WO2010013702A1 - 生物由来の生理活性物質の測定方法及び測定装置 - Google Patents
生物由来の生理活性物質の測定方法及び測定装置 Download PDFInfo
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- WO2010013702A1 WO2010013702A1 PCT/JP2009/063405 JP2009063405W WO2010013702A1 WO 2010013702 A1 WO2010013702 A1 WO 2010013702A1 JP 2009063405 W JP2009063405 W JP 2009063405W WO 2010013702 A1 WO2010013702 A1 WO 2010013702A1
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- active substance
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Classifications
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
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- G01N2021/825—Agglutination
Definitions
- 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 LAL, such as endotoxin or ⁇ -D-glucan.
- LAL such as endotoxin or ⁇ -D-glucan.
- the present invention relates to a measuring method and a measuring apparatus.
- Endotoxin is a lipopolysaccharide present in the cell wall of Gram-negative bacteria and is the most typical pyrogenic substance. 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.
- LAL Limulus amoebocyteeblysate
- endotoxin serine protease activated by endotoxin exists in the blood cell extract of horseshoe crab (hereinafter also referred to as “LALLA: Limulus amoebocyteeblysate”).
- LAL reacts with endotoxin
- the coagulogen present in LAL is hydrolyzed into coagulin by the enzyme cascade by serine protease activated according to the amount of endotoxin, and an insoluble gel forms. Generated.
- endotoxin can be detected with high sensitivity.
- ⁇ -D-glucan is a polysaccharide (polysaccharide) that forms a cell membrane characteristic of fungi. By measuring ⁇ -D-glucan, it is effective for screening a wide range of fungal infections including rare fungi as well as common clinical fungi such as Candida, Spergillus, and Cryptococcus.
- ⁇ -D-glucan In the measurement of ⁇ -D-glucan, ⁇ -D-glucan can be detected with high sensitivity by utilizing the property that the blood cell extract component of horseshoe crab coagulates (gel coagulation) with ⁇ -D-glucan. .
- predetermined physiologically active substances biologically active substances derived from organisms
- a sample to be detected or a concentration measurement of a predetermined physiologically active substance hereinafter simply referred to as “measurement of a predetermined physiologically active substance”
- LAL concentration measurement of a predetermined physiologically active substance
- a turbidimetric method that measures and analyzes the turbidity of a sample accompanying the formation of a gel due to the reaction between LAL and a specific physiologically active substance over time, or a colorimetric method that uses a synthetic substrate that is hydrolyzed by an enzyme cascade
- a mixed solution of a measurement sample and LAL is generated in a glass measurement cell that has been subjected to dry heat sterilization. Then, the gelation of the mixed solution is optically measured from the outside.
- the turbidimetric method it may take a very long time for LAL to gel, particularly in a sample having a low concentration of a predetermined physiologically active substance.
- a method capable of measuring a predetermined physiologically active substance in a short time is required.
- the mixture of the measurement sample and LAL is stirred using, for example, a magnetic stirrer to form gel fine particles, and the intensity of laser light scattered by the gel particles or the mixture is transmitted.
- a laser scattering particle measurement method or a stirring turbidimetric method capable of measuring the presence of a predetermined physiologically active substance in a sample in a short time from the intensity of light has been proposed.
- the present invention has been devised in view of the above-mentioned problems, and the object of the present invention is to provide a measurement method capable of reducing the measurement time in the detection or concentration measurement of biologically-derived physiologically active substances, and It is to provide a measuring device used.
- the inventor can directly detect the coagulin itself (coagulin monomer), which is the final product of the protease cascade, and the extremely minute aggregate (coagulin aggregate) formed by the association thereof. I thought it would lead to shortening. Then, light is irradiated to a mixed liquid of the sample for measuring a predetermined physiologically active substance and LAL to collide with particles in the mixed liquid to generate scattered light, and from the increasing rate of scattered light detected by the light receiving element, The greatest feature is to detect or measure the concentration of a predetermined physiologically active substance.
- the present invention provides an increase rate of scattered light detected by a light receiving element when light is irradiated to a mixture of a predetermined physiologically active substance and LAL to collide with particles in the mixture to generate scattered light. Is based on the new insight obtained by the inventor's earnest research that it depends on the concentration of a predetermined physiologically active substance.
- the present invention is based on a water-soluble protein coagulogen, which is based on a turbidimetric method that does not use a special reagent, such as a colorimetric method, but occurs very early in the gelation reaction of LAL with a predetermined physiologically active substance.
- the differential method similar to the colorimetric method is applied to the determination by detecting the coagulin monomer changed to hydrophobicity and the oligomer in which several monomers are aggregated with scattered light.
- the present invention detects the physiologically active substance in the sample by reacting a biologically active substance derived from an organism present in the sample with LAL, which is a blood cell extract of horseshoe crab, or the physiologically active substance.
- LAL which is a blood cell extract of horseshoe crab
- a method for measuring biologically active substances derived from living organisms After mixing the sample and LAL, light is incident on the mixed solution of the sample and LAL and the intensity of the scattered light of the incident light by the mixed solution is obtained. It is a method for measuring a biologically active substance derived from an organism, wherein the biologically active substance in the sample is detected or its concentration is measured from the increasing rate of the intensity of the scattered light.
- the physiologically active substance in the sample may be detected or the concentration measured from the increase rate before the increase rate of the intensity of the scattered light undergoes a predetermined sudden change.
- the generation of a coagulin monomer and an oligomer in which several monomers are aggregated is detected by scattered light.
- the scattering particles are very small and have a size equal to or smaller than the wavelength of the incident light, the scattered light at this time is considered to be mainly based on Rayleigh scattering.
- the scattered light changes mainly to those based on Mie scattering.
- the increase rate of the scattered light intensity before the predetermined sudden change is detected, that is, the increase rate of the scattered light mainly based on Rayleigh scattering.
- the rate of increase in scattered light from the coagulin monomer that occurs at the very beginning of the LAL gelation reaction with a predetermined physiologically active substance and the oligomer in which several monomers are aggregated.
- the present invention detects weak scattered light from extremely small particles as described above, it is desirable that the output density of incident light be as high as possible. It has been newly found that good detection is possible if the output density is 50 mW / mm 2 or more. Therefore, in the present invention, the output density of light incident on the mixture is preferably 50 mW / mm 2 or more. This output density may be adjusted by the output of the light source, or may be adjusted by reducing the incident light diameter to a smaller value.
- the wavelength of light incident on the mixture may be 300 nm or more and 800 nm or less. That is, the scattered light intensity of Rayleigh scattering shows dependence on the wavelength of incident light, and it has been found that shorter wavelengths are more advantageous for detection.
- an extremely short wavelength may adversely affect the function of LAL, and there may be a disadvantage that a material such as an optical element needs to correspond to a short wavelength. Under such circumstances, it is possible to realize good measurement by using a wavelength in the above range.
- the mode value may be the intensity of scattered light in the period.
- the rate of increase in the intensity of scattered light from extremely small diameter particles occurring at the very early stage of the gelation reaction of LAL by a predetermined physiologically active substance is measured.
- impurities such as undissolved reagent, micrometer-level fine particles remaining in the reagent manufacturing method, and minute bubbles accompanying stirring of the sample.
- the scattered light from these contaminants is very small but very powerful, so the weak signals scattered from the coagulin monomer and the minute coagulin aggregates are buried in the scattered light from these contaminants. In some cases, measurement may be impossible.
- a plurality of scattered light intensities acquired during a predetermined period are sampled and compared, and a filter is applied that uses the minimum value or the mode value of the histogram as the scattered light intensity in the period. It was to be. Since the frequency of intense scattered light from contaminants is low, the scattering value from the contaminants can be selected by selecting the minimum value or the mode value of the histogram from the scattered light intensity sampled during a given period. The influence of light can be removed.
- the mixed solution may be stirred when the intensity of the scattered light is acquired.
- the sample When the mixture is allowed to stand without stirring, as in the case of the turbidimetric method, the sample eventually gels and an increase in scattered light is observed, but the coagulin monomer in the initial reaction and the minute coagulin It may be difficult to detect an increase in scattered light due to aggregates.
- stirring the mixed solution it is possible to efficiently perform the reaction homogenization, the promotion of the reaction, the rapid mobilization of the produced coagulin monomer to the oligomer, and the like.
- the undissolved reagent in the liquid mixture as described above, micrometer-level fine particles, minute bubbles, etc. remaining in the reagent manufacturing process stay in the scattering region, leading to an inadvertent increase in scattered light intensity. It can suppress that measurement accuracy falls.
- the stirring speed of the mixed solution may be 300 rpm or more and 3000 rpm or less.
- the stirring speed of the mixed solution within the above range, mixing of micrometer level fine particles and bubbles can be satisfactorily suppressed, and inhibition of the coagulin monomer aggregation process can be avoided.
- the biologically active substance derived from the organism may be endotoxin or ⁇ -D-glucan.
- endotoxin the most typical pyrogen
- endotoxin-contaminated fluids, injections, blood, etc. can enter the human body and prevent side effects. it can.
- ⁇ -D-glucan can be detected or measured more accurately, and a wide range of fungal infections including rare fungi as well as common clinical fungi such as Candida, Spergillus, and Cryptococcus can be obtained. Screening can be performed more accurately.
- the present invention can detect the physiologically active substance in the sample by reacting a biologically active substance derived from an organism present in the sample with LAL, which is a blood cell extract of horseshoe crab.
- An apparatus for measuring a biologically active substance derived from a living organism that measures a concentration An admixture holding means for holding an admixture of the sample and LAL so that light can enter, and for allowing a reaction between the physiologically active substance and LAL to proceed;
- a light incident means for making light incident on the mixed liquid in the mixed liquid holding means;
- a light receiving means for receiving scattered light of the incident light in the mixture and converting it into an electrical signal;
- this measuring apparatus it becomes possible to detect a predetermined physiologically active substance such as endotoxin or ⁇ -D glucan or measure the concentration in a shorter time.
- the derivation means has a concentration of the physiologically active substance in the sample from the increase rate after the sample and LAL are mixed in the admixture holding means and before the increase rate causes a predetermined sudden change. May be derived. Then, the increase rate of scattered light from the coagulin monomer and the oligomer in which several monomers are aggregated can be obtained more accurately, and the detection of the predetermined physiologically active substance and the measurement of the concentration can be performed more accurately. It becomes possible.
- the output density of the light incident by the light incident means may be 50 mW / mm 2 or more.
- the wavelength of the light incident by the light incident means may be 300 nm or more and 800 nm or less. According to this, it is possible to realize more efficient and good measurement.
- a plurality of electrical signals converted by the light receiving means are sampled and compared in a predetermined period, and a minimum value filter for outputting the minimum value or a mode value for outputting the mode value of the histogram. You may make it further provide a frequent value filter. According to this, the influence of scattered light from various contaminants on the mixed solution can be removed, and the detection or concentration measurement of the predetermined physiologically active substance can be performed more accurately.
- the admixture holding means may have a stirring means for stirring the admixture.
- the stirring speed of the mixed solution by the stirring means is 300 rpm or more and 3000 rpm or less. According to this, it is possible to prevent the contamination from stagnating in the scattering region, thereby causing an inadvertent increase in scattered light intensity and reducing the measurement accuracy, and inhibiting the coagulin monomer aggregation process. Can be avoided.
- the biologically active substance derived from the organism may be endotoxin or ⁇ -D-glucan.
- Example 6 shows schematic structure of the measurement system of the predetermined physiologically active substance in the Example of this invention. It is a graph which shows the time change of the scattered light intensity
- Example 10 is a graph in which the relationship between the ⁇ -D-glucan concentration and the rate of increase in the intensity of the initial scattered light is plotted in logarithm in Example 7 of the present invention. It is a schematic diagram for explaining the process of LAL gelation by endotoxin or ⁇ -D-glucan and the detection method thereof.
- factor G when ⁇ -D-glucan binds to factor G in LAL, factor G is activated to become active factor G.
- the activated factor G hydrolyzes the precursor of the clotting enzyme in LAL into a clotting enzyme.
- 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.
- serine proteases such as Christmas factors and thrombin found in mammals.
- Such an enzyme cascade reaction has a very strong amplification action because even a very small amount of activator is activated by linking the subsequent cascade. Therefore, according to the method for measuring a predetermined physiologically active substance using LAL, it is possible to detect a very small amount of the predetermined physiologically active substance on the order of subpicogram / mL.
- a turbidimetric method and a laser light scattering particle measuring method can be mentioned. As shown in FIG. 1, these measurement methods detect the coagulin aggregate produced by the LAL enzyme cascade reaction as turbidity in the former, and the latter as fine particles of gel produced in the system. Highly sensitive measurement is possible.
- the laser light scattering particle measurement method directly measures the gel fine particles generated in the system, so it is more sensitive than the turbidimetric method and generally forcibly agitates the sample consisting of LAL and the specimen. Therefore, the formation of gel can be detected in a shorter time compared to the turbidimetric method.
- a colorimetric method as another method for measuring endotoxin.
- the sample is not turbid by coagulin gel, but is synthesized by using a synthetic substrate that is hydrolyzed by a clotting enzyme and develops color.
- This is a method of reacting LAL containing a substrate with a specimen and measuring the change in absorbance.
- the concentration of the color-developing substance produced in the system is measured. Therefore, compared with the turbidimetric method and the laser light scattering particle measurement method, which measure the gel formation in the sample, the concentration is low.
- the predetermined physiologically active substance can be measured.
- the turbidimetric method is evaluated as being easy to use in the field in that a special reagent is not required unlike the colorimetric method and that the concentration range of the predetermined physiologically active substance that can be measured is wide.
- the turbidimetric method has a problem that it takes a very long time to measure a predetermined physiologically active substance at a low concentration. This is because the turbidimetric method does not look at the amount of coagulin itself that is the end product of the protease cascade, but the process by which the light transmittance is reduced by the gel formed by further association. This is because.
- the concentration of coagulin does not reach a certain level, gelation does not occur. Therefore, it is necessary to wait until the gel is formed in order to detect a predetermined physiologically active substance in the turbidimetric method. Therefore, when the predetermined physiologically active substance concentration is high, the required time and sufficient concentration of coagulin is quickly generated and gelation starts, so the measurement time is shortened, but if the predetermined physiologically active substance concentration is low, the coagulin concentration required for gelation It takes time to reach the point, and the measurement time becomes long.
- the laser light scattering particle measurement method is an improvement from the turbidimetric method in that the sample is stirred and the particle is detected rather than gelled by the laser, and the measurement time is greatly reduced compared to the turbidimetric method. be able to.
- the degree of shortening of the measurement time was not as good as the colorimetric method.
- the turbidimetric method and the laser light scattering particle measurement method are different in physical quantity, they are common in that the point in time when a certain threshold is exceeded is taken as the starting point of the reaction. Called the threshold method).
- this method is referred to as a differential method.
- problems such as the need for special reagents and the narrow measurable concentration range.
- the following method has been completed in order to solve the disadvantages of the various methods described above. That is, the light from the light source is focused and irradiated to a mixture of a predetermined physiologically active substance and LAL, and the coagulin itself (coagulin monomer), which is the final product of the protease cascade, and the extremely It collides with a minute aggregate (coagulin aggregate) to generate scattered light. And the density
- the present invention is based on the turbidimetric method for detecting the gelation of LAL itself, a low concentration of a predetermined physiologically active substance can be rapidly obtained using a normal LAL reagent without using a special reagent. Can be detected.
- a differential method that only needs to detect the rate of increase in the intensity of scattered light within a predetermined time is adopted, so as with the colorimetric method, it is necessary to wait until gelation occurs. Therefore, the measurement time can be shortened.
- a laser or a high-intensity LED is used as the light source of the present invention.
- the light energy of the incident light can be concentrated on the irradiated part, so that scattered light of sufficient intensity can be generated and detected from extremely small particles such as coagulin monomer and minute coagulin aggregates. It is possible.
- the sample is stirred by a stirrer built in the measurement container, and by stirring the sample, the reaction is homogenized, the reaction is accelerated, and the generated coagulin monomer is rapidly oligomerized. Mobilization of people is carried out efficiently.
- the sample is allowed to stand without stirring, as in the case of the turbidimetric method, the sample is finally gelled, so an increase in scattered light is observed.
- coagulin monomer and minute coagulin aggregation at the initial stage of the reaction are observed. It may be difficult to accurately detect an increase in scattered light due to an object.
- a plurality of scattered light intensities acquired in a predetermined period are sampled and compared, and the minimum value or the mode of the histogram is used as a scattered light intensity in the period. By doing so, the influence of impurities is eliminated, and weak scattered light of the target substance is obtained.
- the present invention focuses on the fact that the higher the concentration of endotoxin on which the ratio of the temporal change of the weak scattered light generated from the measurement object obtained acts, the smaller the lower the concentration. Therefore, even a sample having a low endotoxin concentration can be quantified in a short time without waiting for the appearance of aggregated fine particles or gelation. This can be said to be the effect of using the differential method in the present invention as well as the colorimetric method.
- the concentration of the predetermined physiologically active substance is overwhelmingly high, rather than observing the increase rate of weak scattered light from the target substance such as the coagulogen monomer and the coagulogen oligomer sufficiently.
- the target substance such as the coagulogen monomer and the coagulogen oligomer
- micrometer-level coagulogen polymers with which they are further associated may be formed.
- a threshold method may be applied in which the concentration is calculated after a time when the intensity of the scattered light exceeds a certain level.
- FIG. 1 shows a schematic configuration of a measurement system 1 for a predetermined physiologically active substance in the present embodiment.
- the light source 2 used in the measurement system a laser, an ultra-bright LED, or the like is 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 mixed solution of a sample to be measured for a predetermined physiologically active substance and the LAL reagent.
- the light incident on the sample cell 4 is scattered by particles (coagulogen monomer, measurement object such as coagulogen oligomer) in the mixture.
- the emission optical system 5 is arranged on the side of the incident optical axis of the sample cell 4.
- 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.
- the light receiving element 6 is electrically connected to an amplifying circuit 7 that amplifies the electric signal photoelectrically converted by the light receiving element 6.
- a filter 8 for removing noise from the electric signal amplified by the amplifier circuit 7, an increase rate of scattered light is calculated from the electric signal after the noise is removed, and a concentration of a predetermined physiologically active substance is derived.
- a display 10 for displaying the results.
- the scattered light intensity ks is expressed as follows.
- n is the number of particles
- d is the particle diameter
- m is the reflection coefficient
- ⁇ is the wavelength of the incident light. Therefore, the shorter the wavelength in the light source 2, the more advantageous for measurement.
- LAL contains a high concentration of protein, extremely short wavelengths not only adversely affect the function of LAL, but also require special materials that optically transmit these short wavelengths and are practical. is not.
- the wavelength of incident light it is not necessary to specifically limit the wavelength of incident light, but a range of 250 nm to 1200 nm is desirable. Furthermore, the range of 300 nm or more and 800 nm or less is desirable. If the wavelength of incident light is in the range of 300 nm or more and 800 nm or less, it is possible to obtain scattered light sufficiently efficiently, and there is no influence on the function of LAL, and optical materials made of general materials can be used. . Moreover, when narrowing down these light sources to a sample, it is necessary that scattered light from minute particles (coagulogen monomer, measurement object such as coagulogen oligomer) in the mixed solution has sufficient intensity.
- minute particles coagulogen monomer, measurement object such as coagulogen oligomer
- the beam width (beam diameter) of the light incident on the sample cell 4 is preferably 3 mm or less, and more preferably 1 mm or less.
- the beam width of the light incident on the sample cell 4 is preferably 3 mm or less, and more preferably 1 mm or less.
- the light receiving element 6 that receives the scattered light needs to be low in noise and capable of detecting weak scattered light. Accordingly, examples of the light receiving element 6 include a photodiode, a phototransistor, an array in which a large number of these are incorporated, and a photomultiplier.
- a line sensor or an area sensor using a CCD (charge coupled device) or a C-MOS (complementary metal oxide semiconductor device) may be used. Since the intensity of the scattered light obtained by these light receiving elements 6 is extremely weak compared to the intensity of the scattered light obtained from micrometer-level fine particles, usually, at least one amplification circuit 7 such as a resistor or an operational amplifier is provided. It is necessary to incorporate and amplify the above.
- a filter 8 for removing the influence of fine particles contaminating the sample or reagent 1) sampling and comparing the scattered light intensities at several points in the close time (during a predetermined period), the minimum of them A minimum value filter that outputs a value or a mode filter that outputs a mode value of a histogram, and 2) since scattered light of a contaminant is rarely generated compared to generation of scattered light of a target substance, this is applied to an electronic circuit. 3) a digital filter that digitally removes contaminants by acquiring a change in potential over time. By using at least one or more of these filters, it is possible to eliminate the influence of impurities and obtain weak scattered light of the target substance.
- the weak scattered light from the measurement object after passing through the filter 8 may already have a large value at the start of measurement by the amplification device 6 described above. Therefore, it may be used for analysis by removing it as a baseline and further amplifying it as appropriate.
- a dilution series of a predetermined physiologically active substance with a known concentration is prepared, and for each sample, 1) the rate of increase in scattered light intensity obtained as the slope when time is plotted on the horizontal axis and scattered light intensity is plotted on the vertical axis ( Differentiation method), 2) The initial scattered light intensity is subtracted from the scattered light intensity at each time, and the time when the difference exceeds a predetermined threshold (threshold method) is obtained.
- the relational expression (calibration curve) between the concentration of the predetermined physiologically active substance and these values is calculated, and the calibration curve obtained by the differential method and the threshold value method are obtained from the sample whose concentration of the predetermined physiologically active substance is unknown. Apply to one or both of the calibration curves obtained in. Thereby, the concentration of the predetermined physiologically active substance can be measured.
- the sample cell 4 is provided with a magnetic stirrer bar (stirring bar) 11 that rotates by applying electromagnetic force from the outside and stirs the mixed liquid as the sample.
- a measuring system 1 outside the cell 4 is provided with a magnetic stirrer 12.
- the stirring speed is preferably from 100 rpm to 5,000 rpm, and more preferably from 300 rpm to 3,000 rpm. At 2,000 rpm or higher, the suppression of aggregation may be observed, and at 500 rpm or lower, the sample is not sufficiently stirred, and coagulin aggregation may be observed only below the sample.
- the coagulin monomer to be measured by the stirring and fine particles larger than the minute coagulin aggregate are used as the beam of the light source. Since it has an effect of not staying above, it can be made difficult to be affected by variations in data and an increase in apparent scattered light.
- ⁇ Production example 1> Put a stainless steel stirrer ( ⁇ 1mm, length 5mm) in a glass container (outer diameter ⁇ 7mm, length 50mm, hereinafter abbreviated as cuvette), and put some aluminum foil on the cuvette opening. They were further covered with aluminum foil, heat-treated at 250 ° C. for 3 hours, and the glass container was sterilized (dry heat sterilization). Thereby, the endotoxin adhering to the container is thermally decomposed and inactivated.
- An illumination optical system (diameter of the entrance to the sample is 0.2 mm) capable of narrowing down and irradiating the laser beam by appropriately combining a lens with a semiconductor laser (output: 10 mW, wavelength: 655 nm) was created. In this case, the output density of incident light is about 80 mW / mm 2 .
- a sample containing 0.01 EU / mL endotoxin was mixed with LAL (Limulus ES-II Single Test Wako: manufactured by Wako Pure Chemical Industries, Ltd.), and then placed in the cuvette manufactured in Production Example 1.
- a stainless steel stirrer 11 in the sample was rotated by a magnetic stirrer 12 as shown in FIG. 1, and the admixture was set on a stirrable holder. In this example, a cuvette is used as the sample cell 4.
- the sample was stirred at 1,000 rpm. This holder portion is kept at 37 ° C. in order to advance the gel reaction of LAL.
- a photodiode was used as the side-scattering light receiving element 6.
- the scattered light components received are fine particles in the sample (undissolved reagent, fine particles contained in the reagent, minute bubbles) Etc.) containing strong scattered light. Therefore, the scattered light of these contaminants was removed using the minimum value filter as the filter 8, and the time series change of the scattered light was taken.
- the minimum value filter amplifies the optical potential received by the light receiving element 6 as appropriate by the amplifier circuit 7 and then performs analog-digital conversion (10 bits), which is performed 25 times every 20 milliseconds. The method of obtaining the value of was used. Here, 20 milliseconds corresponds to a predetermined period in this embodiment.
- Example 2 A semiconductor laser (output 10 mW, wavelength 655 nm) was appropriately combined with a lens, and an optical system (the diameter of the entrance to the sample was 3.0 mm) capable of irradiating the laser beam in the form of a parallel laser pointer was created. Only the conditions of this light source optical system differed from those of Example 1, and the other conditions were the same, and processing was performed to take a time series change of scattered light. In this case, the output density of incident light is about 0.35 mW / mm 2 .
- Example 1 The results of Example 1 and Example 2 are shown in FIG.
- the horizontal axis represents time (minutes), and the vertical axis represents scattered light intensity obtained from the output of the photodiode.
- Example 1 there is a phase in which scattered light increases from the initial stage of the reaction, as in the portion A surrounded by an ellipse in the figure, and then, through the inflection point, the phase B has a larger inclination.
- the A phase is hardly observed, and the B phase generated with a delay is mainly observed.
- the measurement conditions other than the light source 2 such as the concentration of the endotoxin to be acted on are the same.
- Example 2 since sufficient scattered light cannot be obtained from minute particles, the sample It is believed that changes that are progressing inside cannot be detected.
- the inflection point in the curve of Example 1 corresponds to the point at which a predetermined sudden change occurs in this example.
- the sample is sufficiently irradiated with the light source 2 such as a high-power laser, and sufficient output is obtained. It is necessary to ensure the density.
- Example 3 Under the same conditions as in Example 1, except that the sample was not stirred, the time series change of scattered light was taken. As a result, even when stirring was not performed, it was possible to obtain a state in which the coagulin monomer and minute coagulin aggregates increased with time. However, the acquired data varies greatly, and if there are particles larger than the target microparticles on the beam of the light source 2, the data stays at the same position for a long time. In some cases, the apparent scattered light may be very high even if the process is performed. In such a case, it has been difficult to correctly evaluate the phenomenon in which weak scattered light increases. On the other hand, when the sample was agitated, these large particles did not stay on the beam promptly, but quickly dislodged, so that correct evaluation was possible.
- Example 4 The light source 2 and the incident optical system 3 used in Example 1 are used, and the processing is the same as in Example 1 except that the scattered light filtering method uses an average value filter. Time series changes were taken.
- the average value filter the optical potential received by the light receiving element 6 is appropriately amplified by the amplifier circuit 7 and then analog-digital converted (10 bits). This is performed 25 times every 20 milliseconds, and the average of 25 data obtained is obtained. The method of outputting a value was used.
- the obtained data was greatly influenced by the scattered light of particles larger than the target fine particles, and it was difficult to correctly evaluate the phenomenon in which weak scattered light increased.
- Particles larger than the target fine particles such as coagulin monomer and minute coagulin aggregates are removed from the minute bubbles and reagent remaining in the mixture, and further removed in the reagent manufacturing process. Cell fragments that are not used. Since the appearance of these particles varies depending on the mixture, it is considered that they cannot be removed appropriately as long as the average value method is used.
- a simulated sample of an object to be measured that is, neutral fat (0.0002% intralipid) and polystyrene latex particles ( ⁇ 1 ⁇ m, weight percentage 0.0025%)
- the performance of the following filter 8 was examined using a mixture of Each of the filters 8 amplifies the optical potential received by the light receiving element 6 by the amplifier circuit 7 and then performs analog-to-digital conversion (10 bits). This is performed 25 times every 20 milliseconds. From the obtained 25 data, 1 2) Median filter that outputs the 13th value after sorting in descending order 2) Average filter that outputs average value 3) Minimum value filter that outputs minimum value 4) Maximum value that outputs maximum value It is a value filter.
- the minimum value filter is most suitable for obtaining the minute scattered light of the target substance for the following reason.
- the average value is lower than that of other filters, this is because scattered light of a target substance having a low intensity is mainly output.
- the standard deviation is smaller than that of other filters, this indicates that the standard deviation is not easily affected by scattered light of large particles that are contaminated.
- the maximum value filter outputs not only scattered light of the target substance but also a signal of contaminating fine particles, so that the average value is large and the standard deviation is large.
- the average value filter outputs the average value of both the weak scattered light of the target substance and the scattered light of the contaminating fine particles, it shows an intermediate value between the minimum value filter and the maximum value filter. Also, the median filter has the same result as the average filter, and it cannot be said that the median filter is superior in performance.
- the minimum value filter it may be possible to output a value in a specific order of the smaller one of the sampled data and an average value thereof. For example, a filter that obtains 25 data, performs sorting, and outputs the average value of the remaining 5 data without using the lowest of the lower 6 data works effectively.
- Example 5 A dilution series with various endotoxin concentrations was prepared by the method shown in Example 1, and the relationship between the endotoxin concentration and the initial increase rate of scattered light due to coagulin monomer and minute coagulin aggregates was examined. It was. As a result, it was found that the lower the endotoxin concentration, the smaller the increase rate, and the higher the concentration, the greater the increase rate.
- the horizontal axis represents endotoxin concentration (EU / mL), and the vertical axis represents the initial minute scattered light increase rate (the slope of the initial scattered light increase curve).
- Example 6 In the same manner as in Example 5, the relationship between the endotoxin concentration and the initial increase rate of the scattered light was examined in the same manner as in Example 5 by making a device in which the light receiving element 6 for scattered light was replaced with a CCD area sensor.
- FIG. 4B shows the result.
- the horizontal axis represents endotoxin concentration (EU / mL)
- the vertical axis represents the initial minute scattered light increase rate (the slope of the initial scattered light increase curve).
- FIG. 4 (a) the change over time in the intensity of scattered light is also shown.
- the horizontal axis represents time (minutes) and the vertical axis represents scattered light intensity obtained from the output of the CCD area sensor.
- the initial scattered light linearly increases in the same manner as the phase A in FIG. 2, and then passes through the inflection point to a phase with a larger increase rate (the phase of the ellipse B in FIG. 2). You can see how it changes.
- Example 7 A method equivalent to the method shown in Example 1 was examined using ⁇ -D-glucan instead of endotoxin.
- LAL reagent ⁇ -glucan test Wako (manufactured by Wako Pure Chemical Industries) was used. Dilution series with various concentrations of ⁇ -D-glucan were prepared, and the relationship between the ⁇ -D-glucan concentration, the coagulin monomer, and the initial increase rate of scattered light due to minute coagulin aggregates was examined. As a result, it was found that the lower the ⁇ -D-glucan concentration, the smaller the increase rate, and the higher the concentration, the higher the increase rate.
- the horizontal axis represents the ⁇ -D-glucan concentration (pg / mL)
- the vertical axis represents the initial minute scattered light increase rate (the slope of the initial scattered light increase curve).
- a measurement apparatus in which the measurement system 1 of the predetermined physiologically active substance as shown in FIG. 1 is integrated may be configured.
- the arithmetic unit 9 calculates the concentration of the predetermined physiologically active substance from the calibration curves obtained in FIGS. 3, 4, and 5 and the increase rate obtained from the scattered light from the mixture, 10 may automatically display the result.
- the sample cell 4 corresponds to the liquid mixture holding means
- the light source 2 and the incident optical system 3 correspond to the light incident means
- the emission optical system 5 and the light receiving element 6 correspond to the light receiving means
- the arithmetic unit 9 corresponds to the derivation means.
- the stirrer 11 and the magnetic stirrer 12 correspond to a stirrer.
- a general Limulus reagent used in the turbidimetric method can be used as it is.
- the configuration of the measurement system (measurement device) can be simplified, and multi-channel (8 to 16 ch) is relatively easy. 4) Measurement can be completed in the same time as the colorimetric method using a special reagent. And so on.
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Abstract
Description
前記試料とLALとの混和後において、前記試料とLALとの混和液中に光を入射するとともに該入射光の前記混和液による散乱光の強度を取得し、
前記散乱光の強度の増加率より前記試料中の前記生理活性物質を検出しまたは濃度を測定することを特徴とする生物由来の生理活性物質の測定方法である。
前記試料とLALとの混和液を光の入射可能に保持し、前記生理活性物質とLALとの反応を進行させる混和液保持手段と、
前記混和液保持手段中の混和液に光を入射する光入射手段と、
前記入射光の前記混和液における散乱光を受光し電気信号に変換する受光手段と、
前記受光手段において変換された電気信号から取得される前記散乱光の強度の増加率より前記試料中の前記生理活性物質の濃度を導出する導出手段と、
を備えることを特徴とする生物由来の生理活性物質の測定装置であってもよい。
本発明の光源にはレーザーあるいは高輝度のLEDが用いられ、それをレンズによって絞込んで混和液に照射する。これにより、照射した部分に入射光の光エネルギーを集中させることができるので、コアギュリンモノマー及び、微小なコアギュリン凝集物といった極めて微小な粒子からも充分な強度の散乱光を発生させて検出することが可能である。
また、本発明において試料は測定容器中に内蔵している攪拌子により攪拌しており、この試料の攪拌によって、反応の均一化、反応の促進、生成されたコアギュリンモノマーの速やかなオリゴマーへの動員などが効率よく行なわれる。試料を攪拌せずに静置した場合は比濁法と同様に最終的には試料がゲル化するために散乱光の増加が認められるが、反応初期のコアギュリンモノマー及び、微小なコアギュリン凝集物による散乱光の増加を精度よく検出することは困難な場合がある。
さらに、試料中には試薬の溶け残り、試薬の製法上残存するマイクロメータレベルの微粒子、試料の攪拌に伴う微小な気泡などが存在する。コアギュリンモノマー及び、微小なコアギュリン凝集物などから散乱する微弱な信号は、数は少ないが非常に強力な散乱光を発生させるこれらの夾雑物の散乱光に埋もれてしまうため、そのままでは測定することができない。
本発明は上記のように、取得された測定対象から発生する微弱な散乱光の時間変化の割合が作用させたエンドトキシンの濃度が高いほど大きく、濃度が低いほど小さいことに着目したものである。そのため、エンドトキシン濃度の低い試料であっても、凝集微粒子の出現やゲル化を待つまでもなく、短時間でその濃度を定量することが可能である。これは本発明は比色法と同様微分法を利用することによる効果と言える。
ガラス製の容器(外径φ7mm、長さ50mm。以下、キュベットと略)にステンレス製の攪拌子(φ1mm、長さ5mm)を入れ、キュベット開口部にアルミホイルをして、それを何本かまとめてアルミ箔でさらに覆い、250℃、3時間加熱処理し、ガラス容器を滅菌処理した(乾熱滅菌)。これにより、容器に付着するエンドトキシンは熱分解を受け不活性化される。
半導体レーザー(出力10mW、波長655nm)に適宜レンズを組み合わせて、レーザー光を絞り込んで照射できる照明光学系(試料への入射口の直径は0.2mm)を作成した。この場合の入射光の出力密度は、約80mW/mm2である。0.01EU/mLのエンドトキシンを含有する試料をLAL(リムルスES-II シングルテストワコー:和光純薬製)と混合し、続いて製造例1にて製造したキュベット内部に入れた。これを図1に示したようなマグネチックスターラー12により試料中のステンレス製攪拌子11を回転せしめ、混和液を攪拌可能なホルダ部にセットした。この例ではキュベットが試料セル4として用いられている。
半導体レーザー(出力10mW、波長655nm)に適宜レンズを組み合わせ、レーザー光をレーザーポインタ状に平行光にして照射できる光学系(試料への入射口の直径は3.0mm)を作成した。実施例1とはこの光源光学系の条件のみが異なり、他の条件は同一で処理を行い、散乱光の時系列変化を取った。なお、この場合の入射光の出力密度は、約0.35mW/mm2である。
実施例1と同条件で、ただし、試料の攪拌を行わずに散乱光の時系列変化を取った。その結果、攪拌を行わない場合であっても、コアギュリンモノマー及び、微小なコアギュリン凝集物が時間とともに増加していく様子を得ることができた。しかしながら、取得されるデータはばらつきが大きく、また、光源2のビーム上に目的の微小粒子よりも大きな粒子が存在した場合、同じ位置に長い時間留まってしまうため、データに対し最小値フィルタ処理などを行ったとしても見かけの散乱光が非常に高く出てしまう場合があった。そのような場合は、微弱な散乱光が増加していく現象を正しく評価することが困難であった。これに対し試料を攪拌した場合には、これらの大型粒子は速やかにビーム上に留まることなく、速やかに外れるために正しい評価が可能となった。
実施例1で使用した光源2及び入射光学系3を用い、実施例1とは散乱光のフィルタ処理の方式が平均値フィルタを使用しているほかは同一の条件で処理を行い、散乱光の時系列変化を取った。平均値フィルタには、受光素子6で受光した光電位を増幅回路7で適宜増幅した後にアナログディジタル変換(10ビット)し、これを20ミリ秒ごとに25回行い、得られた25データの平均値を出力するという方法を用いた。
実施例1に示した方法で、エンドトキシン濃度を種々に変化させた希釈系列を作成し、エンドトキシン濃度とコアギュリンモノマー、ならびに、微小なコアギュリン凝集物による散乱光の初期の増加率の関係を調べた。その結果、エンドトキシン濃度が低いほど増加率が小さく、濃度が高いほど増加率が大きいことが分かった。エンドトキシン濃度と初期散乱光の増加率の関係を両対数でプロットすると図3のように直線関係が得られた。横軸はエンドトキシン濃度(EU/mL)、縦軸は初期微小散乱光増加率(初期の散乱光の増加曲線の傾き)を示している。
実施例1に示した方法で、散乱光の受光素子6をホトダイオードから、CCDエリアセンサに置き換えた装置を作り、実施例5と同様にエンドトキシン濃度と散乱光の初期増加率の関係を調べた。図4(b)にはその結果を示す。図4(b)において横軸はエンドトキシン濃度(EU/mL)、縦軸は初期微小散乱光増加率(初期の散乱光の増加曲線の傾き)を示している。
図4(a)においては同時に、散乱光の強度の時間変化についても掲載した。図4(a)において横軸は時間(分)、縦軸はCCDエリアセンサの出力より得られた散乱光強度である。CCDエリアセンサを用いても、図2中のAの相と同様に初期散乱光が直線的に増加した後、屈曲点を経てさらに大きな増加率の相(図2中の楕円Bの相)で変化していく様子が分かる。
実施例1に示した方法と同等の方法で、エンドトキシンの代わりにβ-D-グルカンを用いて検討を行った。LAL試薬としてはβ-グルカン テストワコー(和光純薬製)を用いた。β-D-グルカン濃度を種々にした希釈系列を作成し、β-D-グルカン濃度とコアギュリンモノマー、ならびに、微小なコアギュリン凝集物による散乱光の初期の増加率の関係を調べた。その結果、β-D-グルカン濃度が低いほど増加率が小さく、濃度が高いほど増加率が大きいことが分かった。β-D-グルカン濃度と初期散乱光の増加率の関係を両対数でプロットすると図5のように直線関係が得られた。図5において横軸はβ-D-グルカン濃度(pg/mL)、縦軸は初期微小散乱光増加率(初期の散乱光の増加曲線の傾き)を示している。
2・・・光源
3・・・入射光学系
4・・・試料セル
5・・・出射光学系
6・・・受光素子
7・・・増幅回路
8・・・ノイズ除去フィルタ
9・・・演算装置
10・・・表示器
11・・・攪拌子
12・・・マグネチックスターラー
Claims (16)
- 試料中に存在する生物由来の生理活性物質とカブトガニの血球抽出物であるLALとを反応させることで、前記試料中の前記生理活性物質を検出しまたは前記生理活性物質の濃度を測定する、生物由来の生理活性物質の測定方法であって、
前記試料とLALとの混和後において、前記試料とLALとの混和液中に光を入射するとともに該入射光の前記混和液による散乱光の強度を取得し、
前記散乱光の強度の増加率より前記試料中の前記生理活性物質を検出しまたは濃度を測定することを特徴とする生物由来の生理活性物質の測定方法。 - 前記散乱光の強度の増加率が所定の急変化を起こす前における前記増加率より前記試料中の前記生理活性物質を検出しまたは濃度を測定することを特徴とする請求項1に記載の生物由来の生理活性物質の測定方法。
- 前記混和液に入射する光の出力密度は50mW/mm2以上であることを特徴とする請求項1または2に記載の生物由来の生理活性物質の測定方法。
- 前記混和液に入射する光の波長は300nm以上800nm以下であることを特徴とする請求項1から3のいずれか一項に記載の生物由来の生理活性物質の測定方法。
- 前記散乱光の強度の増加率を取得する際には、所定期間に取得された散乱光の強度を複数個サンプリングして比較し、そのうちの最小値または、ヒストグラムの最頻値を前記期間における散乱光の強度とすることを特徴とする請求項1から4のいずれか一項に記載の生物由来の生理活性物質の測定方法。
- 前記散乱光の強度を取得する際には、前記混和液を攪拌することを特徴とする請求項1から5のいずれか一項に記載の生物由来の生理活性物質の測定方法。
- 前記混和液の攪拌速度は、300rpm以上3000rpm以下であることを特徴とする請求項6に記載の生物由来の生理活性物質の測定方法。
- 前記生物由来の生理活性物質は、エンドトキシンまたはβ-D-グルカンであることを特徴とする請求項1から7のいずれか一項に記載の生物由来の生理活性物質の測定方法。
- 試料中に存在する生物由来の生理活性物質とカブトガニの血球抽出物であるLALとを反応させることで、前記試料中の前記生理活性物質を検出しまたは前記生理活性物質の濃度を測定する、生物由来の生理活性物質の測定装置であって、
前記試料とLALとの混和液を光の入射可能に保持し、前記生理活性物質とLALとの反応を進行させる混和液保持手段と、
前記混和液保持手段中の混和液に光を入射する光入射手段と、
前記入射光の前記混和液における散乱光を受光し電気信号に変換する受光手段と、
前記受光手段において変換された電気信号から取得される前記散乱光の強度の増加率より前記試料中の前記生理活性物質の濃度を導出する導出手段と、
を備えることを特徴とする生物由来の生理活性物質の測定装置。 - 前記導出手段は、前記混和液保持手段における前記試料とLALとの混和後であって前記増加率が所定の急変化を起こす前における前記増加率より前記試料中の前記生理活性物質の濃度を導出することを特徴とする請求項9に記載の生物由来の生理活性物質の測定装置。
- 前記光入射手段により前記混和液に入射される光の出力密度は50mW/mm2以上であることを特徴とする請求項9または10に記載の生物由来の生理活性物質の測定装置。
- 前記光入射手段により前記混和液に入射される光の波長は300nm以上800nm以下であることを特徴とする請求項9から11のいずれか一項に記載の生物由来の生理活性物質の測定装置。
- 前記受光手段において変換された電気信号を、所定期間に複数個サンプリングして比較し、そのうちの最小値を出力する最小値フィルタまたは、ヒストグラムの最頻値を出力する最頻値フィルタをさらに備えることを特徴とする請求項9から12のいずれか一項に記載の生物由来の生理活性物質の測定装置。
- 前記混和液保持手段は、前記混和液を攪拌する攪拌手段を有することを特徴とする請求項9から13のいずれか一項に記載の生物由来の生理活性物質の測定装置。
- 前記攪拌手段による前記混和液の攪拌速度は、300rpm以上3000rpm以下であることを特徴とする請求項14に記載の生物由来の生理活性物質の測定装置。
- 前記生物由来の生理活性物質は、エンドトキシンまたはβ-D-グルカンであることを特徴とする請求項1から15のいずれか一項に記載の生物由来の生理活性物質の測定装置。
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