HK1165003A

HK1165003A - Method and apparatus for measuring biogenous biologically active substances

Info

Publication number: HK1165003A
Application number: HK12105415.2A
Authority: HK
Inventors: 薮崎克己; 原拓也; 杉浦友香
Original assignee: 兴和株式会社
Priority date: 2009-03-13
Filing date: 2010-03-12
Publication date: 2012-09-28

Description

Method and device for measuring physiologically active substance derived from living body

Technical Field

The present invention relates to a measurement method and a measurement apparatus for detecting a physiologically active substance derived from a living organism, which has a property of undergoing gelation by a reaction with LAL, such as endotoxin, β -B-glucan, or measuring the concentration thereof in a sample containing the physiologically active substance.

Background

Endotoxin is a lipopolysaccharide present in the cell wall of gram-negative bacteria, and is the most representative pyrogen. If transfusion, injection, blood, etc. contaminated with the endotoxin enter the human body, serious side effects such as fever, shock, etc. may be caused. Therefore, the above-mentioned drugs and the like need to be managed so as not to be contaminated with endotoxin.

A serine protease activated by endotoxin is present in a blood cell extract of Tachypleus tridentate (hereinafter also referred to as "LAL: Tachypleus tridentate lysate"). When LAL reacts with endotoxin, a serine protease activated depending on the amount of endotoxin generates an enzyme cascade, whereby coagulogen present in LAL is hydrolyzed into coagulin, and the coagulin is associated with one another to form an insoluble gel. Endotoxin can be detected with high sensitivity by utilizing the characteristics of LAL.

In addition, β -D-glucan is a polysaccharide that constitutes a characteristic cell membrane in fungi. The measurement of β -D-glucan is effective not only in fungi commonly seen in clinical use such as Candida (Candida), Aspergillus (Aspergillus), and Cryptococcus (Cryptococcus), but also in screening fungal infections and the like in a wide range including rare fungi.

In the measurement of β -D-glucan, β -D-glucan can be detected with high sensitivity by utilizing the property of the blood cell extract fraction of horseshoe crab that is caused by the coagulation of β -D-glucan (gel coagulation).

As a method for detecting or measuring the concentration of a biologically-derived physiologically active substance (hereinafter, also referred to as a predetermined physiologically active substance) such as endotoxin or β -D-glucan, which can be detected from a limulus blood cell extract component, there is a turbidimetric method in which a mixture obtained by mixing a sample to be subjected to detection or measurement of the concentration of the predetermined physiologically active substance (hereinafter, also referred to as "measurement of the predetermined physiologically active substance") with LAL is left to stand, the reaction of the LAL with the predetermined physiologically active substance causes the formation of a gel, and the turbidity of the sample accompanying the formation of the gel is measured over time to perform analysis.

When a predetermined physiologically active substance is measured by the turbidimetric method, a mixture of the measurement sample and LAL is generated in the glass measurement cell subjected to dry heat sterilization treatment. Then, the gelation of the mixed solution was optically measured from the outside. However, in the turbidimetric method, in particular, in a sample having a low concentration of a predetermined physiologically active substance, it may take a long time for the LAL to gel, and therefore, a method capable of measuring the predetermined physiologically active substance in a short time is required.

In response to this demand, there has been proposed a laser light scattering particle measurement method (hereinafter, simply referred to as a light scattering method) in which a mixture of a measurement sample and LAL is stirred with, for example, a magnetic stirrer to thereby produce gel fine particles, and the presence of a predetermined physiologically active substance in the sample can be measured in a short time from the intensity of laser light scattered by the gel fine particles or the intensity of light transmitted through the mixture; alternatively, a stirring turbidimetric method has been proposed, which is one of the turbidimetric methods, but stirs the measurement sample to make the state of gelation in the mixed solution uniform and to accelerate the reaction. The turbidimetry and the stirred turbidimetry are for detecting light transmittance, and the light scattering method is for detecting generated particles, and they are different in this point, but they are each based on a threshold value method for measuring a time required for the number of particles found from the transmitted light intensity or the scattered light intensity or the number of peaks of the mixed liquid to exceed a threshold value.

In addition, there is also a method of measuring a phenomenon in which a synthetic substrate for a clotting enzyme added to a reagent is added in advance and the synthetic substrate decomposed by the clotting enzyme develops color, or emits fluorescence or light, and a method utilizing color development is widely used as one of important measurement methods in a colorimetric method and a method for quantifying a predetermined physiologically active substance.

The following phenomena were observed in the above measurement methods: in the turbidimetric method and the stirred turbidimetric method, the transmitted light intensity decreases 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 limulus reaction); in the light scattering method, the scattered light intensity or the number of peaks increases (hereinafter, this phenomenon is also referred to as gradual decrease/increase). This gradual decrease/increase has an effect on the time required for the number of particles determined from the intensity of transmitted light, the intensity of scattered light, or the number of peaks to exceed the threshold value in the above-described measurement method, and therefore, the measurement accuracy of the above-described measurement method may be degraded. In the above-described measurement method, the lower the concentration of the predetermined physiologically active substance, the longer the measurement time required for the number of particles obtained from the intensity of transmitted light, or the intensity or the number of peaks of scattered light to exceed the threshold value, and therefore the lower the concentration of the predetermined physiologically active substance, the more likely the concentration of the predetermined physiologically active substance is affected by gradual decrease/increase, and the reaction start time at which gelation or aggregation starts may not be accurately evaluated.

As described above, the means for determining gelation or color development is to use a threshold method or a differential method, and the threshold method is a time point at which a physical quantity that changes due to gelation or color development reaches a predetermined threshold value or more or exceeds the threshold value (hereinafter, simply referred to as a time point at which the physical quantity passes the threshold value) as a reaction start time; the differential method is based on the magnitude of the change in light transmittance or absorbance over a given period of time. When the threshold method is used, it is known that the relationship between the amount of a given physiologically active substance in a sample and the reaction initiation time is a straight line relationship with a negative slope under the log double. In addition, a time-varying curve of a physical quantity such as light transmittance or absorbance that varies due to gelation or color development can be approximated to a logistic curve. Therefore, a very slow change is observed when reacting with a low concentration of a predetermined physiologically active substance, and a rapid change is observed when reacting with a high concentration of a predetermined physiologically active substance. Therefore, when the same threshold value is used to determine the reaction start time in any reaction, the threshold value method has an inconvenience in that the measurement time for a low concentration sample is prolonged.

On the other hand, in the differential method for determining the amount of change in light transmittance or absorbance, although the relationship between the amount of change and the concentration of a predetermined physiologically active substance to be acted on is a straight line relationship, the straight line relationship is limited to a narrow concentration range, and it is impossible to measure a high concentration and a low concentration at the same time.

To solve these problems, an area method using the area of the time curve of light transmittance or absorbance is proposed. In the area method, an area value at each time is recorded, and a time point at which the value becomes equal to or greater than a predetermined threshold value or exceeds the threshold value is set as a reaction start time (detection time) of a predetermined physiologically active substance. However, as described above, practically, the "gradual decrease/increase" in which the light transmittance or absorbance changes at a constant rate can be observed regardless of the LAL reaction. FIG. 21 shows an example of the change of light transmittance with time due to endotoxin reaction. It was found that the light transmittance decreased linearly by a gradual decrease phenomenon from the start of the measurement to about 18 minutes. In this case, the area value linearly increases in the area method regardless of the LAL reaction, and thus a predetermined physiologically active substance may not be accurately measured.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2004-061314

Patent document 2: japanese laid-open patent publication No. 10-293129

Patent document 3: international publication WO2008/038329 pamphlet

Patent document 4: japanese patent laid-open No. 2009-150723.

Disclosure of Invention

The present invention is made in view of the above problems, and has an object to: a measurement method and a measurement device using the measurement method are provided, which can improve the measurement accuracy in the detection or concentration measurement of a biologically-derived physiologically active substance.

The most important feature of the present invention is that, in order to eliminate the effect of gradual decrease/increase in the measurement of the predetermined physiologically active substance, a difference value of a physical quantity that changes due to the reaction between the measurement sample and LAL in a mixture solution of the measurement sample and LAL is continuously obtained, and a time point at which the difference value becomes equal to or greater than a threshold value or exceeds the threshold value is set as a reaction start time point.

More specifically, it is characterized in that: mixing a sample containing a predetermined biologically-derived physiologically active substance with a limulus blood cell extract LAL, and continuously acquiring a predetermined physical amount, which changes due to a reaction between LAL and the physiologically active substance, as a detection value; setting one acquisition time as a reaction start time when a difference between a detection value at the one acquisition time and a detection value at an acquisition time earlier than the one acquisition time by a predetermined time interval or an absolute value of the difference is equal to or greater than a threshold value or exceeds the threshold value; detecting the physiologically active substance in the sample or measuring the concentration thereof based on the reaction initiation time.

In the present invention, in order to eliminate the influence of gradual decrease/increase in the measurement of the predetermined physiologically active substance, a change amount (difference) per predetermined time of the transmittance or the number of gel particles may be obtained from the intensity of transmitted light or scattered light or the number of peaks of the mixed solution of the measurement sample and LAL, and a time when the change amount (difference) exceeds a threshold value may be set as the reaction start time.

In this case, more specifically, the method for measuring a physiologically active substance derived from a living body comprises reacting a physiologically active substance derived from a living body present in a sample with limulus polyphemus blood cell extract LAL to detect the physiologically active substance in the sample or measure the concentration of the physiologically active substance, and is characterized in that: after the sample and the LAL are mixed, incident light is incident on a mixed solution of the sample and the LAL, and the intensity of light transmitted through the mixed solution or light scattered by the mixed solution among the incident light is acquired; a predetermined physical quantity at an acquisition time set at predetermined time intervals, which is obtained from the acquired light intensity, is used as a detection value; setting a time when a difference between a detection value at one acquisition time and a detection value at a previous acquisition time or an absolute value of the difference exceeds a threshold as a reaction start time; detecting the physiologically active substance in the sample or measuring the concentration thereof based on the reaction initiation time.

Here, it is understood that when the gradual decrease/increase occurs, the intensity of transmitted light or the intensity or the number of peaks of scattered light changes substantially linearly with time regardless of the concentration of the predetermined physiologically active substance. In contrast, in the present invention, the intensity of transmitted light or scattered light from the mixed liquid is obtained. Then, the intensity of light acquired at the acquisition time set at predetermined time intervals or a value obtained by processing the intensity of light acquired at the acquisition time according to the purpose is set as a detection value, and the time when the difference between the detection value at one acquisition time and the detection value at the acquisition time immediately before the one acquisition time (that is, the amount of change in the detection value in a fixed time period) or the absolute value of the difference exceeds a threshold value is set as the reaction start time. Thereby, the influence of gradual decrease/increase on the measurement of a prescribed physiologically active substance can be eliminated.

Therefore, according to the present invention, the reaction start time of the predetermined physiologically active substance in the sample with LAL can be determined with higher accuracy. As a result, the detection of the predetermined physiologically active substance or the measurement of the concentration can be performed with higher accuracy.

In the present invention, the acquired light intensity may be the intensity of light transmitted through the mixed solution, the detection value may be the transmittance of the mixed solution in percentage, and the predetermined time interval may be about 2 minutes, and the reaction start time may be a time at which the absolute value of the difference between the transmittance at the one acquisition time and the transmittance at the one acquisition time exceeds 1.

This case relates to a turbidimetric method in which, in light incident on a mixed solution of a predetermined physiologically active substance and LAL, transmitted light of the mixed solution is obtained, and transmittance is obtained. As described above, in the turbidimetric method, the reaction between the physiologically active substance and LAL is regulated to make the mixed solution turbid, and the transmittance of the mixed solution decreases with time. Here, the shape of the transmittance decrease curve in the turbidimetric method differs 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 sharply decreases. On the other hand, when the concentration of the predetermined physiologically active substance is low, the transmittance gradually decreases.

When the concentration of the predetermined physiologically active substance is high and the transmittance is rapidly decreased, if the acquisition interval of the detection values is set to be long, the range of each decrease of the detection values becomes too large, and sufficient data cannot be secured, so that it is difficult to perform high-precision measurement. On the other hand, if the acquisition interval of the detection values is set to be too short when the concentration of the predetermined physiologically active substance is low and the transmittance is gradually decreased, the decrease width of each detection value is too small, and the difference cannot be detected with sufficient accuracy. As described above, in the present invention, it is preferable to adjust the time interval at which the detection value is obtained according to the concentration of the predetermined physiologically active substance in the sample to be measured.

In contrast, as a result of intensive studies by the present inventors, it was found that when the transmittance is expressed as a percentage and the acquisition interval of the detected value (transmittance) is set to about 2 minutes, the decrease curve of the transmittance can be obtained with high accuracy in a wider range of the concentration of the predetermined physiologically active substance in the present invention. In this case, it is found that the concentration of the predetermined physiologically active substance can be measured with high accuracy by setting the time when the decrease in transmittance within 2 minutes exceeds 1% as the reaction start time.

Therefore, in the present invention, when the transmitted light from the mixed solution of the predetermined physiologically active substance and LAL is acquired, the transmittance is calculated, and the difference (change amount) in transmittance at predetermined time intervals is acquired, the predetermined time intervals are set to about 2 minutes, and the reaction start time is set to a time at which the decrease in transmittance within 2 minutes exceeds 1%. This makes it possible to detect or measure the concentration of a predetermined physiologically active substance with higher accuracy in a wider range of samples.

In the present invention, the intensity of the acquired light is the intensity of light scattered by the mixed solution, the detection value is the number of particles scattering light entering the mixed solution, which is derived from a predetermined number of peaks in the intensity of the scattered light, the predetermined time interval is about 100 seconds, and the reaction start time is a time at which the difference between the number of particles at the one acquisition time and the number of particles at the previous acquisition time exceeds 200.

This case relates to a light scattering method in which scattered light from a mixed solution of a predetermined physiologically active substance and LAL is obtained from light incident on the mixed solution, and the number of (gel) particles in the mixed solution is obtained from the number of peaks in the scattered light (satisfying predetermined conditions for improving measurement accuracy). In the light scattering method as described above, when the mixed solution is stirred when the predetermined physiologically active substance reacts with LAL, gel particles are generated in the mixed solution, and the size and number of the gel particles increase as the reaction proceeds. Therefore, the number of peaks observed in scattered light from the mixed solution increases as the reaction proceeds.

The curve shape of the increase in the number of peaks of light scattering in the light scattering method varies depending on the concentration of a predetermined physiologically active substance. For example, when the concentration of the predetermined physiologically active substance is high, the number of peaks increases sharply, 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 preferable that the light scattering method adjusts the time interval for acquiring the detection value in accordance with the concentration of the predetermined physiologically active substance in the sample to be measured.

In contrast, as a result of intensive studies by the present inventors, it was found that, in the light scattering method, when the acquisition interval of the detected value (the number of scattering particles) is about 100 seconds, an increase curve of the number of peaks can be obtained with higher accuracy in a wider range of concentration of the predetermined physiologically active substance. In this case, the time at which the increase in the number of scattering particles exceeds 200 in 100 seconds is used as the reaction start time, whereby the concentration of the predetermined physiologically active substance can be measured with high accuracy.

Therefore, in the present invention, when scattered light from a mixed solution of a predetermined physiologically active substance and LAL is acquired, the number of scattered particles is detected, and a difference (amount of change) in the number of scattered particles at predetermined time intervals is acquired, the predetermined time intervals are set to about 100 seconds, and a time at which the increase in the number of scattered particles in 100 seconds exceeds 200 is set as a reaction start time. This makes it possible to detect a predetermined physiologically active substance or measure the concentration of the substance with higher accuracy and in a wider range of samples.

As described above, the present invention first proposes a difference method described later, in which a difference value between the light transmittance and the number of light scattering particles at 2 points separated by a predetermined time interval is recorded at each time, and a time point at which the difference value passes a predetermined threshold value is regarded as a detection time. The differential method is different from the differential method in that the amount of change itself is not related to the concentration of the predetermined physiologically active substance, but the time required to pass through the threshold value is related to the concentration of the predetermined physiologically active substance, and therefore, the problem of the narrow measurable concentration range seen in the differential method can be solved. Further, even with respect to a change in linear light transmittance or absorbance that occurs regardless of the LAL reaction, the change is easily eliminated and the measurement accuracy can be improved by obtaining a difference value and making the change constant.

However, in the differential method, when a predetermined physiologically active substance having a low concentration whose change curve gradually changes is measured, it is difficult to obtain a large differential value necessary for measurement, and measurement becomes difficult. Therefore, there is a strong demand for establishing a highly accurate measurement method that can measure a predetermined physiologically active substance in a wide concentration range and is not affected by changes in light transmittance, absorbance, and the like that are unrelated to the LAL reaction.

Therefore, in the present invention, in order to provide a technique for obtaining higher measurement accuracy in a measurement method of a biologically-derived physiologically active substance, a method can be employed which is based on the time at which a physical quantity that changes by the reaction between a limulus blood cell extract LAL and the biologically-derived physiologically active substance passes through a threshold.

The present invention in this case relates to a differential method in which a difference value of a physical quantity that changes due to a reaction between a sample containing a predetermined physiologically active substance and LAL at two times separated by a predetermined time interval is continuously obtained, and a time when the difference value passes a predetermined threshold value is determined as a reaction start time. The biggest characteristics of the invention are as follows: the time interval between the two times is not constant, and can be changed according to the time, so that the actual reaction start time can be obtained even in the measurement of the low concentration sample.

More specifically, the method for measuring a biologically-derived physiologically active substance is carried out as follows: mixing a sample containing a predetermined biologically-derived physiologically active substance with the limulus blood cell extract LAL, and continuously obtaining a predetermined physical amount that changes as a result of the reaction between the LAL and the physiologically active substance as a detection value; setting one acquisition time as a reaction start time when a difference between a detection value at the one acquisition time and a detection value at an acquisition time earlier than the one acquisition time by a predetermined time interval or an absolute value of the difference is equal to or greater than a threshold value or exceeds the threshold value; detecting the physiologically active substance in the sample or measuring the concentration thereof based on the reaction initiation time; the method is characterized in that: the predetermined time interval is changed according to the one acquisition time.

That is, in the measurement of the predetermined physiologically active substance, when a gradual increase/decrease that changes without relation to the reaction between LAL and the predetermined physiologically active substance is observed, it is considered that the influence of the gradual increase/decrease can be removed by acquiring a difference in detection values generated at constant time intervals, and the measurement accuracy of the predetermined physiologically active substance can be improved. However, particularly in the measurement of a predetermined physiologically active substance at a low concentration, the amount of change in the detected value itself is small, and if the time interval for obtaining the difference is narrow, a sufficient difference value cannot be obtained, and as a result, it may be difficult to measure the predetermined physiologically active substance.

In contrast, in the present invention, the time interval for acquiring the difference may be changed according to the acquisition timing of the detection value. That is, when the difference value is extremely small, which has been difficult to measure until now, the time interval for acquiring the difference is set to be long, so that the difference can pass through the threshold value at least at the actual time. Thus, the measurement of the predetermined physiologically active substance can be performed with high accuracy regardless of whether the concentration of the predetermined physiologically active substance in the measurement sample is high or low.

The physical quantity may be optical intensity such as light transmittance, absorbance, scattered light intensity, light scattering particle number, fluorescence intensity, or chemiluminescence intensity, or electrical engineering intensity such as viscosity or conductivity of the sample.

In the present invention, the intensity of light transmitted through the mixed liquid or light scattered by the mixed liquid among the incident light is continuously detected simultaneously with the incident light on the mixed liquid of the LAL and the sample, and any one of the light transmittance, the absorbance, the scattered light intensity, the number of light-scattering particles, the fluorescence intensity, and the chemiluminescence intensity obtained from the continuously detected intensity of the light may be used as the detection value.

Thus, the physical quantity that changes due to the reaction between the LAL and the physiologically active substance after mixing the LAL with the sample can be continuously obtained by a non-contact method, and the measurement of the predetermined physiologically active substance can be performed more easily and with high accuracy.

In the present invention, the predetermined time interval may be further extended so as to further reduce the one acquisition time.

That is, the time interval for obtaining the difference is defined as a function of the elapsed time from the start of the measurement. Thus, when the concentration of the predetermined physiologically active substance is low, the difference between the detection values obtained at the two acquisition times is small, and the difference value does not pass the threshold value at a later time, the difference value can be relatively increased by extending the time interval. As a result, even when the concentration of the predetermined physiologically active substance is low, the difference value can easily pass through the threshold value, and the measurement start time can be obtained within the actual measurement time.

In the present invention, a plurality of series of acquisition times at which the predetermined time interval is set to a constant value may be provided, and the predetermined time interval may be different from each other among the plurality of series, and the series to be used may be switched according to the one acquisition time.

Here, for example, a series of 1 minute, a series of 6 minutes, and a series of 30 minutes at a predetermined time interval may be set. Then, the series of used acquisition times may be switched according to the acquisition time of the physical quantity. For example, when the concentration of the predetermined physiologically active substance is low, the difference between the detection values obtained at the two acquisition times is small, and the difference value does not pass the threshold value at a later time, a series having a long time interval may be used. In this way, the difference value can be relatively increased. As a result, even when the concentration of the predetermined physiologically active substance is low, the difference value can easily pass through the threshold value, and the measurement start time can be obtained within the actual measurement time.

In the present invention, the series used may be a series in which the difference between the detected value at the one acquisition time and the detected value at the acquisition time that is earlier than the one acquisition time by a predetermined time interval or the absolute value of the difference is the largest.

That is, at each time of acquiring the physical quantity, the series having the largest difference value of the detected values is selected from the plurality of series, and the difference value of the series is compared with the threshold value. In this way, the maximum difference value can always be compared with the threshold value at each acquisition time. Therefore, the time required for the difference value to pass through the threshold value can be shortened as much as possible. As a result, the prescribed physiologically active substance can be measured more efficiently, while the problem that the measurement cannot be performed because the difference value does not pass the threshold value can be eliminated.

In the present invention, the acquisition time is changed, the difference or the absolute value of the difference between the detection value of the one acquisition time and the detection value of the acquisition time that is earlier than the one acquisition time by the predetermined time interval is acquired, the value at a predetermined level (rank) when the values are arranged in order of magnitude is used as the reference difference value, and when the value obtained by subtracting the reference difference value from the absolute value of the difference or the difference is equal to or more than the threshold value or exceeds the threshold value, the one acquisition time may be used as the reaction start time.

Here, when the gradual decrease/increase occurs in the detected values of the physical quantity after the start of measurement, it is considered that the influence of the gradual decrease/increase on the measurement is reduced by subtracting the difference or the absolute value of the difference obtained in the initial period after the start of measurement from the difference or the absolute value of the difference between the detected value at the one acquisition time and the detected value at the acquisition time that is earlier than the one acquisition time by the predetermined time interval.

However, when the detection value, or the difference between the detection value and the detection value or the absolute value of the difference is small, it is difficult to obtain the value itself to be subtracted with high accuracy, and it is sometimes difficult to eliminate the influence of gradual decrease/increase with high accuracy. In the present invention, the acquisition time is changed, the difference or the absolute value of the difference between the detection value at a plurality of one acquisition time and the detection value at an acquisition time that is earlier than the one acquisition time by a predetermined time interval is acquired, the value at a predetermined level when the values are arranged in order of magnitude is used as the reference difference value, the reference difference value is subtracted from the difference or the absolute value of the difference, and the acquired value is compared with the threshold value.

For example, the difference or the absolute value of the difference obtained in the past may be arranged in order of magnitude, and the 3 rd smallest value among the last 5 data may be used as the reference difference value. Thus, even if the detected value, or the difference between the detected value and the detected value, or the absolute value of the difference is small, the reliability of the value to be subtracted can be improved, and the influence of gradual decrease/increase on the measurement can be reduced more accurately.

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

Thus, endotoxin, which is the most representative pyrogen, can be detected or measured more accurately, and adverse side effects caused by infusion, injection of drugs, blood, and the like contaminated with endotoxin into the human body can be suppressed. Also, detection or concentration measurement of β -D-glucan can be more accurately performed, and screening of fungal infection can be more accurately performed not only in fungi commonly found in clinical use such as Candida, Aspergillus (Aspergillus), and Cryptococcus, but also in a wide range including rare fungi.

Further, the present invention may be a measurement device for a physiologically active substance derived from a living organism, the measurement device being provided with:

a mixed solution holding means for allowing a reaction in a mixed solution of a sample containing a predetermined biologically-derived physiologically active substance and limulus blood cell extract LAL to proceed while allowing incident light to be held;

a stirring device for stirring the mixed solution in the mixed solution holding device;

a light incidence device for incidence light to the mixed liquid in the mixed liquid holding device;

a light receiving device that receives the transmitted light or the scattered light of the incident light from the mixed liquid and converts the received light into an electrical signal;

a determination device for determining a reaction start time of the physiologically active substance in the sample with the LAL from the electric signal converted by the light receiving device;

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 device is characterized in that: the determination device determines, as the reaction start time, a time at which a difference between a detection signal value at one acquisition time and a detection signal value at a previous acquisition time or an absolute value of the difference exceeds a threshold value, by using a signal obtained by applying a predetermined operation to the electrical signal or the electrical signal as a detection signal value, at acquisition times set at predetermined time intervals.

According to the measurement device for a predetermined physiologically active substance of the present invention, the light receiving means receives transmitted light or scattered light from a mixed solution of the predetermined physiologically active substance and LAL, and converts the received light or scattered light into an electric signal. Then, in the judging device, the reaction start timing in the mixed liquid is judged from the converted electric signal. The determination device acquires detection signal values at predetermined time intervals from the obtained electric signal, and determines a time when a change amount of the detection signal values at the predetermined time intervals exceeds a threshold value as a reaction start time.

According to the measuring device of the present invention, the influence of gradual decrease/increase on the measurement of a prescribed physiologically active substance can be automatically eliminated. Therefore, the detection or concentration measurement of the predetermined physiologically active substance can be performed with higher accuracy.

In this case, when the detection signal value is the transmittance of the liquid mixture expressed as a percentage, the predetermined time interval may be about 2 minutes and the threshold value may be 1. When the detection signal value is the number of particles that scatter light incident on the liquid mixture, the predetermined time interval may be about 100 seconds and the threshold may be 200 seconds. Thus, a higher measurement accuracy can be obtained for a wider range of concentrations of the predetermined physiologically active substance.

In the device for measuring a predetermined physiologically active substance according to the present invention, the predetermined time interval and/or the threshold value may be variable. In this way, the acquisition time interval of the detection signal value and the threshold value for determining the reaction start time can be optimized according to the desired concentration of the predetermined physiologically active substance, and higher measurement accuracy can be obtained for the desired concentration.

The present invention may also be a device for measuring a biologically-derived physiologically active substance, comprising:

the determination device determines, as the reaction start time, a time at which a difference between a detection signal value at one acquisition time and a detection signal value at a previous acquisition time or an absolute value of the difference is equal to or greater than a threshold value or exceeds the threshold value, using a signal obtained by applying a predetermined operation to the electrical signal or the electrical signal as the detection signal value, at acquisition times set at predetermined time intervals. The device is characterized in that: the determination device may change the predetermined time interval based on the one acquisition time.

In this case, the determination device may extend the predetermined time interval to a time after the one acquisition time.

The determination device may be provided with a plurality of series of acquisition times at which the predetermined time interval is set to a constant value, the predetermined time intervals of the plurality of series being different from each other, and the series to be used may be switched according to the one acquisition time.

The series used may be a series in which the difference or absolute value of the difference between the detected value at the one acquisition time and the detected value at the acquisition time that is earlier than the one acquisition time by a predetermined time interval is the largest.

Further, the acquisition time is changed, the difference or the absolute value of the difference between the detection value of the one acquisition time and the detection value of the acquisition time that is earlier than the one acquisition time by the predetermined time interval is acquired, the value of a predetermined level when the values are arranged in order of magnitude is used as a reference difference value, the reference difference value is subtracted from the absolute value of the difference or the difference, and the time when the acquired value is equal to or more than the threshold value or exceeds the threshold value is determined as the reaction start time.

The physiologically active substance derived from a living organism may be endotoxin or β -D-glucan.

The present invention may also be a program for carrying out the above-described method for measuring a biologically-derived physiologically active substance.

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

In the present invention, higher measurement accuracy can be obtained when the physiologically active substance is detected or its concentration is measured by utilizing the reaction between the physiologically active substance derived from a living body such as endotoxin and β -D-glucan and LAL. In addition, a higher measurement accuracy can be obtained in a method for measuring a physiologically active substance derived from a living body, which is based on the time at which a physical quantity that changes due to a reaction between the physiologically active substance derived from a living body and LAL passes through a threshold value.

Drawings

Fig. 1 is a schematic configuration diagram showing a turbidimetric measurement apparatus in example 1 of the present invention.

Fig. 2 is a graph showing a temporal change in light transmittance for explaining the gradual decrease in embodiment 1 of the present invention.

FIG. 3 is a graph showing the relationship between endotoxin concentration and endotoxin detection time obtained by a conventional thresholding method.

FIG. 4 is a graph showing the relationship between endotoxin concentration and endotoxin detection time obtained by the differential method in example 1 of the present invention.

Fig. 5 is a view showing a schematic configuration of a light scattering particle measuring apparatus in example 2 of the present invention.

Fig. 6 is a graph showing a temporal change in the number of detected particles for explaining the gradual increase in embodiment 2 of the present invention.

FIG. 7 is a graph showing the relationship between endotoxin concentration and endotoxin detection time obtained by the thresholding method and the difference method in example 2 of the present invention.

FIG. 8 is a graph showing that the change with time in absorbance obtained by a normal differential method differs depending on the endotoxin concentration.

FIG. 9 is a graph showing that the change with time of the absorbance difference value obtained by a normal difference method differs depending on the endotoxin concentration.

FIG. 10 is a graph showing that the change with time of the absorbance difference value obtained by the time-function difference method in example 4 of the present invention is different depending on the endotoxin concentration.

FIG. 11 is a flowchart of a measuring procedure for endotoxin measurement by the time-function difference method according to example 4 of the present invention.

Fig. 12 is a flowchart of a subroutine for detection determination in the measurement routine pertaining to embodiment 4 of the present invention.

FIG. 13 is a graph showing that the change with time of the absorbance difference value obtained by the multi-series difference method in example 5 of the present invention is different depending on the endotoxin concentration.

FIG. 14 is a flowchart of the endotoxin measurement procedure 2 of example 5 of the present invention by the multi-series differential method.

FIG. 15 is a graph showing a linear comparison of calibration curves for endotoxin assays by stirred turbidimetry in various differential methods.

FIG. 16 is a graph showing the linearity of a calibration curve for endotoxin measurement by colorimetry in the multi-series differential method in example 7 of the present invention.

FIG. 17 is a graph showing the linearity of a calibration curve of a beta-D-glucan measurement by a stirred turbidimetric method in the multi-series differential method in example 8 of the present invention.

FIG. 18 is a graph showing the linearity of a calibration curve of endotoxin measurement using the multi-series difference method in endotoxin measurement by the LAL bead method in example 9 of the present invention.

FIG. 19 is a graph showing the linearity of a calibration curve for endotoxin assay when dynamic update is performed on a value subtracted from a difference value at each time point as a background value in a case where gradual decrease/increase is observed by a multi-series difference method in example 10 of the present invention.

Fig. 20 is a flowchart showing a procedure of a reference difference value operator according to embodiment 10 of the present invention.

FIG. 21 is a graph showing an example of the change with time of light transmittance due to endotoxin reaction when gradually decreasing/increasing was observed.

FIG. 22 is a schematic diagram illustrating the gelation process of LAL caused by endotoxin or β -D-glucan and a method for detecting the same.

Detailed description of the preferred embodiments

The reaction of LAL with endotoxin to form a gel was carefully studied. That is, as shown in FIG. 22, endotoxin binds to the serine protease, factor C, in LAL, and the factor C is activated to form active factor C, which hydrolyzes and activates the other serine protease, factor B, in LAL to form active factor B. The activated factor B immediately hydrolyzes a coagulase precursor in LAL to form a coagulase, and the coagulase hydrolyzes coagulogen in LAL to produce coagulin. Then, the produced coagulans associate with each other to further produce an insoluble gel, and LAL is all involved therein to form a gel.

Similarly, β -D-glucan binds to factor G in LAL, and factor G is activated to form active factor G, which hydrolyzes the coagulase precursor in LAL to form coagulase. As a result, coagulin was produced in the same manner as in the reaction of endotoxin with LAL, and the produced coagulin were associated with each other to produce an insoluble gel.

This series of reactions is analogous to the process of fibrin gel formation mediated by serine proteases such as Christmas factor or thrombin found in mammals. The enzyme cascade reaction is as follows: even a very small amount of the activating factor can be activated by cascade after it occurs, and thus has a very strong amplification effect. Therefore, according to the measurement method of a predetermined physiologically active substance using LAL, an extremely small amount of the predetermined physiologically active substance on the order of subpicogram/mL can be detected.

As reagents for quantifying endotoxin and β -D-glucan, limulus reagents using a limulus blood cell extract (LAL: limulus amebocyte lysate) as a raw material and reagents in which a synthetic substrate that is hydrolyzed by a clotting enzyme and increases in either color intensity, fluorescence intensity or chemiluminescence intensity is added to the limulus reagents are used. In addition, a reagent mixture of a recombinant of factor C (recombinant factor C) in limulus reagent and a synthetic substrate thereof (whether by means of coloration, fluorescence, chemiluminescence, or the like) may be used. Further, a reagent mixture of a recombinant of factor G (recombinant factor G) in the limulus reagent and a synthetic substrate thereof (whether by means of coloration, fluorescence, chemiluminescence, or the like) may be used.

Various physical quantities are considered for quantifying endotoxin and β -D-glucan. The type of the reagent and the type of the measuring apparatus can be selected according to the physical quantity. The physical quantity may be, for example, a change in an optical physical quantity such as light transmittance or turbidity, scattered light intensity, the number of light-scattering particles, absorbance, fluorescence intensity, or chemiluminescence intensity of the sample, or a change in a physical quantity such as viscosity or conductivity of the sample due to gelation of the sample. For the detection of these physical quantities, optical instruments such as a turbidimeter, an absorption photometer, a light scattering photometer, a laser scattering particle measuring instrument, a fluorometer, and a photon counter, and dedicated measuring devices using these instruments can be used. In addition, a viscometer, a conductivity meter, and a dedicated measuring device using them can be used.

As described above, the measurement method capable of quantifying a predetermined physiologically active substance such as endotoxin and β -D-glucan includes various methods such as the aforementioned turbidimetric method, stirred turbidimetric method and light scattering method. As shown in FIG. 22, these measurement methods can be carried out by measuring, with high sensitivity, the association of coagulans formed by the enzyme cascade reaction of LAL, which is detected by the turbidity of the sample, and which is detected by the fine particles of the gel formed in the system.

The turbidimetric method is evaluated to be easy to use on site in that no special reagent is required and the concentration range of a predetermined physiologically active substance that can be measured is wide. On the other hand, 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 observe the amount of the formation of coagulin itself, which is the final product of the protease cascade, but the process of the decrease in light transmittance due to the gel formed by further association thereof.

That is, since gelation does not occur if the concentration of the coagulin is not higher than a certain level, the turbidimetry must wait until the formation of a gel in order to detect a predetermined physiologically active substance. Therefore, when the predetermined physiologically active substance concentration is high, the necessary and sufficient coagulin concentration is rapidly produced and gelation starts, and thus the measurement time becomes short, but when the predetermined physiologically active substance concentration is low, it takes time to reach the coagulin concentration necessary for gelation, and the measurement time becomes long. In this regard, in the stirred turbidimetric method, a mixture solution of a predetermined physiologically active substance and LAL is stirred to promote the reaction between both substances, thereby shortening the measurement time.

In addition, the light scattering method is improved over the turbidimetric method in terms of stirring the sample and detecting particles by laser light instead of gel, and the measurement time can be significantly shortened compared to the turbidimetric method. In the turbidimetry, the stirred turbidimetry, and the light scattering, although physical quantities observed are different, a point in time exceeding a certain threshold is captured as a starting point of the reaction, and these are common at this point (this method is conveniently referred to as a threshold method).

In any of the above-mentioned measurement methods, a decrease in the light transmittance of the mixed solution is observed in the turbidimetric method and the stirred turbidimetric method immediately after the start of the measurement, and an increase in the number of gel particles in the mixed solution, i.e., a so-called gradual decrease/increase phenomenon, is observed in the light scattering method, regardless of the state of the limulus reaction. The reason for this is not clear, and for example, one of the reasons for denaturation of protein, which is caused by a change in the pH of the mixed solution due to the dissolution of carbon dioxide gas or the like in the mixed solution, is considered.

In the turbidimetric stirring method and the light scattering method, the liquid mixture is stirred by a stirrer incorporated in the measurement cell, and the stirring makes the limulus reaction uniform, thereby promoting the reaction. In this stirring, when the rotation axis of the stirring is not appropriate or the bottom surface shape of the measuring vessel is not appropriate, the inclination of gradual decrease/increase tends to occur, and the reason for this is under intense investigation.

When the concentration of the predetermined physiologically active substance in the measurement sample is high, the gelation of the liquid mixture proceeds and the agglutination determination is completed before being affected by the gradual decrease/increase, and therefore the risk of lowering the measurement accuracy due to the gradual decrease/increase is relatively small. However, when the concentration of the predetermined physiologically active substance in the measurement sample is low, since it takes a long time to measure the concentration, the change curve of the light transmittance or the number of gel particles exceeds the threshold value earlier than the actual value due to the influence of gradual decrease/increase, and the accuracy of determination at the time of reaction initiation may be lowered.

In the present embodiment, in the detection or concentration measurement of a predetermined physiologically active substance, a method of determining the reaction start time based on the light transmittance of the liquid mixture of the sample and the LAL or the number of gel particles itself exceeding a threshold value in preparation for the occurrence of the gradual decrease/increase phenomenon is not employed. But the following new method is adopted: the light transmittance or the number of gel particles is acquired at constant time intervals, and the time at which the amount of change in the light transmittance or the number of gel particles in the time intervals exceeds a threshold value is determined as the reaction start time. This threshold value is of course different from the case where the turbidity or the number of gel particles itself is compared as a threshold value. Thus, even if the gradual decrease/increase occurs, the influence thereof can be eliminated, and the determination of the reaction start time of the predetermined physiologically active substance and the LAL can be performed with higher accuracy. (this method is conveniently referred to as a differential method)

Further, it is possible to find the time when the turbidity of the liquid mixture or the amount of change in the number of gel particles abruptly changes. Therefore, the measurement accuracy can be improved even for a high concentration sample and for a low concentration sample. In addition, the influence of the gradual decrease/increase can be eliminated by changing only the analysis process of the data obtained when the predetermined physiologically active substance is measured.

The embodiments of the present invention are given in detail below, but the present invention is not limited to the embodiments shown below. In the following embodiments, the case where the physiologically active substance is endotoxin will be described, and the same case can be considered when the physiologically active substance is β -D-glucan.

Here, the detection target differs depending on the endotoxin measurement performed by the above-mentioned measurement methods. In the turbidimetric method and the stirred turbidimetric method, endotoxin reacts with the LAL reagent to detect the turbidity of the mixed solution when the mixed solution is gelled or aggregated. Therefore, the transmitted light of the mixed solution is obtained, and the time when the light transmittance of the mixed solution is lower than a predetermined threshold value is determined as the reaction start time of endotoxin and LAL.

In the turbidimetric method and the stirred turbidimetric method, when a tendency of decrease in the light transmittance of the liquid mixture is observed regardless of the state of the limulus reaction, first, a moving average of 2 minutes is taken to eliminate noise of the detection value. Next, since the data immediately after the start of the measurement were unstable, the light transmittance 2 minutes after the start of the measurement was assumed to be 100%. Then, the difference in light transmittance (%) originally obtained every 10 seconds at a predetermined time interval Δ T was obtained, and the first time when the square value of the difference continuously exceeded 13 times was used as the reaction start time. The time interval Δ T of the difference is preferably from 1 minute to 5 minutes, particularly preferably about 2 minutes. In the case of a low concentration sample, the amount of change in light transmittance is small, and therefore, it is preferable to set the time interval Δ T to 5 minutes. Here, the square of the difference in the time interval Δ T of the light transmittance (%) is because it can increase the change when the difference value is about 1 to improve the resolution. It is not necessary to square the difference.

On the other hand, the light scattering method is different in that light scattered by aggregates (gel particles) in the mixed solution is obtained, although the process of gelation or aggregation of the mixed solution is observed in the same manner as the turbidimetric method and the stirred turbidimetric method. That is, the limulus reagent is mixed with the sample which is initially transparent and does not contain the gel particles, whereby 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 in the scattered light can be obtained as the number of gel particles. Then, a threshold value is set for an integrated value of the number of gel particles detected within a certain period of time, and a time when the number of detected particles exceeds the threshold value is set as a reaction start time.

When the number of gel particles detected by the visible light scattering method tends to increase regardless of the state of the limulus reaction, the number of particles increased, which is a difference in the detection value (the number of gel particles) at a constant time interval Δ T, is obtained. Regarding the determination of the reaction start time of the limulus reaction, the initial time at which the difference in the number of gel particles continuously exceeds the threshold 200 10 times is adopted as the reaction start time in consideration of the influence of noise. In order that the differential time interval Δ T may respond to a sample having a high endotoxin concentration, it is preferably 30 seconds to 200 seconds, particularly preferably about 100 seconds.

In the above measurement, when the endotoxin concentration is low, the value of the time interval Δ T for obtaining the detection value needs to be set relatively large, and when the endotoxin concentration is high, the value needs to be set relatively small. This is because the lower the endotoxin concentration is, the less the amount of change in turbidity and the number of gel particles after mixing the sample with the LAL reagent is, and therefore, a sufficient time interval is necessary to ensure a sufficiently large difference in the detection values before and after the time interval Δ T.

On the other hand, if the endotoxin concentration is high, the limulus reaction between endotoxin and LAL proceeds relatively rapidly, and therefore it is considered that the aggregation starts during the set time interval Δ T. Therefore, by sequentially detecting changes in the detected values of the turbidity or the number of gel particles of the liquid mixture, roughly estimating early whether the endotoxin concentration is low or high, and changing the time interval Δ T depending on the roughly estimated value, the endotoxin concentration can be measured with higher accuracy. Therefore, the above-described rough estimation algorithm can be adopted as needed.

< production example 1>

A stirrer (phi 1 mm, length 5 mm) made of stainless steel was placed in a glass vessel (phi 7 mm in outer diameter, length 50 mm, hereinafter referred to simply as cuvette). The open part of the cuvette was covered with an aluminum foil, and 20 cuvettes were further covered with an aluminum foil together, and the cuvette was subjected to a dry heat treatment by heating at 250 ℃ for 3 hours. Thus, endotoxin adhering to the cuvette is decomposed by heat and inactivated.

[ example 1]

FIG. 1 shows a schematic configuration of a turbidimetric apparatus 1 as an endotoxin measuring apparatus of the present example. In the turbidimetric apparatus 1 of the present example, endotoxin was measured by a stirred turbidimetric method. In this example, the sample containing the prepared dilution series of endotoxins was transferred to the cuvette 2 as a mixed liquid holding device manufactured in manufacturing example 1. The heat retainer 5 is provided so as to surround the periphery of the well 2. The incubator 5 is provided with a not-shown electric heating wire inside, and the cuvette 2 can be maintained at about 37 ℃ by supplying electricity to the electric heating wire. A stirrer 3 made of stainless steel is arranged in the small pool 2. The stirrer 3 is rotated in the cuvette 2 by the action of a stirrer 4 provided at the lower portion of the cuvette 2. That is, the agitator 4 includes a motor 4a and a permanent magnet 4b provided on an output shaft of the motor 4 a. Then, the motor 4a is energized to rotate the permanent magnet 4 b. Since the magnetic field from the permanent magnet 4b rotates, the stirrer 3 made of stainless steel rotates by the rotating magnetic field. The stirrer 3 and the stirrer 4 correspond to a stirring device. In this example, the rotation speed of the stirrer 3 was set to 1000 rpm.

The turbidimetric measurement apparatus 1 is provided with a light source 6 as a light incident device and a light receiving element 9 as a light receiving device. The light emitted from the light source 6 passes through the aperture 7 and then enters the sample in the cuvette 2 through the entrance hole 5a provided in the heat retainer 5. The light transmitted through the sample in the cuvette 2 is emitted from an exit hole 5b provided in the heat retainer 5, and is irradiated to the light receiving element 9 through the diaphragm 8. 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 an arithmetic device 10 as a determination means and a derivation means. The computing unit 10 can determine the reaction start time and derive the endotoxin concentration based on a program (algorithm) installed in advance. In addition, the turbidimetric apparatus 1 may include a display device for displaying the derived endotoxin concentration.

Fig. 2 shows the gradual decrease observed in this example. This gradual decrease is a phenomenon that occurs significantly 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 mixture. Then, as the reaction between endotoxin and LAL proceeded, a state was observed in which the slope further increased after the inflection point.

In the conventional stirred turbidimetric method, the reaction start time is defined as a time point at which the light transmittance is less than 95%. However, when the decrease occurs, the time when the light transmittance is lower than the threshold value of 95% may be abnormally early due to the influence of the decrease in the baseline. Alternatively, the value of the light transmittance may be lower than the threshold value of 95% before the curve of decrease in light transmittance reaches the inflection point. Thus, the decrease may lower the accuracy of determining the reaction start time.

The results are shown in FIG. 3, which is a graph plotting endotoxin concentration on the horizontal axis and 95% time below the threshold value in the conventional method on the vertical axis by the conventional threshold method. It is known that if a log-log is taken, the graph is a straight line. The correlation coefficient in the results shown in fig. 3 was observed to have an absolute value of 0.975. The japanese pharmacopoeia stipulates a condition that "the absolute value of the correlation coefficient must be 0.980 or more", and the results shown in fig. 3 do not satisfy this condition.

Fig. 4 shows the results of the case where the differential method of the present embodiment is employed. In this case, the time interval Δ T is 2 minutes, and the threshold value for determining the reaction start time is when the absolute value of the change amount of the light transmittance (%) exceeds 1%. As is clear from the graph, the absolute value of the correlation coefficient of the relationship between the endotoxin concentration and the endotoxin detection time was 0.987 by the differential method. That is, by adopting the difference method, a result having a stronger correlation is obtained, linearity is improved, and the above-mentioned condition in the japanese pharmacopoeia can be satisfied. As described above, in the present embodiment, the influence of gradual decrease can be eliminated by merely changing the program (algorithm) in the arithmetic device 10 of the turbidimetric measurement apparatus 1, and therefore the measurement accuracy of the turbidimetric measurement apparatus 1 is improved.

In this embodiment, the value of the light transmittance corresponds to the detection value and the detection signal value. In addition, in the turbidimetric measurement apparatus 1 of the present embodiment, the time interval Δ T and the threshold value are adjustable in the apparatus. Whereby the above-described rough estimation algorithm can be implemented more easily.

[ example 2]

Next, as example 2, measurement by the light scattering method will be described. FIG. 5 shows a schematic configuration of a light-scattering particle measuring apparatus 11 as an endotoxin measuring apparatus in this embodiment. The light source 12 used in the light scattering particle measuring apparatus 11 is a laser light source, and besides, an ultra high brightness LED or the like may be used. The light irradiated by the light source 12 is concentrated in the incident optical system 13 and is incident into the sample cell 14. The sample cell 14 holds a mixture of a sample for endotoxin assay and a LAL reagent. The light incident on the cuvette 14 is scattered by particles (measurement objects such as a coagulogen monomer and a coagulogen oligomer) in the mixed solution.

An exit optical system 15 is disposed on the side of the incident optical axis of the sample cell 14. A light receiving element 16 is disposed on an extension of the optical axis of the emission optical system 15, and the light receiving element 16 receives scattered light scattered by particles in the mixed liquid in the sample cell 14 and concentrated by the emission optical system 15, and converts the scattered light into an electric signal. The light receiving element 16 is electrically connected to an amplification circuit 17, a filter 18, an arithmetic device 19, and a display 20, wherein the amplification circuit 17 amplifies an electric signal photoelectrically converted by the light receiving element 16; the filter 18 is for removing noise from the electric signal amplified by the amplifying circuit 17; an arithmetic device 19 for calculating the number of gel particles from the number of peaks of the electric signal from which the noise has been removed, and further determining the reaction start time and deriving the endotoxin concentration; the display 20 displays the results.

In addition, a stirrer 21 is installed in the sample cell 14, the stirrer 21 rotates by applying an electromagnetic force from the outside and stirs a mixed solution as a sample, and a stirrer 22 is installed outside the sample cell 14. By these, stirring or no stirring can be performed, and the stirring speed can be adjusted.

Here, the sample cell 14 is the cuvette manufactured in manufacturing example 1, and corresponds to the mixed solution holding apparatus of the present example. The light source 12 and the incident optical system 13 correspond to a light incident device. The stirrer 21 and the stirrer 22 correspond to a stirring device. The emission optical system 15 and the light receiving element 16 correspond to a light receiving device. The arithmetic device 19 corresponds to a judgment device and a derivation device.

Fig. 6 shows an example of detecting a temporal change in the number of particles when the light scattering method is used. In this example, endotoxin was measured by incubating the sample at 37 ℃ under stirring in the same manner as in the stirring turbidimetric apparatus. The assay device specifically used PA-200 manufactured by Koshiki Kaisha. As is clear from B in FIG. 6, the light scattering method confirmed a phenomenon in which the number of detection (gel) particles increased regardless of the reaction state of endotoxin and LAL. This reaction exceeds the threshold value (the number of detected particles 200) before the "rapid increase in the number of particles" that should be detected, and therefore it is difficult to accurately determine the number of particles.

Next, the measurement method of the present example using the differential method will be described with reference to the light scattering method. In the arithmetic unit 19, first, every 1 second, histogram data of a peak for 1 second is created from the electric signal from which the noise has been removed. The total number of particles detected in 1 second is then calculated with the help of the histogram data. Then, in order to smooth the variation of the particle number, the total particle number calculated every 1 second is subjected to the moving accumulation for 10 seconds. The first histogram data acquisition time when the difference (increase) between the 10-second movement accumulated value obtained by the movement accumulation and the movement accumulated value obtained by advancing only the time interval Δ T (=100 seconds) continuously 10 times (10 seconds) exceeds the threshold 200 is determined as the reaction start time.

Fig. 7 shows the results of plotting the endotoxin concentration on the horizontal axis and the reaction start time on the vertical axis by applying the threshold method and the difference method to the light scattering method. When the absolute value of the correlation coefficient is 0.943 and the linearity is severely interrupted when the number of particles is simply to be detected, a strong correlation is obtained because the absolute value of the correlation coefficient is 0.999 when the time interval Δ T is set to 100 seconds and the increase of particles in 100 seconds exceeds the threshold 200 as the reaction start time. As described above, the algorithm to be detected with the particle increment of 100 seconds is considered to be a more stable algorithm than the conventional algorithm to be detected with the number of particles. As described above, in the present embodiment, by merely changing the program (algorithm) in the arithmetic unit 19 of the light scattering particle measuring apparatus 11, the influence of the gradual increase can be eliminated, and the measurement accuracy of the light scattering particle measuring apparatus 11 can be improved.

In the present embodiment, the value of the number of detection particles (the number of gel particles) corresponds to the detection value and the detection signal value. In addition, in the light scattering particle measuring apparatus 11 of the present embodiment, the time interval Δ T and the threshold value can be adjusted in the apparatus. Whereby the above-described rough estimation algorithm can be implemented more easily.

In the present embodiment, as described above, the arithmetic device 19 calculates the total particle number detected in 1 second from the histogram data, adds the total particle number by moving for 10 seconds, compares the movement accumulated value for 10 seconds with the movement accumulated value obtained by advancing only the time interval Δ T, and determines the first data acquisition time when the difference (increase amount) exceeds the threshold value for 10 consecutive times (10 seconds) as the reaction start time. However, this measurement method is merely an example, and the detection time of the histogram data, whether or not the shift accumulation is performed, the shift accumulation time, the number of times the difference (increase amount) at the reaction start time is judged to exceed the threshold value, and the like are not limited to the values of the present embodiment, and they may be changed as appropriate.

[ example 3]

Endotoxin was measured using a toxinometer (manufactured by Wako pure chemical industries, Ltd.) which is an apparatus used for a conventional turbidimetric method. In the toxinometer (turbidimetry) in which the sample was not stirred, a gradual decrease in the baseline, whose cause was unknown, was observed in the temporal change of the light transmittance. Therefore, the measurement result of toxinometer using the threshold method in which more than 95% of the time points are used as the reaction start time is also affected by gradual decrease. Therefore, in order to improve the measurement accuracy, the reanalysis is performed by the differential method in the same manner. As a result, the linearity of the regression line increases, the influence on endotoxin measurement due to the detection error of the differential method can be suppressed, and improvement is observed.

In the above embodiments, the fact that the difference exceeds the threshold value does not necessarily mean that the state where the difference is smaller than the threshold value is changed to the state where the difference is larger than the threshold value. For example, it is needless to say that the difference in the tendency to decrease changes from a state larger than the threshold value to a state smaller than the threshold value.

However, in the case of the above-mentioned differential method, when the time interval for obtaining the absorbance or the number of gel particles is made constant, it is difficult to obtain a sufficiently large differential value in the measurement of a predetermined physiologically active substance at a low concentration in which the change curve of the absorbance or the number of gel particles gradually progresses as described above, and therefore, the measurement may be difficult in a practical time. The following is a description of the case of a corresponding method incorporating this problem.

In the present invention, in the differential method, the time interval for obtaining the difference in absorbance or the number of gel particles is further changed depending on the time of obtaining. More specifically, the time interval at which the difference in absorbance or gel particle number is obtained is defined as a function of time from the start of measurement, and the interval is changed over time, or a plurality of series having different time intervals are prepared in advance. In the following description, unless otherwise specified, endotoxin measurement by the reaction of LAL with endotoxin will be described as an example.

In the measurement of a predetermined physiologically active substance, it is indispensable to use a reagent not containing the substance, preparation water, and a test device not containing the substance. In the preparation of a reagent for dissolution or dilution series of endotoxin, water for injection (produced by Otsuka pharmaceutical Co., Ltd.) was used in which an endotoxin was mixed in an extremely small amount. In addition, consumables such as pipette tips use individually packaged materials labeled as endotoxin-free. Since the measurement vessel is made of glass, a vessel subjected to a conventional endotoxin inactivation treatment (dry heat treatment) is used.

Production example 2 (glass vessel for measurement)

A stirrer (4.5 mm, 0.7 mm thick) made of stainless steel for stirring was placed in the glass container (φ 6 mm) for measurement, and the opening of the container was covered with aluminum foil. 20 glass containers gathered in a bundle and further covered with aluminum foil were taken as 1 pack, a plurality of the packs were gathered and added to a metal dry heat sterilization can, which was capped and subjected to a dry heat treatment at 250 ℃ for 3 hours.

Hereinafter, in the differential method, an example in which the time interval for obtaining the difference in absorbance or the number of gel particles is changed according to the time of obtaining will be described. In the following examples, absorbance is used as a physical quantity that changes due to a reaction between LAL and a predetermined physiologically active substance. However, the present invention is not limited to the substances, reagents and physical quantities of the measurement target exemplified in the following examples. Hereinafter, the method of obtaining the difference value at the time interval defined by the time function is referred to as a "time function difference method", and the method of obtaining the difference value using a plurality of series having different time intervals is referred to as a "multi-series difference method".

First, in the present embodiment, the turbidimetric apparatus used for obtaining absorbance is an apparatus equivalent to the apparatus shown in fig. 1.

< comparative example (ordinary difference method with constant time intervals) >

In the present embodiment, first, in order to verify the effects of the time-function difference method and the multi-series difference method, endotoxin measurement by a normal difference method with a constant time interval was performed as a comparison target. Limulus reagent A limulus ES-II single test manufactured by Tachypleus tridentatus and Wako pure chemical industries was used. Dilution series of endotoxin concentrations of 1.0, 0.1, 0.01, 0.001 EU/mL were prepared and reacted with limulus reagent in cuvette 2. The absorbance was recorded and analyzed by using a turbidimetric apparatus 1 (Absorbance measuring apparatus (EX-100, manufactured by Kyowa Kagaku Co., Ltd.).

The difference value is obtained by the method for making the time interval constant. The time interval was set to 3 minutes, and the difference was recorded over time, and the time at which the absorbance difference exceeded the threshold was used as the reaction initiation time (detection time). The threshold value used is a value of 0.003. The absorbance change curve of each sample is shown in FIG. 8, and the time-dependent change curve of the absorbance difference is shown in FIG. 9.

As is clear from an examination of FIG. 9, in the case of the conventional differential method in which the time interval at the time of obtaining the absorbance difference value does not change, endotoxin can be measured at a concentration of 1.0 to 0.001 EU/mL, and at a lower concentration of 0.0001 EU/mL, the absorbance difference value is not a sufficiently large value and does not exceed the threshold value, and therefore, detection is impossible.

< example 4 (time function difference method) >

Next, as example 4, a time function difference method in which a time interval when a time difference is obtained is defined by a time function will be described. Here, when endotoxin at a low concentration is reacted with LAL, the change in absorbance is slow, and therefore, a function in which the time interval is expanded with time must be used. Specifically, the time interval may change linearly (linear function) from the time of measurement start, or may change by a univariate polynomial definition such as a quadratic function or a cubic function. Alternatively, an exponential function, a logarithmic function, or the like may be used. In practice, the absorbance data obtained by the turbidimetric apparatus 1 is often sampled at fixed time intervals, for example, at 1 second intervals, and therefore, in this case, a discontinuous time interval function obtained by combining the above-mentioned functions with the fixed time intervals inherent to the apparatus can be obtained.

Here, the difference value change in absorbance was obtained by a time function difference method using each absorbance change curve data obtained in the above comparative examples. The turbidimetric apparatus 1 used for measurement is of a type that outputs data at 1 second intervals, and therefore cannot define a time function as a continuous function. Here, the time interval I (minutes) for calculating the difference value is defined in the form of a discontinuous function of equation (1).

I＝floor (T/10)+1 (1)

Where T is the time (minutes) elapsed since the start of the assay.

In addition, the function floor (x) represents a floor function (lower integer function). In addition, here, the threshold value is made a constant value of 0.01. Under the present conditions, the difference value change in absorbance was obtained by the time function difference method, and as a result, a time-dependent curve of the absorbance difference value for each dilution series of samples is shown in fig. 10. As is clear from FIG. 10, when the endotoxin concentration was as low as 0.001 EU/mL or 0.0001 EU/mL, the absorbance difference curve was larger than that of the comparative example shown in FIG. 9, and both of them could determine the reaction initiation time within the actual measurement time.

In this example, the difference in absorbance is also approximately constant at the initial stage of measurement, i.e., when there is no gradual decrease/increase in the reaction between LAL and endotoxin, and therefore, the initial value of the difference can be stored and subtracted from the difference at each time as a background value. In this way, the effect of the gradual decrease/increase on the assay can be reduced.

However, in the present embodiment, the time interval varies as a function of time, and therefore, even if the gradual decrease/increase is a linear variation, the time interval at which the difference is obtained gradually expands, and thus, the value of the difference caused by the gradual decrease/increase also expands as a function of time. Therefore, the effect of the gradual decrease/increase on the measurement cannot be completely removed. This can be handled as follows: and storing the difference value at the early stage of the reaction as a background value, multiplying the background value by a coefficient which is a ratio of the time interval at each acquisition time to the time interval at the early stage, and subtracting the obtained value from the difference value at each acquisition time.

In addition, the threshold value of the difference value in the present embodiment may be a constant that does not depend on the time from the start of the measurement as described above, or may be a threshold value that changes according to a time function. In fact, when reacting a predetermined physiologically active substance with LAL, if the concentration of the predetermined physiologically active substance is low, the change in absorbance is very slow, and therefore, when using a time function, the absolute value of the threshold value can be set to decrease with time. The time function at this time can be expected and utilized as a linear function, a univariate polynomial, or the like.

Alternatively, when a graph showing the change in the absorbance difference value with time at each endotoxin concentration as shown in FIG. 9 is obtained in advance, the threshold value may be defined by the obtained curve by connecting, for example, 20% of the peak of the curve for each endotoxin concentration. Thus, even when the endotoxin concentration is low, the difference value can be made to exceed the threshold more reliably. In addition, the threshold value may be defined by a curve inversely proportional to the elapsed time from the start of measurement.

FIG. 11 is a flowchart showing a measuring procedure for endotoxin measurement by the time-function difference method. This program is executed by the arithmetic device 10 at the same time as the measurement is started. In executing this routine, first, an initialization operation is performed in S101, and the value of the variable T, which is the time from the start of measurement, is reset. Subsequently, the process proceeds to S102, where the photoelectric signal data according to the intensity of light received by the light receiving element 9 is taken into the arithmetic device 10. Subsequently, in S103, the time interval I (minute) for calculating the difference value is calculated based on the T value at the current time point by the formula (1).

Next, in S104, it is determined whether or not the current time is a storage time of the initial value of absorbance. In this example, the storage time of the initial value of absorbance is set to 1 second after the start of measurement. If an affirmative determination is made in S104, the process proceeds to S105. On the other hand, if a negative determination is made in S104, the process proceeds to S106. In S105, an initial value (reference light intensity) of the photoelectric signal data corresponding to the light intensity received by the light receiving element 9 is stored. In S106, it is determined whether or not the current time is a preset sampling time. The determination of the sampling time is made by determining whether or not the sampling time has elapsed from the sampling interval calculated in S103. If an affirmative determination is made here, the process proceeds to S107. On the other hand, if a negative determination is made here, the process returns to the step before the processing in S102.

When an affirmative determination is made in S106, that is, when the current time point is the sampling time, the data sequence in the memory in the arithmetic device 10 is updated in S107. That is, the data obtained in S102 is collected in the memory as the latest data. Next, in S108, it is determined whether or not the current time is a predetermined determination time. Here, the preset determination time is a time for comparing a difference value between the latest data and the previous data with a preset threshold value to determine whether or not the difference value is a reaction start time, and may be set equal to or completely independent of the sampling time. When a negative determination is made in S108, the process returns to the state before the process in S102. On the other hand, if an affirmative determination is made in S108, the process proceeds to S109.

The light transmittance or absorbance is calculated in S109. The light transmittance can be calculated by dividing the latest data in the data series by the data in the state of no sample taken in advance. In addition, the absorbance can be calculated by subtracting the calculated light transmittance from 1. Next, a difference value is calculated in S110. In this embodiment, the following calculation may be performed: the value of light transmittance or absorbance calculated from the data of the previous sampling time in the data series is subtracted from the value of light transmittance or absorbance calculated from the latest data.

In S111, a reference difference value is calculated. In this embodiment, the reference difference value is calculated as follows: the light transmittance or absorbance calculated from the data sampled at the 1 st sampling time is subtracted from the light transmittance or absorbance calculated from the data sampled at the 2 nd sampling time after the start of the measurement. The reference difference value is a background value for eliminating the effect of gradual decrease/increase in the present assay.

In S112, detection determination is performed. Here, basically, it is determined whether or not a value obtained by subtracting the reference difference value calculated in S111 from the difference value calculated in S110 is larger than a preset threshold value, and when it is determined that the value is larger than the threshold value 5 times in succession, it is determined that the reaction start time is detected, and when it is equal to or smaller than the threshold value, it is determined that the reaction start time has not been detected. When it is determined that the reaction start time is detected, the value of the reaction start time and the detected information are stored in the memory of the arithmetic device 10. The details of this process are described below.

The value of the timer T is updated in S113. And it is determined in S114 whether or not the reaction start timing has been detected. When it is determined in S112 that the reaction start time has been detected and the detected information has been stored, the present routine is terminated. On the other hand, if information that has not been picked up is stored in S114, the process returns to the processing in S102.

Next, fig. 12 shows a subroutine for performing the detection determination of S112 in the above-described measurement routine. When this subroutine is executed, first, in S1001, it is determined whether or not a value obtained by subtracting the reference difference value calculated in S111 from the difference value calculated in S110 is larger than a preset threshold value. If an affirmative determination is made here, the process proceeds to S1002. On the other hand, if a negative determination is made, the process proceeds to S1007.

In S1002, it is determined whether or not the condition of S1001 is satisfied in the previous 4 determinations. If an affirmative determination is made here, the process proceeds to S1003. On the other hand, if a negative determination is made, the process proceeds to S1007. That is, in S1002, it is determined whether a state in which the value obtained by subtracting the reference difference value calculated in S111 from the difference value calculated in S110 is larger than a preset threshold value is satisfied continuously 5 times.

Next, in S1003, it is determined that the reaction start time is detected. On the other hand, in S1007, it is determined that the reaction start time is not detected. In S1004, the time T when the reaction start time is determined to be detected in S1003 is set as the detection time (reaction start time), and the detection time is displayed and recorded in the memory of the arithmetic device 10.

In S1005, the endotoxin concentration is calculated from a map (corresponding to a calibration curve) storing the relationship between the endotoxin concentration and the reaction start time obtained in advance, and the calculated value is displayed and recorded in the memory of the computing unit 10. In S1006, the detection completion determination is stored in the memory of the arithmetic device 10. In S1008, the detection determination is not completed and stored in the memory of the arithmetic device 10. When the processing in S1006 or S1008 is finished, the present routine is finished and the routine proceeds to the processing in S113 of the measurement routine.

The flowchart of the measurement routine shown in fig. 11 and the flowchart of the subroutine shown in fig. 12 are examples of the routine for performing the measurement of the present embodiment, and are not intended to limit the routines to those shown in these flowcharts. In S1002 of fig. 12, it is determined whether or not a state in which the value obtained by subtracting the reference difference value calculated in S111 from the difference value calculated in S110 is larger than a preset threshold value is satisfied 5 times in succession, which is a measure for improving the measurement accuracy, and the number of times the state should be satisfied may be, for example, 1 time other than 5 times.

< example 5 (Multi-series Difference method) >

Next, as example 5 of the present embodiment, a case where a plurality of series with different execution intervals are prepared in advance is examined. In this case, the number of series must be 2 or more. There is no upper limit to the number of series, and if the measurement is to be performed for a longer time, more series can be prepared, thereby enabling the measurement to be performed with higher accuracy. The number of series that can be prepared in practice is limited by the size and processing power of the computer memory used in the analysis. In addition, if there are many channels of the device that can be measured, it is necessary to prepare these series at the same time. Therefore, the number of series is preferably 30 or less, and more preferably 10 or less per channel.

The time interval allocated to each series is arbitrary, and may be allocated at equal intervals (linear function) of 5 seconds, 10 seconds, 15 seconds, 20 seconds, or the like, or may be allocated to exponentially increasing time intervals of 1 second, 3 seconds, 10 seconds, and 30 seconds, for example. In the case where the change in absorbance does not include the gradual decrease/increase in the difference values obtained at the time intervals, the difference value at the initial stage of the measurement is preferably 0. In addition, since the sampling interval of each series does not change when the change in absorbance includes gradual decrease/increase, an initial value of the difference value is stored in each series and may be subtracted from the difference value at each time as a background value. In this way, the effect of the gradual decrease/increase on the assay can be completely removed in each series.

In this example, the absorbance difference value was obtained by a multi-series difference method using each absorbance change curve data obtained in the comparative example. The number of the series used for obtaining the difference values was 3 series (series names: S1, S2, S3), and each series had a sequence capable of maintaining the absorbance of 60 data. The sampling interval of the data of each series is based on first-in first-out (FIFO: method of discarding the oldest data in the series and adding a new data), and the calculation of absorbance and the update operation of data (deletion of the oldest data and recording of the newest data into the series) are performed every 1 second in S1, every 6 seconds in S2, and every 30 seconds in S3.

The value of difference in absorbance Δ ABS was calculated by calculating the difference in value between both ends of the sequence in each series as shown in the following formula (2).

ΔABS=A[60]-A[1] (2)

Wherein A60 represents absorbance data of No. 60 (latest) in the sequences contained in each series, and A1 represents absorbance data of No. 1 (oldest) in the sequences contained in each series. The difference in absorbance was calculated for all series S1-S3 according to equation (2). Initial values P1, P2, and P3 of absorbance obtained for the first time in each series were obtained. The time of acquisition is: p1 is the time point from the start of 1 minute, P2 is the time point from the start of 6 minutes, P3 is the time point from the start of 30 minutes.

Since the absorbance difference values obtained under the above conditions include a gradual decrease/increase in some degree, a value obtained by subtracting the initial value of absorbance from the absorbance difference value at each time in each series is recorded over time, and the time at which the absorbance difference value in any series exceeds the threshold value is regarded as the reaction start time (detection time). The time-dependent change in the absorbance difference of each sample is shown in FIG. 13. As is clear from FIG. 13, the absorbance difference curve at low endotoxin concentrations of 0.001 EU/mL and 0.0001 EU/mL is larger than that of the comparative example shown in FIG. 9, and both can determine the reaction initiation time within the actual measurement time.

In this embodiment, as described above, the threshold value of the difference value may be a constant value, which is not dependent on the time from the start of the measurement, or may be a threshold value that changes according to a time function. In fact, since the change in absorbance when a predetermined physiologically active substance reacts with LAL is very slow if the concentration of the predetermined physiologically active substance is low, the absolute value set as the threshold value decreases with time when using a time function. The time function at this time can be expected and utilized as a linear function, a univariate polynomial, or the like.

When a plurality of series having different time intervals are prepared in advance, the timing at which the time exceeds the threshold value differs for each series. In this case, the first time of the series of passes is used as a method for determining the reaction start time. Or various determination methods such as taking an average value of 2 series that have passed first, etc. are considered. However, since only 1 series is considered to exceed the threshold value according to the concentration of the predetermined physiologically active substance, it is preferable to use the value of the series that first exceeds the threshold value as the reaction start time for reliable detection.

In this embodiment, different thresholds may be used for each series, for example, 0.01 for series S1, 0.005 for S2, 0.003 for S3, etc. Thus, in the measurement of a sample having a low endotoxin concentration, the difference value can more surely exceed the threshold value.

FIG. 14 is a flowchart of the measurement procedure 2 for endotoxin measurement by the multi-series differentiation method. This program is executed by the arithmetic device 10 at the same time as the measurement is started. In the present routine, first, in S201, an initialization operation is performed to reset the value of the variable T, which is the time since the start of measurement. Subsequently, the process proceeds to S202, where the photoelectric signal data corresponding to the light intensity received by the light receiving element 9 is taken into the arithmetic device 10.

Next, in S203, it is determined whether or not the current time is the storage time of the initial value in any one of the series of S1, S2, and S3. Here, in the case of S1, the storage time of the initial value is set to 1 second after the start of measurement. In the case of S2, the initial value storage time is set 6 seconds after the start. Here, in the case of S3, the storage time of the initial value is set 30 seconds after the start. If an affirmative determination is made in S203, the process proceeds to S204. On the other hand, if a negative determination is made in S203, the process proceeds to S205.

In S204, the initial value (reference light intensity) of the light intensity of each series is stored. More specifically, when it is determined in S203 that the initial value of the series S1 is stored, the reference light intensity for the series S1 is stored in S204. If it is determined at S203 that the initial value of the series S2 is stored, the reference light intensity for the series S2 is stored at S204. If it is determined at S203 that the initial value of the series S3 is stored, the reference light intensity for the series S3 is stored at S204.

In S205, it is determined whether or not the sampling time is the sampling time used in S1. The sampling time is determined by whether or not the sampling interval (1 second) for S1 set in advance has elapsed with respect to the previous sampling time for S1. If the positive determination is made here, the process proceeds to S210, whereas if the negative determination is made here, the process proceeds to S206.

In S206, it is determined whether or not the sampling time is the sampling time for S2. The sampling time is determined by whether or not the sampling interval (6 seconds) for S2 set in advance has elapsed with respect to the previous sampling time for S2. If the positive determination is made here, the process proceeds to S220, whereas if the negative determination is made here, the process proceeds to S207.

In S207, it is determined whether or not the sampling time is the sampling time for S3. The sampling time is determined by whether or not the sampling interval (30 seconds) for S3 set in advance has elapsed with respect to the previous sampling time for S3. If the positive determination is made here, the process proceeds to S230, whereas if the negative determination is made here, the process proceeds to S208.

The processing of S210 to S215 performed at the sampling timing of the series S1, the processing of S220 to S225 performed at the sampling timing of the series S2, and the processing of S230 to S235 performed at the sampling timing of the series S3 are equivalent to the processing of S107 to S112 of the measurement routine shown in fig. 11. Therefore, detailed description of these processes is omitted here. In this routine, when a negative determination is made in S211 and the process of S215 is completed, the process proceeds to the process of S206. When a negative determination is made in S221 and the process in S225 is completed, the process proceeds to S207. When a negative determination is made in S231 and the process in S235 is completed, the process proceeds to S240, and the value of the timer T is updated.

In S241, it is determined whether any series of series S1-S3 has been detected. When a positive determination is made here, the present routine is completed. On the other hand, if a negative determination is made, the process returns to the processing in S202.

The flowchart of the measurement routine S2 shown in fig. 14 is an example of a routine for performing the measurement of the present embodiment, but is not intended to be limited to the flow shown in these flowcharts.

< example 6 (comparison of difference methods) >

The same measurement data was analyzed by the techniques described in comparative example and examples 4 and 5, and the reaction initiation time of the endotoxin dilution series aqueous solution sample was obtained. Here, the reaction start times obtained by these methods are compared to evaluate the effectiveness of the method according to the present embodiment. It is known that the relationship between endotoxin concentration and reaction initiation time is approximated to a straight line when a log-log graph is used. Here, as shown in fig. 15, the endotoxin concentration (horizontal axis) and the reaction initiation time (detection time (vertical axis)) were each plotted logarithmically. Each figure is presented as an average of 2 measurements.

In the usual differential method according to the comparative example, in which the time interval is not changed, endotoxin can be detected at an endotoxin concentration in the range of 1.0 to 0.001 EU/mL, but at a concentration of 0.0001 EU/mL or less, the difference in absorbance does not reach a sufficiently large value and does not exceed a threshold value, and thus, it cannot be detected. On the other hand, in the time-function difference method and the multiple-series difference method of the present invention, endotoxin can be detected at a very wide concentration range of 1.0 to 0.0001 EU/mL. The linearity (in relation to the approximation) of the plots obtained by the various difference methods is shown in table 1.

[ Table 1]

As shown in fig. 15 and table 1, the limits of the measurable range of the conventional differential method are: the endotoxin concentration of the sample was 0.001 EU/mL. In addition, the correlation coefficient is also lower than that of other methods. This is supported by the large deviation above the approximate formula in the figure, particularly at a concentration of 0.001 EU/mL. On the other hand, in the two difference methods of the present embodiment, the deviation of the graph from the approximate expression is small, and the correlation coefficient is very good. Particularly, the linearity is very good in the multi-series difference method.

< example 7 (example of measurement of limulus reagent for colorimetry) >

In this example, the endotoxin dilution series (1.0 to 0.001 EU/mL) was measured using パイオクラム of Biochemical バイオビジネス as a limulus reagent for colorimetry and using the turbidimetric apparatus 1 (EX-100) used in the above example. Endotoxin was detected by a multi-series differential assay. The conditions such as the number of series, the data sampling interval of each series, the number of elements of the sequence held in each series, and the method of calculating the absorbance difference value were exactly the same as those of example 5. The reaction initiation time (detection time) was determined as the average of the two data numbers, and the relationship with the endotoxin concentration was plotted using a log-log graph, and the result was shown in FIG. 16, which shows extremely high linearity. The approximate expression is expressed by expression (3), and the correlation coefficient (| r |) is 0.9988.

Y=9.0266X^-0.2984 (3)。

< example 8 (measurement of. beta. -D-glucan by means of. beta. -D-glucan measurement reagent)

In this example, a dilution series of beta-D-glucan was measured at a concentration of 30 to 0.5 pg/mL using a limulus reagent (manufactured by Wako pure chemical industries, Ltd.) containing テストワコウ, a drug for in vitro diagnosis. The detection of beta-D-glucan can be performed by the multi-series differential method used in example 5. The number of series was 3, the sampling interval of S1 was every 1 second, hereinafter, S2 was every 6 seconds, and S3 was every 15 seconds. As a result, the difference interval at which the difference was obtained was: 1 minute for S1, 6 minutes for S2, and 15 minutes for S3. The other conditions were the same as the measurement described in example 5. The relationship between the β -D-glucan concentration and the reaction initiation time (detection time) obtained by the measurement was plotted by log double plot, and as shown in fig. 17, extremely high linearity was obtained. The approximate expression is shown as the formula (4), and the correlation coefficient (| r |) is 0.9970.

Y=46.348X^-0.3852 (4)。

< example 9 (example of endotoxin measurement by LAL-bound bead method) >

In this example, endotoxin dilution series measurements were carried out at a concentration of 1.0 to 0.001 EU/mL using the LAL bead-bound method (see, for example, patent document 4). In the LAL-bound bead method, a reagent is prepared by adsorbing or binding a protein contained in LAL to beads (microparticles) dispersed in a previously prepared reagent solution. Then, the sample containing endotoxin is allowed to act on the reagent to cause the microparticles to associate with each other, thereby forming a large aggregate at an early stage, and the formation of the aggregate is detected, whereby endotoxin can be measured.

Endotoxin was assayed by the multi-series differential method used in example 5. The number of series was 3, and the analysis conditions such as the sampling interval in the series S1-S3 were the same as in example 5. On the other hand, in the measurement using LAL-bound beads, since a large amount of beads as light scatterers are contained in the sample, which is originally turbid, is made transparent by aggregation. In this process, it is more appropriate to use the difference in light transmittance for the agglutination determination than to obtain the difference in absorbance, and therefore, unlike in example 5, the present embodiment performs the agglutination determination using the difference in light transmittance. The threshold used for the determination was 2.0 for all series S1-S3. The relationship between the endotoxin concentration and the reaction initiation time (detection time) obtained by the measurement was plotted by log double, and as shown in FIG. 18, extremely high linearity was obtained. The approximate expression is shown in formula (5), and the correlation coefficient (| r |) is 0.9960.

Y=4.307X^-0.294(5)。

< example 10 (example of endotoxin assay using LAL reagent by turbidimetry)

In this example, the present invention was applied to endotoxin measurement using a LAL reagent by turbidimetry. Endotoxin was assayed by the multi-series differential method used in example 5. The number of series was 3, and the analysis conditions such as the sampling interval in the series S1-S3 were the same as in example 5. When the change in absorbance includes gradual decrease/increase, in each series, the initial value of the difference value as the background value is not subtracted from the difference value at each time, but the value to be subtracted is dynamically updated.

FIG. 19 shows an example of endotoxin assay using LAL reagent "パイロテル (manufactured by ケープコッド, sold by Biochemical バイオビジネス)" by turbidimetry. In this example, the endotoxin dilution series of 7 concentrations was measured at the sampling intervals of the above 3 series within the range of 1 to 0.001 EU/mL.

In this embodiment, the absorbance difference value is measured for each sampling time, a plurality of absorbance difference values obtained from past sampling times are rearranged in order of magnitude for each series, and the latter 5 data are updated and recorded. Then, the value of the 3 rd smallest value in each series was used as a reference value for each series, and was subtracted from the absorbance difference value obtained at that time point. Then, the determination of the reaction start time is made by whether or not the subtracted value exceeds a threshold value. Regarding the threshold, the sampling interval was 0.01 when it was 1 second or 6 seconds (in series S1 and S2), and the sampling interval was 30 seconds (in series S3), which was 0.005.

The relationship between the endotoxin concentration and the reaction initiation time (detection time) obtained by the measurement was plotted by log double, and as shown in FIG. 19, extremely high linearity was obtained. パイロテル, the method of the present embodiment is effective in that when the change in absorbance includes a gradual decrease/increase, the value to be subtracted from the difference value at each time is changed at any time, and the determination is performed. The approximate expression is shown in formula (6), and the correlation coefficient (| r |) is 0.9955.

Y=11.191X^-0.239(6)。

Fig. 20 illustrates a reference difference value calculation subroutine in the present embodiment. This reference difference value operator program is a subroutine executed in the processing of S214, S224, and S234 when the measurement program 2 shown in fig. 14 is executed in the present embodiment. In the present procedure, first, in S701, an absorbance difference value is obtained for each sampling time.

In S702, it is determined whether the difference value data of absorbance is 5 data. If an affirmative determination is made here, the process proceeds to S703. On the other hand, if a negative determination is made, the process proceeds to S706. In S703, it is determined whether the value of the absorbance difference at the current time point has entered the last 5 digits. If an affirmative determination is made here, the process proceeds to S704. On the other hand, if a negative determination is made, the process proceeds to S705.

In S704, newly acquired data is added to the next 5 data to update the next 5 data. The process of S704 ends, and the flow proceeds to S705. In S705, the later 3 rd data at this time point is set as the reference difference value. In S706, the reference difference value is set for the minimum data at the time point. When the processing in S705 or S706 is completed, the process proceeds to S707, and in S707, the reference difference value is determined and stored, and is restored to the main routine of the measurement program 2. Here, the flowchart of the reference difference value calculation subroutine shown in fig. 20 is an example of a routine for performing the measurement of the present embodiment, and is not intended to be limited to the routines described in these flowcharts.

In the above examples, the present invention was explained as an example of the stirring turbidimetry using the turbidimetry apparatus 1, but it is needless to say that the present invention may be applied to a turbidimetry not based on stirring, a measurement method other than the stirring turbidimetry, and a measurement device. In the above-described embodiment, an example was described in which the reaction start time is a time when a physical quantity such as absorbance exceeds a threshold value, but a time when a physical quantity such as absorbance becomes equal to or more than a threshold value may be used as the reaction start time. Or the reaction start time is a time when a physical quantity such as the amount of transmitted light, the amount of scattered light, the number of light-scattering particles, the fluorescence intensity, or the chemiluminescence intensity exceeds or exceeds a threshold value.

In the above-described embodiment, when the absorbance is used as the detection value, the time at which the difference in absorbance between the two acquisition times exceeds the threshold value is determined as the reaction start time, and when the light transmittance is used as the detection value, for example, the detection value decreases with time.

In the above embodiment, the average value or the median value of a plurality of data before and after the acquisition time may be actually used as the detection value or the difference value at the acquisition time. The data may be further rearranged in order of size, using a numerical value of a specified rank, or the like. This reduces the influence of noise on the detection value or the difference value at each acquisition time, and enables more accurate measurement. For example, 30 to 40 data in total before and after the acquisition time may be averaged to obtain a detection value or a difference value at the acquisition time.

In the above embodiment, when determining whether or not the difference value exceeds the threshold value, it may be determined that the difference value exceeds the threshold value continuously at a plurality of acquisition times. This can reduce the influence of noise on the determination of the reaction start time, and can improve the accuracy of endotoxin measurement more reliably.

Description of the symbols

1 turbidimetric measuring device

2 glass container

3 stirrer

4 stirrer

4a motor

4b magnet

5 Heat preservation device

5a incident hole

5b exit aperture

6 light source

7 aperture

8 aperture

9 light receiving element

10 arithmetic device

11 measurement system

12 light source

13 incident optical system

14 sample pool

15 emergent optical system

16 light receiving element

17 amplifier circuit

18 noise removing filter

19 arithmetic device

20 display

21 stirrer

22 stirrer

Claims

1. A method for measuring a physiologically active substance derived from a living organism, comprising:

mixing a sample containing endotoxin or β -D-glucan, which is a physiologically active substance derived from a living organism, with a limulus blood cell extract LAL, and continuously obtaining, as a detection value, any one of light transmittance, absorbance, scattered light intensity, the number of light-scattering particles, fluorescence intensity, and chemiluminescence intensity, which changes due to a reaction between the LAL and the physiologically active substance, after the mixing;

setting one acquisition time as a reaction start time when a difference between a detection value at the one acquisition time and a detection value at an acquisition time that is earlier than the one acquisition time by an acquisition time or a concentration of the physiologically active substance in the sample or at a constant time interval or an absolute value of the difference is equal to or more than a threshold value;

detecting the physiologically active substance in the sample or determining the concentration thereof based on the reaction initiation time.

2. The method for assaying a physiologically active substance derived from a living organism according to claim 1, which comprises detecting the physiologically active substance in a sample or measuring the concentration of the physiologically active substance by reacting the physiologically active substance derived from a living organism present in the sample with Limulus polyphemus blood cell extract LAL,

the measurement method is characterized in that:

after the sample and the LAL are mixed, the intensity of light transmitted through the mixed solution or light scattered by the mixed solution among the incident light is obtained while the light is incident on the mixed solution of the sample and the LAL,

using any one of light transmittance, absorbance, scattered light intensity, number of light scattering particles, fluorescence intensity, and chemiluminescence intensity at the time of acquisition set at the time interval, which is obtained from the intensity of the light obtained as described above, as a detection value,

the reaction start time is set to be a time when the difference between the detected value at one acquisition time and the detected value at the previous acquisition time or the absolute value of the difference is equal to or greater than a threshold value or exceeds the threshold value.

3. The method for measuring a physiologically active substance derived from a living organism according to claim 2, characterized in that:

the intensity of the light obtained is the intensity of the light transmitted through the mixed liquid,

the measurement value is a transmittance of the mixed solution expressed in percentage,

the time interval was made to be 2 minutes,

the reaction start time is set to a time at which the absolute value of the difference between the transmittance at the one acquisition time and the transmittance at the previous acquisition time exceeds 1.

4. The method for measuring a physiologically active substance derived from a living organism according to claim 2, characterized in that:

the intensity of the acquired light is the intensity of light scattered by the mixed liquid,

the detection value is a particle number derived based on the number of peaks in the scattered light intensity and scattering light incident on the mixed solution,

the time interval was made to be 100 seconds,

the reaction start time is set to a time point at which the difference between the number of particles at the one acquisition time and the number of particles at the previous acquisition time exceeds 200.

5. The method for measuring a physiologically active substance derived from a living organism according to claim 1, wherein: changing the time interval in accordance with the one acquisition instant.

6. The method for measuring a physiologically active substance derived from a living organism according to claim 5, wherein:

simultaneously and continuously detecting, with respect to light incident on a mixed solution of the LAL and the sample, an intensity of light transmitted through the mixed solution or light scattered by the mixed solution among the incident light,

any one of light transmittance, absorbance, scattered light intensity, the number of light scattering particles, fluorescence intensity, and chemiluminescence intensity obtained from the continuously detected light intensity is used as a detection value.

7. The method for measuring a physiologically active substance derived from a living organism according to claim 5 or 6, characterized in that: the time interval is extended so that the one acquisition instant is further back.

8. The method for measuring a physiologically active substance derived from a living organism according to claim 5 or 6, characterized in that:

a plurality of series in which the time intervals are set to a constant acquisition time are provided, the time intervals of the plurality of series being different from each other,

the series used is switched according to the one acquisition time.

9. The method for measuring a physiologically active substance derived from a living organism according to claim 8, characterized in that: the series used is a series in which the difference between the detected value at the one acquisition time and the detected value at the acquisition time that is earlier than the one acquisition time by the time interval or the absolute value of the difference is the largest.

10. The method for measuring a physiologically active substance derived from a living organism according to claim 8, characterized in that:

changing the acquisition time, and acquiring a difference between the detection values at the plurality of one acquisition times and the detection value at an acquisition time earlier than the one acquisition time by the time interval or an absolute value of the difference; taking the difference or the value at the central level when the absolute values of the differences are arranged according to the magnitude sequence as a reference difference value; and when the value obtained by subtracting the reference difference value from the difference or the absolute value of the difference is equal to or more than the threshold value or exceeds the threshold value, the one obtaining time is used as the reaction starting time.

11. An assay device for a physiologically active substance derived from a living organism, said assay device being provided with:

a mixed solution holding means for holding a mixed solution of a sample containing a physiologically active substance endotoxin or β -D-glucan derived from a living organism and a limulus blood cell extract LAL to be allowed to enter an incident light while allowing a reaction in the mixed solution to proceed;

a stirring device that stirs the mixed liquid in the mixed liquid holding device;

a light incidence device that enters light into the mixed liquid in the mixed liquid holding device;

a light receiving device that receives the transmitted light or scattered light of the incident light from the mixed liquid and converts the received light into an electric signal;

a determination device that determines a reaction start timing of the physiologically active substance in the sample with the LAL from the electric signal converted in the light-receiving device;

a deriving means that derives the presence or concentration of the physiologically active substance in a sample from a relationship between the reaction start time and the concentration of the physiologically active substance, which is obtained in advance by measuring the reaction start time of the sample containing the physiologically active substance at a known concentration;

the device is characterized in that: the determination device determines, as the reaction start time, a time at which a difference between a detection signal value at one acquisition time and a detection signal value at a previous acquisition time or an absolute value of the difference is equal to or greater than a threshold value or exceeds the threshold value, by using a signal obtained by applying an operation of calculating any one of light transmittance, absorbance, scattered light intensity, the number of light-scattering particles, fluorescence intensity, and chemiluminescence intensity to the electric signal or by using the electric signal as a detection signal value, at an acquisition time set at a time interval that is changed or constant depending on the acquisition time or the concentration of the physiologically active substance in the sample.

12. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 11, wherein:

the detection signal value is the transmittance of the mixed solution expressed in percentage,

the time interval is 2 minutes and the time interval is,

the threshold is 1.

13. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 11, wherein:

the detection signal value is the number of particles that scatter light incident on the mixed solution,

the time interval is 100 seconds and,

the threshold is 200.

14. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 11, wherein: the time interval and/or the threshold value is made variable.

15. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 11, wherein: the determination device changes the time interval according to the one acquisition time.

16. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 15, wherein: the determination device extends the time interval to further reduce the one acquisition time.

17. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 16, wherein:

the determination device sets a plurality of series of acquisition timings at which the time intervals are set to be constant, the time intervals of the plurality of series being different from each other,

the series used is switched according to the one acquisition time.

18. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 17, wherein: the series used is a series in which the difference between the detected value at the one acquisition time and the detected value at the acquisition time that is earlier than the one acquisition time by the time interval or the absolute value of the difference is the largest.

19. The apparatus for measuring a physiologically active substance derived from a living organism according to claim 17, wherein:

changing the acquisition time, and acquiring a difference between the detection values at the plurality of one acquisition times and the detection value at an acquisition time earlier than the one acquisition time by the time interval or an absolute value of the difference; taking the difference or the value at the central level when the absolute values of the differences are arranged according to the magnitude sequence as a reference difference value; and determining the moment when the value obtained by subtracting the reference difference value from the difference or the absolute value of the difference is more than or equal to the threshold value or exceeds the threshold value as the reaction starting moment.