01, 25 μl of the above-mentioned diluent was added, and the negative slope of each diluent was calculated by the above method. Finally, the data was plotted on the X-axis as the fibrinogen concentration and the negative slope to obtain the Y-axis, and the presence or absence of the correlation was examined based on whether or not the prepared graph showed linearity.

FIG. 6 shows a correlation diagram between the fibrinogen concentration in the sample and the obtained negative slope. As can be seen from FIG. 6, as the fibrinogen concentration in the sample increases, the negative slope obtained increases, but the linearity as shown in Example 1 cannot be obtained. Therefore, it was found that it is actually difficult to determine the fibrinogen concentration in the sample using the negative slope of the movement signal of the magnetic particles.

Example 2 Correlation between fibrinogen concentration and clotting time when the end point was changed The fibrinogen quantitative drying reagent used was the lyophilized reagent of Example 1, and C was used as an apparatus for quantifying fibrinogen.
Using G01 [A & T Sales], the correlation between the fibrinogen concentration in the sample and the coagulation time when the end point was determined by changing the ratio of attenuation to the peak value of the movement signal of the magnetic particles was examined.

As the end point, a point at which the obtained peak value of the kinetic signal of the magnetic particles is attenuated by 10, 20, 30, and 40% is taken, and the time from the addition of the sample to the end point is defined as the respective coagulation time. did. In addition, these points can be expressed as a viscosity increase 100/90 (about 1.1) times, 100 times.
/ 80 (1.25) times, 100/70 (about 1.43)
Times and 100/60 times (approximately 1.67) times.

The method for examining the correlation between the coagulation time and the fibrinogen concentration was carried out as follows. First, 600m
A dilution series of human plasma from 100 to 600 mg / dl was made using human plasma containing g / dl fibrinogen and fibrinogen deficient human plasma (George King). Next, each of the plasmas in the dilution series was diluted 20-fold with Orlen buffer (manufactured by Sigma). Then, the lyophilized reagent is set in the blood coagulation measuring device CG01, 25 μl of the diluent described above is added, and the coagulation time of each diluent is determined by the above method. Finally, the X axis of the log-log graph was used as the fibrinogen concentration, and the data was plotted as the coagulation time at which the Y-axis was obtained. The presence or absence of the correlation was examined based on whether or not the prepared graph showed linearity.

FIG. 7 shows a correlation diagram between the fibrinogen concentration and the obtained coagulation time. 7A is a graph showing the relationship between the obtained coagulation time and the fibrinogen concentration when the point at which the peak value of the kinetic signal of the magnetic particles is attenuated by 10% is set as the end point, and the graph of FIG. A graph showing the relationship between the obtained coagulation time and the fibrinogen concentration when the point 20% attenuated with respect to the peak value of the movement signal is regarded as the end point. The graph C shows 30% relative to the peak value of the movement signal of the magnetic particles. A graph showing the relationship between the obtained coagulation time and the fibrinogen concentration when the attenuated point is regarded as the end point, and the graph of D shows the results obtained when the point attenuated by 40% with respect to the peak value of the kinetic signal of the magnetic particles is regarded as the end point. 4 is a graph showing the relationship between the obtained coagulation time and the fibrinogen concentration. As can be seen from FIG. 7, when the end point is obtained by changing the rate of decay (viscosity increase) with respect to the peak value of the kinetic signal of the magnetic particles within the viscosity change range of the present invention, the obtained coagulation time, the fibrinogen concentration in the specimen, Are all very good correlations.

Example 3 Reproducibility of coagulation time The fibrinogen quantitative drying reagent used was the lyophilized reagent of Example 1, and the apparatus for quantifying fibrinogen was C.
The reproducibility of coagulation time was examined using G01 [A & T Sales]. In addition, the method of Example 1 was used for the method of detecting the end point and the method of recognizing the coagulation time.

In the test method, first, plasma containing 150 mg / dl and 400 mg / dl of fibrinogen is diluted 20-fold with an Oulen buffer (manufactured by Sigma). Next, the above reagent was set in CG01, 25 μl of the above-mentioned diluent was added, and the coagulation times of the two diluents were determined. This operation was performed five times, and the reproducibility was examined.

Table 1 shows the results. From Table 1,
It is clear that the obtained clotting times are reproducible.

[0055]

[Table 1]

Example 4 Correlation between the conventional method using a solution-type measuring reagent and the method of the present invention The result of quantifying fibrinogen using the fibrinogen quantitative drying reagent and the method of measuring the fibrinogen clotting time of the present invention using 41 human plasma samples. The results were compared with those obtained by quantifying fibrinogen by a conventional method using a reagent in the form of a solution.

For the determination of fibrinogen using a conventional method using a reagent in the form of a solution, the reagent was Datafi fibrinogen (manufactured by Dade) and the measuring device was KC-1.
0 (manufactured by Amerung Co., Ltd.), and quantified by the method indicated in the datasheet of Dataphi Fibrinogen.

Fibrinogen quantification Fibrinogen was quantified using the drying reagent and the method of measuring fibrinogen clotting time of the present invention as follows. The lyophilized reagent used in Example 1 was used as the fibrinogen quantitative drying reagent, and CG01 was used as an apparatus for quantifying fibrinogen.
[A & T Sales] was used. Note that the same method as in Example 1 was used for the method of detecting the end point and the method of recognizing the coagulation time.

First, human plasma was diluted 20-fold with Orlen buffer (Sigma). Next, the lyophilized reagent is set in CG01, 25 μl of the diluent described above is added, and the coagulation time of each diluent is determined by the above method. Finally, using the graph shown in FIG. 4 of Example 1 as a calibration curve, a method of finding the concentration of fibrinogen in plasma from the obtained clotting time was used.

FIG. 8 shows a correlation diagram between the fibrinogen quantitative value determined by using the fibrinogen quantitative drying reagent and the fibrinogen coagulation time measuring method of the present invention and the fibrinogen quantitative value determined by the conventional method using a reagent in the form of a solution. .

From FIG. 8, it can be seen that the quantitative value of fibrinogen determined by using the fibrinogen quantitative drying reagent and the fibrinogen coagulation time measuring method of the present invention and the quantitative value of fibrinogen quantitatively determined by the conventional method using a reagent in the form of a solution are in good agreement. It is clear that the correlation is high.

Comparative Example 2 Correlation between fibrinogen concentration and coagulation time when the end point is changed The fibrinogen quantitative drying reagent used is the lyophilized reagent of Example 1, and the apparatus for quantifying fibrinogen is C
Using G01 [A & T Sales] to determine the fibrinogen concentration in the sample and the coagulation time when the point of the decay rate outside the viscosity change range of the present invention is set as the end point with respect to the peak value of the movement signal of the magnetic particles. The correlation and reproducibility were examined.

As the end point, a point at which the peak value of the obtained kinetic signal of the magnetic particles is attenuated by 3%, 30% and 60% is taken, and the time from the addition of the sample to the end point is defined as the respective coagulation time. And These points are respectively 100/97 (approximately 1.03) times the viscosity increase and 100/70.
(Approximately 1.43) times and 100/40 (2.5) times.

The method for examining the correlation between the coagulation time and the fibrinogen concentration was carried out as follows. First, 800m
g / dl human plasma containing fibrinogen and fibrinogen-deficient human plasma (George King) were used for 50,90,100,200,400,600,
A dilution series of seven types of human plasma at 800 mg / dl was prepared. Next, each of the plasmas in the dilution series was diluted 20-fold with Orlen buffer (manufactured by Sigma). Then, the lyophilized reagent is set in the blood coagulation measuring device CG01, 25 μl of the diluent described above is added, and the coagulation time of each diluent is determined by the above method. In addition, each plasma dilution was measured three times. Finally, the data is plotted as the coagulation time (average value of three measurements) on the X-axis of the log-log graph as the fibrinogen concentration and the Y-axis is obtained, and the correlation is determined based on whether or not the prepared graph shows linearity. Was examined. FIG.
FIG. 2 shows a correlation diagram between the fibrinogen concentration and the obtained coagulation time. The graph of FIG. 8A is a graph showing the relationship between the obtained coagulation time and the fibrinogen concentration when the point at which the peak value of the kinetic signal of the magnetic particles is attenuated by 3% is taken as the end point, and the graph of FIG. A graph showing the relationship between the obtained clotting time and the fibrinogen concentration when the point at which the signal has attenuated by 30% with respect to the peak value of the movement signal is defined as the end point,
The graph C shows the relationship between the obtained coagulation time and the fibrinogen concentration when the point at which the peak value of the motion signal of the magnetic particles is attenuated by 60% is set as the end point.
The point “•” in the figure is the average value of the three measurements, and the vertical line in the figure is the amplitude of the measurement values of the three measurements.

From FIG. 9, it can be seen that when the peak value of the kinetic signal of the magnetic particles is 3% attenuated, which is outside the range of the viscosity change of the present invention, when the end point is used, the sensitivity is low and the simultaneous reproducibility is poor. It can be seen that there is a problem that accurate fibrinogen quantification cannot be performed. Similarly, when a point at which the peak value of the movement signal of the magnetic particles is attenuated by 60% is set as the end point, although the sensitivity is high, the plasma of less than 200 mg / dl cannot have the end point and the clotting time cannot be obtained. It can be seen that there is a disadvantage that the range is narrow.

FIG. 9 shows that the sensitivity is high, the quantitative range is wide,
In addition, a sample having good reproducibility means that the kinetic signal of the magnetic particles within the viscosity change range defined in the present invention is 3 to the peak value.
It is clear that the end point is the point where 0% attenuation has occurred.

[Brief description of the drawings]

FIG. 1 is an example of a typical reaction slide used for a fibrinogen quantitative drying reagent.

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

FIG. 3 is a diagram showing an end point analysis method when a clotting time corresponding to the amount of fibrinogen is obtained using a fibrinogen quantitative drying reagent.

FIG. 4 is a diagram showing the relationship between the clotting time and the plasma fibrinogen concentration obtained using the endpoint analysis method of FIG. 3;

FIG. 5 is a diagram illustrating a method of calculating a negative slope of a motion signal of a magnetic particle.

FIG. 6 is a diagram showing a correlation between a negative slope obtained by the method of FIG. 5 and a fibrinogen concentration in plasma.

FIG. 7 is a graph showing the correlation between the obtained coagulation time and the fibrinogen concentration when the end point is obtained by changing the ratio of attenuation to the peak value of the movement signal of the magnetic particles.

FIG. 8 is a correlation diagram between the quantitative value of fibrinogen by the method of the present invention and the quantitative value of fibrinogen by the conventional solution method.

FIG. 9 is a diagram showing the correlation between the obtained coagulation time and the fibrinogen concentration when a point having an attenuation ratio different from that of the present invention with respect to the peak value of the movement signal of the magnetic particles is set as the end point.

[Explanation of symbols]

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

Claims (1)

(57) [Claims] 1. A sample is added and mixed to a fibrinogen quantitative dry reagent containing a protein having thrombin activity, magnetic particles, an amino acid or a salt or saccharide thereof,
Next, the change in the viscosity of the fibrinogen quantitative drying reagent after the addition of the sample is monitored, the elapsed time from the addition of the sample to the increase in the viscosity to a specific viscosity is measured, and the measured elapsed time is measured. A method for determining the amount of fibrinogen in a sample to be evaluated as an index representing the amount of fibrinogen in a sample, wherein the specific viscosity is 1.05 to the minimum value of the viscosity of the fibrinogen quantitative drying reagent after sample addition.
A method for determining the amount of fibrinogen in a sample, characterized in that the viscosity is multiplied by a specific magnification within a range of 2 times.