JP2012255679A - Method and apparatus for measuring bio-based physiological active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the accuracy of a measurement by performing the measurement without incurring coagulation or gelatinization which is not derived from a bio-based physiological substance, caused by stirring a mixture solution of an AL reagent and a sample when measuring the concentration of the physiological substance in the sample according to a light scattering method (including an AL bound beads method).SOLUTION: An AL reagent and a sample containing a bio-based physiological substance are mixed, light is made incident to a mixture solution and on the basis of the intensity of scattered light or transmitted light, the coagulation or gelatinization of protein caused by a reaction of the AL and the bio-based physiological substance in the mixture solution is detected. In place of stirring a mixture solution 7, an incident position of incident light is moved using a galvano mirror device 5 or the like.

Description

The present invention contains a biologically active substance derived from an organism having a property of gelling by reaction with a blood cell extract of horseshoe crab (hereinafter, also referred to as “AL: Amoebocyte lysate”) such as endotoxin and β-D-glucan. The present invention relates to a measurement method and a measurement apparatus for detecting the physiologically active substance in a sample or measuring the concentration thereof.

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

  As a semi-quantitative test method for detecting endotoxin contamination such as injections, the following method has been used in the past. That is, a sample heated to 37 ° C. is injected into the ear vein of a healthy rabbit weighing 1.5 kg or more, and the body temperature is measured at intervals of 30 minutes or less until 3 hours after the injection. The average value of the body temperature of the rabbit measured twice at 30 minute intervals between 40 minutes before the injection and the injection is taken as the subject body temperature, and the difference between the subject body temperature and the maximum body temperature is taken as the body temperature rise.

  The above-mentioned body temperature rise is measured using three rabbits, and the following determination is made based on the sum of the three body temperature rises. First, if the total body temperature rise of 3 animals is 1.3 ° C. or less, the exothermic substance is negative, and if it is 2.5 ° C. or more, the exothermic substance is positive. If the total body temperature rise of 3 animals is greater than 1.3 ° C and less than 2.5 ° C, a test with 3 animals is added, and the total body temperature rise of 6 animals is less than 3.0 ° C. If the temperature is 4.2 ° C. or higher, the pyrogen substance is positive. If the total temperature rise is greater than 3.0 ° C and less than 4.2 ° C, a test with 3 animals is added. If the body temperature rise of 9 animals is less than 5.0 ° C, fever occurs. If the temperature is 5.0 ° C. or higher, the exothermic substance is positive.

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

  By the way, in AL, there exists a serine protease activated by endotoxin, β-D-glucan and the like. When AL reacts with endotoxin or β-D-glucan, the coagulogen present in AL is hydrolyzed to coagulin by an enzyme cascade by serine protease activated according to the amount thereof. Associate and produce an insoluble gel. Endotoxin and β-D-glucan can be detected with high sensitivity using the characteristics of AL. In recent years, a method using AL for detection or concentration measurement of endotoxin has been devised using this fact.

As a method for detecting or measuring the concentration of biologically active substances derived from organisms (hereinafter also referred to as predetermined physiologically active substances) such as endotoxin and β-D-glucan, detection of predetermined physiologically active substances or A mixture of a sample to be measured for concentration (hereinafter simply referred to as “measurement of a predetermined physiologically active substance”) and a reagent produced based on AL (AL reagent) is allowed to stand, and after a certain period of time. There is a semi-quantitative gelation method in which a container is turned over to determine whether or not the sample has gelled depending on whether or not the sample sags, and whether or not the sample contains endotoxin at a certain concentration or higher.

  Alternatively, the mixture of the sample to be measured for the predetermined physiologically active substance and AL is allowed to stand in a state where the temperature is maintained at 37 ° C., and the sample accompanying the generation of the gel by the reaction between AL and the predetermined physiologically active substance is prepared. There is a turbidimetric method that measures and analyzes turbidity over time. In this turbidimetric method, after the start of measurement, the time when the transmittance has dropped below a certain value is defined as the gel time. Quantification uses the fact that gelation time is correlated with the amount of endotoxin in the sample. That is, the amount of endotoxin in the specimen is calculated from a calibration curve prepared by measuring a plurality of standard samples mixed with a known amount of endotoxin in advance and the gelation time obtained by the measurement.

  When measuring a predetermined physiologically active substance by the turbidimetric method described above, a mixed solution of the measurement sample and AL is generated in a glass measurement cell subjected to dry heat sterilization. And the liquid mixture is left still and the gelation is optically measured from the outside. On the other hand, there is a stirring turbidimetric method in which a sample mixed with an AL reagent is measured by the turbidimetric method while stirring. In this stirring turbidimetric method, the sample is stirred during the measurement by rotating the stirring bar contained in the glass measurement cell. Then, similarly to the turbidimetric method, the endotoxin amount in the specimen is calculated by the calibration curve method. In the stirring turbidimetric method, measurement can be performed more rapidly and stably than the turbidimetric method by stirring the sample during measurement.

  In addition, there is a method for measuring the phenomenon in which a synthetic substrate for the coagulation enzyme added in the reagent is preliminarily prepared and the synthetic substrate decomposed by the coagulation enzyme develops color, or fluorescence, and further emits light. Is called a colorimetric method, and is widely used as one of important measurement methods for a predetermined physiologically active substance. In the colorimetric method, there is an end point-colorimetric method that utilizes the fact that there is a correlation between the concentration of a predetermined physiologically active substance and the amount of liberated chromophore after a certain reaction time, There is a kinetic-colorimetric method that uses the fact that there is a correlation between the time required for the light absorption or transmittance of the admixture to reach a certain value, or the rate of change with time of color development.

  Further, there is an AL-bound bead method in which a mixed solution containing fine particles having AL bound to the surface (hereinafter also referred to as AL-bound beads) together with a measurement sample and AL is generated and measured by the stirring turbidimetric method. In this AL-bound bead method, turbidity of the sample accompanying the aggregation of AL-bound beads caused by the reaction between the sample and AL and the AL-bound beads is measured and analyzed over time. In this AL-bound bead method, the time when the transmittance increases to a certain value or more after the start of measurement is defined as the gel time. Then, similarly to the stirring turbidimetric method, the endotoxin amount in the specimen is calculated by the calibration curve method. The AL-bound bead method can measure more rapidly than the turbidimetric method and the stirring turbidimetric method described above. This is mainly due to the fact that in the AL-bound bead method, AL-bound beads that are 10 to 100 times larger than coagulin agglutinate, and the change in transmittance becomes sharp.

On the other hand, the mixture of the measurement sample and AL is stirred using, for example, a magnetic stirrer to form gel fine particles. From the intensity of the laser light scattered by the gel particles, the presence of a predetermined physiologically active substance in the sample A laser light scattering particle measurement method (hereinafter also simply referred to as a light scattering method) has been proposed. Gel particles are generated by the reaction between the sample and AL, and when the reaction proceeds and they aggregate with each other, a spike-like peak signal is detected in the scattered light. In the light scattering method, the time point when these peak signals are detected at a certain frequency or higher is set as the aggregation start time. Similar to the gelation time, the aggregation start time has a correlation with the endotoxin amount in the sample, and this is used to calculate the endotoxin amount in the sample by the calibration curve method. The light scattering method can measure more rapidly and with higher sensitivity than the turbidimetric method and the stirring turbidimetric method described above. This is mainly due to the fact that the light scattering method can detect this at the initial stage of gelation, that is, when small gel particles are generated. As the light scattering method, for example, a method of detecting a physiologically active substance such as endotoxin from the increasing rate of the intensity of scattered light and measuring the concentration has been proposed (for example, see Patent Document 1).

  In the light scattering method, the sample is stirred during measurement by rotating the stirring bar contained in the glass sample cell, but the purpose of stirring is different from that of the stirring turbidimetric method. The reason why stirring is necessary in the light scattering method is that the generated gel particles need to be detected as a peak signal across the laser beam. In other words, when stirring is not performed in the light scattering method, for example, when the target particles remain at the same position on the beam for a long time, the apparent scattered light is detected as a peak signal only when it is very high. There is a possibility that the number of gel particles cannot be measured accurately. By stirring, the gel particles do not stay on the beam, but quickly move from the optical axis, and when the particles cross the beam, a scattered light peak signal is generated and the number of gel particles can be measured for the first time.

  Here, as in the light scattering method described above, when the mixture of the AL reagent and the sample is stirred with a stirrer in a glass sample cell, the shape of the reaction curve (number of gel particles (light scattering method)) changes. As a result, there is a disadvantage that the accuracy of the gelation time or the aggregation start time obtained by the measurement is lowered (for example, see Non-Patent Document 1). In order to avoid these inconveniences, each measurement method has been devised to determine the gelation time or aggregation start time so that even if there are some changes in the reaction curve, the measurement results will not be affected. (For example, refer to Patent Document 2).

  However, especially when measuring endotoxin in dilute plasma preparations or water for injection itself, the above-mentioned workaround may not function completely, and erroneous results may be obtained. The cause of the change in the shape of the reaction curve due to agitation has not been identified, and no direct measures have been taken.

JP 2010-32436 A JP 2010-216878 A International Publication No. WO2008 / 038329 Pamphlet JP 2009-150723 A

Takahashi et al., “Problems in clinical application of endotoxin measurement using endotoxin scattering photometry”, 14th Endotoxemia Lifesaving Treatment Study Group Program, Abstracts, p. 29, 2010

  The object of the present invention is to optically measure aggregation or gelation (without stirring the sample) caused by the reaction between AL, which is a blood cell extract of horseshoe crab, and a biologically active substance derived from a living organism. It is to provide a technique for obtaining higher measurement accuracy in a method for measuring biologically active substances derived from living organisms.

  More specifically, when the concentration of a biologically active substance derived from an organism in a sample is measured by a light scattering method (including the AL-bound bead method), the physiologically active substance resulting from stirring of the mixture of the AL reagent and the sample An object is to perform measurement without causing aggregation or gelation not derived from the above, thereby improving the measurement accuracy of the above measurement.

  In the present invention, an AL reagent and a sample containing a biologically active substance are mixed, light is incident on the mixed solution, and based on the intensity of the scattered light or transmitted light, the AL in the mixed solution is derived from the biological material. It detects protein aggregation or gelation caused by reaction with a physiologically active substance. And, the greatest feature is to move the incident position of incident light instead of stirring the admixture.

In more detail, a sample containing AL, which is a blood cell extract of horseshoe crab, and a biologically active substance derived from a predetermined organism is mixed, and protein aggregation resulting from the reaction between AL and the physiologically active substance in the produced mixed liquid or A method for measuring a biologically active substance derived from a living organism by detecting gelation to detect the physiologically active substance in the sample or measuring the concentration of the physiologically active substance,
Injecting light into the mixed solution of the sample and AL and moving the incident position related to the incidence,
Based on the intensity of scattered or transmitted light from the mixture,
The physiologically active substance in the sample is detected or the concentration is measured.

Here, it has become clear that the cause of the phenomenon that the reaction curve between the sample and the AL reagent is changed by stirring the admixture is as follows. That is, when measuring a sample with a very low content of biologically-derived physiologically active substance, (1) the AL reagent and / or the proteins contained in the sample are aggregated by stirring, or (2) stirring. It is found that the shearing stress (physical stimulus) accompanying cleaving causes the proclotting enzyme to be cleaved and activated, changing to the clotting enzyme, and generating aggregates not derived from biologically active substances derived from living organisms. It was.

  Aggregation not derived from biologically active substances derived from living organisms is detected as a signal similar to the generation of gel particles in the light scattering method, thus affecting the shape of the reaction curve (time course) and reducing the measurement accuracy. , There was a risk of erroneous measurement. On the other hand, if we should avoid stirring, this is not the case. If the stirring is stopped, the measurement principle that the generated gel particles cross the laser beam incident on the sample and the scattered light signal exhibits a peak shape as described above does not hold. It will return to the measurement method.

  On the other hand, the present inventors invented a technique for measuring the intensity of scattered light or transmitted light without stirring the admixture that causes the measurement accuracy to be lowered, but without losing the action of stirring. did. That is, according to the present invention, the intensity of the scattered light or transmitted light is acquired while the measurement light is incident into the mixed solution of the sample and the AL reagent and the incident position of the incident light is moved. And based on the intensity | strength change of the acquired scattered light or transmitted light, it decided to detect the bioactive substance in a sample, or measure a density | concentration.

According to this, when detecting or measuring the concentration of biologically active substances derived from living organisms, (1) the AL reagent and / or the proteins contained in the sample are aggregated by stirring, or (2) the stirring is performed. The pro-clotting enzyme is cleaved and activated by the accompanying shear stress (physical stimulus) and changed to the clotting enzyme, preventing the generation of aggregates not derived from biologically active substances derived from living organisms. it can. In addition, when target gel particles are present on the beam, it is possible to suppress the inconvenience of staying at the same position for a long time and generating a peak signal only by the appearance of very high apparent scattered light. As a result, it is possible to obtain higher measurement accuracy in a biologically-derived physiologically active substance measurement method that optically measures aggregation or gelation resulting from the reaction between AL and biologically-derived physiologically active substance. . In addition, as a method of moving the incident position of incident light, the incident light incident direction can be changed by using a galvanometer mirror, a Nipkow disk, a MEMS resonant mirror, or the like.

  In the present invention, the incident position may be moved by allowing light to enter and scan the mixture of the sample and AL. According to this, scattered light or transmitted light from the liquid mixture can be acquired while continuously moving the incident position, and apparent scattered light is extremely high due to the stay of the gel particles more reliably. It is possible to suppress the inconvenience of not forming a peak signal.

In the present invention, the change in the intensity of the scattered light or transmitted light with respect to the intensity of the scattered light or transmitted light when the AL and the sample are mixed or after a predetermined time has passed exceeds a predetermined threshold. From the time until
The physiologically active substance in the sample may be detected or the concentration may be measured.

  That is, when the reaction between the physiologically active substance and AL in the mixture proceeds and the number and size of the fine particles increase, the average intensity of the scattered light or transmitted light obtained while moving the incident position changes. To do. Therefore, from the time until the change in the intensity of the scattered light or transmitted light exceeds the predetermined threshold with respect to the intensity of the scattered light or transmitted light after mixing the AL with the sample or after a predetermined time has elapsed, It is possible to detect or measure the concentration of the physiologically active substance. Thereby, the physiologically active substance in the sample can be detected or the concentration can be measured more reliably. Here, the predetermined threshold is when the intensity of the scattered light or transmitted light exceeds the value of the scattered light or transmitted light after mixing the AL with the sample or after a predetermined time has passed. Is a value of change in light intensity that can be determined that the reaction between the physiologically active substance and AL in the admixture has progressed and the number and size of the fine particles have increased (aggregation start time). Also good.

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

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

The present invention also includes mixing a sample containing AL, which is a blood cell extract of horseshoe crab, and a physiologically active substance derived from a predetermined organism, with a protein resulting from the reaction between AL and the physiologically active substance in the resulting mixture. An apparatus for measuring a biologically active substance derived from a living body, which detects the physiologically active substance in the sample or measures the concentration of the physiologically active substance by detecting aggregation or gelation,
A light incident means for making light incident on the mixed liquid of the sample and AL and moving an incident position related to the incidence after mixing the sample and AL;
A light receiving means for receiving scattered light or transmitted light from the admixture and converting it into an electrical signal;
Derivation means for detecting or deriving the concentration of the physiologically active substance in the sample based on the intensity of the scattered light or transmitted light obtained from the electrical signal;
It is also possible to use a biologically active substance measuring apparatus derived from a living body.

In this case, the light incident means may move the incident position by causing the light to enter and scan the mixed liquid of the sample and AL. Further, the derivation means has a change in intensity of the scattered light or transmitted light that exceeds a predetermined threshold with respect to the intensity of the scattered light or transmitted light when the AL and the sample are mixed or after a predetermined time has elapsed since the mixing. From this time, the physiologically active substance in the sample may be detected or the concentration may be derived. The biologically active substance derived from the organism may be endotoxin or β-D-glucan.

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

  In the present invention, when the concentration of a biologically active substance derived from a living organism in a sample is measured by a turbidimetric method, a light scattering method, or an AL binding bead method, the concentration is caused by stirring of a mixture of the AL reagent and the sample. Measurement can be performed without causing aggregation or gelation not derived from the physiologically active substance, thereby improving the measurement accuracy of the measurement.

It is a figure which shows schematic structure of the light-scattering particle | grain measuring apparatus in Example 1 of this invention. It is a figure for demonstrating the action | operation of the galvanometer mirror apparatus in the light-scattering particle | grain measuring apparatus in Example 1 of this invention. It is a figure for demonstrating the scattered light intensity information obtained by the light-scattering particle | grain measuring apparatus in Example 1 of this invention. It is a figure which shows schematic structure of the light-scattering particle | grain measuring apparatus in Example 2 of this invention. It is a figure which shows schematic structure of the light-scattering particle | grain measuring apparatus in Example 3 of this invention. It is a figure which shows schematic structure of the light-scattering particle | grain measuring apparatus in Example 4 of this invention. It is the schematic for demonstrating the process which AL gelatinizes by an endotoxin or (beta) -D-glucan, and its detection method. It is a figure which shows schematic structure of the conventional light-scattering particle | grain measuring apparatus.

  EMBODIMENT OF THE INVENTION Below, the form for implementing this invention is demonstrated, using drawing. In the following description, endotoxin will be described as an example of the predetermined physiologically active substance. However, the following description is applicable to other physiologically active substances such as β-D-glucan.

[Example 1]
The process by which AL and endotoxin react to form a gel (hereinafter also referred to as the Limulus reaction) is well investigated. That is, as shown in FIG. 7, when endotoxin binds to factor C which is a serine protease in AL, factor C is activated to become active factor C, and active factor C is another serine protease in AL. Factor B is hydrolyzed and activated to obtain activated factor B. This activated factor B immediately hydrolyzes the precursor of the clotting enzyme in AL to form a clotting enzyme, and this clotting enzyme hydrolyzes the coagulogen in AL to produce coagulin. And it is thought that the produced coagulin associates with each other to further generate an insoluble gel, and the entire AL is involved and gelled.

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

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

  Reagents for quantifying endotoxin and β-D-glucan include Limulus reagent made from horseshoe crab blood cell extract (AL: Amoebocyte lysate), and coloring intensity and fluorescence intensity hydrolyzed by coagulase to Limulus reagent. Or a reagent with a synthetic substrate that increases either the chemiluminescence intensity is used. Moreover, a mixed reagent of a recombinant of factor C (recombinant factor C) in a Limulus reagent and its synthetic substrate (regardless of coloring, fluorescence, chemiluminescence) may be used. Furthermore, it is also possible to use a mixed reagent of a recombinant of factor G (recombinant factor G) in a Limulus reagent and its synthetic substrate (regardless of coloring, fluorescence, chemiluminescence).

  The physical quantity that changes due to the reaction between a predetermined physiologically active substance such as endotoxin or β-D-glucan and AL may be selected according to the type of reagent. Detects the light transmittance of the sample or changes in optical physical quantities such as turbidity, scattered light intensity, number of light scattering particles, absorbance, fluorescence intensity, chemiluminescence intensity, or viscosity of the sample as the sample gels, What is necessary is just to detect the change of physical quantities, such as electrical conductivity. For the detection of these physical quantities, turbidimeters, absorptiometers, light scattering photometers, laser light scattering particle measuring instruments, fluorescent photometers, photon counters, and other optical instruments, as well as dedicated measuring devices that apply them, are used. Can be used. Moreover, you may use a viscometer, an electrical conductivity meter, and the measuring apparatus for exclusive use which applied these.

  Examples of the measurement method for quantifying the predetermined physiologically active substance include the stirring turbidimetric method and the light scattering method as described above. As shown in FIG. 7, these measurement methods detect coagulin associated with AL enzyme cascade reaction by detecting the turbidity of the former as the sample and the latter as the number of gel particles generated in the system. Highly sensitive measurement is possible.

  Next, a conventionally used light scattering particle measuring apparatus will be briefly described with reference to FIG. In the conventional light scattering particle measuring apparatus 101, the light emitted from the light source 102 is focused by the incident optical system 103 and enters the sample cell 104. The sample cell 104 holds a mixed solution of the sample to be measured for endotoxin and the AL reagent. The light incident on the sample cell 104 is scattered by the admixture.

  An emission optical system 105 is disposed on the side of the incident optical axis of the sample cell 104. Further, on the extension of the optical axis of the emission optical system 105, there is a light receiving element 106 that receives the scattered light that is scattered by the particles in the mixed liquid in the sample cell 104 and narrowed by the emission optical system 105 and converts it into an electrical signal. Has been placed. The light receiving element 106 includes an amplifying circuit 107 that amplifies the electric signal photoelectrically converted by the light receiving element 106, a filter 108 for removing noise from the electric signal amplified by the amplifying circuit 107, and the electric power after the noise is removed. A calculation device 109 that calculates the number of gel particles from the number of signal peaks, further determines the gelation detection time to derive the endotoxin concentration, and a display 110 that displays the result are electrically connected.

  Further, the sample cell 104 is provided with a stirrer 111 that rotates by applying electromagnetic force from the outside and stirs the mixed liquid as a sample, and a stirrer 112 is provided outside the sample cell 104. ing. Thus, it is possible to adjust the presence / absence of stirring and the stirring speed.

In the above light scattering particle measuring apparatus 101, the appearance time (gelation detection time = gelation time) of the coagulin gel particles, which is the final stage of the Limulus reaction, is measured and established between the endotoxin concentration and the gelation detection time. The endotoxin concentration in the sample is calculated using the calibration relationship. However, actually, the measurement results tend to deviate from the calibration curve in a low concentration region, such as when measuring an endotoxin solution or water for injection prepared at a low concentration. Then, when the determination is made as it is using the above calibration curve, the measured value of endotoxin concentration becomes higher than the original concentration, and there is a possibility that false positive determination occurs.

  The inventors found that the measurement results deviate from the calibration curve in the low concentration region described above, because the non-endotoxin-derived aggregates were generated by stirring the mixed solution of the sample and the AL reagent, and the reaction curve of the Limulus reaction changed. I found out that it was the cause. The inventors have also found that the reason why non-endotoxin-derived aggregates are produced by stirring the mixed solution of the sample and the AL reagent is as follows.

  That is, (1) When measuring a sample with a very low endotoxin concentration, the proteins in the AL reagent, the proteins contained in the sample, or the protein in the AL reagent and the sample are mixed by stirring. Aggregation with contained proteins. (2) The pro-clotting enzyme is cleaved by the shear stress (physical stimulus) accompanying agitation and is pseudo-activated and changed to a clotting enzyme. Alternatively, it has been found that these are both phenomena (1) and (2). In addition, this non-endotoxin-derived agglutination is detected as a signal similar to gel particles in the stirring turbidimetric method and the light scattering method. It has been confirmed by experiments that it causes a decrease.

  In order to solve the above inconvenience, in this embodiment, the mixed solution of the sample and the AL reagent is not stirred, but instead, the incident light incident on the sample cell is scanned and the incident position (measurement) is measured. Change the point), obtain the intensity of scattered light from the moving incident position (measurement point), and detect endotoxin in the sample or measure the concentration of endotoxin by temporal change of the intensity of the scattered light It was.

  In FIG. 1, schematic structure of the light-scattering particle | grain measuring apparatus 1 in a present Example is shown. In the light scattering particle measuring apparatus 1, a laser light source is used as the light source 2, but an ultra-high brightness LED or the like may be used. Incident light emitted from the light source 2 is narrowed by the lens system 3 and then enters the mirror 5 b of the galvanometer mirror device 5. The galvanometer mirror device 5 includes a motor 5a and a mirror 5b fixed to the output shaft 5c of the motor 5a.

  Although the incident light is reflected by the mirror 5b, the angle θ1 of the mirror 5b is changed by driving the motor 5a as shown in FIG. 2, and therefore the angle θ2 of the incident light after being reflected by the mirror 5b. Changes by controlling the motor 5a. In the present embodiment, the output shaft 5c of the motor 5a is arranged in the horizontal direction, and the motor 5a is controlled to swing the mirror 5b, so that the incident light after being reflected by the mirror 5b is Reciprocates in the vertical direction (perpendicular to the paper surface in the figure). Since the incident light is incident on the sample cell 6 in which the mixed solution 7 of the sample to be measured for endotoxin and the AL reagent is held, the incident light passes through the mixed solution 7 in the sample cell 6 in the height direction. It is controlled to scan in the direction (perpendicular to the page).

Incident light is incident on the admixture 7 in the sample cell 6 and is scattered by fine particles (coagulogen monomer, measurement object such as coagulogen oligomer) in the admixture. The scattered light is received by the light receiving element 11 through the lens system 8, the mask 9, and the lens system 10. The light receiving element 11 is composed of, for example, a photodiode (PD). A signal processing circuit 12 is electrically connected to the light receiving element 11, and the scattered light intensity converted into an electric signal in the light receiving element 11 is amplified and filtered by the signal processing circuit 12 and then the arithmetic unit 13. Is input. In the arithmetic unit 13, the presence / absence of endotoxin or the concentration of endotoxin is calculated based on the scattered light intensity signal. Further, the value of the scattered light intensity and the calculation result are recorded. The arithmetic device 13 may be constituted by a personal computer. The scattered light intensity value and calculation result recorded by the calculation device 13 are appropriately displayed on the display device 16.

  In the above, the light incident means includes the light source 2, the lens system 3, and the galvanometer mirror device 5. The light receiving means includes the light receiving element 11. The derivation means includes the arithmetic device 13.

  Next, the scattered light intensity data obtained by one scan of incident light will be described with reference to FIG. As shown in FIG. 3A, the mask 9 provided in the optical path blocks light in the periphery of the scanning range, and the range in which the light receiving element 11 receives scattered light during scanning is regulated. Data obtained by the light receiving element 11 is, for example, as shown in FIGS. FIG. 3B shows a scattered light intensity signal when the AL reagent is mixed with the sample (when the sample cell 6 is set in a sample cell holder (not shown)). FIG. 3C shows a scattered light intensity signal obtained after a certain amount of time has elapsed after mixing the sample and the AL reagent. The scattered light intensity corresponding to the mask portion is the background level A, and the difference between the background level A and the scattered light intensity B or C from the mixture 7 becomes the actual scattered light intensity.

  In the present embodiment, the time when the AL reagent is mixed into the sample (the time when the sample cell 6 is mixed and set in the sample cell holder (not shown)) is set as the measurement start time. Scanning of incident light by the galvanometer mirror device 5 is continuously performed after the start of measurement, and scattered light intensity data is accumulated in the arithmetic device 13 each time. When a sample containing endotoxin is mixed with an AL reagent, a coagulin monomer is formed by the reaction between endotoxin and AL, and eventually coagulates and crosslinks with each other to form a gel network that gels. Go.

  Therefore, in this embodiment, when time elapses from the stage of FIG. 3 (b) from the start of measurement, the state shown in FIG. 3 (c) is obtained. The peaks in the waveforms shown in FIGS. 3B and 3C indicate the scattered light signal from the aggregate that has become larger due to aggregation. The actual scattered light intensity value in FIGS. 3B and 3C (the value obtained by subtracting the background level A from the scattered light intensity B in FIG. 3B or the scattered light intensity in FIG. 3C). The value obtained by subtracting the background level A from the light intensity C) indicates the intensity of the scattered light due to coagulin monomer aggregation, and increases with time.

  3B and 3C, the size of the monomer aggregation is smaller than the wavelength of light, so the aggregation is not yet recognized as particles and the scattered light is considered to be due to Rayleigh scattering. . Therefore, at this stage, rather than the number and height of the peaks increasing, the scattered light intensity increases on average over time. In this example, it is necessary that the difference between the scattered light intensity B at the start of measurement obtained in FIG. 3B and the subsequent scattered light intensity C obtained in FIG. 5C exceeds the threshold value. The endotoxin concentration is determined from the measured time (aggregation start time).

  More specifically, a calibration curve was prepared in advance by determining the relationship between the aggregation start time and endotoxin concentration, and the endotoxin concentration (presence / absence of presence) was determined based on the aggregation start time and calibration curve in the actually measured sample. Measure).

As described above, in the present invention, after mixing the sample and the AL reagent and starting the measurement, instead of stirring the mixture, the incident light for measurement is scanned to determine the incident position (measurement point). I decided to move it. Therefore, the inconvenience that the target gel particles stay on the beam for a long time and the apparent scattered light is only very high and no peak signal is formed can be suppressed, and the measurement accuracy can be improved. it can. At the same time, it is possible to prevent the occurrence of aggregation derived from non-endotoxin due to stirring of the mixed solution, and it is possible to suppress a decrease in measurement accuracy due to a change in the shape of the reaction curve (time course).

  Moreover, in the conventional light scattering method, since the scattered light from the said particle was acquired when the gel particle became a size of about several tens of μm, a peak was observed in the scattered light from the particle from the start of measurement. There was an inconvenience that some time was required before starting. On the other hand, in the present invention, the degree of change in scattered light when the coagulin monomer of about several nanometers aggregates to a size of about several tens to several hundreds of nanometers (length shorter than the wavelength). Therefore, it is possible to measure the endotoxin concentration in the sample specimen at an earlier stage than before.

  In the above example, the time when the AL reagent was mixed in the sample (the time when the sample cell 6 was mixed and set in the sample cell holder (not shown)) was set as the start of measurement. I can't. The time after elapse of a predetermined time (for example, 10 seconds, 1 minute, etc.) from the time when the AL reagent is mixed with the sample may be set as the measurement start time. For this, it is possible to define a convenient time for performing the measurement.

[Example 2]
Next, a second embodiment of the present invention will be described. In this embodiment, an example in which incident light is scanned two-dimensionally by two galvanometer mirror devices will be described. In the present embodiment, only differences from the other embodiments will be described, and the same components as those in the other embodiments will be denoted by the same reference numerals and description thereof will be omitted.

  In FIG. 4, schematic structure of the light-scattering particle | grain measuring apparatus 20 in a present Example is shown. The difference between the light scattering particle measuring device 20 and the light scattering particle measuring device 1 shown in Example 1 is that the light scattering particle measuring device 20 includes a galvanometer mirror device 15 in addition to the galvanometer mirror device 5, and the total The point is that two galvanometer mirrors are provided.

  The galvanometer mirror device 5 has a function of scanning incident light in the height direction of the sample cell 6 (perpendicular to the paper surface) as in the first embodiment. On the other hand, the galvanometer mirror device 15 has a function of scanning incident light in the width direction of the sample cell 6 (direction parallel to the paper surface). Therefore, by controlling the galvanometer mirror device 5 and the galvanometer mirror device 15 in synchronization, it is possible to scan incident light two-dimensionally in the width direction and height direction of the sample cell 6. Thereby, the incident light can be scanned over the entire mixture 7 in the sample cell 6, and the incident position (measurement point) can be moved in a wider range. As a result, information on the average scattered light intensity from a wider range can be obtained, and the measurement accuracy can be improved.

Example 3
Next, Embodiment 3 of the present invention will be described. In this embodiment, an example in which a Niipou disc is used to scan incident light will be described. FIG. 7 shows a schematic configuration of the light scattering particle measuring device 30 in the present embodiment. In the light scattering particle measuring device 30 in the present embodiment, the Nipkow disk device 25 is used as a mechanism for scanning incident light instead of the galvanometer mirror device. In this embodiment, only differences from the other embodiments will be described, and the same components as those in the other embodiments will be denoted by the same reference numerals and description thereof will be omitted.

In the nipou disc device 25, the motor 25a rotates the nipou disc 25b provided with pinholes (not shown) distributed in a spiral shape. Since the Niipou disk 25b is provided with a plurality of spiral pinholes, the measurement point can be scanned two-dimensionally in the sample cell 6 by rotating the pinhole disk 25b. The drive control of the Niipou disc device 25 is performed based on a command from the arithmetic device 13. The rotation angle information of the Niipou disc 25b is detected by a sensor such as a rotary encoder (not shown) and is input to the arithmetic unit 13.

  In the present embodiment, the light emitted from the light source 2 passes through the lens system 3 and is condensed and condensed on the incident end face 21 a of the optical fiber 21. Incident light propagates through the optical fiber 21 and exits from the exit end face 21 b of the optical fiber 21. The light emitted from the optical fiber 21 is narrowed by the lens system 22. Then, after passing through the aperture 23, the beam diameter is further expanded by being converted into parallel light by the lens system 24. This expanded incident light irradiates the pinhole distribution region in the Niipou disc 25b. Incident light that has passed through the pinhole on the Nipkow disk 25 b is collected by the lens system 27 into the mixture 7 in the sample cell 6. Since the position of the pinhole irradiated with the enlarged incident light apparently moves with the rotation of the Nipkow disc 25b, the incident position (measurement point) where the incident light is condensed in the mixture 7 also moves. Become. The processing of the scattered light scattered in the admixture 7 is the same as that in the first embodiment, and the description thereof is omitted here.

  As described above, in this embodiment, the Nipkow disk device 25 is used to scan the incident light in the mixture 7 in the sample cell 6. Therefore, the incident light can be scanned two-dimensionally with a simpler mechanism and simple control, and the incident position (measurement point) can be changed two-dimensionally in the liquid mixture 7.

Example 4
Next, a fourth embodiment of the present invention will be described. In the present embodiment, an example in which a two-dimensional scanning device using MEMS (Micro Electro Mechanical Systems) is used to scan incident light in the mixture 7 in the sample cell 6 will be described. In this embodiment, only differences from the other embodiments will be described, and the same components as those in the other embodiments will be denoted by the same reference numerals and description thereof will be omitted.

  In FIG. 6, schematic structure of the light-scattering particle | grain measuring apparatus 40 in a present Example is shown. In the light scattering particle measuring apparatus 40, incident light emitted from the optical fiber 21 is focused by a lens system 31 and irradiated onto a mirror 32a of a DMD (Digital Micromirror Device) 32 as an example of a MEMS (Micro Electro Mechanical Systems). Is done. The mirror 32a is coupled to the base 32b by a horizontal hinge 32c and a vertical hinge 32d, and can be tilted in the horizontal direction and the vertical direction by electrostatic attraction generated by voltage application from the outside.

  The DMD 32 is driven based on a command signal from the arithmetic unit 13 to control the tilt of the mirror 32a and to control the direction of light reflected from the mirror 32a. The light reflected from the mirror 32 a is narrowed down by the lens system 33 and collected in the mixture 7 in the sample cell 6. And the incident position (measurement point) which is the condensing point can be changed two-dimensionally by control of the mirror 32a, and incident light can be scanned two-dimensionally.

  As described above, in this embodiment, the DMD 32 is used to scan the incident light in the mixture 7 in the sample cell 6. Since the DMD 32 can move at a very high speed, incident light can be scanned two-dimensionally at a very high speed by a simple mechanism and simple control.

  In addition, although an example in which the mirror 32a exists alone has been described with reference to FIG. 6, a DMD 32 in which a plurality of mirrors 32a are arranged on the base 32b may be used. In this case, by controlling the plurality of mirrors 32a independently, it becomes possible to scan a plurality of incident lights in the mixed liquid 7 at a time with multiple channels. As a result, more scattered light intensity information can be acquired at once, and the measurement can be speeded up.

  In the above embodiment, the example of detecting endotoxin or measuring the concentration based on the intensity of scattered light by the mixture 7 in the sample cell 6 has been described, but the intensity of transmitted light is used instead of the intensity of scattered light. Based on this, endotoxin detection or concentration measurement may be performed. That is, in this case, as the reaction between endotoxin and AL proceeds, it is detected that the transmitted light decreases as the scattered light increases, and the time until the decrease in the transmitted light exceeds a predetermined threshold value. Based on the above, endotoxin may be detected or the concentration may be measured. All of the above examples are applicable when endotoxin detection or concentration measurement is performed based on the intensity of transmitted light.

  In the above-described embodiments, the example in which the incident position is moved by scanning the incident light has been described. However, the method for moving the incident position of the incident light is not limited to scanning. For example, incident light may be incident intermittently at random positions. Even in such a method and apparatus, it is possible to obtain the same effect as in the above embodiment.

DESCRIPTION OF SYMBOLS 1 ... Light-scattering particle measuring device 2 ... Light source 3 ... Lens system 5 ... Galvanometer mirror device 6 ... Sample cell 7 ... Mixture 8 ... Lens system 9 ... Mask DESCRIPTION OF SYMBOLS 10 ... Lens system 11 ... Light receiving element 12 ... Signal processing circuit 13 ... Arithmetic device 15 ... Galvanometer mirror device 16 ... Display device 20 ... Light-scattering particle measuring device 21 ... Optical fiber 22 ... Lens system 23 ... Aperture 24 ... Lens system 25 ... Nipou disk device 27 ... Lens system 30 ... Light scattering particle measuring device 31 ... Lens system 32 ...・ DMD
33 ... Lens system 40 ... Light scattering particle measuring device

Claims (8)

Samples containing AL, which is a blood cell extract of horseshoe crab, and a biologically active substance derived from a predetermined organism are detected, and protein aggregation or gelation resulting from the reaction between AL and the physiologically active substance in the resulting mixture is detected. A method for measuring a biologically active substance derived from an organism, wherein the physiologically active substance in the sample is detected or the concentration of the physiologically active substance is measured,
Injecting light into the mixed solution of the sample and AL and moving the incident position related to the incidence,
Based on the intensity of scattered or transmitted light from the mixture,
A method for measuring a biologically active substance derived from an organism, wherein the physiologically active substance in the sample is detected or the concentration is measured.   2. The method for measuring a biologically active substance derived from an organism according to claim 1, wherein the incident position is moved by causing light to enter and scan the mixed solution of the sample and AL. From the time until the change in the intensity of the scattered light or transmitted light exceeds the predetermined threshold with respect to the intensity of the scattered light or transmitted light when the AL and the sample are mixed or after a predetermined time has elapsed since the mixing,
The method for measuring a biologically active substance derived from an organism according to claim 2, wherein the physiologically active substance in the sample is detected or the concentration is measured.   The method for measuring a biologically active substance of biological origin according to any one of claims 1 to 3, wherein the biologically active substance of biological origin is endotoxin or β-D-glucan. Samples containing AL, which is a blood cell extract of horseshoe crab, and a biologically active substance derived from a predetermined organism are detected, and protein aggregation or gelation resulting from the reaction between AL and the physiologically active substance in the resulting mixture is detected. A biologically-derived physiologically active substance measuring device for detecting the physiologically active substance in the sample or measuring the concentration of the physiologically active substance,
A light incident means for making light incident on the mixed liquid of the sample and AL and moving an incident position related to the incidence after mixing the sample and AL;
A light receiving means for receiving scattered light or transmitted light from the admixture and converting it into an electrical signal;
Derivation means for detecting or deriving the concentration of the physiologically active substance in the sample based on the intensity of the scattered light or transmitted light obtained from the electrical signal;
An apparatus for measuring biologically active substances derived from living organisms, comprising:   6. The biologically active substance measuring apparatus according to claim 5, wherein the light incident means moves the incident position by causing light to enter and scan the mixed solution of the sample and AL. .   The derivation unit is configured to change the intensity of the scattered light or transmitted light with respect to the intensity of the scattered light or transmitted light when the AL is mixed with the sample or after a predetermined time has elapsed after the mixing exceeds a predetermined threshold. The biologically active substance measuring apparatus according to claim 6, wherein the biologically active substance in the sample is detected or the concentration is derived from time.   The biologically active substance-derived biologically active substance measuring apparatus according to any one of claims 5 to 7, wherein the biologically active substance derived from an organism is endotoxin or β-D-glucan.

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