JP5421622B2 - Measuring method and measuring apparatus for biologically active substances derived from living organisms - Google Patents

Description

  The present invention is to detect or measure the concentration of a physiologically active substance in a sample containing a biologically active substance derived from an organism having a property of gelling by reaction with LAL, such as endotoxin or β-D-glucan. The present invention relates to a measuring method and a measuring apparatus.

  Endotoxin is a lipopolysaccharide present in the cell wall of Gram-negative bacteria and is the most typical pyrogen. If an infusion solution, injection drug, blood, or the like contaminated with this endotoxin enters the human body, it may cause serious side effects such as fever and shock. For this reason, it is obliged to manage the above drugs so that they are not contaminated by endotoxin.

  By the way, in the blood cell extract of horseshoe crab (hereinafter also referred to as “LAL: Limulus amoebocyte lysate”), there exists a serine protease activated by endotoxin. When LAL reacts with endotoxin, the coagulogen present in LAL is hydrolyzed into coagulin by the enzyme cascade by serine protease activated according to the amount of endotoxin, and an insoluble gel forms. Generated. Using this LAL characteristic, endotoxin can be detected with high sensitivity.

Β-D-glucan is a polysaccharide (polysaccharide) that constitutes a cell membrane characteristic of fungi. By measuring β-D-glucan, it is effective for screening a wide range of fungal infections including not only common fungi such as Candida, Aspergillus , cryptococcus but also rare fungi.

  Also in the measurement of β-D-glucan, it is possible to detect β-D-glucan with high sensitivity by utilizing the property that the blood cell extract component of horseshoe crab is coagulated (gel coagulation) by β-D-glucan. .

  As a method for detecting or measuring the concentration of a biologically active substance derived from a living organism (hereinafter also referred to as a predetermined physiologically active substance) such as endotoxin or β-D-glucan that can be detected by a blood cell extract component of horseshoe crab, a predetermined physiological activity is used. A mixture of the sample to be detected or the concentration measurement (hereinafter simply referred to as “measurement of the predetermined physiologically active substance”) and LAL is allowed to stand, and the reaction between LAL and the predetermined physiologically active substance is allowed to stand. There is a turbidimetric method for measuring and analyzing the turbidity of a sample accompanying the formation of a gel over time.

  When measuring a predetermined physiologically active substance by the turbidimetric method described above, a mixed solution of a measurement sample and LAL is generated in a glass measurement cell that has been subjected to dry heat sterilization. Then, the gelation of the mixed solution is optically measured from the outside. However, in the turbidimetric method, it may take a very long time for the LAL to gel, particularly in a sample having a low concentration of the predetermined physiologically active substance, so that it is possible to measure the predetermined physiologically active substance in a short time. There is a need for a method.

On the other hand, the mixture of the measurement sample and LAL is stirred using, for example, a magnetic stirrer to form gel fine particles, and the intensity of laser light scattered by the gel particles or the light transmitted through the mixture A sample of a laser light scattering particle measurement method (hereinafter also simply referred to as a light scattering method) that can measure the presence of a predetermined physiologically active substance in a sample in a short time, or a measurement sample that is a form of turbidimetry A stirring turbidimetric method has been proposed in which the mixture is homogenized to make the gelation state in the admixture uniform and the reaction promoted. The turbidimetric method and the stirred turbidimetric method detect the light transmittance, and the light scattering method is different in that the generated particles are detected, but in both cases, the intensity of the transmitted light from the admixture or the scattered light is different. The threshold method for measuring the time until the number of particles obtained from the intensity or the number of peaks exceeds the threshold is used as the basis for the determination.

  Here, in the above-described measurement method, in the turbidimetric method and the stirring turbidimetric method, the transmitted light intensity is increased immediately after the start of measurement regardless of the state of the reaction between the predetermined physiologically active substance and LAL (hereinafter also referred to as the Limulus reaction). In the light scattering method, a phenomenon in which the intensity of scattered light or the number of peaks increases is observed (hereinafter, this phenomenon is also referred to as a gradual decrease / increase). This gradual decrease / increase affects the time until the number of particles obtained from the intensity of transmitted light or the intensity of scattered light or the number of peaks in the above measurement method exceeds the threshold value. May be reduced. In the above measurement method, the lower the concentration of the predetermined physiologically active substance, the longer the measurement time until the number of particles obtained from the intensity of transmitted light or the intensity or peak number of scattered light exceeds the threshold value. The lower the concentration of, the more likely it is to be affected by a gradual decrease / increase, and the reaction start time at which gelation or aggregation has started may not be evaluated correctly.

JP 2004-061314 A JP-A-10-293129 International Publication No. WO2008 / 038329 Pamphlet

  The present invention has been devised in view of the above-described problems, and the object of the present invention is to provide a measurement method capable of further improving the measurement accuracy in the detection or concentration measurement of biologically-derived physiologically active substances. And it is providing the measuring apparatus using the same.

  In the present invention, in order to exclude the influence of the gradual decrease / increase in the measurement of the above-mentioned predetermined physiologically active substance, the transmittance is determined from the intensity or peak number of transmitted light or scattered light from the mixture of the measurement sample and LAL. Alternatively, the greatest feature is that the amount of change (difference) per certain time with respect to the number of gel particles is acquired, and the time at which the amount of change (difference) exceeds the threshold is set as the reaction start time.

More specifically, by reacting a biologically active substance derived from an organism present in a sample with LAL which is a blood cell extract of horseshoe crab, the physiologically active substance in the sample is detected or the concentration of the physiologically active substance is determined. A method for measuring biologically active substances derived from living organisms,
After mixing the sample and LAL, light is incident on the mixed solution of the sample and LAL, and the intensity of the light transmitted through the mixed solution or the light scattered by the mixed solution of the incident light is determined. Acquired,
A predetermined physical quantity at an acquisition time set at a predetermined time interval obtained from the acquired light intensity is set as a detection value,
The difference between the detection value at one acquisition time and the detection value at an earlier acquisition time or the time when the absolute value of the difference exceeds a threshold is set as the reaction start time,
The physiologically active substance in the sample is detected or the concentration is measured based on the reaction start time.

Here, when the above-described gradual decrease / increase occurs, the intensity of transmitted light, the intensity of scattered light, and the number of peaks change substantially linearly with time regardless of the concentration of the predetermined physiologically active substance. I understand. On the other hand, in this invention, the intensity | strength of the transmitted light or scattered light from the said liquid mixture is acquired. Then, detection at one acquisition time using the intensity of the light acquired at the acquisition time set at a predetermined time interval or the value obtained by processing the acquired light intensity at the acquisition time according to the purpose as a detection value The reaction start time is defined as the difference between the value and the detected value at the previous acquisition time (that is, the amount of change in the detected value over a fixed time) or the time when the absolute value of the difference exceeds the threshold. Thereby, it becomes possible to cancel the influence of the gradual decrease / increase on the measurement of the predetermined physiologically active substance.

  Therefore, according to the present invention, the reaction start time between the predetermined physiologically active substance in the sample and LAL can be determined with higher accuracy. As a result, the predetermined physiologically active substance can be detected or the concentration can be measured with higher accuracy.

In the present invention, the intensity of the acquired light is the intensity of light transmitted through the admixture,
The detected value is the transmittance of the admixture expressed as a percentage,
The predetermined time interval is about 2 minutes,
The reaction start time may be a time when the absolute value of the difference between the transmittance at the one acquisition time and the transmittance at the previous acquisition time exceeds 1.

  In this case, the present invention relates to a turbidimetric method for acquiring the transmittance by acquiring the transmitted light of the mixed liquid out of the light incident on the mixed liquid of the predetermined physiologically active substance and LAL. In the turbidimetric method, as described above, the admixture becomes turbid due to the reaction between the predetermined physiologically active substance and LAL, and the transmittance of the admixture decreases with time. Here, the shape of the transmittance decrease curve in the turbidimetric method varies depending on the concentration of the predetermined physiologically active substance. For example, when the concentration of the predetermined physiologically active substance is high, the transmittance decreases sharply. On the other hand, when the concentration of the predetermined physiologically active substance is low, the transmittance gradually decreases.

  If the concentration of the predetermined physiologically active substance is high and the transmittance sharply decreases, if the detection value acquisition interval is set long, the amount of decrease in each detection value is too large and a sufficient number of data is secured. This makes it difficult to measure with high accuracy. On the other hand, when the concentration of the predetermined physiologically active substance is low and the transmittance gradually decreases, if the detection value acquisition interval is set too short, the one-time decrease width of each detection value is too small, and the difference is sufficiently accurate. It cannot be detected. Thus, in the present invention, it is desirable to adjust the detection value acquisition time interval according to the concentration of the predetermined physiologically active substance in the sample to be measured.

  On the other hand, when the transmittance is expressed as a percentage according to the inventors' earnest research, when the acquisition interval of the detection value (transmittance) is about 2 minutes, a wider predetermined physiologically active substance in the present invention It has been found that it is possible to obtain a transmittance decrease curve with high accuracy in the concentration range of. In that case, it has been found that the concentration of the predetermined physiologically active substance can be measured with high accuracy by setting the reaction start time at the time when the amount of decrease in transmittance over 1 minute exceeds 1%.

  Therefore, in the present invention, when the transmitted light from the mixture of the predetermined physiologically active substance and LAL is acquired to calculate the transmittance, and the transmittance difference (change amount) at a predetermined time interval is acquired, the predetermined The time interval was about 2 minutes, and the reaction start time was defined as the time when the decrease in transmittance in 2 minutes exceeded 1%. Thereby, it is possible to detect a predetermined physiologically active substance or measure the concentration with a higher accuracy for a wider range of samples.

In the present invention, the intensity of the acquired light is the intensity of light scattered by the admixture,
The detected value is the number of particles that scatter the light incident on the admixture derived based on a predetermined number of peaks in the intensity of the scattered light,
The predetermined time interval is about 100 seconds,
The reaction start time may be a time when the difference between the number of particles at the one acquisition time and the number of particles at the previous acquisition time exceeds 200.

  In this case, out of the light incident on the mixture of the predetermined physiologically active substance and LAL, the scattered light from the mixture is acquired and mixed from the number of peaks in the scattered light (satisfying a predetermined condition for improving measurement accuracy). The present invention relates to a light scattering method for obtaining the number of (gel) particles in a liquid. As described above, in the light scattering method, gel particles are generated in the mixture by stirring the mixture during the reaction between the predetermined physiologically active substance and LAL, and the size and number of the gel particles increase as the reaction proceeds. I will do it. Therefore, as the reaction proceeds, the number of peaks observed in the scattered light from the mixture increases.

  Further, the shape of the increase curve of the number of peaks of scattered light in the light scattering method varies depending on the concentration of the predetermined physiologically active substance. For example, when the concentration of the predetermined physiologically active substance is high, the number of peaks increases steeply, whereas when the concentration of the predetermined physiologically active substance is low, the number of peaks increases gradually. Therefore, as in the case of the turbidimetric method, it is desirable to adjust the detection value acquisition time interval in the light scattering method according to the concentration of the predetermined physiologically active substance in the sample to be measured.

  On the other hand, according to the inventors' diligent research, in the light scattering method, when the acquisition interval of the detection value (the number of scattered particles) is set to about 100 seconds, it is higher in a wider concentration range of the predetermined physiologically active substance. It has been found that it is possible to obtain an increasing curve of the number of peaks with accuracy. In that case, it has been found that the concentration of the predetermined physiologically active substance can be measured with high accuracy by setting the reaction start time at the time when the increase in the number of scattered particles in 100 seconds exceeds 200.

  Therefore, in the present invention, when scattered light from a mixture of a predetermined physiologically active substance and LAL is acquired to detect the number of scattered particles, and the difference (change amount) in the number of scattered particles at a predetermined time interval is acquired. The predetermined time interval is about 100 seconds, and the reaction start time is defined as the time when the increase in the number of scattered particles in 100 seconds exceeds 200. Thereby, it is possible to detect a predetermined physiologically active substance or measure the concentration with a higher accuracy for a wider range of samples.

  In the present invention, the biologically active substance derived from the organism may be endotoxin or β-D-glucan.

In this way, endotoxin, the most typical pyrogen, can be detected or measured more precisely, and endotoxin-contaminated fluids, injections, blood, etc. can enter the human body and prevent side effects. it can. Similarly, β-D-glucan can be detected or measured more accurately, and a wide range of fungal infections including not only common fungi such as Candida, Aspergillus , cryptococcus but also rare fungi can be detected. Screening can be performed more accurately.

In addition, the present invention holds a mixed solution of a sample containing a physiologically active substance derived from a predetermined organism and LAL, which is a blood cell extract of horseshoe crab, so that light can enter, and the mixed solution holding that promotes the reaction in the mixed solution Means,
Stirring means for stirring the mixed liquid in the mixed liquid holding means;
A light incident means for making light incident on the mixed liquid in the mixed liquid holding means;
A light receiving means for receiving transmitted light or scattered light in the admixture of the incident light and converting it into an electrical signal;
Determination means for determining a reaction start time between the physiologically active substance and LAL in the sample from the electrical signal converted in the light receiving means;
Deriving means for deriving the presence or concentration of the physiologically active substance in the sample from a predetermined relationship between the reaction start time and the concentration of the physiologically active substance,
The determination means uses a signal obtained by adding a predetermined calculation to the electric signal at the acquisition time set at a predetermined time interval or the electric signal as a detection signal value, and the detection signal value at one acquisition time The biologically active substance measuring device derived from a living body may be characterized in that the reaction start time is determined based on the difference between the detection signal value at the acquisition time or the time when the absolute value of the difference exceeds a threshold value.

  According to the measuring apparatus for a predetermined physiologically active substance in the present invention, the transmitted light or scattered light from the mixture of the predetermined physiologically active substance and LAL is received by the light receiving means and converted into an electrical signal. Then, the reaction start time in the mixed liquid is determined by the determination means from the converted electric signal. The determination means acquires the detection signal value at a predetermined time interval based on the obtained electrical signal, and determines the reaction start time at the time when the amount of change in the detection signal value at the predetermined time interval exceeds a threshold value.

  According to the measuring apparatus of the present invention, the influence of the gradual decrease / increase on the measurement of a predetermined physiologically active substance can be automatically canceled. Therefore, it becomes possible to detect the predetermined physiologically active substance or measure the concentration with higher accuracy.

  In this case, when the detection signal value is the transmittance of the mixed liquid expressed as a percentage, the predetermined time interval may be about 2 minutes and the threshold value may be 1. Further, when the detection signal value is the number of particles that scatter light incident on the mixture, the predetermined time interval may be about 100 seconds and the threshold value may be 200. By doing so, it becomes possible to obtain higher measurement accuracy for a wider range of predetermined physiologically active substance concentrations.

  In the measurement apparatus for a predetermined physiologically active substance according to the present invention, the predetermined time interval and / or threshold value may be variable. Then, the detection signal value acquisition time interval and the threshold for determining the reaction start time can be optimized according to the expected concentration of the predetermined physiologically active substance. High measurement accuracy can be obtained. Furthermore, in the present invention, the biologically active substance derived from the organism may be endotoxin or β-D-glucan.

  The means for solving the above-described problems of the present invention can be used in combination as much as possible.

  In the present invention, a higher measurement can be performed when detecting or measuring the concentration of the physiologically active substance by utilizing the reaction between a biologically active substance derived from an organism such as endotoxin or β-D-glucan and LAL. Accuracy can be obtained.

It is a figure which shows schematic structure of the turbidimetric measuring apparatus in Example 1 of this invention. It is a graph which shows the time change of the light transmittance for demonstrating the gradual reduction in Example 1 of this invention. It is a graph which shows the relationship between the endotoxin density | concentration obtained by the conventional threshold value method, and endotoxin detection time. It is a graph which shows the relationship between the endotoxin density | concentration obtained by the difference method, and endotoxin detection time in Example 1 of this invention. It is a figure which shows schematic structure of the light-scattering particle | grain measuring apparatus in Example 2 of this invention. It is a graph which shows the time change of the number of detected particles for demonstrating the gradual rise in Example 2 of this invention. It is a graph which shows the relationship between the endotoxin density | concentration obtained by the threshold value method and the difference method, and endotoxin detection time in Example 2 of this invention. It is the schematic for demonstrating the process in which LAL gelatinizes with an endotoxin or (beta) -D-glucan, and its detection method.

  The process by which LAL and endotoxin react to form a gel is well examined. That is, as shown in FIG. 8, when endotoxin binds to factor C, which is a serine protease in LAL, factor C is activated to become active factor C. Active factor C is another serine protease in LAL. A certain factor B is hydrolyzed and activated to obtain an activated factor B. This activated factor B immediately hydrolyzes the precursor of the clotting enzyme in LAL to form a clotting enzyme, and this clotting enzyme hydrolyzes the coagulogen in LAL to produce coagulin. And it is thought that the produced coagulin associates with each other to further produce an insoluble gel, and the entire LAL is entrained in this to gel.

  Similarly, when β-D-glucan binds to factor G in LAL, factor G is activated to become active factor G. Active factor G hydrolyzes the precursor of the coagulation enzyme in LAL and coagulates. Enzyme. As a result, similarly to the reaction between endotoxin and LAL, coagulin is produced, and the produced coagulin associates with each other to further produce an insoluble gel.

  This series of reactions is similar to the fibrin gel formation process mediated by serine proteases such as Christmas factors and thrombin found in mammals. Such an enzyme cascade reaction has a very strong amplification action because even a very small amount of activator is activated by linking the subsequent cascade. Therefore, according to the method for measuring a predetermined physiologically active substance using LAL, it is possible to detect a very small amount of the predetermined physiologically active substance on the order of subpicogram / mL.

  Examples of the measuring method capable of quantifying a predetermined physiologically active substance such as endotoxin and β-D-glucan include the turbidimetric method, the stirring turbidimetric method, and the light scattering method as described above. As shown in FIG. 8, these measurement methods detect the coagulin aggregate produced by the LAL enzyme cascade reaction as turbidity in the former, and the latter as gel fine particles produced in the system. Highly sensitive measurement is possible.

  The turbidimetric method has an evaluation that it is easy to use in the field in that no special reagent is required and the concentration range of the predetermined physiologically active substance that can be measured is wide. On the other hand, however, the turbidimetric method has a problem that it takes a very long time to measure a predetermined physiologically active substance at a low concentration. This is because the turbidimetric method does not look at the amount of coagulin itself that is the end product of the protease cascade, but the process by which the light transmittance is reduced by the gel formed by further association. This is because.

  That is, if the coagulin concentration does not reach a certain level or more, gelation does not occur. Therefore, in order to detect a predetermined physiologically active substance in the turbidimetric method, it is necessary to wait until the gel is formed. Therefore, when the predetermined physiologically active substance concentration is high, the required time and sufficient concentration of coagulin is quickly generated and gelation starts, so the measurement time is shortened, but if the predetermined physiologically active substance concentration is low, the coagulin concentration required for gelation It takes time to reach the point, and the measurement time becomes long. In that respect, in the stirring turbidimetric method, the reaction of both is promoted by stirring a mixture of a predetermined physiologically active substance and LAL to shorten the measurement time.

In addition, the light scattering method is an improvement from the turbidimetric method in that the sample is stirred and the particle is detected instead of gelled by a laser, and the measurement time can be greatly reduced compared to the turbidimetric method. . The turbidimetric method and the stirred turbidimetric method are different from the light scattering method in that the physical quantity being viewed is different, but is common in that the point in time when a certain threshold is exceeded is regarded as the starting point of the reaction (hereinafter referred to as this method). Is called a threshold method for convenience).

  Here, in any of the above-described measurement methods, the light transmittance of the mixed solution is reduced in the turbidimetric method and the stirring turbidimetric method immediately after the start of measurement, regardless of the state of the Limulus reaction, and in the light scattering method, the mixed solution is mixed. A gradual decrease / increase phenomenon was observed in which the number of gel particles increased. Although the cause of this is not clarified, for example, it is considered that one of the causes is that protein is denatured when carbon dioxide or the like dissolves in the mixture and the pH of the mixture changes.

  In the stirring turbidimetric method and the light scattering method, the admixture is stirred by a stirrer built in the measurement container, and this stirring makes the state of the Limulus reaction uniform or accelerates the reaction. Yes. During this stirring, if the rotation axis of stirring is inappropriate or the bottom shape of the measurement container is inappropriate, there is a tendency to gradually decrease / rise. It is in.

  When the concentration of the specified physiologically active substance in the measurement sample is high, the mixture solution gels before being affected by the gradual decrease / increase, and the aggregation determination is completed. Therefore, the measurement accuracy is affected by the gradual decrease / increase. The risk of lowering is relatively low. However, when the concentration of the predetermined physiologically active substance in the measurement sample is low, it takes a long time to measure the concentration. Therefore, the change curve of the light transmittance and the number of gel particles gradually sets the threshold earlier than it actually is due to the effect of gradual decrease / increase. And the accuracy of the determination of the reaction start time may be reduced.

  In the present embodiment, in preparation for the phenomenon of a gradual decrease / increase in the detection or concentration measurement of a predetermined physiologically active substance, the light transmittance and the number of gel particles themselves in the mixed liquid of the sample and LAL have threshold values. The method of determining the reaction start time based on exceeding is not adopted. A new method is adopted in which the light transmittance and the number of gel particles are acquired at regular time intervals, and the time at which the amount of change in the light transmittance and the number of gel particles in the time interval exceeds the threshold is determined as the reaction start time. . Naturally, this threshold value is different from that when the turbidity or the number of gel particles is compared with the threshold value. As a result, even if a gradual decrease / increase occurs, the influence can be removed, and the reaction start time between the predetermined physiologically active substance and LAL can be determined with higher accuracy. (Hereafter, this method is called the difference method for convenience.)

  In addition, this makes it possible to determine the time when the turbidity of the admixture and the amount of change in the number of gel particles change rapidly. Therefore, it is possible to improve the measurement accuracy for a low concentration sample as well as a high concentration sample. In addition, according to this, the influence of the gradual decrease / increase can be removed by changing only the analysis process of the data obtained at the time of measuring the predetermined physiologically active substance.

  Details of the embodiment of the present invention will be described below. However, this invention is not limited to the form shown below. In the following embodiments, the case where the predetermined physiologically active substance is endotoxin will be described, but the same mode can be considered when the predetermined physiologically active substance is β-D-glucan.

  Here, the detection target in the case of measuring endotoxin by each of the measurement methods described above is different. In the turbidimetric method and the stirring turbidimetric method, endotoxin acts on the LAL reagent to detect turbidity of the mixed solution when the mixed solution gels or aggregates. Therefore, the transmitted light of the mixed solution is obtained, and the time when the light transmittance in the mixed solution falls below a preset threshold is determined as the reaction start time of endotoxin and LAL.

  In the turbidimetric method and the stirring turbidimetric method, when the light transmittance of the mixed solution tends to decrease regardless of the state of the Limulus reaction, first take a 2-minute moving average to remove the noise of the detected value. . Next, since the data is unstable immediately after the start of measurement, the light transmittance after 2 minutes from the start of measurement is set to 100%. And the difference in the predetermined time interval (DELTA) T of the light transmittance (%) originally acquired every 10 seconds is taken, and the first time when the square value exceeds 1 continuously 3 times is set as the reaction start time. adopt. The difference time interval ΔT is preferably 1 minute to 5 minutes, particularly about 2 minutes. In the case of a low-concentration sample, since the amount of change in light transmittance is small, the time interval ΔT is preferably 5 minutes. Here, the reason why the difference of the light transmittance (%) in the time interval ΔT is squared is to increase the change and improve the resolution when the difference value is around 1. Therefore, it is not always necessary to square the difference.

  On the other hand, the light scattering method observes the process of gelling or agglomeration of the admixture, similar to the turbidimetric method and the stirring turbidimetric method, but the point is that the light scattered by the agglomerates (gel particles) in the admixture is obtained. Different. That is, when a Limulus reagent is mixed in a sample that is initially transparent and does not contain gel particles, aggregation occurs and the number of gel particles increases. Since the frequency of occurrence of peaks in the scattered light increases as the number of gel particles increases, the number of peaks of the scattered light is acquired as the number of gel particles. Then, a threshold is provided for the integrated value of the number of gel particles detected in a certain time, and the reaction start time is defined as the time when the number of detected particles exceeds the threshold.

  When the number of gel particles detected in the light scattering method tends to increase regardless of the state of the Limulus reaction, the difference between the detected values (number of gel particles) at a certain time interval ΔT, that is, the number of increased particles is obtained. Regarding the determination of the reaction start time of the Limulus reaction, the first time when the difference in the number of gel particles exceeds the tenth consecutive threshold value 200 is adopted as the reaction start time in consideration of the influence of noise. The difference time interval ΔT is preferably 30 seconds to 200 seconds, and more preferably about 100 seconds, so that it can be applied to a sample having a high endotoxin concentration.

  In the above measurement, the value of the time interval ΔT for obtaining the detection value needs to be set relatively large when the endotoxin concentration is low and relatively small when the endotoxin concentration is high. This is because the lower the endotoxin concentration, the smaller the amount of change in the turbidity and the number of gel particles after mixing the sample and the LAL reagent, so that a sufficiently large difference can be secured in the detected values before and after the time interval ΔT. This is because a sufficient time interval is required.

  On the other hand, when the endotoxin concentration is high, the Limulus reaction between endotoxin and LAL proceeds relatively quickly, and it is considered that aggregation has started during the set time interval ΔT. Therefore, the turbidity of the admixture or the change in the detection value of the number of gel particles is sequentially detected, and an early estimation is made as to whether the endotoxin concentration is low or high, and the time interval ΔT is divided into cases based on the approximate value. By changing the above, it is possible to measure the endotoxin concentration with higher accuracy. Therefore, the above estimation method may be adopted as necessary.

[Production Example 1]
A stainless steel stirrer (φ1 mm, length 5 mm) was placed in a glass container (outer diameter φ7 mm, length 50 mm, hereinafter abbreviated as cuvette). The opening of the cuvette was covered with aluminum foil, and 20 of the cuvettes were covered together with aluminum foil, heat-treated at a temperature of 250 ° C. for 3 hours, and the cuvette was dry-heat treated. Thereby, endotoxin adhering to the cuvette is inactivated by thermal decomposition.

[Example 1]
In FIG. 1, schematic structure of the turbidimetric measuring apparatus 1 as an endotoxin measuring apparatus of a present Example is shown. In the turbidimetric measuring apparatus 1 of the present embodiment, endotoxin is measured by the stirring turbidimetric method. In this example, the prepared sample containing diluted endotoxin is transferred to the cuvette 2 as the admixture holding means manufactured in Production Example 1. A warmer 5 is provided so as to surround the cuvette 2. A heating wire (not shown) is provided inside the incubator 5, and the cuvette 2 is kept at about 37 ° C. by energizing the heating wire. The cuvette 2 is provided with a stainless steel stirring bar 3. The stirring bar 3 rotates in the cuvette 2 by the action of the stirrer 4 installed at the lower part of the cuvette 2. That is, the stirrer 4 includes a motor 4a and a permanent magnet 4b provided on the output shaft of the motor 4a. And the permanent magnet 4b rotates by supplying with electricity to the motor 4a. Since the magnetic field from the permanent magnet 4b rotates, the stainless steel stirring bar 3 rotates by the action of the rotating magnetic field. The stirrer 3 and the stirrer 4 correspond to stirring means. In this embodiment, the rotation speed of the stirrer 3 was 1000 rpm.

  The turbidimetric measurement apparatus 1 is provided with a light source 6 as a light incident means and a light receiving element 9 as a light receiving means. The light emitted from the light source 6 passes through the aperture 7 and then enters the sample in the cuvette 2 through the incident hole 5 a provided in the incubator 5. The light transmitted through the sample in the cuvette 2 is emitted from the emission hole 5 b provided in the heat retaining device 5, passes through the aperture 8, and is irradiated on the light receiving element 9. The light receiving element 9 outputs a photoelectric signal corresponding to the intensity of the received light. The output of the photoelectric signal is input to the arithmetic unit 10 as a determination unit and a derivation unit. In the arithmetic unit 10, the reaction start time is determined and the endotoxin concentration is derived in accordance with a program (algorithm) stored in advance. In addition to this, the turbidimetric measurement apparatus 1 may include a display device that displays the derived endotoxin concentration.

  FIG. 2 shows the gradual decrease observed in this example. This gradual decrease is a phenomenon that appears remarkably in the portion A shown in the figure. This is a phenomenon in which the baseline of light transmittance decreases immediately after the start of measurement, regardless of the reaction state of endotoxin and LAL in the mixed solution. Then, it was confirmed that when the reaction between endotoxin and LAL progressed, it continued to change with a larger slope through an inflection point.

  In the conventional stirring turbidimetric method, the reaction start time is defined as the time when the light transmittance falls below 95%. However, when a gradual decrease occurs, the time when the light transmittance falls below the threshold value of 95% may become abnormally early due to the effect of the decrease in the baseline. Alternatively, the light transmittance value may fall below the threshold value of 95% before the light transmittance decrease curve reaches the inflection point. As described above, the accuracy of the determination of the reaction start time may be reduced due to the gradual decrease.

  FIG. 3 shows the results of plotting using the conventional threshold method, with the endotoxin concentration plotted on the horizontal axis and the time below the threshold of 95% of the conventional method plotted on the vertical axis. It is known that this graph rides a straight line when the logarithm is taken. When looking at the correlation coefficient in the result shown in FIG. 3, the absolute value was 0.975. According to the Japanese Pharmacy Law, the condition that “the absolute value of the correlation coefficient must be 0.980 or more” is imposed, but the result shown in FIG. 3 does not satisfy this condition.

  In FIG. 4, the result at the time of using the difference method which concerns on a present Example is shown. In this case, the time interval ΔT was 2 minutes, and the threshold for determining the reaction start time was that when the absolute value of the amount of change in light transmittance (%) exceeded 1%. As can be seen from the figure, by applying the difference method, the absolute value of the correlation coefficient in the relationship between the endotoxin concentration and the endotoxin detection time was 0.987. That is, a result with stronger correlation was obtained by applying the difference method, the linearity was improved, and the conditions in the above Japanese pharmacy method could be satisfied. As described above, in this embodiment, the influence of the gradual decrease can be removed only by changing the program (algorithm) in the arithmetic unit 10 of the turbidimetric measurement apparatus 1, and the measurement accuracy of the turbidimetric measurement apparatus 1 can be improved. Can be improved.

  In this embodiment, the light transmittance value corresponds to the detection value and the detection signal value. Further, in the turbidimetric measurement apparatus 1 according to the present embodiment, the time interval ΔT and the threshold value may be adjustable in the apparatus. As a result, the above-described estimation method can be executed more easily.

[Example 2]
Next, as Example 2, measurement using a light scattering method will be described. In FIG. 5, schematic structure of the light-scattering particle | grain measuring apparatus 11 as an endotoxin measuring apparatus in this Embodiment is shown. A laser light source is used as the light source 12 used in the light scattering particle measuring apparatus 11, but an ultra-high brightness LED or the like may be used. The light emitted from the light source 12 is narrowed by the incident optical system 13 and enters the sample cell 14. The sample cell 14 holds a mixture of a sample to be measured for endotoxin and the LAL reagent. The light incident on the sample cell 14 is scattered by particles (coagulogen monomer, measurement object such as coagulogen oligomer) in the mixture.

  An emission optical system 15 is arranged on the side of the incident optical axis of the sample cell 14. In addition, on the extension of the optical axis of the output optical system 15, there is a light receiving element 16 that receives scattered light that is scattered by particles in the mixed liquid in the sample cell 14 and is narrowed by the output optical system 15 and converts it into an electric signal. Has been placed. The light receiving element 16 includes an amplifying circuit 17 that amplifies the electric signal photoelectrically converted by the light receiving element 16, a filter 18 for removing noise from the electric signal amplified by the amplifying circuit 17, and the electricity after the noise is removed. An arithmetic unit 19 that calculates the number of gel particles from the number of signal peaks, further determines the reaction start time and derives the endotoxin concentration, and a display 20 that displays the result are electrically connected.

The sample cell 14 is provided with a stirrer 21 that rotates by applying an electromagnetic force from the outside and stirs the mixed liquid as a sample, and a stirrer 22 is provided outside the sample cell 14. ing. Thus, it is possible to adjust the presence / absence of stirring and the stirring speed.

  Here, the sample cell 14 is the cuvette manufactured in Manufacturing Example 1 and corresponds to the admixture holding means of this embodiment. The light source 12 and the incident optical system 13 correspond to light incident means. The stirrer 21 and the stirrer 22 correspond to stirring means. The emission optical system 15 and the light receiving element 16 correspond to light receiving means. The arithmetic unit 19 corresponds to determination means and derivation means.

  FIG. 6 shows an example of a temporal change in the number of detected particles when the light scattering method is used. In this example, the endotoxin was measured by keeping the temperature at 37 ° C. while stirring the sample in the same manner as the stirring turbidimetric apparatus. Specifically, PA-200 manufactured by Kowa was used as the measuring device. As can be seen from FIG. 6B, the light scattering method also confirmed a phenomenon in which the number of detected (gel) particles increased irrespective of the reaction state between endotoxin and LAL. This reaction exceeded the threshold value (detected particle number 200) before “abrupt increase in the number of particles” that should be detected, making accurate determination difficult.

  Next, the measurement process in the present embodiment in which the difference method is applied to the light scattering method will be described. In the arithmetic unit 19, first, histogram data of a peak for 1 second is created every second from the electric signal after the noise is removed. Then, the total number of particles detected per second is calculated from the histogram data. Further, in order to smooth the variation in the number of particles, the total number of particles calculated every second is moved and added for 10 seconds. The moving addition value for 10 seconds obtained by moving addition is compared with the same value obtained previously by the time interval ΔT (= 100 seconds), and the difference (increase) is continuously thresholded 10 times (10 seconds). The first histogram data acquisition time when it exceeds 200 is determined as the reaction start time.

In FIG. 7, the threshold method and the difference method are applied to the light scattering method, and the endotoxin concentration is plotted on the horizontal axis.
The results of plotting the reaction start time on the vertical axis are shown. In the case where the number of particles is simply detected, the absolute value of the correlation coefficient is 0.943 and the linearity is greatly lost. On the other hand, the time interval ΔT is set to 100 seconds, and the amount of increase in particles for 100 seconds is set to the threshold 200. In the case of exceeding the reaction start time, the absolute value of the correlation coefficient was 0.999 and a strong correlation was obtained. From the above, it can be considered that the algorithm whose detection target is the amount of increase in particles in 100 seconds is a more stable algorithm compared to the conventional algorithm whose target is the number of particles. As described above, in this embodiment, the influence of the gradual increase can be removed only by changing the program (algorithm) in the arithmetic unit 19 of the light scattering particle measuring device 11, and the measurement of the light scattering particle measuring device 11 can be performed. Accuracy can be improved.

  In this embodiment, the value of the number of detected particles (number of gel particles) corresponds to the detected value and the detected signal value. Further, in the light scattering particle measuring apparatus 11 according to the present embodiment, the time interval ΔT and the threshold value may be adjustable in the apparatus. As a result, the above-described estimation method can be executed more easily.

  In the present embodiment, as described above, the calculation device 19 calculates the total number of particles detected in one second from the histogram data. The total number of particles is moved and added for 10 seconds, and the moving addition value for 10 seconds is calculated. Compared with the same value obtained before time interval ΔT, the reaction start time was determined based on the first data acquisition time when the difference (increase) exceeded the threshold value 10 times (10 seconds) continuously. However, this measurement process is only an example, and the number of times that the difference (increase) exceeds the threshold when determining the detection time of histogram data, the presence or absence of movement addition or movement addition time, and the reaction start time, etc. The values are not limited to the example values, and can be changed as appropriate.

Example 3
Endotoxin was measured using a toxinometer (Wako Pure Chemical Industries, Ltd.), which is a conventional device for turbidimetry. Even in the toxinometer (turbidimetric method) in which the sample was not stirred, a gradual decrease in the baseline of unknown cause was confirmed in the temporal change in light transmittance. Accordingly, the toxinometer using the threshold method in which the reaction start time is set to a time point exceeding 95% is also affected by a gradual decrease. Therefore, in order to improve the measurement accuracy, reanalysis was similarly performed using the difference method. As a result, the linearity of the regression line increased, and the influence on the endotoxin measurement due to detection error was suppressed by the difference method, and an improvement was seen.

  In the above embodiments, the difference exceeding the threshold does not necessarily mean that the difference changes from a state smaller than the threshold to a larger state. For example, it is a matter of course that the difference that tends to decrease includes a change from a state larger than a threshold value to a smaller state.

DESCRIPTION OF SYMBOLS 1 ... Turbidity measuring device 2 ... Glass container 3 ... Stirrer 4 ... Stirrer 4a ... Motor 4b ... Magnet 5 ... Insulator 5a ... Incident hole 5b ... Exit hole 6 ... Light source 7 ... Aperture 8 ... Aperture 9 ... Light receiving element 10 ... Calculating device 11 ... Measurement system 12 ... Light source 13 ... Incoming optical system 14 ... Sample cell 15 ... Output optical system 16 ... Light receiving element 17 ... Amplifying circuit 18 ... Noise removal filter 19 ... Arithmetic device 20 ... Display device 21 ... Stir bar 22 ... Stirrer

Claims (9)

An organism for detecting the physiologically active substance in the sample or measuring the concentration of the physiologically active substance by reacting a biologically active substance derived from an organism present in the sample with LAL which is a blood cell extract of horseshoe crab A method for measuring a physiologically active substance derived from
After mixing the sample and LAL, light is incident on the mixed solution of the sample and LAL, and the intensity of the light transmitted through the mixed solution or the light scattered by the mixed solution of the incident light is determined. Acquired,
A predetermined physical quantity at an acquisition time set at a predetermined time interval obtained from the acquired light intensity is set as a detection value,
The difference between the detection value at one acquisition time and the detection value at an earlier acquisition time or the time when the absolute value of the difference exceeds a threshold is set as the reaction start time,
A method for measuring a biologically active substance derived from an organism, wherein the physiologically active substance in the sample is detected or the concentration is measured based on the reaction start time. The intensity of the acquired light is the intensity of light transmitted through the admixture,
The detected value is the transmittance of the admixture expressed as a percentage,
The predetermined time interval is about 2 minutes,
The biological origin according to claim 1, wherein the reaction start time is defined as a time when an absolute value of a difference between the transmittance at the one acquisition time and the transmittance at the previous acquisition time exceeds 1. Method for measuring physiologically active substances of The intensity of the acquired light is the intensity of light scattered by the admixture,
The detected value is the number of particles that scatter the light incident on the admixture derived based on a predetermined number of peaks in the intensity of the scattered light,
The predetermined time interval is about 100 seconds,
2. The biological activity of biological origin according to claim 1, wherein the reaction start time is a time when the difference between the number of particles at the one acquisition time and the number of particles at the previous acquisition time exceeds 200. Method for measuring substances.   The method for measuring a biologically active substance of biological origin according to any one of claims 1 to 3, wherein the biologically active substance of biological origin is endotoxin or β-D-glucan. A mixed liquid holding means for holding a mixed liquid of a sample containing a physiologically active substance derived from a predetermined organism and LAL that is a blood cell extract of horseshoe crab so that light can enter, and for allowing a reaction in the mixed liquid to proceed;
Stirring means for stirring the mixed liquid in the mixed liquid holding means;
A light incident means for making light incident on the mixed liquid in the mixed liquid holding means;
A light receiving means for receiving transmitted light or scattered light in the admixture of the incident light and converting it into an electrical signal;
Determination means for determining a reaction start time between the physiologically active substance and LAL in the sample from the electrical signal converted in the light receiving means;
Deriving means for deriving the presence or concentration of the physiologically active substance in the sample from a predetermined relationship between the reaction start time and the concentration of the physiologically active substance,
The determination means uses a signal obtained by adding a predetermined calculation to the electric signal at the acquisition time set at a predetermined time interval or the electric signal as a detection signal value, and the detection signal value at one acquisition time An apparatus for measuring a biologically active substance derived from a living body, characterized in that the reaction start time is determined based on a difference from a detection signal value at the acquisition time or an absolute value of the difference exceeding a threshold value. The detection signal value is the transmittance of the admixture expressed as a percentage,
The predetermined time interval is about 2 minutes;
6. The biologically active substance measuring apparatus according to claim 5, wherein the threshold value is 1. The detection signal value is the number of particles that scatter light incident on the admixture,
The predetermined time interval is about 100 seconds;
6. The biologically active physiologically active substance measuring apparatus according to claim 5, wherein the threshold value is 200.   6. The biologically active physiologically active substance measuring apparatus according to claim 5, wherein the predetermined time interval and / or the threshold value are variable.   The biologically active substance measuring apparatus according to any one of claims 5 to 8, wherein the biologically active substance derived from an organism is endotoxin or β-D-glucan.

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