JP2012154815A - Method and apparatus for measuring biogenous physiologically active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the accuracy of a measurement when measuring the density of a biogenous physiologically active substance in a specimen by a stirring turbidimetric method, a light scattering method, or an AL-bound beads method, by suppressing aggregation and gelation that is not derived from the physiologically active substance but caused by stirring of liquid mixture.SOLUTION: When a protein aggregation derived from a reaction between AL and a biogenous physiologically active substance is detected by mixing AL and a specimen including the biogenous physiologically active substance while stirring the liquid mixture, a heat-treated predetermined protein is previously added to the liquid mixture so as to suppress the protein aggregation or gelation that is not derived from the reaction between the AL and the biogenous physiologically active substance in the liquid mixture.

Description

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

  Endotoxin is a lipopolysaccharide present in the cell wall of Gram-negative bacteria and is the most typical pyrogen. If an infusion solution, injection drug, blood, or the like contaminated with this endotoxin enters the human body, it may cause serious side effects such as fever and shock. For this reason, it is obliged to manage the above drugs so that they are not contaminated by endotoxin. 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, a blood cell extract of horseshoe crab (hereinafter also referred to as “AL: Amoebocyte lysate”).
) Include serine proteases activated by endotoxin, β-D-glucan, and the like. When AL reacts with endotoxin or β-D-glucan, coagulogen present in AL is hydrolyzed into coagulin by an enzyme cascade by serine protease activated according to the amount of AL. 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.

  There is also 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 the 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.

  Furthermore, 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.

  Even in the light scattering method, the sample is stirred during the measurement by rotating the stirring bar contained in the glass sample cell, but the purpose of stirring is different from the stirring turbidimetric method. The peak signal is detected when the gel particles cross the laser beam incident on the specimen. The gel particles are moved by stirring so that the gel particles can cross the laser beam. 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.

On the other hand, there is a method for measuring a phenomenon in which a synthetic substrate for a coagulation enzyme added in a reagent is previously stored, 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, or the concentration of a predetermined physiologically active substance. 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.

  Here, when the mixture of the AL reagent and the sample is stirred with a stirrer in a glass sample cell as in the stirring turbidimetric method and the light scattering method, the reaction curve (transmittance / absorbance with respect to time (stirring ratio) is used. (Turbidity method) or the number of gel particles (light scattering method)) changes, and the accuracy of the gelation time or aggregation start time obtained by measurement is reduced (for example, , See Non-Patent Document 1.) In order to avoid these problems, 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 are not affected. (For example, refer to Patent Document 1).

  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 2010-216878 A JP 2004-061314 A JP-A-10-293129 International Publication No. WO2008 / 038329 Pamphlet JP 2009-150723 A

Manabu Takahashi, Shigehiro Shibata, Yasushi Suzuki, Masahiro Oga, Naoya Matsumoto, Tatsuyori Shodoshima, Junya Inada, Shigetatsu Endo, “Problems in Endotoxin Measurement Using Endotoxin Scattering Photometry”, Endotoxemia Lifesaving Journal of Therapeutic Research Society, Vol. 14, No. 1, p. 111-119, 2010

  An object of the present invention is to provide a method for measuring a biologically active substance derived from a living organism by optically measuring aggregation or gelation resulting from the reaction between AL, which is a blood cell extract of horseshoe crab, and the biologically active substance derived from the living organism. In other words, a technique for obtaining higher measurement accuracy is provided.

  More specifically, it occurs due to the stirring of the mixture of the AL reagent and the sample when the concentration of the biologically active substance derived from the organism in the sample is measured by the stirring turbidimetric method, the light scattering method, or the AL binding bead method. An object of the present invention is to improve the measurement accuracy of the above measurement by suppressing aggregation or gelation not derived from the physiologically active substance.

The present invention mixes an AL reagent and a sample containing a biologically active substance derived from a living organism, stirs the mixed solution, and aggregates or gels a protein resulting from the reaction between the AL and the biologically active substance derived from the living organism in the mixed solution. When detecting crystallization, by adding a predetermined protein that has been heat-treated in advance to the mixture, protein aggregation or gelation is not caused by the reaction between AL in the mixture and the biologically active substance derived from the organism. The greatest feature is suppression.

More specifically, a mixed solution of a sample containing AL, which is a blood cell extract of horseshoe crab, and a biologically active substance derived from a predetermined organism is generated, and while stirring the mixed liquid, the AL and the physiologically active substance in the mixed liquid are mixed. This is a method for measuring a biologically active substance derived from a living body, wherein the biologically active substance in the sample is detected or the concentration of the physiologically active substance is measured by detecting protein aggregation or gelation caused by the reaction of And
When the agitation is performed, a predetermined protein that has been heat-treated in advance is added to the admixture to suppress aggregation or gelation that is not derived from the physiologically active substance in the admixture. .

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 the biologically active substance derived from this organism is detected as a signal similar to the generation of gel particles in both the stirring turbidimetric method and the light scattering method, so that the shape of the reaction curve (time course) is obtained. There was a risk that the measurement accuracy would be reduced, resulting in erroneous measurement. On the other hand, the inventors' diligent research has suppressed the induction of aggregation or gelation not derived from the above-mentioned biologically active substances by adding a predetermined heat-treated protein when mixing the sample and the AL reagent. It was found that it was possible. Furthermore, it was confirmed by experiments that the addition of this heat-treated predetermined protein to the sample had no effect on the measurement of aggregation or gelation derived from biologically active substances derived from living organisms.

  According to the present invention, when measuring aggregation or gelation in a mixed solution of a sample having a low biological bioactive substance content and an AL reagent with stirring, the bioactive bioactive substance by stirring is used. Aggregation or gelation which is not caused can be suppressed. As a result, it is possible to improve accuracy in measurement of biologically active substances derived from living organisms.

  In the present invention, the predetermined protein subjected to the heat treatment in advance may be at least one of albumin, globulin, and lysozyme. These proteins have been confirmed to be able to suppress the induction of aggregation or gelation not derived from biologically active substances derived from living organisms by being added at the time of mixing the sample and the AL reagent. Aggregation or gelation not caused by biologically active substances derived from living organisms can be suppressed. In addition, it is thought that many types, such as protein which exists in human plasma, animal and plant, or an insect protein, can be used as a protein which can be used for this invention, for example. For convenience of explanation, this document will focus on albumin.

  In the present invention, when the predetermined protein subjected to the heat treatment is albumin, the final concentration when added to the mixture may be 0.015% or more and 10% or less. This can more effectively suppress aggregation or gelation that is not caused by a biologically active substance derived from a living organism due to stirring. The final concentration is preferably 0.03% or more and 5% or less. More desirably, it is 0.05% or more and 0.5% or less.

In the present invention, the temperature of the heat treatment previously applied to a predetermined protein is desirably 70 ° C. or higher. The heat treatment time is preferably 10 minutes or longer. In addition, as the heat treatment previously applied to a predetermined protein, an autoclave sterilization treatment may be performed, or a heat treatment using a device such as an aluminum block heater may be performed. By these heat treatments, aggregation or gelation that is not caused by biologically active substances derived from organisms due to stirring can be more reliably suppressed than when a protein that is not subjected to heat treatment is added.

  The temperature of the autoclave sterilization is generally 120 ° C. or higher. By setting it as this temperature range, the aggregation or gelation which is not caused by the biologically active substance derived from the organism by stirring can be suppressed more reliably. More specifically, the autoclave sterilization temperature is preferably 120 ° C. or higher and 300 ° C. or lower, more preferably 120 ° C. or higher and 260 ° C. or lower.

  Moreover, when heat processing is performed by the said autoclave sterilization process, it is good for the time of heat processing to be 10 minutes or more. By setting it as this time range, the aggregation or gelation which is not attributed to the biologically active substance derived from the organism by stirring can be suppressed more reliably. In more detail, it is desirable to set it as 10 minutes or more and 120 minutes or less. By setting this time range, it is possible to more reliably suppress aggregation or gelation that is not caused by biologically active substances derived from organisms due to stirring, and more reliably biologically active substances derived from organisms that contaminate proteins. Can be inactivated.

  In addition, even when heating at 95 ° C., which is lower than 120 ° C., using an aluminum block heater, by heating for 10 minutes or more, aggregation or gelation not caused by biologically active substances derived from organisms by stirring is achieved. The inhibitory effect has been confirmed. Therefore, when heat treatment is performed using an aluminum block heater, the temperature of the heat treatment is preferably 95 ° C. or higher. More specifically, the temperature may be 95 ° C. or higher and 260 ° C. or lower. The heating time in the heat treatment is preferably 10 minutes or more. More specifically, it may be 10 minutes or more and 120 minutes or less. By adopting the heat treatment conditions as described above, aggregation or gelation that is not caused by a biologically active substance derived from a living body by stirring can be more reliably suppressed.

  However, in the present invention, the above conditions are not particularly limited as long as the protein concentration, the heating temperature, and the heating time can suppress the aggregation or gelation not caused by the biologically active substance derived from the organism by stirring more effectively. . For example, by performing autoclave sterilization for a long time even at a lower temperature, such as at 90 ° C. for 3 hours, the aggregation or gelation not caused by biologically active substances derived from organisms due to stirring is suppressed, and the protein is It is possible to sufficiently inactivate physiologically active substances derived from contaminated organisms.

  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.

In addition, the present invention includes AL, which is a blood cell extract of horseshoe crab, generates a mixed solution with a sample containing a biologically active substance derived from a predetermined organism, and agitates the mixed liquid while causing the physiologically active substance to A reagent for measuring a biologically active substance derived from a living organism for detecting the physiologically active substance in the sample or measuring the concentration of the physiologically active substance by detecting aggregation or gelation of the protein A reagent for measuring a biologically active substance derived from a living organism, characterized in that a predetermined protein that has been heat-treated in advance is added, and aggregation or gelation not derived from the physiologically active substance in the mixed solution can be suppressed. Also good. In that case, the predetermined protein subjected to the heat treatment in advance may be at least one of albumin, globulin, and lysozyme.

In addition, the present invention maintains a mixed solution of a sample containing a physiologically active substance derived from a predetermined organism and AL, which is a blood cell extract of horseshoe crab, in a state where light can be incident and promotes a reaction in the mixed solution. A mixture holding means;
Stirring means for stirring the mixed liquid in the mixed liquid holding means;
A light incident means for making light incident on the mixed liquid in the mixed liquid holding means;
A light receiving means for receiving transmitted light or scattered light in the admixture of the incident light and converting it into an electrical signal;
Determination means for determining the reaction start time of the physiologically active substance and AL in the sample from the electrical signal converted in the light receiving means;
Measurement of a biologically active substance derived from an organism, comprising: a derivation 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. A device,
An apparatus for measuring a biologically active substance derived from a living organism, further comprising protein addition means for adding a predetermined protein that has been previously heat-treated to the admixture when the admixture is agitated by the agitation means. There may be.

  According to this apparatus, when the mixed solution of the sample and the AL reagent is stirred, a predetermined protein that has been previously heat-treated can be automatically added to the mixed solution. Protein aggregation or gelation not derived from the physiologically active substance in can be suppressed.

  Also in this apparatus, the protein added by the protein addition means may be at least one of albumin, globulin, and lysozyme. When the protein to be added is albumin, the final concentration when added to the mixture is preferably 0.015% or more and 10% or less. Moreover, you may perform an autoclave sterilization process, for example as heat processing performed beforehand to the protein added. In that case, the temperature of the autoclave sterilization treatment may be 120 ° C. or more, and the time of the autoclave sterilization treatment may be 10 minutes or more. Moreover, you may perform the heat processing using apparatuses, such as an aluminum block heater, as the heat processing given beforehand to the protein added, for example. In that case, the temperature of the heat treatment is preferably 95 ° C. or higher. The heating time in the heat treatment is preferably 10 minutes or more. Furthermore, the biologically active substance derived from a living body 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 stirring turbidimetric method, a light scattering method, or an AL binding bead method, By suppressing aggregation or gelation not derived from a physiologically active substance, the measurement accuracy of the above measurement can be improved.

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 graph which shows the relationship between the endotoxin density | concentration in a sample and gelation detection time, and a calibration curve conventionally. It is a graph which shows the relationship between the density | concentration of the human serum albumin (HSA) added to the liquid mixture of a sample and AL reagent, and gelation detection time. It is a figure which shows the calibration curve and correlation coefficient when HSA is added and when HSA is not added. It is a graph which shows the relationship between stirring rotation speed and gelation detection time. It is a figure which shows schematic structure of the turbidimetric 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 turbidimetric measuring apparatus in Example 2 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.

<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. 9, 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. 9, these measurement methods detect the coagulin aggregation produced by the enzyme cascade reaction of AL as the turbidity of the sample, and the latter as the number of gel fine particles produced in the system. This enables highly sensitive measurement.

  Next, a light scattering particle measuring apparatus used for acquiring scattered light in this embodiment will be described. 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. In FIG. 1, schematic structure of the light-scattering particle | grain measuring apparatus 1 as an endotoxin measuring apparatus in a present Example is shown. Although a laser light source is used as the light source 2 used in the light scattering particle measuring apparatus 1, an ultra-high brightness LED or the like may be used. The light emitted from the light source 2 is focused by the incident optical system 3 and enters the sample cell 4. The sample cell 4 holds a mixture of a sample to be measured for endotoxin and the AL reagent. The light incident on the sample cell 4 is scattered by particles (coagulogen monomer, measurement object such as coagulogen oligomer) in the mixture.

  An emission optical system 5 is arranged on the side of the incident optical axis of the sample cell 4. In addition, on the extension of the optical axis of the output optical system 5, there is a light receiving element 6 that receives scattered light that is scattered by particles in the mixed liquid in the sample cell 4 and is narrowed by the output optical system 5 and converts it into an electrical signal. Has been placed. The light receiving element 6 includes an amplifying circuit 7 for amplifying the electric signal photoelectrically converted by the light receiving element 6, a filter 8 for removing noise from the electric signal amplified by the amplifying circuit 7, and the electricity after the noise is removed. A calculation device 9 for calculating the number of gel particles from the number of signal peaks, further determining the gelation detection time to derive the endotoxin concentration, and a display 10 for displaying the result are electrically connected.

  Further, the sample cell 4 is provided with a stirrer 11 that rotates by applying electromagnetic force from the outside and stirs the mixed liquid as a sample, and a stirrer 12 is provided outside the sample cell 4. ing. Thus, it is possible to adjust the presence / absence of stirring and the stirring speed.

  Here, the sample cell 4 corresponds to the admixture holding means of the present embodiment. The light source 2 and the incident optical system 3 correspond to light incident means. The stirrer 11 and the stirrer 12 correspond to stirring means. The emission optical system 5 and the light receiving element 6 correspond to a light receiving means. The arithmetic device 9 corresponds to determination means and derivation means.

  In the light scattering particle measuring apparatus 1 described above, 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. For example, the gelation detection time when measuring endotoxin solutions of 0.01, 0.001, and 0.0001 EU / mL (1 EU / mL≈100 to 200 pg / mL) is 20 (depending on the type and lot of the AL reagent). It becomes about -30, 40-60 and 80-150 minutes, and a linear calibration curve is obtained on the log-log graph.

  However, in actuality, as shown in FIG. 2, even when measuring an endotoxin solution or water for injection prepared at a concentration lower than 0.0001 EU / mL, gelation was detected in about 80 to 150 minutes, and low In the concentration region, the measurement results tended to deviate from the calibration curve. Therefore, if the determination is made as it is using the above calibration curve, the measured value of endotoxin concentration becomes higher than the original concentration, which may cause false positive determination. The AL reagent used for the measurement in FIG. 2 is ES-II Single Test Wako (manufactured by Wako Pure Chemical Industries).

This is because (1) the protein is denatured and insoluble aggregates are generated by stimulation of stirring by the stirrer 11 and the stirrer 12, and (2) the coagulation enzyme is activated and coagulin gel is generated by stimulation of stirring. It has been clarified by the inventors' investigation that it is caused by the formation of aggregates (gel particles) not derived from endotoxin. Therefore, in order to accurately measure a low concentration of endotoxin of 0.0001 EU / mL or less and prevent false positive determination, it is necessary to suppress aggregation that is not derived from the endotoxin.

  In contrast, the inventors' diligent research has revealed that addition of human serum albumin (HSA) can suppress aggregation not derived from endotoxin. In this example, an endotoxin-free human serum albumin (HSA) solution (autoclaved) was used as an endotoxin detection extract (Wako 293-51601: manufactured by Wako Pure Chemical Industries). The inhibitory effect was investigated. In this measurement, ES-II Single Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the AL reagent. In the above, the autoclave sterilization treatment corresponds to a heat treatment performed in advance.

  The result of the measurement is shown in FIG. The horizontal axis in FIG. 3 is the HSA concentration in the mixture, and the vertical axis is the gelation detection time. As can be seen from FIG. 3, when HSA was included, the gelation detection time tended to be delayed as compared with the case where HSA was not included. In particular, when the concentration of HSA is 0.125% to 0.25%, the gelation detection time is remarkably delayed. Thereby, it was suggested that aggregation not derived from endotoxin can be suppressed by adding an appropriate amount of HSA such as an HSA concentration of 0.015% or more and 10% or less.

  As described above, it was recognized that aggregation not derived from endotoxin was suppressed by adding HSA to the mixture, and the gelation detection time of the endotoxin solution was measured with HSA added. A calibration curve was created. FIG. 4 shows calibration curves when HSA is added and when HSA is not added. FIG. 4A shows a calibration curve when HSA is added, and FIG. 4B shows a calibration curve when HSA is not added. In this measurement, ES-II single test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the AL reagent. 4A, 10 μL of endotoxin detection extract (Wako293-51601: manufactured by Wako Pure Chemical Industries, Ltd.) is added to the AL reagent together with 200 μL of each concentration of endotoxin solution, and the HSA concentration in the mixture is determined. The content was 0.24%.

  As can be seen from FIG. 4, when HSA is added, the endotoxin concentration is in the range of 0.01 to 0.0001 EU / mL, and the gelation detection time when HSA is added does not become slower than when it is not added. It was. This indicates that HSA does not inhibit the Limulus reaction. When HSA was added, a calibration curve having a good linearity with a correlation coefficient of 0.992 in the range of 1 to 0.00001 EU / mL could be obtained. This is considered to be an effect of suppressing aggregation not derived from endotoxin by addition of HSA. In the above, human serum albumin (HSA) solution (autoclaved) was used as an extract for endotoxin detection, and the effect of suppressing aggregation caused by endotoxin was investigated. The same effect was confirmed by heating (Albuminer, CSL Behring). Moreover, since globulin or lysozyme is a protein having properties similar to albumin, it is considered that the same effect as described above can be obtained when these are used.

Next, since the aggregation not derived from the endotoxin is considered to be caused by the stirring of the mixed solution by the stirrer 11 and the stirrer 12, it was studied to reduce the number of rotations of stirring with respect to the mixed solution. Usually, the gelation detection time of water for injection was measured while changing the number of revolutions of stirring at 1000 rpm between 600 and 1200 rpm. The measurement results are shown in FIG. In FIG. 5, the horizontal axis represents the stirring rotation speed of the stirrer 11. The vertical axis represents the gelation detection time. As can be seen from the figure, the gelation detection time was not significantly delayed even when the stirring rotation speed of the stirrer 11 was changed. As a result, it was found that the effect of reducing the number of stirring revolutions cannot be expected as a measure for suppressing aggregation not derived from endotoxin.

  In the above embodiment, the example in which the present invention is applied to the case where the endotoxin concentration is measured by the light scattering method using the light scattering particle measuring apparatus 1 is described. However, the present invention is applied to the light scattering. Of course, not only by law. For example, you may apply when measuring an endotoxin density | concentration by the stirring turbidimetric method. Below, the turbidimetric measuring apparatus used for the stirring turbidimetric method will be described.

  FIG. 6 shows a schematic configuration of a turbidimetric measurement apparatus 21 as another example of the endotoxin measurement apparatus of the present embodiment. In this turbidimetric measuring device 21, endotoxin is measured by a stirring turbidimetric method. Also in the turbidimetric measurement device 21, the prepared sample containing diluted series of endotoxin is transferred to a measuring glass container (hereinafter referred to as cuvette) 22 as a mixed liquid holding means. A warmer 25 is provided so as to surround the cuvette 22. A heating wire (not shown) is provided inside the heat insulator 25, and the cuvette 22 is kept at about 37 ° C. by energizing the heating wire. The cuvette 22 is provided with a stainless stirrer 23. The stirrer 23 rotates in the cuvette 22 by the action of a stirrer 24 installed at the lower part of the cuvette 22. That is, the stirrer 24 includes a motor 24a and a permanent magnet 24b provided on the output shaft of the motor 24a. And the permanent magnet 24b rotates by supplying with electricity to the motor 24a. Since the magnetic field from the permanent magnet 24b rotates, the stainless steel stirrer 23 rotates by the action of the rotating magnetic field. The stirrer 23 and the stirrer 24 correspond to a stirring means.

  The turbidimetric measurement device 21 is provided with a light source 26 as a light incident means and a light receiving element 29 as a light receiving means. The light emitted from the light source 26 passes through the aperture 27 and then enters the sample in the cuvette 22 through the incident hole 25 a provided in the heat insulator 25. The light transmitted through the sample in the cuvette 22 is emitted from the emission hole 25 b provided in the heat insulator 25, passes through the aperture 28, and is irradiated on the light receiving element 29. The light receiving element 29 outputs a photoelectric signal corresponding to the intensity of the received light. The output of the photoelectric signal is input to the arithmetic unit 30 as a determination unit and a derivation unit. In the arithmetic unit 30, the reaction start time is determined and the endotoxin concentration is derived according to a program (algorithm) stored in advance. In addition, the turbidimetric measurement device 21 may be included including a display device that displays the derived endotoxin concentration.

  Table 1 shows the case where non-heated HSA is added to the mixture of the sample and the AL reagent, and the case where HSA that has been heat-treated in advance in an autoclave sterilization process (120 ° C., 15 to 120 minutes) is added. The gelation detection time is shown. The HSA concentration in the mixed solution is 0.25% in any case. As can be seen from Table 1, the gelation detection time tended to be delayed when the preheated HSA was added than when the non-heated HSA was added. By this, it was shown that aggregation or gelation not caused by biologically active substances derived from organisms due to stirring can be more reliably suppressed by adding HSA that has been previously heat-treated.

Table 2 also shows the case where a non-heated human serum albumin preparation (Albminer) is added to the mixture of the sample and the AL reagent, and the heat treatment (95 ° C., 5 to 3) using an aluminum block heater.
0 min) shows the gelation detection time in the case of adding the albumin that has been previously heat-treated. Also in this case, the albumin concentration in the mixed solution is 0.25% in any case. As can be seen from Table 2, also in this case, the gelation detection time tended to be delayed when the albumin that had been previously heat-treated was added, compared to when the non-heated albumin was added. Thus, it was shown that aggregation or gelation not caused by a biologically active substance derived from a living body by stirring can be more reliably suppressed by adding albumin that has been heat-treated in advance.

<Example 2>
In Example 1 described above, the example in which HSA is added to the mixed solution of the sample and the AL reagent using the general light scattering particle measurement device 1 and the turbidimetric measurement device 21 has been described. Therefore, a special light scattering particle measuring device and a turbidimetric measuring device that automatically add HSA to the mixed solution of the sample and the AL reagent may be used.

  FIG. 7 shows a light scattering particle measuring device 31 provided with a protein addition device 13 for automatically adding a protein such as HSA to a mixed solution of a sample and an AL reagent. The difference from the light scattering particle measuring apparatus 1 described with reference to FIG. 1 is that it has a protein reservoir 13a and a protein addition tube 13b. Prior to the measurement, the protein storage unit 13a stores the heat-treated HSA solution in a precisely measured amount. When the mixed solution of the sample and the AL reagent is set at the measurement position at the time of measurement, a switch (not shown) is turned on, and air pressure acts on the protein reservoir 13a by, for example, the small pump 13c. Then, the HSA solution stored in the protein storage unit 13a is added into the cuvette via the protein addition tube 13b.

  According to this example, aggregation that is not caused by endotoxin, which occurs when the mixture is stirred, can be reduced more easily, and the measurement accuracy of endotoxin measurement can be improved. The protein adding device 13 corresponds to protein adding means.

  Of course, the protein addition apparatus 13 in the present embodiment is not limited to the one having the above-described configuration. For example, an HSA solution that has been sterilized by autoclaving may be discharged by a liquid discharge device capable of precisely controlling the discharge amount when the mixed solution of the sample and the AL reagent is set at the measurement position at the time of measurement. . Alternatively, set a dropper that sucks up the required amount of the HSA solution that has been sterilized by autoclaving, and add the HSA solution to the mixture in the glass sample cell 4 by compressing the flexible pump part of the dropper. You may make it do.

  It is desirable that the heat-treated HSA solution be added to the mixed solution of the sample and the AL reagent before the stirring by the stirrer 11 is started. By this, the aggregation which does not originate in endotoxin which arises when stirring a liquid mixture more reliably can be suppressed. However, even when the stirring by the stirrer 11 is started or after stirring is started, the heat-treated HSA solution is added to the mixed solution of the sample and the AL reagent to suppress aggregation not derived from endotoxin. An effect can be obtained.

Similarly, FIG. 8 shows a turbidimetric measurement device 41 including a protein addition device 33 that automatically adds a protein such as HSA to a mixed solution of a sample and an AL reagent. The difference from the turbidimetric measurement device 21 described in FIG. 6 is that a protein addition device 33 including a syringe 33a, a piston 33b, and a piston pressing portion 33c is provided. Here, a necessary amount of the HSA solution that has been autoclaved is sucked into the syringe 33a. When the cuvette 22 that holds the mixed solution of the sample and the AL reagent at the time of measurement is set at the measurement position, a switch (not shown) is turned on, and the piston pressing portion 33c presses the piston 33b, and the HSA solution in the syringe 33a. Is added into the cuvette 22. Even with such a turbidimetric measurement device 41, aggregation that is not caused by endotoxin, which occurs when the mixture is stirred, can be reduced more easily, and the measurement accuracy of endotoxin measurement can be improved.

  Of course, the protein addition apparatus 33 in the present embodiment is not limited to the apparatus having the above-described configuration, and any apparatus may be used as long as it has an equivalent function.

DESCRIPTION OF SYMBOLS 1 ... Light-scattering particle measuring device 2 ... Light source 3 ... Incident optical system 4 ... Sample cell 5 ... Outgoing optical system 6 ... Light receiving element 7 ... Amplifying circuit 8 ... Noise removal filter 9 ... arithmetic device 10 ... display 11 ... stirrer 12 ... stirrer 13 ... protein addition device 21 ... turbidimetric measurement device 22 ... glass container (cuvette) )
23 ... Stirrer 24 ... Stirrer 24a ... Motor 24b ... Magnet 25 ... Insulator 25a ... Incident hole 25b ... Emission hole 26 ... Light source 27 ... Aperture 28 ... Aperture 29 ... Light receiving element 30 ... Arithmetic unit 31 ... Light scattering particle measuring device 33 with protein addition device ... Protein addition device 41 ... Ratio with protein addition device Turbidity measuring device

Claims (19)

Resulting from the reaction between AL and the physiologically active substance in the mixed solution while producing a mixed liquid containing AL, which is a blood cell extract of horseshoe crab, and a biologically active substance derived from a predetermined organism and stirring the mixed liquid A method for measuring a biologically active substance derived from a living body, wherein the biologically active substance in the sample is detected or the concentration of the physiologically active substance is measured by detecting aggregation or gelation of the protein,
When the agitation is performed, a predetermined protein that has been heat-treated in advance is added to the admixture to suppress aggregation or gelation that is not derived from the physiologically active substance in the admixture. A method for measuring biologically active physiological substances.   The method for measuring a biologically active substance derived from an organism according to claim 1, wherein the predetermined protein subjected to the heat treatment is at least one of albumin, globulin and lysozyme. The predetermined protein subjected to the heat treatment in advance is albumin,
The biologically-derived physiological activity according to claim 1 or 2, wherein the final concentration when a predetermined protein that has been heat-treated in advance is added to the mixed solution is 0.015% or more and 10% or less. Method for measuring substances.   The method for measuring a biologically active substance derived from a living body according to any one of claims 1 to 3, wherein the heat treatment is a heat treatment, and the treatment temperature is 95 ° C or higher.   The method for measuring a biologically active biological substance according to any one of claims 1 to 4, wherein the heat treatment is a heat treatment, and the treatment time is 10 minutes or more.   The heat treatment is an autoclave sterilization treatment, the treatment temperature of the autoclave sterilization treatment is 120 ° C or higher, and the time of the autoclave sterilization treatment is 10 minutes or more. A method for measuring a biologically active substance derived from the organism described above.   The method for measuring a biologically active substance of biological origin according to any one of claims 1 to 6, wherein the biologically active substance of biological origin is endotoxin or β-D-glucan. An agglutination or gel of a protein caused by the physiologically active substance is produced while producing a mixed liquid with a sample containing a biologically active substance derived from a predetermined organism, containing AL which is a blood cell extract of horseshoe crab, and stirring the mixed liquid A reagent for measuring a biologically active substance derived from a living body for detecting the physiologically active substance in the sample or measuring the concentration of the physiologically active substance by detecting oxidization,
A reagent for measuring a biologically active substance derived from a living organism, wherein a predetermined protein that has been previously heat-treated is added so that aggregation or gelation not derived from the physiologically active substance in the mixed solution can be suppressed.   9. The reagent for measuring a biologically active substance derived from a living body according to claim 8, wherein the predetermined protein subjected to the heat treatment is at least one of albumin, globulin, and lysozyme.   The reagent for measuring a biologically active substance of biological origin according to claim 8 or 9, wherein the heat treatment is a heat treatment, and the treatment temperature is 95 ° C or higher.   The reagent for measuring a biologically active substance derived from an organism according to any one of claims 8 to 10, wherein the heat treatment is a heat treatment, and the treatment time is 10 minutes or more.   The biological treatment according to claim 8 or 9, wherein the heat treatment is an autoclave sterilization treatment, a treatment temperature of the autoclave sterilization treatment is 120 ° C or higher, and a time of the autoclave sterilization treatment is 10 minutes or more. Reagent for measuring physiologically active substances. A mixed liquid holding means for holding a mixed liquid of a sample containing a physiologically active substance derived from a predetermined organism and AL, which is a blood cell extract of horseshoe crab, in a state where light can be incident, and for allowing a reaction in the mixed liquid to proceed;
Stirring means for stirring the mixed liquid in the mixed liquid holding means;
A light incident means for making light incident on the mixed liquid in the mixed liquid holding means;
A light receiving means for receiving transmitted light or scattered light in the admixture of the incident light and converting it into an electrical signal;
Determination means for determining the reaction start time of the physiologically active substance and AL in the sample from the electrical signal converted in the light receiving means;
Measurement of a biologically active substance derived from an organism, comprising: a derivation 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. A device,
An apparatus for measuring a biologically active substance derived from a living body, further comprising protein addition means for adding a predetermined protein that has been previously heat-treated to the admixture when the admixture is agitated by the agitation means.   The biologically active substance measuring apparatus according to claim 13, wherein the protein added by the protein adding means is at least one of albumin, globulin, and lysozyme.   The protein added by the protein addition means is albumin, and the final concentration when the predetermined protein that has been pre-heated to the admixture is added by the protein addition means is 0.015% or more and 10% or less. The biologically active substance-measuring device for living organisms according to claim 13 or 14,   The biological heat-active substance measuring apparatus according to any one of claims 13 to 15, wherein the heat treatment is a heat treatment, and a treatment temperature thereof is 95 ° C or higher.   The biological heat-active substance measuring apparatus according to any one of claims 13 to 16, wherein the heat treatment is a heat treatment, and the treatment time is 10 minutes or more.   The heat treatment is an autoclave sterilization treatment, the treatment temperature of the autoclave sterilization treatment is 120 ° C or higher, and the time of the autoclave sterilization treatment is 10 minutes or more. The biologically active substance measuring apparatus according to the description.   The biologically active substance measuring apparatus according to any one of claims 13 to 18, wherein the biologically active substance derived from an organism is endotoxin or β-D-glucan.

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