JP2012215461A - Measuring method and measuring device of physiologically active substance derived from organisms - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve measurement accuracy of measurement when measuring the concentration of a physiologically active substance derived from an organism in a sample by an agitation turbidimetric method, a light scattering method or an AL coupling bead method, by suppressing coagulation or gelatinization not derived from the physiologically active substance, which results in agitation of mixed liquid.SOLUTION: When mixing AL and a sample containing the physiologically active substance derived from an organism and detecting the coagulation of protein resulting in reaction of the AL and the physiologically active substance in the mixed liquid while agitating the mixed liquid, by adding a prescribed surfactant to the mixed liquid, the coagulation or gelatinization of the protein that does not result in the reaction of the AL and the physiologically active substance in the mixed liquid is suppressed.

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 in the glass sample cell, but the purpose of stirring is different from that of 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). There is also a method of suppressing aggregation that is not derived from endotoxin by adding heat-treated albumin to a mixture of the AL reagent and the sample. This is mainly due to the fact that the heat-treated albumin suppresses the activation of the coagulation enzyme by the stimulation of stirring.

  However, especially when endotoxin in a diluted plasma preparation or water for injection itself is measured under various conditions with different types, manufacturers, and lots of the AL reagent, the above-mentioned workaround may not function sufficiently. This is thought to be due to the fact that the shape of the reaction curve changes due to stirring not only due to the activation of the coagulation enzyme by stirring stimulation but also to further measures.

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, the addition of a surfactant to the admixture will suppress the protein aggregation or gelation that is not caused by the reaction of AL in the admixture with the biologically active substance derived from the organism. It is characterized by.

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 stirring is performed, a predetermined surfactant is added to the mixed solution to suppress aggregation or gelation that is not derived from the physiologically active substance in the mixed solution.

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. In contrast, the inventors' diligent research has shown that the addition of a surfactant during mixing of the sample and the AL reagent can suppress the induction of aggregation or gelation not derived from the biologically active substance derived from the organism. It was issued. In particular, it has been found that the addition of this surfactant can suppress aggregation of proteins contained in the AL reagent and / or sample. Furthermore, it was confirmed by experiments that the addition of this surfactant to a sample has 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. In particular, since the surfactant can suppress aggregation of proteins contained in the AL reagent and / or the sample, even when heat-treated albumin cannot be expected to be effective, aggregation or non-aggregation due to biologically active substances derived from organisms caused by stirring Gelation 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 surfactant is an anionic surfactant, for example, pure soap (fatty acid sodium), pure soap (fatty acid potassium), alpha sulfo fatty acid ester sodium, linear alkylbenzene sulfonate sodium , Sodium alkyl sulfate ester, sodium alkyl ether sulfate ester, sodium alpha olefin sulfonate, and sodium alkyl sulfonate. Nonionic surfactants such as Tween (trade name), Triton X (trade name), Pluronic (trade name), polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan mono Stearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene-p-isooctylphenol, polyethylene oxide / polypropylene oxide block polymer, sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid Alkanolamide, polyoxyethylene alkyl ether, and polyoxyethylene alkyl phenyl ether may be used. Further, an emulsion type antifoaming agent in which silicone oil or vegetable oil is emulsified in a nonionic surfactant may be used. Also, zwitterionic surfactants such as sodium alkylamino fatty acid, alkylbetaine, and alkylamine oxide may be used. Further, a cationic surfactant, for example, an alkyltrimethylammonium salt or a dialkyltrimethylammonium salt may be used. These surfactants have been confirmed to be able to suppress the induction of aggregation or gelation not derived from biologically-derived physiologically active substances by being added when the sample and the AL reagent are mixed. It is possible to suppress aggregation or gelation that is not caused by biologically-derived physiologically active substances.

  In the present invention, the final concentration when a predetermined surfactant is added to the mixture may be 0.0001% 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 0.0005% or more and 5% or less. More preferably, it is 0.005% or more and 0.5% or less.

  In the present invention, the addition of heat-treated albumin is used in combination with the addition of the surfactant of the present invention in order to suppress agglomeration or gelation that is not caused by biologically-derived physiologically active substances due to stirring. May be.

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

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

In addition, the present invention 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 an organism, characterized in that a predetermined surfactant is added and aggregation or gelation not derived from the physiologically active substance in the mixed solution can be suppressed. In this case, the predetermined surfactant is an anionic surfactant such as pure soap (fatty acid sodium), pure soap (fatty acid potassium), alpha sulfo fatty acid ester sodium, linear alkylbenzene sulfonic acid. Sodium, sodium alkyl sulfate ester, sodium alkyl ether sulfate ester, sodium alpha olefin sulfonate, sodium alkyl sulfonate may be used. Nonionic surfactants such as Tween (trade name), Triton X (trade name), Pluronic (trade name), polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan mono Stearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene-p-isooctylphenol, polyethylene oxide / polypropylene oxide block polymer, sucrose fatty acid ester sorbitan fatty acid ester,
Polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether may be used. Further, an emulsion type antifoaming agent in which silicone oil or vegetable oil is emulsified in a nonionic surfactant may be used. Also, zwitterionic surfactants such as sodium alkylamino fatty acid, alkylbetaine, and alkylamine oxide may be used. Further, a cationic surfactant, for example, an alkyltrimethylammonium salt or a dialkyltrimethylammonium salt may be used.

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 physiologically active substance derived from a living organism, further comprising a surfactant adding means for adding a predetermined surfactant to the mixed liquid when the mixed liquid is stirred by the stirring means. May be.

  According to this apparatus, when the mixed solution of the sample and the AL reagent is stirred, a predetermined surfactant can be automatically added to the mixed solution, and the physiological function in the mixed solution can be more easily performed. Aggregation or gelation of proteins that are not derived from active substances can be suppressed.

  Also in this apparatus, the surfactant added by the surfactant addition means is an anionic surfactant such as pure soap (fatty acid sodium), pure soap (fatty acid potassium), alpha sulfo fatty acid ester sodium, Linear sodium alkylbenzene sulfonate, sodium alkyl sulfate ester, sodium alkyl ether sulfate, sodium alpha olefin sulfonate, sodium alkyl sulfonate may be used. Nonionic surfactants such as Tween (trade name), Triton X (trade name), Pluronic (trade name), polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan mono Stearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene-p-isooctylphenol, polyethylene oxide / polypropylene oxide block polymer, sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid Alkanolamide, polyoxyethylene alkyl ether, and polyoxyethylene alkyl phenyl ether may be used. Further, an emulsion type antifoaming agent in which silicone oil or vegetable oil is emulsified in a nonionic surfactant may be used. Also, zwitterionic surfactants such as sodium alkylamino fatty acid, alkylbetaine, and alkylamine oxide may be used. Further, a cationic surfactant, for example, an alkyltrimethylammonium salt or a dialkyltrimethylammonium salt may be used. The final concentration when a predetermined surfactant is added to the mixed solution is preferably 0.0001% or more and 10% or less. Desirably, it is 0.0005% or more and 5% or less. More preferably, it is 0.005% or more and 0.5% or less. 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 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 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 at the time of using the conventional (reagent-free) AL reagent, and a calibration curve. It is a graph which shows a reaction curve at the time of adding Tween20 in a liquid mixture in Example 1 of this invention. It is a graph which shows the relationship between the density | concentration of Tween20 added to the liquid mixture of the sample and AL reagent in Example 1 of this invention, and gelation detection time. It is a graph which shows a reaction curve at the time of adding Triton X-100 to a liquid mixture in Example 1 of this invention. It is a graph which shows the relationship between the density | concentration of Triton X-100 added to the liquid mixture of the sample and AL reagent in Example 1 of this invention, and gelation detection time. It is a graph which shows the relationship between the endotoxin density | concentration in a sample, and a gel detection time, and a calibration curve at the time of adding Tween20 in a liquid mixture in Example 1 of this invention. It is a graph which shows a reaction curve at the time of adding APB, Tween20, and APB and Tween20 to a liquid mixture. It is a graph which shows a reaction curve at the time of adding APB and Tween20 to a liquid mixture. 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.

<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. 1, 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. 1, these measurement methods detect the coagulin association 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. 2, 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.

  FIG. 3 shows the relationship between the endotoxin concentration and the gelation detection time and the calibration curve. In FIG. 3, a mixed solution in which 100 μL of the AL reagent and 100 μL of a sample containing 10 to 0.0001 EU / mL endotoxin were mixed was used for the measurement. When the endotoxin concentration in the sample was 10 to 0.01 EU / mL, the lower the endotoxin concentration in the sample, the clearer the gelation detection time was. Regardless of concentration, gelation detection time was observed within 14-30 minutes. As a result, it was difficult to accurately measure endotoxin of 0.01 EU / mL or less. The linearity of the calibration curve was not sufficient because the correlation coefficient was 0.974.

  This is because the protein is denatured and insoluble aggregates are generated by stimulation of stirring by the stirrer 11 and the stirrer 12, and aggregates (gel particles) not derived from the endotoxin are generated. Therefore, in order to accurately measure a low concentration of endotoxin of 0.01 EU / mL or less, it is necessary to suppress aggregation that is not derived from the endotoxin. On the other hand, the inventors' diligent research has revealed that aggregation not derived from endotoxin can be suppressed by adding a surfactant such as Tween. In this example, Tween 20 and Triton X-100 were used as surfactants, and the effect of suppressing aggregation not derived from endotoxin was investigated. Both Tween 20 and Triton X-100 have low endotoxin content and could be used as they are. However, if they contain a large amount of endotoxin, the endotoxin may be inactivated by autoclave sterilization in advance.

The results of the above measurement will be described below. A mixture of 100 μL of AL reagent and 100 μL of Tween 20 (0%, 0.001%, 0.01%, 0.1%, 1%) (ie, Tw
The final concentration of een20 is 0%, 0.0005%, 0.005%, 0.05%, and 0.5%). The horizontal axis in FIG. 4 is the reaction time, and the vertical axis is the number of gel particles. When Tween 20 is 0% (that is, not added) and 0.0005%, the number of gel particles increases sharply with the passage of time from the start of the reaction, and decreases again with a peak at about 80 to 100 minutes. This is because immediately after the reaction, the protein is denatured by the stimulation of stirring by the stirrer 11 and the stirrer 12 to produce insoluble aggregates, and aggregates (gel particles) not derived from this endotoxin are measured. It is done.

On the other hand, when Tween 20 is contained in an amount of 0.005% or more, it can be seen that a sharp increase in the number of gel particles immediately after the start of the reaction is suppressed, and the number of gel particles to be measured stably increases. From the above, when the surfactant is Tween or an equivalent product, if the final concentration is 0.005% or more and 0.5% or less, a sharp increase in the number of gel particles immediately after the start of the reaction is surely suppressed. Can be expected. Considering that the generation of aggregates not derived from endotoxin varies depending on the type, manufacturer and lot of the AL reagent, generally, if the surfactant is 0.0001% or more and 10% or less, the difference in degree is Even if it exists, an effect of suppressing a sharp increase in the number of gel particles immediately after the start of the reaction can be expected.

  Next, the gelation detection time obtained as a result of the above measurement is shown in FIG. When Tween 20 was contained in an amount of 0.005% or more, the gelation detection time tended to be delayed. Thereby, it was shown that aggregation not derived from endotoxin can be suppressed by adding an appropriate amount of Tween20.

  Similarly, FIG. 6 shows a reaction curve when Triton X-100 is used as a surfactant in place of Tween20. The horizontal axis in FIG. 6 is the reaction time, and the vertical axis is the number of gel particles. When Triton X-100 is 0% (that is, not added) and 0.0005%, the number of gel particles sharply increases with the passage of time from the start of the reaction due to the formation of aggregates (gel particles) not derived from endotoxin. Increased. In contrast, when Triton X-100 was contained in an amount of 0.005% or more, a sharp increase in the number of gel particles immediately after the start of the reaction was suppressed. The gelation detection time obtained as a result is shown in FIG. When Triton X-100 was contained in an amount of 0.005% or more, the gelation detection time tended to be delayed.

Thereby, it was shown that aggregation not derived from endotoxin can also be suppressed by adding an appropriate amount of Triton X-100. From the above, the surfactant is Triton
In the case of X or an equivalent product, if the final concentration is 0.005% or more and 0.5% or less, an effect of reliably suppressing a sharp increase in the number of gel particles immediately after the start of the reaction can be expected. Considering that the generation of aggregates not derived from endotoxin varies depending on the type, manufacturer and lot of the AL reagent, generally, if the surfactant is 0.0001% or more and 10% or less, the difference in degree is Even if it exists, an effect of suppressing a sharp increase in the number of gel particles immediately after the start of the reaction can be expected.

  As described above, it was confirmed that aggregation not derived from endotoxin was suppressed by adding a surfactant such as Tween 20 or Triton X-100 to the mixture, and thus AL containing 0.01% of Tween 20 was observed. Reagents were prepared. A mixture (that is, the final concentration of Tween 20 was 0.005%) in which 100 μL of this AL reagent and 100 μL of a sample containing 10 to 0.0001 EU / mL endotoxin were mixed was used for the measurement. FIG. 8 shows the relationship between the obtained endotoxin concentration and the gelation detection time, and the calibration curve.

  As can be seen by comparing the calibration curve when Tween 20 is not added shown in FIG. 3 and FIG. 8, first, when Tween 20 is added, the endotoxin concentration in the sample is in the range of 10 to 0.01 EU / mL. Thus, the gelation detection time when Tween 20 was added did not become slower than when it was not added. This indicates that Tween 20 does not inhibit the Limulus reaction. When Tween 20 was added, a calibration curve having a good linearity with a correlation coefficient of 0.993 in the range of 10 to 0.0001 EU / mL could be obtained. This is considered to be an effect of suppressing aggregation not derived from endotoxin by addition of Tween20.

  Furthermore, in order to investigate the action mechanism in which Tween suppresses aggregation that is not derived from endotoxin, an addition experiment of amidinophenyl benzoate (hereinafter sometimes referred to as APB) was conducted. APB is one of the protease inhibitors and inhibits the activity of the clotting enzyme in the Limulus reaction. Reaction curves when using another AL reagent and mixing a mixture of AL reagent 100 μL and 0.1 mM APB 100 μL and a mixture of AL reagent 100 μL and 0.1% Tween 20 100 μL As shown in FIG. FIG. 9 also shows a reaction curve when a mixed solution in which 100 μL of AL reagent containing 0.1 mM APB and 100 μL of 0.1% Tween 20 were mixed was used for the measurement.

  As can be seen from FIG. 9, when APB was not added, a sharp increase in the number of particles not derived from endotoxin was observed from the beginning of the measurement. The degree of increase in the number of particles is larger than in FIGS. 4 and 6 because the AL reagent is different. Moreover, even when APB was added, the increase in the number of gel particles was not suppressed. This indicates that the sharp increase in the number of gel particles not derived from endotoxin is not due to activation of the coagulation enzyme by agitation stimulation, but is measuring aggregates formed by protein aggregation. ing. When only Tween 20 and both APB and Tween 20 were added, the monotonous increase in the number of gel particles not derived from the endotoxin was remarkably suppressed. That is, it was shown that Tween 20 exhibits a great inhibitory effect on the aggregation of proteins.

  Reaction curves when another AL reagent is used and a mixture of 100 μL of AL reagent and 100 μL of 0.1 mM APB and a mixture of 100 μL of AL reagent and 100 μL of 0.1% Tween 20 are used for measurement. Is shown in FIG. As can be seen from FIG. 10, when APB is not added using this AL reagent, no sharp increase in the number of particles is observed from the beginning of the measurement, and the number of particles not derived from endotoxin is observed after about 100 minutes. An increase was observed. The reason for the increase in the number of particles compared to FIGS. 4, 6 and 9 is that the AL reagent is different. When APB was added, the increase in the number of gel particles was clearly suppressed. This indicates that the increase in the number of gel particles not derived from the endotoxin is due to the activation of the coagulation enzyme by stimulation of stirring. However, even when Tween 20 was added, the increase in the number of gel particles not derived from the endotoxin was not suppressed. That is, it was shown that Tween 20 does not have an inhibitory effect on the activation of the clotting enzyme by stirring stimulation, unlike the heat-treated albumin.

  Thus, Tween has a different mechanism of action from heat-treated albumin, which is considered to suppress the activation of coagulation enzymes mainly due to stimulation of stirring, and suppresses aggregation of proteins due to stimulation of stirring. Therefore, the effect of suppressing aggregation that is not derived from endotoxin can be expected even for an AL reagent that cannot be expected with heat-treated albumin. In addition, unlike natural albumin, Tween can be chemically synthesized, so it is easy to eliminate endotoxin contamination in the initial process. In fact, Tween did not inhibit the Limulus reaction (FIG. 8), and the addition of Tween slowed the gelation detection time (FIGS. 4 and 5), indicating that the endotoxin content of Tween used in this example was sufficient. Is low.

  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. 11 shows a schematic configuration of a turbidimetric measurement device 21 as another example of the endotoxin measurement device 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.

  In the above embodiment, examples of using Tween 20 or Triton X-100 as the surfactant to be added to the admixture have been described. However, the surfactant may be polyoxyethylene sorbitan even if the trade name is different. Even if monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene-p-isooctylphenol, etc. Good. Other nonionic surfactants such as Pluronic, polyethylene oxide / polypropylene oxide block polymer, sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide, polyoxyethylene alkyl ether, polyoxyethylene Even if alkylphenyl ether is used, the same effect can be expected. In addition, if it is a surfactant, even if it is not a nonionic one, it is thought that the effect of suppressing the aggregation of proteins can be obtained, so an emulsion type in which silicone oil or vegetable oil is emulsified in a nonionic surfactant An antifoaming agent may be used. Also, anionic surfactants such as pure soap (fatty acid sodium), pure soap (fatty acid potassium), alpha sulfo fatty acid ester sodium, linear alkylbenzene sulfonate sodium, alkyl sulfate sodium, alkyl ether sulfate sodium , Sodium alpha olefin sulfonate and sodium alkyl sulfonate may be used. In addition, zwitterionic surfactants such as sodium alkylamino fatty acid, alkylbetaine, and alkylamine oxide may be used. Moreover, you may use cationic surfactant, for example, alkyl trimethyl ammonium salt, dialkyl trimethyl ammonium salt. Moreover, you may mix and use various above-mentioned surfactants.

<Example 2>
In Example 1 described above, a surfactant such as Tween 20 or Triton X-100 is added to the mixed solution of the sample and the AL reagent using the general light scattering particle measuring device 1 and the turbidimetric measuring device 21. Although an example of addition has been described, in order to carry out the present invention, a special light scattering particle measuring apparatus for automatically adding a surfactant such as Tween or Triton X to a mixed solution of a sample and an AL reagent, and A turbidimetric measurement device may be used.

FIG. 12 shows a light scattering particle measuring device 31 including a surfactant addition device 13 that automatically adds a surfactant such as Tween or Triton X 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. 2 is that it has a surfactant reservoir 13a and a surfactant addition tube 13b. Prior to measurement, the surfactant storage unit 13a stores a surfactant such as Tween or Triton X 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 surfactant reservoir 13a by, for example, the small pump 13c. Then, a surfactant such as Tween or Triton X stored in the surfactant storage unit 13a is added into the cuvette via the surfactant 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 surfactant adding device 13 corresponds to a surfactant adding means.

Of course, the surfactant addition device 13 in the present embodiment is not limited to the above-described configuration. For example, by using a liquid discharge device that can precisely control the discharge amount, a surfactant such as Tween or Triton X is discharged when the mixed solution of the sample and the AL reagent is set at the measurement position during measurement. Also good. Alternatively, set a dropper or pipette that sucks up the necessary amount of surfactant such as Tween or Triton X, and compress the flexible pump part to remove the surfactant such as Tween or Triton X. Alternatively, it may be added to the mixed solution in the glass sample cell 4.

The timing of adding a surfactant such as Tween or Triton X to the mixed solution of the sample and the AL reagent is preferably 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 the stirring is started, a surfactant such as Tween or Triton X is not added to the endotoxin by adding a surfactant such as Tween or Triton X to the mixed solution of the sample and the AL reagent. The effect of suppressing aggregation can be obtained.

Similarly, FIG. 13 shows Tween and Triton X for the mixture of the sample and the AL reagent.
A turbidimetric measurement device 41 having a surfactant addition device 33 for automatically adding a surfactant such as is shown. The difference from the turbidimetric measurement device 21 described with reference to FIG. 11 is that a surfactant adding device 33 including a syringe 33a, a piston 33b, and a piston pressing portion 33c is provided. Here, a necessary amount of a surfactant such as Tween or Triton X is sucked into the syringe 33a. Then, 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, the piston pressing portion 33c presses the piston 33b, and Tween and Triton
A surfactant such as X 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.

  It should be noted that the surfactant addition device 33 in the present embodiment is not limited to the one having the above-described configuration, and any device 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 device 11 ... stirrer 12 ... stirrer 13 ... surfactant addition device 21 ... turbidimetric measuring 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 provided with surfactant addition device ... Surfactant addition device 41 ... Surfactant Turbidity measuring device with addition device

Claims (13)

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 surfactant is added to the mixed solution to suppress aggregation or gelation not derived from the physiologically active substance in the mixed solution. Method for measuring physiologically active substance.   The method for measuring a biologically active substance derived from an organism according to claim 1, wherein the predetermined surfactant is a nonionic surfactant. The predetermined surfactant is Tween, Triton X, Pluronic, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene Sorbitan trioleate, polyoxyethylene-p-isooctylphenol, polyethylene oxide / polypropylene oxide block polymer, sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl It contains one kind or two or more kinds of ethers according to claim 1. A method of measuring biologically active substances derived from living organisms.   The biological concentration according to any one of claims 1 to 3, wherein a final concentration when the predetermined surfactant is added to the admixture is 0.0001% or more and 10% or less. Method for measuring physiologically active substance.   The method for measuring a biologically active substance derived from a living body according to any one of claims 1 to 4, wherein the biologically active substance derived from the living organism 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 an organism, wherein a predetermined surfactant is added, and aggregation or gelation not derived from the physiologically active substance in the mixed solution can be suppressed.   The reagent for measuring a biologically active substance derived from an organism according to claim 6, wherein the predetermined surfactant is a nonionic surfactant. The predetermined surfactant is Tween, Triton X, Pluronic, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene Sorbitan trioleate, polyoxyethylene-p-isooctylphenol, polyethylene oxide / polypropylene oxide block polymer, sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl 7. One type of ether, or two or more types are included. Reagent for measuring biologically active biological 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-derived physiologically active substance, further comprising a surfactant adding means for adding a predetermined surfactant to the mixed liquid when the mixed liquid is stirred by the stirring means.   10. The biologically active physiologically active substance measuring apparatus according to claim 9, wherein the surfactant added by the surfactant adding means is a nonionic surfactant. The surfactant added by the surfactant adding means is Tween, Triton.
X, Pluronic, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, polyoxyethylene-p-iso Octylphenol, polyethylene oxide / polypropylene oxide block polymer, sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, or two or more The measuring apparatus for biologically active substances derived from organisms according to claim 9, comprising:   12. The final concentration when a predetermined surfactant is added to the mixed solution by the surfactant addition means is 0.0001% or more and 10% or less. An apparatus for measuring a biologically active substance derived from an organism according to Item.   The biologically active substance measuring apparatus according to any one of claims 9 to 12, wherein the biologically active substance derived from an organism is endotoxin or β-D-glucan.

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