WO2013062013A1 - 生物由来の生理活性物質の測定方法及び、それに用いられる微粒子及び抽出液 - Google Patents
生物由来の生理活性物質の測定方法及び、それに用いられる微粒子及び抽出液 Download PDFInfo
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- WO2013062013A1 WO2013062013A1 PCT/JP2012/077509 JP2012077509W WO2013062013A1 WO 2013062013 A1 WO2013062013 A1 WO 2013062013A1 JP 2012077509 W JP2012077509 W JP 2012077509W WO 2013062013 A1 WO2013062013 A1 WO 2013062013A1
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- endotoxin
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- active substance
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
Definitions
- the present invention relates to a measuring method 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 ⁇ -glucan. And it is related with the microparticles used for it.
- a biologically active substance derived from a living organism that adheres to a medical device is extracted and adsorbed to fine particles, and then the fine particles are mixed with a Limulus reagent (AL reagent) that is a reagent for measuring the physiologically active substance, and the mixture is stirred under stirring conditions.
- AL reagent Limulus reagent
- the present invention relates to a method for measuring a biologically active substance derived from a living body in a medical device, which measures the physiologically active substance by detecting aggregation of AL by an optical technique, and an extract used therefor.
- 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. On the other hand, measuring endotoxin in the blood of septic patients may contribute to the prevention and treatment of severe endotoxin shock.
- ⁇ -glucan is a polysaccharide (polysaccharide) that constitutes a cell membrane characteristic of fungi. By measuring ⁇ -glucan, it is effective for screening of a wide range of fungal infections including rare fungi as well as common fungi such as Candida, Aspergillus and Cryptococcus.
- AL Amoebocyte lysate
- coagulogen present in AL is hydrolyzed into coagulin and associated by an enzyme cascade by serine protease activated according to their amount, An insoluble gel is produced.
- endotoxin and ⁇ -glucan can be detected with high sensitivity.
- a method using AL for detection or concentration measurement of endotoxin has been devised using this fact.
- predetermined physiologically active substances such as endotoxin and ⁇ -glucan
- measurement of predetermined physiologically active substances The following is known as a method of performing the above.
- a turbidimetric method for measuring a reaction in which a predetermined physiologically active substance acts on AL and the sample becomes turbid a reaction in which a synthetic substrate added by an enzyme in AL activated by the action of a predetermined physiologically active substance decomposes and develops color
- a turbidity of AL itself and the color developed by the synthetic substrate when a low concentration of a predetermined physiologically active substance acts are very weak, the measurement lower limit of the physiologically active substance in these measurement methods is 0.001 EU / mL (EU: Endotoxin unit).
- dialysate lacks measurement sensitivity with conventional turbidimetric and colorimetric methods there are cases in which dialysate lacks measurement sensitivity with conventional turbidimetric and colorimetric methods.
- the endotoxin concentration of ultrapure dialysate and replacement dialysate is less than 0.001 EU / mL.
- the turbidimetric method is developed to stir the sample (stirring turbidimetric method), and the gel particles formed in the stirred sample are detected by the light scattering method (light Scattering method), a method in which AL is bonded onto fine particles and the aggregation reaction is enhanced (AL binding bead method) have been reported.
- a technique for measuring a predetermined physiologically active substance of 0.0001 EU / mL or less within a practical time has been reported.
- AL gels by a chain reaction of a plurality of proteolytic enzymes (serine protease). Therefore, if the sample contains a substance that inhibits or enhances the serine protease of AL (interfering substance), the sample cannot be diluted sufficiently or the interfering substance must be removed to accurately quantify the predetermined physiologically active substance. There was a case that could not be done.
- an amino acid, a polypeptide adsorbing a predetermined physiologically active substance, a lipid A binding peptide selected from a random peptide library, etc. are bound on a bead-shaped carrier and mixed with a sample containing the predetermined physiologically active substance.
- a batch method has been proposed in which a predetermined physiologically active substance is fixed to a carrier.
- a carrier to which a predetermined physiologically active substance is immobilized is reacted with AL, and the turbidity change of AL due to gelation or the action of the predetermined physiologically active substance is measured.
- the batch method there is a pyrosep method in which pyrosep capable of binding endotoxin is bound to cellulose or agarose beads to form a column, and a sample containing a large amount of endotoxin is passed through this (see Non-Patent Document 1). ).
- the column after passing through the sample is washed with an alkaline buffer to dissociate endotoxin, then reacted with AL, and endotoxin is quantified by kinetic turbidimetry.
- 0.5 EU / mL endotoxin can be detected in about 60 minutes.
- a random peptide library method (Patent Document 1).
- lipid A which is an active center of endotoxin
- a random peptide library method is converted into 200-400 mesh (50-100 ⁇ m) Propionyl chloride functionalized silica. Bind to gel (Sigma Aldrich).
- the endotoxin-containing sample is mixed with the silica gel after the above binding, and the endotoxin is adsorbed on the beads. And after washing
- the binding carriers used in the above methods are all large, have low light scattering properties, and are fragile materials (agarose, cellulose, silica gel).
- measurement methods that enable short-time measurement such as the AL-bound bead method, require a binding carrier with a high refractive index and high strength to optically measure a predetermined physiologically active substance with stirring of the sample. Is done. Therefore, it is difficult to apply the above method to the AL-bound bead method and the like, and it has been difficult to significantly reduce the measurement time of a predetermined physiologically active substance by the above method.
- the medical device refers to a device installed in the patient body during treatment, an injection device such as a syringe or a needle, a filter tube or a bag in contact with an injection drug or an infusion solution.
- Patent Document 5 it is known that endotoxin adhering to medical devices is difficult to extract with water (Patent Document 5).
- Patent Document 5 a method of extracting endotoxin from a medical device with an extract containing an amine-containing compound or albumin as appropriate and measuring the endotoxin concentration after extraction in the extract is effective.
- extraction of endotoxin adhering to the instrument depends on the size of the instrument, a large amount of extract must be used, so that endotoxin is diluted by the extract. Then, since endotoxin is dilute, there are problems that measurement becomes difficult and detection time becomes long.
- the present invention has been devised in view of the above-mentioned problems, and the object of the present invention is to contain a sample having a very low concentration of biologically active substance derived from living organisms and a component having an interference action on AL. It is another object of the present invention to provide a technique that can detect a biologically active physiologically active substance or measure its concentration with higher accuracy or in a shorter period of time. Another object of the present invention is to provide a measurement method capable of measuring a biologically active substance derived from a living organism adhering to a medical device more accurately or in a shorter time, and an extract used for the measurement method.
- the present invention disperses fine particles having affinity for a predetermined physiologically active substance in a sample containing the predetermined physiologically active substance, and adsorbs the predetermined physiologically active substance on the fine particles, and then collects the fine particles.
- the greatest feature is that agglutination is induced by reacting with AL, and agglutination is detected by an optical method to measure a predetermined physiologically active substance.
- a mixture of an AL reagent containing AL which is a blood cell extract of horseshoe crab, and a sample containing a physiologically active substance derived from a predetermined organism is generated, and the mixture is mixed with the AL in the mixture while stirring the mixture.
- the biologically active substance in the sample is detected or the concentration of the physiologically active substance is measured.
- fine particles containing a material having a low optical transparency or a high refractive index and sufficient strength against mechanical external force are mixed with a sample containing a predetermined physiologically active substance.
- a predetermined physiologically active substance is adsorbed on the fine particles.
- the sample at that time may have a characteristic that the concentration of the predetermined physiologically active substance is extremely low and / or a characteristic that includes an interfering substance that affects the reaction of AL.
- the fine particles adsorbed with the predetermined physiologically active substance are separated and recovered by operations such as natural sedimentation, centrifugation, and filter filtration, and the fine particles are washed with a liquid that does not contain the predetermined physiologically active substance as necessary.
- a predetermined physiologically active substance is measured by causing the microparticles to react with AL to induce an agglutination reaction and acquiring optical characteristics such as light transmittance, intensity of light scattered light, and number of peaks.
- the predetermined physiologically active substance adhering to the medical device is adsorbed onto the fine particles having an affinity for the predetermined physiologically active substance, and then the fine particles are separated and recovered and reacted with AL to cause an agglutination reaction,
- the aggregation may be detected by an optical technique to measure a predetermined physiologically active substance.
- an AL reagent containing AL which is a blood cell extract of horseshoe crab, and an extract containing a predetermined physiologically active substance extracted from a medical device is generated, and the mixture is stirred while the mixture is stirred.
- the predetermined physiologically active substance in the extract is detected or the concentration of the predetermined physiologically active substance is measured
- a method for measuring a predetermined physiologically active substance in a medical device The fine particles capable of adsorbing the predetermined physiologically active substance on the surface are dispersed in the extract to separate the fine particles from the extract after the predetermined physiologically active substance is adsorbed on the surface of the fine particles, Producing a mixture of the AL reagent and the fine particles having a predetermined physiologically active substance adsorbed on the surface thereof; Aggregation or gelation of the fine particles in the mixed solution of the AL reagent and the fine particles may be detected by an optical technique.
- the predetermined physiologically active substance adhering to the medical device is extracted into an extract in which fine particles having an affinity for the predetermined physiologically active substance are dispersed and adsorbed onto the fine particles.
- the fine particles adsorb a predetermined physiologically active substance whose main component or one of the components is a material having low optical transparency or high refractive index and sufficient strength against mechanical external force. It is a fine particle.
- the fine particles adsorbed with the predetermined physiologically active substance are separated and recovered by natural sedimentation, centrifugation, filter filtration or the like, and the fine particles are washed with a liquid not containing the predetermined physiologically active substance as necessary.
- microparticles are reacted with AL to cause an agglutination reaction, and optical properties such as light transmittance and intensity / peak number of scattered light are obtained to measure a predetermined physiologically active substance attached to a medical device. I do.
- the fine particles include pyrosep, polymyxin B, polylysine, polyornithine, polyarginine, polyhistidine, aminosilane, chitosan, anti-endotoxin antibody, anti-endotoxin aptamer, material in random peptide library, alumina, titania, silica, zirconia, It may be made of one or a plurality of materials selected from metal oxides such as hydroxyapatite, natural or synthetic minerals such as kaolin, montmorillonite, manganese oxide, and mica.
- an adsorbing material that adsorbs a predetermined physiologically active substance an organic compound with high selectivity that has an excellent ability to adsorb a predetermined physiologically active substance such as endotoxin, or a predetermined physiologically active substance such as endotoxin that is not highly selective. Any inorganic substance having excellent adsorption ability may be used.
- the inorganic substance include metal oxides such as alumina, titania, silica, zirconia, and hydroxyapatite, and natural or synthetic minerals such as kaolin, montmorillonite, manganese oxide, and mica.
- the above-mentioned organic compound may be granulated if possible, or may be granulated by electrostatic adsorption or chemical bonding on fine particles of an organic or inorganic binding carrier. That is, in the present invention, the fine particles may be formed by binding an adsorption material that selectively adsorbs the physiologically active substance on the surface of the carrier fine particles.
- the adsorption material in this case is one or more selected from Pyrosep, polymyxin B, polylysine, polyornithine, polyarginine, polyhistidine, aminosilane, chitosan, anti-endotoxin antibody, anti-endotoxin aptamer, and materials in a random peptide library. It may be a material.
- organic carrier fine particles include fine particles of polystyrene latex, polyethylene, nylon, cellulose, agarose, polyvinyl alcohol, acrylic resin, and the like.
- Inorganic carrier fine particles include simple substances such as alumina, titania, silica, zirconia, and hydroxyapatite, or composite fine particles thereof (for example, silica alumina and alumina titania), kaolin, montmorillonite, manganese oxide, mica, etc. Natural or synthetic mineral fine particles.
- the material itself is a fine particle, or the adsorbent material is bonded to the carrier fine particle
- at least one kind of fine particles may be selected and used. You may mix and use the above different materials. In selecting materials, the following two points should be noted. First, for detection of fine particles, particles having a low light transmittance (including colored particles) and particles having a high refractive index even if the light transmittance is high are more efficient. Next, under agitation, fragile particles cannot withstand the external force caused by agitation, and the particles are destroyed. Therefore, the particles need to have sufficient strength.
- the fine particle carrier is preferably a highly refractive and hard ceramic fine particle such as alumina or titania. Ceramic fine particles can be easily sterilized by dry heat, so that mixing of endotoxin can be prevented, and since they have an appropriate specific gravity, the fine particles can be easily collected by centrifugation.
- fine particles are desired to be able to be separated and recovered by natural sedimentation or simple operation. It is difficult to collect fine particles at very small particle sizes.
- the particle size is large, (1) the dispersibility of the fine particles in the water for use or the extract is low. (2) The adsorption effective surface area of fine particles is low. (3) It is difficult for the predetermined physiologically active substance to enter the particles. For this reason, the adsorption ability of the predetermined physiologically active substance is lowered. (4) When it is necessary to wash the fine particles after the predetermined physiologically active substance is adsorbed, the cleaning effect is low because it is difficult to discharge water inside the fine particles.
- the particle diameter of the fine particles is preferably 5 nm to 50 ⁇ m, and more preferably 10 nm to 20 ⁇ m.
- the particles may be concentrated by selecting or combining at least one of them. Considering shortening of the concentration time, simplicity, and improvement of the concentration rate, a method of precipitating fine particles by centrifugation is desirable.
- a surfactant may be added to the fine particles.
- the fine particles may be formed so that the entire surface is covered with the adsorption material.
- a material capable of adsorbing the predetermined physiologically active substance is used as a carrier.
- the material capable of adsorbing the predetermined physiologically active substance crosslinks between the plurality of carrier particles, and the fine particles locally aggregate.
- a device for preventing the fine particles from aggregating or a device for dispersing the agglomerated fine particles may be added.
- Examples of the former include addition of a substance having a surface active action, and coating of the entire surface of the carrier fine particles by binding an excessive amount of a material capable of adsorbing a predetermined physiologically active substance to the carrier fine particles. Further, as the latter, it is possible to exemplify dispersing fine particles that have been aggregated by applying mechanical impact or vibration, or by grinding with a mill or a homogenizer.
- the fine particles may be made of a material having affinity for coagulogen in the AL reagent.
- the fine particles may be formed to include a material having affinity for coagulogen, and the coagulogen may be bound in advance.
- the AL-bound bead method enables high-speed agglutination reaction by binding the coagulogen in the microparticles, especially the coagulogen in the AL, causing the coagulogen on the microparticles to change to coagulin and causing rapid aggregation.
- a coagulogen may be immobilized on the fine particles in advance, if necessary.
- a substance having affinity for AL or coagulogen may be immobilized in advance on a part of the fine particles.
- the microparticles themselves may have affinity for both endotoxin and coagulogen.
- alumina and titania have high affinity with endotoxin, but at the same time have high affinity with protein. Therefore, these fine particles can be used for the purpose of the present invention.
- some organic materials that adsorb predetermined physiologically active substances such as endotoxins have high affinity with proteins.
- proteins for example, polylysine, polyornithine, polyarginine and the like adsorb proteins electrostatically.
- the microparticles By binding these organic materials to the microparticles, the microparticles have an affinity for both the predetermined physiologically active substance and the coagulogen.
- the fine particles it is possible to shorten the measurement time by concentrating endotoxin and allowing the fine particles to participate in the agglutination reaction.
- the fine particles after separation from the sample or the extract may be removed by washing.
- the sample or extract contains a substance (interfering substance) that inhibits or enhances the serine protease of AL, as described above, the sample or extract is fully diluted or the interfering substance is removed. Otherwise, accurate measurement of the predetermined physiologically active substance may not be possible.
- the interference is obtained by washing the fine particles separated from the sample or the extract. Material was removed.
- the AL reagent may be dispersed with beads adsorbed on the surface of a predetermined protein contained in the extract of blood cells of horseshoe crab.
- an AL reagent in which a protein contained in AL is bound or adsorbed on the bead is prepared, and a sample containing a predetermined physiologically active substance is allowed to act on this AL reagent.
- beads are associated with each other to form a larger aggregate at an early stage, and aggregation or gelation is promoted in a mixed solution of the AL reagent and the sample or the extract. Therefore, if the AL reagent mixed with the fine particles adsorbed on the surface of the predetermined physiologically active substance according to the present invention is dispersed with beads by the AL-bound bead method, the synergistic effect of the present invention and the AL-bound bead method can be obtained. It is possible to perform the measurement of the predetermined physiologically active substance in a shorter time.
- the biologically active substance derived from the organism may be endotoxin or ⁇ -glucan.
- endotoxin the most typical pyrogen
- endotoxin-contaminated fluids, injections, blood, etc. can enter the human body and prevent side effects. it can.
- ⁇ -glucan detection or concentration measurement can be performed more accurately, and screening of a wide range of fungal infections including rare fungi as well as common clinical fungi such as Candida, Aspergillus, Cryptococcus, etc. It becomes possible to carry out accurately.
- the present invention may be a fine particle capable of adsorbing the physiologically active substance on the surface and used for the method for measuring any one of the above-mentioned biologically active substances.
- the present invention also provides an extract for extracting a predetermined physiologically active substance from a medical device in the method for measuring a biologically active substance derived from a living body in the medical device described above, wherein the predetermined physiologically active substance can be adsorbed on a surface. It may be an extract used for a method for measuring a biologically active substance derived from a living body in a medical device, characterized in that fine particles are dispersed in advance.
- the present invention it is possible to measure biologically active substances derived from living organisms in a state where the biologically active substances derived from living organisms in the sample are concentrated and adsorbed on the microparticles.
- the physiologically active substance derived therefrom can be measured.
- a high-speed aggregation reaction is induced, and it becomes possible to measure a low concentration biologically active substance in a short time.
- the substance can be removed by washing the microparticles separated and recovered from the sample as appropriate, and the biologically active substance derived from living organisms can be measured with higher accuracy. It can be carried out.
- the present invention it is possible to extract a predetermined physiologically active substance adhering to a medical device and concentrate and adsorb the predetermined physiologically active substance to fine particles that can participate in the agglutination reaction.
- the predetermined physiologically active substance adhering to the medical device can be measured to a lower concentration than before.
- a large amount of extract is required to extract a predetermined physiologically active substance from a medical device, but in the present invention, the predetermined physiologically active substance adsorbed on the fine particles can be concentrated together with the fine particles by centrifugation or the like. Therefore, it is possible to avoid the inconvenience that it cannot be measured due to dilution with the amount of the extract.
- the substance can be removed by washing the microparticles separated and recovered from the sample as appropriate, and the organism attached to the medical device with higher accuracy.
- the physiologically active substance derived therefrom can be measured.
- endotoxin or ⁇ -glucan will be described as an example of the predetermined physiologically active substance, but the following explanation can be applied to physiologically active substances derived from other organisms.
- fine particles in the present embodiment (1) inorganic fine particles capable of adsorbing endotoxin and coagulogen in AL although having low selectivity for endotoxin, and (2) organic adsorption having high selectivity and high affinity for endotoxin.
- Fine particles in which the material is bonded to inorganic carrier fine particles are conceivable. In the former case, since the selectivity to endotoxin is low, components contained in the sample bind endotoxin more strongly than inorganic fine particles, or components other than endotoxin in the sample bind to inorganic fine particles. In such a case, the function as an endotoxin adsorbing material is reduced. On the other hand, since the latter has a high binding specificity of endotoxin, it can fully exhibit the ability as an adsorbing material.
- the carrier fine particles ceramic fine particles such as alumina and titania, which are inorganic materials having affinity for both endotoxin and coagulogen in AL, can be used.
- an organic adsorbing material capable of adsorbing endotoxin selectively or with high affinity may be bound to the surface of these ceramic fine particles.
- polymyxin B When binding organic endotoxin adsorbing material to alumina or titania fine particles, polymyxin B, polylysine, polyarginine, polyhistidine, polyornithine, anti-endotoxin / lipid A antibody, anti-endotoxin / lipid A nucleic acid aptamer, random peptide A peptide for endotoxin / lipid A selected from the library may be used. Since alumina and titania have high affinity with proteins and nucleic acids as described above, it is possible to bind the adsorbing material without using chemical bonds.
- adsorbing materials dissociate when they are electrostatically bonded to alumina or titania, and the binding ability to a predetermined physiologically active substance such as endotoxin or glucan decreases due to the binding.
- a functional group capable of chemically bonding with an organic compound on the surface of alumina or titania and chemically bond the adsorbing material through the functional group.
- the functional group to be introduced in this case include a silane coupling agent.
- an aminosilane coupling agent having an amino group and capable of chemically bonding with the carboxyl group of the adsorbing material may be used.
- the epoxy silane coupling agent which has an epoxy group and can chemically bond with the amino group in the adsorbing material may be used.
- an isocyanato group or an isothiocyanato silane coupling agent that has an isocyanato group or an isothiocyanato group and can chemically bond with an amino group in the adsorbent may be used.
- the chemical substance used here may be any substance that can be chemically bonded to a part of the adsorbent and can be bonded to an inorganic compound such as titania or alumina, and the silane coupling agent exemplified here. It is not limited to.
- endotoxin adsorbing materials when these endotoxin adsorbing materials are bound to fine particles, a small amount of an AL component is added as necessary, or the endotoxin adsorbing material and the AL component are first bound and then the other is allowed to act. It is good to combine them.
- the fine particles to which the endotoxin adsorbing material is bound and the fine particles to which AL or coagulogen is bound may be prepared separately and mixed at an appropriate mixing ratio.
- organic adsorbent material binds to inorganic carrier fine particles, if the affinity of organic adsorbent material to inorganic carrier fine particles is strong, the adsorbent material may become a crosslinking agent (crosslinking reaction), In some cases, some or all of the fine particles may aggregate due to the neutralization of the charge. Therefore, it is preferable to react an excessive amount of the organic adsorbing material with the inorganic carrier fine particles. It is also effective to add a substance having a surface active action in the preparation process.
- the substance having a surface active action is a substance that does not interfere with the AL reaction or has a small interference action.
- nonionic surfactants such as Triton, Tween, Nonidet, or Pluronic, and albumin that is frequently used as an AL stabilizer or endotoxin extractant when measuring endotoxin are used.
- the substance having a surface active action is not limited to the above specific examples.
- the particles aggregate even after such a device, it is better to loosen them by applying a strong impact after the particles are precipitated, or to make them fine by using a mill or a homogenizer.
- a mill or a homogenizer for example, as in Production Example 3, endotoxin-adsorbing fine particles prepared by mixing excess polylysine with alumina fine particles and binding them, and then removing them by washing and centrifuging are excellent in photorefractive properties and particles. Since the diameter is small, it can be used in the endotoxin measurement method according to the present invention.
- the dispersion containing the prepared microparticles is mixed with a sample, adsorbed for an appropriate time, and then precipitated by a method such as centrifugation. . If the sample component has an interference effect on the AL reaction, the microparticles are washed with an appropriate water (such as water for injection) containing no endotoxin at this point. In washing, the fine particles in the mixed solution of water and fine particles are precipitated by a method such as centrifugation, and the supernatant is removed. Then, the remaining fine particles are resuspended with water, and precipitated again by centrifugation, and the supernatant is removed. By repeating this operation as appropriate, the fine particles can be washed.
- an appropriate water such as water for injection
- the fine particles can be stabilized during the endotoxin adsorption reaction.
- endotoxin since endotoxin is dispersed by the surface active action, endotoxin can be efficiently adsorbed to the fine particles.
- a component having a surfactant activity when mixing fine particles adsorbed with endotoxin with AL, it is preferable to add a component having a surfactant activity. This is because if the microparticles themselves or the endotoxin-adsorbing material bound on the microparticles are rapidly bound to the coagulogen in AL, an agglutination reaction unrelated to the agglutination reaction due to endotoxin may occur.
- Example 1 As the specific procedure of measurement is as in Example 1, 100 ⁇ L of the fine particle dispersion prepared in Production Example 3 is mixed with 1 mL of sample and reacted at room temperature. Then, the fine particles are separated and collected from the mixed solution by centrifugation and dispersed in 50 ⁇ L of the dispersion. The dispersed fine particles and 50 ⁇ L of the AL reagent are placed in the glass container produced in Production Example 1 and reacted, and the agglutination reaction at that time is optically measured to measure endotoxin.
- Agglomeration reaction in the mixture of fine particles and AL reagent is a light transmittance measuring device having a stirring mechanism, for example, stirring turbidimetry device EX-100 (manufactured by Kowa Co., Ltd.) or time-resolved particle size distribution analysis using laser light scattering. It can be measured by an apparatus EX-300 (manufactured by Kowa Co., Ltd.). Moreover, you may use the microplate reader which has the stirring function of a sample and can measure a light transmittance or scattered light.
- ⁇ Production Example 2> (Inorganic fine particles having endotoxin adsorption ability) Silica fine particles SFP-20M (primary particle size: 20 nm) manufactured by Denki Kagaku Kogyo Co., Ltd., alumina fine particles ASFP-20 (20 nm) manufactured by Denki Kagaku Kogyo Co., Ltd., and alumina fine particles AA03 manufactured by Sumitomo Chemical Co., Ltd. (400 nm)
- titania fine particles AEROXIDE P25 (20 nm) manufactured by Nippon Aerosil Co., Ltd. are subdivided into glass tubes and the openings of the glass tubes are covered with aluminum foil.
- the part was further covered with aluminum foil and subjected to a dry heat treatment at 250 ° C. for 30 minutes to completely inactivate endotoxin which may be mixed. This was dispersed in a dispersion to a prescribed concentration to prepare fine particles having no endotoxin selectivity but having an adsorption ability.
- ⁇ Production Example 3> (Alumina fine particles combined with an organic material having endotoxin adsorption ability)
- the alumina fine particle ASFP-20 manufactured by Electrochemical Industry Co., Ltd. prepared in Production Example 2 was added to 0.1% poly-L-lysine (Poly-LK) (hereinafter abbreviated so that the fine particle concentration was 10 mg / mL). And may be referred to as PLK)) and dispersed in an aqueous solution (manufactured by Sigma-Aldrich). The mixture was slowly stirred at room temperature for 2 hours to adsorb poly-L-lysine onto the alumina fine particles.
- Poly-LK poly-L-lysine
- a fine particle (PLK-ASFP) having a selective endotoxin adsorption ability was prepared by being dispersed to 25 mg / mL.
- ⁇ Production Example 4> (silica fine particles combined with an organic material having endotoxin adsorption ability) Disperse silica fine particle SFP-20M manufactured by Electrochemical Industry Co., Ltd. prepared in Production Example 2 in 0.1% poly-L-lysine aqueous solution (manufactured by Sigma-Aldrich) so that the fine particle concentration is 10 mg / mL. It was. The mixture was slowly stirred at room temperature for 2 hours to adsorb poly-L-lysine onto the silica fine particles.
- Fine particles (PLK-SFP) having selective endotoxin adsorption ability by dispersing were prepared.
- AL reagent ES-II multi-reagent manufactured by Wako Pure Chemical Industries, Ltd.
- ⁇ Production Example 6> (titania fine particles combined with an organic material having endotoxin adsorption ability) It was dispersed in 0.1% poly-L-lysine aqueous solution (manufactured by Sigma-Aldrich) so that the concentration of titania fine particles AEROXIDE P25 manufactured by Nippon Aerosil Co., Ltd. prepared in Production Example 2 was 10 mg / mL. The poly-L-lysine was bound onto the titania microparticles by gently stirring at room temperature for 2 hours.
- the fine particles (PLK-P25) having selective endotoxin adsorption ability were prepared by dispersing to a concentration of 0.25 mg / mL.
- ⁇ Production Example 7> (Alumina fine particles bound with coagulogen)
- the alumina fine particle ASFP-20M manufactured by Electrochemical Industry prepared in Production Example 2 was dispersed in distilled water for injection so as to have a concentration of 30 mg / mL.
- 500 ⁇ L of P25 dispersion was added to 500 ⁇ L of AL reagent (Wako Pure Chemicals ES-II multi-reagent) and mixed well, followed by heat treatment at 80 ° C. for 20 minutes. Thereafter, it was washed once with water for injection and dispersed in 1 mL of water for injection to obtain alumina fine particles (AL-ASFP) bonded with AL.
- AL-ASFP alumina fine particles
- poly-L-lysine was used as an endotoxin adsorbing material.
- -D / L-lysine and ⁇ -poly-L-lysine in which the ⁇ -position amino group and carboxyl group are amide bonds (not narrowly defined peptide bonds). Since the L-form is easily decomposed by the enzyme of the cell, the D-form that is difficult to be decomposed may be used as necessary.
- any of poly-L-lysine and poly-D-lysine may be used as the adsorption material.
- Example 1 Endotoxin concentration by inorganic fine particles and quantification by aggregation reaction
- SFP-20M sica
- AA03 alumina
- AEROXIDE P25 titanium
- the fine particles were dispersed to a concentration of 1 mg / mL.
- Aggregation was measured by the stirring turbidimetric method (EX-100). As shown in FIG. 2, the endotoxin adsorbed with the fine particles of any material reacts with AL to cause an agglutination reaction, and the detection time was shortened as the concentration of the endotoxin acted was higher.
- inorganic fine particles can efficiently adsorb various organic substances, and endotoxin and coagulogen in AL are also bound.
- AL coagulogen
- AL quickly binds to the surface of the fine particles to which endotoxin is not adsorbed, and then the endotoxin on the fine particles is factor C in the AL. It is considered that the activity of AL increases, coagulogen changes to coagulin, and aggregation reaction involving fine particles occurs.
- Example 2 (Examination of adsorption conditions)
- the endotoxin-adsorbing microparticles of the present invention can concentrate endotoxin from an endotoxin-containing sample and mix with the AL reagent.
- the endotoxin concentration action was evaluated by reacting with a volume of endotoxin aqueous solution.
- Alumina fine particles described in Production Example 1 (ASFP-20, 20 ⁇ L of 1 mg / mL) and poly-L-lysine as an endotoxin adsorption material described in Production Example 4 were bound (adsorbed) to various endotoxin aqueous solutions.
- Silica fine particles (PLK-SFP, 20 mg / mL, 10 mg / mL) mixed with 0.01EU / mL endotoxin-containing solution were allowed to react at room temperature for 20 minutes.
- FIG. 3 shows the result. From the figure, it can be seen that the endotoxin detection time is shortened as the amount of the endotoxin aqueous solution in which any of the endotoxin-adsorbing fine particles acted is increased. Thus, it was shown that endotoxin can be concentrated from a large amount of liquid by these fine particles.
- microparticles are coupled with coagulin, which is produced as a result of endotoxin adsorbed (captured) on the microparticles activating AL by binding coagulogens in the AL on the microparticles at the very beginning of the aggregation of AL. It was revealed that the fine particles were cross-linked to induce a steep agglomeration reaction.
- This agglutination reaction is similar to the patent document (Japanese Patent Laid-Open No. 2009-150723), and the agglutination reaction mainly consisting of fine particles occurs earlier than the reaction in which AL itself aggregates due to endotoxin. It is possible to measure endotoxin at a lower concentration.
- endotoxin adsorption reaction was performed at various temperatures, and then the fine particles adsorbed with endotoxin were separated and recovered and dispersed in a dispersion (50 ⁇ L). Then, this was reacted with an AL reagent (manufactured by Wako Pure Chemical Industries, Ltd., ES-II multi-reagent, 50 ⁇ L), the aggregation reaction was measured with EX-100, and the endotoxin detection time was compared.
- the fine particles used were alumina fine particles (PLK-ASFP) bonded with poly-L-lysine described in Production Example 3.
- the activated endotoxin is 1 mL at a concentration of 0.01 EU / mL.
- the reaction time was 30 minutes.
- the results are shown in FIG.
- the detection time was shorter as the temperature was lower.
- the adsorption reaction between endotoxin and fine particles is a physical adsorption reaction with an adsorbent material that is highly selective for endotoxin (the reaction is faster at lower temperatures, because the normal adsorption reaction generates heat of adsorption). I understand that.
- Example 3 (Example in which endotoxin adsorbing material previously adsorbed endotoxin is combined with fine particles)
- silica fine particles bonded with poly-L-lysine as an endotoxin-adsorbing material described in Production Example 4 are mixed in an endotoxin-containing solution, and the fine particles are precipitated by centrifugation.
- the suspension was resuspended in an aqueous ionic surfactant Triton X-100 solution, which was mixed with the AL reagent and reacted.
- a water-soluble endotoxin adsorbent and a sample containing endotoxin are mixed, and endotoxin is adsorbed on the endotoxin adsorbing material.
- silica fine particles which are carrier fine particles
- the endotoxin adsorbing material in a state where endotoxin is adsorbed is combined with the silica fine particles. According to this, endotoxin-adsorbing fine particles can be prepared more easily.
- the coagulogen quickly binds to the surface of the microparticles where the endotoxin is not adsorbed. Thereafter, endotoxin on the microparticles binds to C-factor in AL, the activity of AL increases, and coagulogen changes to coagulin to cause an aggregation reaction involving microparticles. Then, when the microparticles adsorbed with endotoxin are reacted with AL, a two-stage action occurs, and the aggregation of the microparticles also progresses smoothly. Therefore, it is difficult to accurately detect the start point of the aggregation. There is a possibility.
- microparticles adsorbing endotoxin hold coagulogen on the particle in advance, they can be incorporated into the AL aggregate at the initial stage of aggregation, and the aggregation reaction can be induced in a shorter time. I understood.
- FIG. 6 shows the reaction curve.
- FIG. 6 (a) shows the results of normal aggregation (reaction of 50 ⁇ L of a solution in which PLK-P25 is directly dispersed in an endotoxin aqueous solution of each concentration and 50 ⁇ L of AL).
- FIG. 6B shows the result in this example.
- the detection time can be greatly shortened by using the AL-bound beads in combination with the endotoxin-adsorbing fine particles of the present invention.
- the detection time is shortened by using both endotoxin-adsorbing fine particles and a reagent obtained by adding AL-ASFP to AL, compared with the case where it is used alone. I was able to confirm.
- highly sensitive measurement can be performed in a shorter time.
- An interfering substance that inhibits or enhances the AL reaction may be included in a sample for measuring endotoxin.
- blood contains a serine protease cascade very similar to AL.
- thrombin and antithrombin III in blood act directly on the protease activity of AL and interfere with the AL reaction. Therefore, when measuring endotoxin in blood, a heating dilution method is used in which plasma is diluted about 10 times with a buffer containing a surfactant and heated to suppress the action of interfering substances.
- the above-described antithrombin III is used as an antithrombotic injection drug. In this case, antithrombin III binds to the serine protease active center of thrombin to neutralize the function of thrombin and prevent the coagulation system from being enhanced.
- the sample containing antithrombin III is one of the most difficult samples for measuring endotoxin.
- antithrombin cannot suppress the interference action unless it is highly diluted.
- endotoxin itself was diluted to a measurable range or less, and it was difficult to measure by a normal measurement method.
- the endotoxin is adsorbed from the sample and concentrated, and at the same time, the interfering substance can be removed by washing the reacted fine particles as necessary.
- the measurement of most types of samples was possible according to the present invention, if endotoxin of a sample containing antithrombin III, which is one of the most difficult samples, can be measured. .
- Antithrombin III (Neuart, manufactured by Mitsubishi Tanabe Seiyaku, 50 U / mL) was diluted 10-fold with distilled water for injection, and endotoxin was added thereto so that the concentration became 0.01 EU / mL.
- the titania fine particle dispersion P25, 0.3 mg / mL, 0.2% Triton X-100
- 100 ⁇ L of a titania fine particle dispersion (PLK-P25, 0.3 mg / mL, 0.2% Triton X-100) was added. And it was made to react for 30 minutes at room temperature, and endotoxin was made to adsorb
- each fine particle was separated and collected by centrifugation.
- a dispersion (ATIII-P25-non-washing and ATIII-PLK-P25-non-washing) in which each precipitate was dispersed in 0.02% Triton X-100 aqueous solution (50 ⁇ L) was prepared.
- the precipitates dispersed in 0.02% Triton X-100 aqueous solution (1 mL) were centrifuged again to precipitate, and the supernatant was removed and dispersed again in the same aqueous solution three times.
- Microparticles washed (ATIII-P25-wash and ATIII-PLK-P25-wash) were prepared.
- a dispersion of titania fine particles described in Production Example 2 (P25, 0.3 mg / mL, 0.2% Triton X-100) in 0.01 EU / mL endotoxin aqueous solution not containing ATIII, or Production Example 6 100 ⁇ L of titania fine particle dispersion (PLK-P25, 0.3 mg / mL, 0.2% Triton X-100) to which poly-L-lysine was bound, and then allowed to react at room temperature for 30 minutes, respectively. Adsorbed on the fine particles.
- the use of the endotoxin-adsorptive aggregated microparticles in the present invention can eliminate the interference effect by washing the microparticles after adsorbing endotoxin even in the sample containing antithrombin III, which is the most difficult to measure. It was. Further, it was found that the fine particles to be used are those in which an adsorbing material having high endotoxin selectivity is bound on the fine particles (they should not be limited to PLK-P25 exemplified in this example).
- the endotoxin was able to be measured without being affected by the interference substance by combining the endotoxin-adsorbing aggregated fine particles of the present invention with a washing operation.
- it is desirable that the fine particles are as small as possible within a range where they can be easily precipitated. This is because when the fine particles are large, the specific surface area is small and the endotoxin adsorption sites are small, so that the endotoxin adsorption efficiency is lowered.
- the liquid is held inside the particles because of the large particle size, and the antithrombin held inside the particles even after washing operation This is because interfering substances such as III cannot be easily removed. Since the fine particles of the present invention have a primary particle size of about 10 ⁇ m at the maximum, cleaning is easy with little retention of liquid inside the particles.
- endotoxin in blood is considered easier than measuring endotoxin in antithrombin III.
- the antithrombin III preparation is produced by concentrating the antithrombin III in the blood, and thus the inhibitory action of antithrombin III on AL is orders of magnitude greater than the inhibitory action of blood.
- no example was shown here, even when measuring endotoxin in blood, by combining the endotoxin-adsorptive agglomerated fine particles of the present invention with a washing operation, it can be easily performed without being affected by interfering substances in blood. It is assumed that endotoxin can be measured.
- Example 7 (Endotoxin concentration action by various endotoxin adsorbing materials)
- an endotoxin adsorbing material a polymer containing a primary or secondary amine in the side chain is often used.
- the first amino acid begins with an amino acid having an amine in any side chain of arginine, lysine or histidine. Since one amino group of an amino acid is used when a peptide bond is formed, poly-L-arginine, poly-polyamino acid homopolymers in which amino groups remain in the side chain are used in addition to poly-L-lysine. Whether L-histidine or poly-L-ornithine had an adsorption concentration effect was examined. Further, polyethyleneimine known to have a high amino group content was also examined in the same manner.
- amino acid homopolymers made by Sigma Aldrich were used, and polyethyleneimine made by Wako Pure Chemicals. Diluted with distilled water for injection to 0.1% (polyethyleneimine is 1%), mixed with 10 mg / mL titania nanoparticle (AEROXIDE P-25) dispersion at a volume ratio of 9: 1, and 1 hour The reaction was performed at room temperature. Next, the reaction solution was centrifuged, the supernatant was removed, and washing by a method of resuspending in distilled water for injection was performed a total of three times to remove unreacted adsorbent material.
- AEROXIDE P-25 titania nanoparticle
- Example 8> Measurement of ⁇ -glucan
- curdlan manufactured by Wako Pure Chemical Industries
- the AL reagent originally reacts with endotoxin and ⁇ -glucan.
- Endotoxin-specific reagents are known that have a mechanism that inhibits the action of glucan by adding a large amount of an analog of ⁇ -glucan to the reagent, or that in which a protein to which ⁇ -glucan is bound is removed.
- Limulus HS-T (Wako Pure Chemical Industries), which is an AL reagent that is not endotoxin-specific, was used.
- the curdlan was dissolved in distilled water for injection to prepare a 10-fold dilution series from 10 ng / mL to 1 pg / mL. These dilution series (50 ⁇ L) and HS-T (50 ⁇ L) were mixed in the glass container produced in Production Example 1, and the aggregation reaction was measured by EX-100 (ordinary method).
- the method of reacting the curdlan with the titania fine particles bound with polylysine produced in Production Example 6 after adsorbing and concentrating with AL was performed as follows. That is, 100 ⁇ L of the fine particle dispersion of Production Example 6 was added to a 1 mL aqueous solution of curdlan, and the curdlan was adsorbed on the fine particles by shaking with a vortex mixer for 10 minutes.
- FIG. 8 shows the results of plotting logarithm with the concentration of curdlan on the horizontal axis and the detection time of curdlan on the vertical axis. As shown in the figure, either method showed high linearity even at a wide curdlan concentration, but it took a very long time to detect when measured by the usual method. On the other hand, in the present invention, the detection time was greatly shortened, and even a low concentration (0.001 ng / mL) could be detected in a practical time of less than 30 minutes.
- the polylysine used as the adsorbent here has not been reported as a selective adsorbent for glucan, but the mechanism by which the glucan is adsorbed to the fine particles by polylysine will be discussed below.
- Glucan usually has a high degree of hydrogen bonding between alcohols in adjacent chains, so it is poorly soluble in water. However, it is known that hydrogen bonds are broken by alkali treatment at this time. Therefore, a mechanism can be considered in which the amino group of polylysine having a very high amino group density acts as an alkali, cleaves the hydrogen bond of glucan, forms a hydrogen bond with glucan alcohol, and adsorbs glucan.
- both polylysine and glucan are high polymers, a mechanism may be considered in which polylysine on the fine particles entangles the glucan by stirring and is adsorbed and fixed on the particles.
- a mechanism may be considered in which polylysine on the fine particles entangles the glucan by stirring and is adsorbed and fixed on the particles.
- details are unknown for now.
- the same adsorbing material can be used for measuring any substance, not only endotoxin but also glucan can be adsorbed to the fine particles by polylysine.
- the fine particles in the present embodiment are also (1) inorganic fine particles capable of adsorbing endotoxin and coagulogen in AL, although the selectivity to endotoxin is low, and (2) selectivity and affinity for endotoxin.
- Fine particles obtained by binding a highly adsorbing organic adsorbing material to inorganic carrier fine particles are conceivable. In the former case, since the selectivity to endotoxin is low, depending on the material of the medical device, the component contained in the material of the medical device binds endotoxin more strongly than the inorganic fine particles, or the material of the medical device When these components bind to inorganic fine particles, the function as an endotoxin adsorbing material is reduced.
- the dispersion containing the prepared microparticles is used as an extract, and this is brought into contact with a medical device as appropriate. Then, after endotoxin is extracted from the medical device for an appropriate time and simultaneously adsorbed onto the fine particles, the fine particles are precipitated by a method such as centrifugation. Since the extraction operation varies greatly depending on the structure and material of the medical device to be evaluated, an optimum extraction method may be determined in advance and appropriately determined.
- the target is a filtration filter (the one with coarse eyes)
- the extract containing the endotoxin-adsorbing microparticles is passed through these instruments several times as necessary to extract the endotoxin adhering to the microparticles. What is necessary is just to make it adsorb
- the filter is fine and the fine particles cannot pass or if the structure is complicated and difficult to pass the extract, put the fine particles that adsorb endotoxin inside the tool or immerse the tool in the extract. Measures may be taken. And the extraction efficiency of the endotoxin from a tool may be improved by processes, such as shaking and an ultrasonic treatment, as needed.
- the extraction of endotoxin from a medical device is not performed with an extract in which fine particles that adsorb endotoxin are dispersed, but an aqueous solution of an endotoxin-adsorbing organic material having a high affinity for endotoxin may be used as the extract. In this case, after extracting endotoxin, these endotoxin-adsorptive organic materials may be mixed with carrier fine particles that can be bound, and the organic material adsorbed with endotoxins may be bound onto the carrier fine particles.
- the fine particles adsorbed with endotoxin are washed with an appropriate water containing no endotoxin (water for injection, etc.). Specifically, washing can be performed by carrying out the steps of resuspending the fine particles adsorbed with endotoxin with their water, precipitating them again by centrifugation, and removing the supernatant. It is natural that the cleaning effect is improved by repeating the above steps a plurality of times as necessary.
- the fine particle is stabilized during the endotoxin adsorption reaction, and the endotoxin is dispersed by the surface-active action and efficiently adsorbed to the fine particle. It becomes possible to make it.
- the microparticles adsorbed with endotoxin with the AL reagent, if the microparticles themselves or the endotoxin-adsorbing material bound on the microparticles binds rapidly to coagulogen in AL, aggregation is independent of the aggregation reaction by endotoxin. A reaction may occur. In order to suppress such rapid bonding, it is desirable to add a component having a surface active action to the dispersion in which the precipitated fine particles are resuspended.
- the optical transmittance measurement method is made of glass having a diameter of 6 mm and a length of 50 mm, and a stainless steel stirrer ( ⁇ 0.75 mm, length of 3. mm) for stirring the sample. 5 mm) was used.
- a dedicated container made of glass having a diameter of 7 mm and a length of 50 mm and having a stainless stirrer ( ⁇ 1 mm and a length of 5 mm) for stirring the sample was used. Covering the opening of these glass containers with aluminum foil, and further packaging 20 pieces of aluminum foil in small portions into an iron dry heat treatment can, heat-treating at 250 ° C. for 3 hours to thermally decompose endotoxin .
- Titania fine particles AEROXIDE P25 (20 nm) manufactured by Nippon Aerosil Co., Ltd. were subdivided into glass tubes. Then, cover the opening of the glass tube with aluminum foil, and put several pieces together in a glass beaker. Cover the opening with aluminum foil and heat-treat at 250 ° C. for 30 minutes, possibly causing contamination. There are endotoxins completely inactivated. This was dispersed in a dispersion liquid to a prescribed concentration to prepare aggregated fine particles having no endotoxin selectivity but having adsorption ability.
- ⁇ Production Example 10> (titania fine particles on which an organic material having endotoxin adsorption ability is immobilized) It was dispersed in 0.1% poly-L-lysine aqueous solution (manufactured by Sigma-Aldrich) so that the concentration of titania fine particles AEROXIDE P25 manufactured by Nippon Aerosil Co., Ltd. prepared in Production Example 9 was 10 mg / mL. The poly-L-lysine was adsorbed onto the titania fine particles by slowly stirring at room temperature for 2 hours. Thereafter, the supernatant was removed by centrifugation, and washed 3 times with distilled water for injection containing no endotoxin.
- poly-L-lysine aqueous solution manufactured by Sigma-Aldrich
- agglomerated fine particles with selective endotoxin adsorption ability by being dispersed in the nonionic surfactant Triton X-100 (0.2%) so that the concentration of the beads of the material is 0.2 mg / mL. (PLK-P25) was prepared.
- Eppendorf tube in which endotoxin was physically adsorbed on the inner wall was prepared.
- Eppendorf tubes are made of polypropylene, and polypropylene and polyethylene are known to adsorb endotoxins because the materials are hydrophobic (see, for example, Non-Patent Documents 2 and 3).
- poly-L-lysine was used as an endotoxin adsorbing material.
- -D / L-lysine and ⁇ -poly-L-lysine in which the ⁇ -position amino group and carboxyl group are amide bonds (not narrowly defined peptide bonds). Since the L-form is easily decomposed by the enzyme of the cell, the D-form that is difficult to be decomposed may be used as necessary.
- any of poly-L-lysine and poly-D-lysine may be used as the adsorption material.
- Example 9> Endotoxin extraction and concentration by endotoxin-adsorbing fine particles and measurement by aggregation reaction
- the endotoxin measurement method using AL is a calibration curve method for creating a calibration curve based on measurement of endotoxin dilution series of known concentrations. Therefore, it is necessary to prepare a calibration curve for each of the cases where the endotoxin-adsorbing fine particles are not used and the case where they are used.
- the endotoxin detection time when no fine particles were added was 118.5 minutes. This exceeded the detection time at the lowest concentration of 0.001 EU / mL in the dilution series in Table 2, and strictly speaking, it cannot be quantified below the measurement sensitivity of 0.001 EU / mL or less (by extrapolation of the calibration curve). It was 0.00074EU / mL when calculated.
- the detection time was 13.9 minutes, and when the fine particles of Production Example 10 were used, the detection time was 14.3 minutes. This is a detection time located between two samples each having a high concentration in the dilution series. When the concentration is calculated by applying a calibration curve, 0.0110 EU / mL is obtained when the fine particles of Production Example 9 are used, and Production Example 10 When the fine particles were used, the amount was 0.0099 EU / mL.
- the present invention capable of extracting endotoxin adhering to a tool in a minute amount and adsorbing it onto the fine particle, and concentrating the fine particle by centrifugation or the like to measure the endotoxin concentration,
- the concentration can be easily measured in a short time.
- FIG. 10A The measurement flow shown in FIG. 10 (a) describes the case where endotoxin-adsorbing fine particles are not used, and the measurement flow shown in FIG. 10 (b) describes the case where endotoxin-adsorbing fine particles are used.
- FIG. 10A 1 mL of water for injection is added to the Eppendorf tube 1 with endotoxin attached. Then, endotoxin 1a attached to the Eppendorf tube 1 is dispersed in the water 100 for injection.
- endotoxin dispersed in the extract 10 is preferentially adsorbed on the endotoxin-adsorbing fine particles, a larger part of the endotoxin adhering to the Eppendorf tube 1 is transferred from the Eppendorf tube 1 to the endotoxin-adsorbing fine particles. Move to the surface of 10a.
- the extract 10 in which the endotoxin-adsorbed microparticles 10a are dispersed is centrifuged, and the supernatant is removed to separate the endotoxin-adsorbed microparticles 10a.
- the endotoxin in the extract 10 is concentrated on the separated fine particles 10a. This is redispersed in 60 ⁇ L of a 0.02% Triton X-100 aqueous solution.
- 50 ⁇ L of the dispersion 12 and 50 ⁇ L of the AL reagent are mixed in the glass container 5 using the micropipettors 3 and 4, and the aggregation reaction is measured.
- the change in the light transmittance obtained as a result is shown by a curve B in FIG.
- the blood cell extract of horseshoe crab in the present invention (hereinafter also referred to as “AL: Amoebocyte lysate”) is derived from its origin (Limulus Amebocyte Lysate) or TAL (Tachypleus from Asian horseshoe crab). Amebocyte Lysate) etc.
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Abstract
Description
前記生理活性物質を表面に吸着可能な微粒子を前記試料中に分散させることで前記生理活性物質を前記微粒子の表面に吸着させた後に前記試料から前記微粒子を分離し、
前記AL試薬と前記生理活性物質が表面に吸着された前記微粒子との混和液を生成し、
前記AL試薬と前記微粒子との混和液における前記微粒子の凝集またはゲル化を光学的手法により検出することを特徴とする。
所定生理活性物質を表面に吸着可能な微粒子を前記抽出液中に分散させることで所定生理活性物質を前記微粒子の表面に吸着させた後に前記抽出液から前記微粒子を分離し、
前記AL試薬と所定生理活性物質が表面に吸着された前記微粒子との混和液を生成し、
前記AL試薬と前記微粒子との混和液における前記微粒子の凝集またはゲル化を光学的手法により検出することとしてもよい。
光透過率測定法ではφ6mm、長さ50mmのガラス製で、試料を攪拌するためのステンレス製の攪拌子(φ0.75mm、長さ3.5mm)を内在した専用の容器を使用した。一方、レーザー散乱粒子計測法では、φ7mm、長さ50mmmのガラス製で、試料を攪拌するためのステンレス製の攪拌子(φ1mm、長さ5mmを)を内在した専用の容器を使用した。これらのガラス容器の開口部をアルミ箔で覆い、さらに、20本ずつアルミ箔で小分けに梱包したものを鉄製の乾熱処理缶に入れ、250℃で3時間加熱処理して、エンドトキシンを熱分解した。
電気化学工業株式会社製のシリカ微粒子SFP-20M(一次粒径:20nm)、電気化学工業株式会社製のアルミナ微粒子ASFP-20(同20nm)、住友化学株式会社製のアルミナ微粒子AA03(同400nm)、ならびに、日本アエロジル株式会社製のチタニア微粒子AEROXIDE P25(同20nm)はガラスチューブに小分けにしてガラスチューブの開口部をアルミ箔で覆い、さらに、数本をまとめてガラスビーカーに入れて、その開口部をさらにアルミ箔で覆い、250℃で30分間乾熱処理をして、混入の恐れがあるエンドトキシンを完全に失活させた。これを規定の濃度になるように分散液に分散させてエンドトキシン選択性はないが吸着能力を持つ微粒子を調製した。
製造例2で調製した電気化学工業株式会社製のアルミナ微粒子ASFP-20を微粒子の濃度が10mg/mLになるように、0.1%ポリ-L-リジン(Poly-L-K(以降、短縮してPLKと表記することもある))水溶液(シグマ・アルドリッチ製)に分散させた。室温で2時間ゆっくりと攪拌してポリ-L-リジンをアルミナ微粒子上に吸着させた。その後、遠心して上清を除去し、エンドトキシンを含まない注射用蒸留水で3回洗浄して、最後に非イオン性界面活性剤のPluronic F68水溶液(30%)に素材のビーズの濃度換算で0.25mg/mLになるように分散させて選択的なエンドトキシン吸着能力を有する微粒子(PLK-ASFP)を調製した。
製造例2で調製した電気化学工業株式会社製のシリカ微粒子SFP-20Mを微粒子の濃度が10mg/mLになるように、0.1%ポリ-L-リジン水溶液(シグマ・アルドリッチ製)に分散させた。室温で2時間ゆっくりと攪拌してポリ-L-リジンをシリカ微粒子上に吸着させた。その後、遠心分離して上清を除去し、エンドトキシンを含まない注射用蒸留水で3回洗浄して、最後に注射用蒸留水に素材の微粒子の濃度換算で1.0mg/mLになるように分散させて選択的なエンドトキシン吸着能を有する微粒子(PLK-SFP)を調製した。
製造例3の方法で調製したポリ-L-リジンを結合させたアルミナ微粒子(PLK-ASFP、濃度:10mg/mL)0.5mLに対して、AL試薬(和光純薬製ES-IIマルチ試薬)50μLを加え、60℃で10分間加熱処理した。その後、エンドトキシンを含まない注射用蒸留水で1回洗浄してエンドトキシン吸着素材及びALと結合した微粒子(PLK=LAL-ASFP)を調製した。
製造例2で調製した日本アエロジル株式会社製のチタニア微粒子AEROXIDE P25の濃度が10mg/mLになるように、0.1%ポリ-L-リジン水溶液(シグマ・アルドリッチ製)に分散させた。室温で2時間ゆっくりと攪拌してポリ-L-リジンをチタニア微粒子上に結合させた。その後、遠心分離して上清を除去し、エンドトキシンを含まない注射用蒸留水で3回洗浄して、最後に非イオン性界面活性剤のTriton X-100(0.2%)に素材の微粒子の濃度換算で0.25mg/mLになるように分散させて選択的なエンドトキシン吸着能を持つ微粒子(PLK-P25)を調製した。
製造例2で調製した電気化学工業製のアルミナ微粒子ASFP-20Mの濃度が30mg/mLになるように、注射用蒸留水に分散させた。AL試薬(和光純薬製ES-IIマルチ試薬)500μLに対してP25の分散液を500μL加えてよく混合させ、80℃で20分加熱処理をした。その後、1回注射用水で洗浄して注射用水1mLに分散させてALを結合したアルミナ微粒子(AL-ASFP)を得た。
製造例2で調製した微粒子を用いてエンドトキシン吸着作用を評価した。高濃度のエンドトキシン(100EU/mL)、あるいは、低濃度のエンドトキシン(0.01EU/mL)の水溶液1mLに、SFP-20M(シリカ)、AA03(アルミナ)、AEROXIDE P25(チタニア)のいずれかを1mg分散させて、微粒子の濃度を1mg/mLとした。これを室温で30分攪拌し、その後、遠心分離(15,000xg、5分)により微粒子を沈殿させ、上清50μLとAL50μLを混合させて凝集反応を惹起し、攪拌比濁法(興和EX-100)により測定した。結果を図1に示す。図1が示すようにアルミナとチタニアはエンドトキシンを吸着し、高低いずれの濃度においても初期濃度の100分の1以下になるまでエンドトキシンを吸着除去した。しかしながら、シリカはエンドトキシンを吸着せず、対照の結果(初期濃度)と変わらなかった。
本発明のエンドトキシン吸着微粒子がエンドトキシン含有試料からエンドトキシンを濃縮し、且つ、AL試薬と混和した際に凝集に参加する(巻き込まれる)ことが可能かどうかを調べるため、エンドトキシン吸着微粒子を用い、種々の容量のエンドトキシン水溶液と反応させて、エンドトキシン濃縮作用を評価した。種々の容量のエンドトキシン水溶液に対して製造例1に記載のアルミナ微粒子(ASFP-20、1mg/mLを20μL)ならびに、製造例4に記載の、エンドトキシン吸着素材としてポリ-L-リジンを結合(吸着固定)させたシリカ微粒子(PLK-SFP、10mg/mLを20μL)を0.01EU/mLのエンドトキシン含有溶液に混和させ、室温で20分間反応させた。
実施例2における図3の説明では、製造例4に記載の、エンドトキシン吸着素材としてポリ-L-リジンを結合させたシリカ微粒子をエンドトキシン含有溶液に混和させ、遠心により微粒子を沈殿させた後に、非イオン性界面活性剤TritonX-100水溶液に再懸濁させ、これをAL試薬と混和して反応させた。それに対し、本実施例では、まず、水溶性のエンドトキシン吸着素剤とエンドトキシンを含む試料とを混和し、エンドトキシン吸着素材にエンドトキシンを吸着させる。そして、その後に、混和液に担体微粒子であるシリカ微粒子を添加することにより、エンドトキシンが吸着した状態のエンドトキシン吸着素材をシリカ微粒子と結合させる。これによれば、エンドトキシン吸着微粒子をより容易に作成することが可能である。
上記の実施例では、微粒子自体(微粒子がエンドトキシンとコアギュロゲンの両方を吸着(両方と結合)できる場合)と、微粒子上にエンドトキシンを吸着する吸着素材を結合したもの(吸着素材がエンドトキシンとコアギュロゲンに親和性を持つ場合)をエンドトキシンを含有する試料に作用させた。そして、エンドトキシンの測定時には、エンドトキシンを吸着した微粒子とALとを反応させた。それに対し、本実施例では、エンドトキシン吸着素材と結合した微粒子に、さらにコアギュロゲンを予め結合させておくこととした。これにより、微粒子がALと反応する時点で初めてAL(コアギュロゲン)と結合(吸着)する場合と比較して、短時間でエンドトキシンを測定することが可能になることが期待できる。
ところで、特許文献(特開2009-150723号公報)に記載されている、LAL結合ビーズ(以下、AL結合ビーズという。)により、エンドトキシンの短時間での測定が可能になることは先述のとおりである。このAL結合ビーズと、本発明におけるエンドトキシン吸着微粒子とを併用して使用することで、さらに短時間でエンドトキシンを測定できる可能性がある。これを確認するため、製造例6のポリ-L-リジンを固定化したチタニア微粒子(PLK-P25)100μLをエンドトキシンを含有する試料(1mL)と反応させた。その後、混和液を遠心して微粒子を回収し、Triton X-100(0.02%、50μL)に分散させた。そして、製造例7に記載のコアギュロゲンを固定化したアルミナ微粒子(AL-ASFP)を予め10分の1量添加させておいたAL試薬(和光純薬製ES-IIマルチ試薬)50μLと反応させた。
エンドトキシンの測定を行う試料の中に、AL反応を阻害または亢進する干渉物質を含んでいる場合がある。例えば、血液にはALと非常に類似したセリンプロテアーゼカスケードが含まれている。特に血液中のトロンビンやアンチトロンビンIIIはALのプロテアーゼ活性に直接作用してAL反応に干渉する。従って、血液中のエンドトキシンを測定する場合には、血漿を界面活性剤入りのバッファで10倍程度に希釈し加熱して干渉物質の作用を抑える加熱希釈法が利用される。一方、上記したアンチトロンビンIIIは抗血栓の注射薬剤として使用される。この場合、トロンビンのセリンプロテアーゼ活性中心にアンチトロンビンIIIが結合してトロンビンの機能を中和し、凝固系が亢進するのを妨げる。
エンドトキシンの吸着素材としては、側鎖に1級、あるいは、2級アミンを含むポリマーを用いることが多い。また、ランダムペプチドライブラリから選択されたペプチドも、最初のアミノ酸はアルギニン、リジン、ヒスチジンのいずれかの側鎖にアミンを有するアミノ酸から始まるものである。アミノ酸のアミノ基はペプチド結合をする際に1つは使用されてしまうので、側鎖にアミノ基が残存するポリアミノ酸ホモポリマーとして、ポリ-L-リジンのほかに、ポリ-L-アルギニン、ポリ-L-ヒスチジン、ポリ-L-オルニチンについて吸着濃縮効果があるかを調べた。また、アミノ基の含有量が高いことが知られているポリエチレンイミンについても同様に調べた。
実施例7までは微粒子によるエンドトキシンの濃縮と微粒子の凝集について説明した。実施例8ではβグルカンによる凝集反応について説明する。直鎖β-1,3グルカンの一つであるカードラン(和光純薬製)を使用して評価を行なった。AL試薬は本来エンドトキシンとβグルカンに反応することが知られている。エンドトキシン特異的試薬はβグルカンのアナログを大量に試薬に加えてグルカンが作用するのを阻害する機序を有するもの、あるいは、βグルカンが結合するタンパク質を除去したものが知られている。本実施例ではエンドトキシン特異的ではないAL試薬であるリムルスHS-T(和光純薬)を用いた。
本発明を医療用具に対して実施する場合についても、光透過率測定法ではφ6mm、長さ50mmのガラス製で、試料を攪拌するためのステンレス製の攪拌子(φ0.75mm、長さ3.5mm)を内在した専用の容器を使用した。一方、レーザー散乱粒子計測法では、φ7mm、長さ50mmmのガラス製で、試料を攪拌するためのステンレス製の攪拌子(φ1mm、長さ5mmを)を内在した専用の容器を使用した。これらのガラス容器の開口部をアルミ箔で覆い、さらに、20本ずつアルミ箔で小分けに梱包したものを鉄製の乾熱処理缶に入れ、250℃で3時間加熱処理して、エンドトキシンを熱分解した。
日本アエロジル株式会社製のチタニア微粒子AEROXIDE P25(同20nm)はガラスチューブに小分けにした。そして、ガラスチューブの開口部をアルミ箔で覆い、さらに、数本をまとめてガラスビーカーに入れて、その開口部をさらにアルミ箔で覆い、250℃で30分間乾熱処理をして、混入の恐れがあるエンドトキシンを完全に失活させた。これを規定の濃度になるように分散液に分散させてエンドトキシン選択性はないが吸着能力を持つ凝集微粒子を調製した。
製造例9で調製した日本アエロジル株式会社製のチタニア微粒子AEROXIDE P25の濃度が10mg/mLになるように、0.1%ポリ-L-リジン水溶液(シグマ・アルドリッチ製)に分散させた。室温で2時間ゆっくりと攪拌してポリ-L-リジンをチタニア微粒子上に吸着させた。その後、遠心して上清を除去し、エンドトキシンを含まない注射用蒸留水で3回洗浄した。最後に非イオン性界面活性剤のTriton X-100(0.2%)に素材のビーズの濃度換算で0.2mg/mLになるように分散させて選択的なエンドトキシン吸着能を持つ凝集性微粒子(PLK-P25)を調製した。
エンドトキシン水溶液(10EU/mL、1mL)をオートクレーブ処理をした1.5mL容量のエッペンドルフチューブに入れ、1昼夜静置し、溶液中のエンドトキシンの一部をチューブの内壁に吸着結合させた。チューブ内の溶液を全て排水し、注射用蒸留水を1mL入れてボルテックスミキサー(アズワン製 TUBE MIXER TRIO HM-1F)で最大回転速度にて10秒間攪拌を行った。その後チューブ内の溶液を全て排出した。この操作を合計2回行い、チューブ内でチューブ内壁に吸着せずに残存するエンドトキシンの溶液を除去した。これにより、エンドトキシンが物理的に内壁に吸着したエッペンドルフチューブを調製した。なお、エッペンドルフチューブはポリプロピレン製であり、ポリプロピレンやポリエチレンは素材が疎水性であるがゆえにエンドトキシンを吸着することが知られている(例えば、非特許文献2、3参照。)。
ALを使用したエンドトキシンの測定法は、既知濃度のエンドトキシン希釈系列の測定に基づく検量線を作成する検量線法である。従って、エンドトキシン吸着性微粒子を用いない場合と用いる場合の各々の検量線を作成する必要がある。微粒子を用いない場合はエンドトキシン希釈系列(0.1、0.01、0.001EU/mL)水溶液50μLとAL試薬50μLを製造例8のチューブ内に投入して攪拌比濁計測装置(興和製EX-100)で凝集反応を測定した。
1a・・・エンドトキシン
3・・・マイクロピペッター
4・・・マイクロピペッター
5・・・ガラス容器
10・・・抽出液
10a・・・エンドトキシンが吸着した微粒子
12・・・分散液
100・・・注射用水
100a・・・エンドトキシン
Claims (16)
- カブトガニの血球抽出物であるALを含むAL試薬と所定の生物由来の生理活性物質を含む試料の混和液を生成し、該混和液を攪拌しつつ、該混和液におけるALと前記生理活性物質との反応に起因する蛋白質の凝集またはゲル化を光学的手法により検出することで、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、
前記生理活性物質を表面に吸着可能な微粒子を前記試料中に分散させることで前記生理活性物質を前記微粒子の表面に吸着させた後に前記試料から前記微粒子を分離し、
前記AL試薬と前記生理活性物質が表面に吸着された前記微粒子との混和液を生成し、
前記AL試薬と前記微粒子との混和液における前記微粒子の凝集またはゲル化を光学的手法により検出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。 - 前記所定の生物由来の生理活性物質を含む試料は、医療用具から抽出した生物由来の生理活性物質を含む抽出液であ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。
- 前記微粒子は、パイロセップ、ポリミキシンB、ポリリジン、ポリオルニチン、ポリアルギニン、ポリヒスチジン、アミノシラン、キトサン、抗エンドトキシン抗体、抗エンドトキシンアプタマー、ランダムペプチドライブラリ中の素材、アルミナ、チタニア、シリカ、ジルコニア、ハイドロキシアパタイトなどの金属酸化物質、カオリン、モンモリロナイト、酸化マンガン、雲母などの天然あるいは合成鉱物より選定された一または複数の素材からな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。
- 前記微粒子は、担体微粒子の表面上に前記生理活性物質を選択的に吸着する吸着素材を結合させて形成され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。
- 前記吸着素材は、パイロセップ、ポリミキシンB、ポリリジン、ポリオルニチン、ポリアルギニン、ポリヒスチジン、アミノシラン、キトサン、抗エンドトキシン抗体、抗エンドトキシンアプタマー、ランダムペプチドライブラリ中の素材、より選定された一または複数の素材であることを特徴とする請求項4に記載の生物由来の生理活性物質の測定方法。
- 前記担体微粒子は、ポリスチレンラテックス、ポリエチレン、ナイロン、セルロース、アガロース、ポリビニルアルコール、アクリル樹脂、アルミナ、チタニア、シリカ、ジルコニア、ハイドロキシアパタイトなどの金属酸化物質、カオリン、モンモリロナイト、酸化マンガン、雲母などの天然あるいは合成鉱物より選定された一または複数の素材からなることを特徴とする請求項4または5に記載の生物由来の生理活性物質の測定方法。
- 前記微粒子の径は5nm以上50μm以下であることを特徴とする請求項1から6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。
- 前記微粒子には、界面活性剤が添加された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。
- 前記微粒子は、前記吸着素材によって表面全体が覆われるように形成されたことを特徴とする請求項4に記載の生物由来の生理活性物質の測定方法。
- 前記微粒子は前記AL試薬中のコアギュロゲンと親和性を有する素材からなることを特徴とする請求項1から9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。
- 前記微粒子はコアギュロゲンと親和性を有する素材を含んで形成され、予めコアギュロゲンが結合されたことを特徴とする請求項1から10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。
- 前記試料に、前記ALと前記生理活性物質との反応に影響を及ぼす干渉物質が含まれている場合に、前記試料から分離した後の前記微粒子を洗浄することで、前記干渉物質を除去することを特徴とする請求項1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方法。
- 前記AL試薬には、カブトガニの血球の抽出物に含まれる所定の蛋白質が表面に吸着されたビーズを分散させたことを特徴とする請求項1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グルカンであることを特徴とする請求項1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方法。
- 前記生理活性物質を表面に吸着可能な微粒子であって、
請求項1から14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に用いられる微粒子。 - 請求項2に記載の生物由来の生理活性物質の測定方法において医療用具から前記生理活性物質を抽出する抽出液であって、前記生理活性物質を表面に吸着可能な前記微粒子が予め分散されたことを特徴とする、医療用具における生物由来の生理活性物質の測定方法に用いられる抽出液。
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CN109001455B (zh) * | 2018-06-15 | 2021-09-10 | 天津一瑞生物科技股份有限公司 | 一种肺泡灌洗液样本处理液及处理检测方法 |
CN111982763B (zh) * | 2020-08-17 | 2021-05-14 | 上海普康药业有限公司 | 一种辅酶 q10 的粒度及粒度分布测定方法 |
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CN112462076A (zh) * | 2020-11-12 | 2021-03-09 | 天津华龛生物科技有限公司 | 应用于微载体的内毒素含量的检测方法 |
Also Published As
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EP2772760A1 (en) | 2014-09-03 |
EP2772760A4 (en) | 2015-08-26 |
JPWO2013062013A1 (ja) | 2015-04-02 |
US20150031568A1 (en) | 2015-01-29 |
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