WO2016153058A1 - 接合菌の測定方法及び接合菌測定用の試薬キット - Google Patents
接合菌の測定方法及び接合菌測定用の試薬キット Download PDFInfo
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- WO2016153058A1 WO2016153058A1 PCT/JP2016/059741 JP2016059741W WO2016153058A1 WO 2016153058 A1 WO2016153058 A1 WO 2016153058A1 JP 2016059741 W JP2016059741 W JP 2016059741W WO 2016153058 A1 WO2016153058 A1 WO 2016153058A1
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- zygomycetes
- acid
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- acid treatment
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
- G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
- G01N2400/28—Chitin, chitosan
Definitions
- the present invention relates to a specific fungal measurement means, specifically, a method for measuring a zygomycete, a preparation for a sample for measuring a zygomycete, a method for preparing a sample for measuring a zygomycete, and a method for measuring a zygomycete
- the present invention relates to a reagent kit and the like.
- Mycosis is an infectious disease caused by fungus colonizing an animal's skin or organ. Fungi are defined as a general term for organisms belonging to the fungus kingdom (Fungi). As mycosis, dermatomycosis (superficial mycosis), deep dermatomycosis (deep superficial mycosis), and deep mycosis (systemic mycosis, visceral mycosis) are known. Dermatomycosis is a mycosis confined to the epidermis, and ringworm, folding screen, skin candidiasis and the like are known.
- Deep cutaneous mycosis is a mycosis confined to the dermis and subcutaneous tissue, and sporotricosis, entomofutrasis and the like are known. Deep mycosis is a mycosis limited to organs, and aspergillosis, candidiasis, cryptococcosis, mucormycosis, and the like are known.
- zygomycosis infectious diseases caused by zygomycota are called zygomycosis.
- mucormycosis which is a mycosis caused by a zygomycete belonging to Mucoromycotina, and entomofutosis
- a mycosis caused by a zygomycete belonging to Entomophthoromycotina entomophthoramycosis
- chitosan and chitin exist in the cell walls of fungi. More specifically, chitin and (1 ⁇ 3) - ⁇ -D-glucan (hereinafter “ ⁇ -glucan”) are present on the cell walls of fungi belonging to Ascomycota, Basidiomycota, and Chytridiomycota. In contrast, the cell walls of fungi belonging to Zygomycota are known to contain a large amount of chitosan instead of chitin and little ⁇ -glucan.
- Non-Patent Document 1 visceral mycosis such as mucormycosis (deep mycosis) is increasing against the background of the widespread immunosuppressive treatment for cancer and blood diseases. It has been pointed out. Further, in this document, mucormycosis is a fulminant mycosis in which the death rate of infected persons reaches approximately 60%, and the proportion of persons infected with mucormycosis among the mycoses is increasing. It has been pointed out. Furthermore, it is pointed out in this document that there is no serological auxiliary diagnostic method useful for early diagnosis as a problem of mucormycosis.
- Patent Document 1 describes a microquantitative method for carbohydrates containing chitosan using an anti-carbohydrate antibody. It is described that it can be used for high-sensitivity detection of quality. However, regarding the measurement of chitosan using a biological sample as a measurement object, no substantial description with specific means is recognized at all.
- Patent Document 2 as a method for detecting and identifying a zygomycosis-causing bacterium, a set of a sense primer and an antisense primer specific for the DNA of the target zygomycosis-causing bacterium was used, using the DNA in the sample as a template. A method for detecting zygomycosis-causing bacteria by PCR is described. In the examples of this document, protease K treatment is used as a pretreatment of the specimen.
- An object of the present invention is to provide means capable of truly measuring zygomycetes. Moreover, the subject of this invention is providing the means which implement
- sample pretreatment methods include surfactant addition, organic solvent addition, metal salt addition, enzyme (nuclease, protease, lipase, glycosidase, etc.) addition, ultrasound and beads
- the present inventors are astonishing that various treatment methods such as a treatment using a homogenizer, a treatment using a homogenizer, a treatment using a column, a treatment using a filtration filter, etc. can be considered.
- measurement of zygomycetes can be performed by a very simple and inexpensive technique of acidifying a sample (acid treatment), thereby completing the present invention.
- a method for measuring zygomycetes wherein zygomycetes are measured using an acid-treated sample as a measurement target.
- the acid treatment is a treatment in which an acidic component and a sample coexist.
- the acidic component is one or more acids selected from the group consisting of oxo acids, organic acids, and inorganic acids.
- oxo acid is one or more acids selected from the group consisting of carboxylic acid, sulfonic acid, sulfuric acid, phosphoric acid, nitric acid, and halogen oxo acids.
- organic acid is a carboxylic acid and / or a sulfonic acid.
- carboxylic acid is one or more acids selected from the group consisting of acetic acid, formic acid, citric acid, and oxalic acid.
- hypohalous acid is one or more acids selected from the group consisting of hypochlorous acid, hypobromous acid, and hypoiodous acid.
- perhalogenic acid is one or more acids selected from the group consisting of perchloric acid, perbromic acid, and periodic acid.
- the animal-derived sample is a blood-derived sample, bronchoalveolar lavage fluid (BALF), biopsy specimen, sputum, cerebrospinal fluid, nasal wipe, pharyngeal wipe, nasal aspirate, or ascites
- BALF bronchoalveolar lavage fluid
- the preparation method according to [29], wherein the treatment for allowing the acidic component and the sample to coexist is the treatment for allowing the preparation agent and the sample described in [23] or [24] to coexist.
- a reagent kit for measuring zygomycetes comprising a reagent containing an acidic component and / or a detection reagent for zygote components as a component.
- a measuring method of zygomycetes (hereinafter referred to as “measuring method of the present invention”) characterized in that zygomycetes are measured using an acid-treated sample as a measurement target. Is done.
- the measurement method of the present invention is, for example, a zygomycote detection method, a zygomycote detection method, a zygomycosis quantification method, a zygomycosis detection method, a zygomycosis detection method, or a zygomycosis quantification method. Can be used.
- the measurement method of the present invention is, for example, for measuring zygomycosis (specimen test) using an acid-treated sample (specimen) as a measurement target and diagnosing whether or not the animal suffers from zygomycosis. It can also be used as a data acquisition method for diagnosing zygomycosis characterized by acquiring data.
- sample preparation agent of this invention As mentioned above, the sample by which the measurement of a mating bacterium is performed in this invention is acid-treated. It is practically simple to perform the acid treatment in the presence of an acidic component in the sample.
- sample preparation of the present invention a preparation for a zygote measurement sample (hereinafter referred to as “sample preparation of the present invention”), which contains an acidic component.
- the agent containing the acidic component can be used as an acid treatment agent for a sample for measuring zygomycetes.
- Both the sample preparation agent and the acid treatment agent of the present invention can be produced by any method including a step of incorporating an acidic component into the agent.
- both the sample preparation agent of the present invention and the acidic component can be suitably used for acid treatment of a sample that performs the measurement method of the present invention.
- sample preparation method of the present invention a method for preparing a sample for measurement of zygomycetes (hereinafter referred to as “sample preparation method of the present invention”), wherein the sample is converted into a sample for measurement of zygomycetes by acid treatment.
- the sample preparation method of the present invention can be suitably used for acid treatment of a sample for performing the measurement method of the present invention.
- the acid treatment is preferably a treatment in which an acidic component and a sample coexist.
- the treatment for allowing the acidic component and the sample to coexist is preferably a treatment for allowing the sample preparation agent of the present invention and the sample to coexist.
- the measurement of the conjugated bacteria is a reagent kit containing at least one of a reagent containing an acidic component and a detection reagent for the conjugated bacterial component as a component (hereinafter referred to as “reagent kit of the present invention”).
- the reagent kit of the present invention can be suitably used for performing the measurement method of the present invention.
- the reagent containing an acidic component is preferably the sample preparation agent of the present invention.
- the detection reagent for the zygote component is an antibody that binds to chitosan.
- the reagent kit of the present invention may further contain other components in addition to the above components.
- the other components can be appropriately selected according to the measurement mode of the zygomycete.
- the reagent kit of the present invention can also be used, for example, as a diagnostic kit for zygomycosis such as mucormycosis or entomophthalosis.
- acid treatment is performed as a pretreatment of a sample for measuring zygomycetes.
- the acid treatment is a treatment for making the sample acidic, and can be performed by subjecting the sample to acidity. It is practically simple that the acid treatment is performed in the presence of an acidic component in the sample.
- the acid-treated sample may be a sample that has been treated to coexist with an acidic component.
- the acidic component may be an organic acid or an inorganic acid.
- the organic acid is preferably a carboxylic acid.
- the carboxylic acid is acetic acid, formic acid or citric acid.
- the inorganic acid is preferably hydrogen halide, sulfuric acid, or phosphoric acid.
- the hydrogen halide is preferably hydrogen chloride (hydrochloric acid). Only 1 type may be used for such an acidic component and it may use it in combination of 2 or more type.
- measurement of zygomycetes can be performed using, for example, a detection reagent for zygomycete components. It is preferable that the detection reagent for the zygote component is an antibody that binds to chitosan.
- the measurement of zygomycetes can be performed by, for example, an immunoassay.
- the immunoassay is preferably an immunoassay using an antibody that binds to chitosan.
- the immunoassay examples include an enzyme immunoassay (Enzyme ImmunoAssay, EIA) method, an immunochromatography method, an immunoblot method, an agglutination method, a turbidimetric method, or a nephelometry method.
- An example of the aggregation method is a latex aggregation method.
- the enzyme immunoassay may be an ELISA (Enzyme-Linked Immunosorbent Assay) method.
- sample for measuring zygomycetes in the present invention is not particularly limited as long as it is a substance that can contain zygomycetes, but is preferably a sample (specimen) derived from an animal including humans, The present invention is suitably used in specimen testing.
- the sample derived from an animal include a sample obtained from an animal body and a sample obtained by collecting a liquid brought into contact with the animal body.
- the liquid to be contacted with the animal body is not particularly limited as long as it is a liquid that is compatible with a living body. Such liquid includes saline.
- the liquid collected after contact with the animal body can be directly subjected to acid treatment as a sample for measuring zygomycetes.
- the sample obtained from the animal body is preferably a blood-derived sample. It is particularly preferred that the blood-derived sample is serum or plasma. It is preferable that the sample obtained by collecting the liquid brought into contact with the animal body is bronchoalveolar lavage fluid (BALF).
- BALF bronchoalveolar lavage fluid
- the animal referred to here is preferably an animal suffering from zygomycosis or an animal that may have suffered from zygomycosis. Examples of zygomycosis include mucormycosis and entomoftorosis.
- the sample on which the measurement of zygomycetes is performed is prepared by performing an acid treatment.
- the sample on which zygomycetes are measured may be a sample that has been neutralized after acid treatment.
- GlcN glucosamine
- GlcNAc N-acetyl-D-glucosamine
- CTS chitosan
- CTS6 chitosan hexamer
- GCTS glycol chitosan
- CTN chitin
- EGCTN ethylene glycol chitin
- CTSpAb anti-chitosan polyclonal antibody
- CTS mAb anti-chitosan monoclonal antibody
- PBS Saline
- the present invention it is possible to measure zygomycetes contained in a sample with high sensitivity.
- the present invention can be used, for example, as a means for realizing serological diagnosis useful for early diagnosis of zygomycosis such as mucormycosis and entomoftorosis.
- a sample for which measurement of zygomycetes is performed is prepared by being subjected to acid treatment as described above.
- the acid treatment is not particularly limited as long as it is a treatment for subjecting the sample to acidity (treatment for bringing the sample into an acidic state). Examples of such means include a cation exchanger, activated carbon (acidic charcoal), or a method in which an acidic component and a sample coexist.
- the acid treatment is preferably simple and inexpensive because it is a treatment in which an acidic component and a sample coexist.
- the coexistence means that the objects can be in contact with each other.
- the coexistence of the acidic component and the sample can be performed by adding the acidic component to the sample, or can be performed by adding the sample to the acidic component.
- the acid treatment is preferably performed in a liquid.
- the liquid on which the acid treatment is performed is preferably water or an aqueous solution.
- Acidic component The acidic component is not particularly limited as long as it is a substance that exhibits acidity when coexisting with water.
- the acidic component may be a compound that liberates hydrogen ions when coexisting with water, or may be a compound that liberates hydrogen ions when ionized. That is, the acidic component may be Arrhenius acid.
- Examples of the acidic component include oxo acid, organic acid, inorganic acid and the like.
- the oxo acid include carboxylic acid, sulfonic acid, sulfuric acid, phosphoric acid, nitric acid, halogen oxo acid and the like.
- Examples of the organic acid include carboxylic acid and sulfonic acid.
- Examples of the carboxylic acid include acetic acid, formic acid, citric acid, oxalic acid and the like.
- Examples of the inorganic acid include hydrogen halide, sulfuric acid, phosphoric acid, nitric acid, halogen oxo acid and the like.
- Examples of the hydrogen halide include hydrogen chloride (hydrochloric acid).
- Examples of the halogen oxo acid include hypohalous acid and perhalogen acid.
- Examples of hypohalous acid include hypochlorous acid, hypobromous acid, hypoiodous acid, and the like.
- Examples of the perhalogen acid include perchloric acid, perbromic acid, and periodic acid.
- the acidic component is preferably a carboxylic acid, and particularly preferably acetic acid, formic acid, or citric acid.
- the acidic component is preferably a hydrogen halide, sulfuric acid, or phosphoric acid, and more preferably a hydrogen halide. It is particularly preferred that the hydrogen halide is hydrogen chloride (hydrochloric acid).
- the acidic component may be, for example, an acid anhydride or a polyoxo acid.
- acid anhydrides include carboxylic acid anhydrides and phosphoric acid anhydrides, and specific examples include acetic anhydride and pyrophosphoric acid.
- polyoxo acid include polyphosphoric acid.
- the acidic component may be a salt as long as it is a substance that exhibits acidity when coexisting with water.
- the salt include ammonium salt and sodium salt, and specific examples include ammonium dihydrogen phosphate, ammonium dihydrogen citrate, sodium dihydrogen phosphate, sodium dihydrogen citrate and the like.
- the shape of the acidic component is not particularly limited, and may be a solid, a liquid, or a gas.
- the liquid acidic component may be prepared by dissolving a solid or gaseous acidic component in a solvent such as water.
- Only one kind of acidic component may be used for acid treatment, or two or more kinds may be used in combination for acid treatment.
- the sample is not particularly limited as long as it is a substance that can contain zygomycetes.
- Examples of the sample include a sample derived from an animal. Specific examples of animals include humans and other animals that can be affected by zygomycosis such as mucormycosis and entomophthalosis, such as mammals such as monkeys, dogs, cats, rabbits, horses, and cows. , Birds and the like.
- the zygomycosis is also a problem of pet-derived infection and the morbidity of the pet itself, and animals other than humans are also important as samples.
- the sample derived from an animal may be a sample obtained from an animal body, or may be a sample obtained by collecting a liquid brought into contact with the animal body.
- samples derived from animals include sputum, cerebrospinal fluid, nasal wiping fluid, throat wiping fluid, nasal aspirate, ascites, bronchoalveolar lavage fluid (BALF), biopsy specimen, and blood-derived sample.
- samples obtained from the animal body include sputum, cerebrospinal fluid, nasal wiping fluid, throat wiping fluid, nasal aspirate, ascites, biopsy specimen, and blood-derived sample.
- samples obtained by collecting the liquid brought into contact with the animal body include bronchoalveolar lavage fluid (BALF).
- BALF Bronchoalveolar lavage fluid
- BALF Bronchoalveolar lavage fluid
- BALF is typically known as a solution obtained by injecting physiological saline or the like into the bronchi to wash the alveoli, and then recovering the alveoli.
- the blood-derived sample may be blood itself or blood containing other components such as an additive such as an anticoagulant, and further, a fraction (plasma or Serum, etc.).
- the blood-derived sample is particularly preferably serum or plasma.
- the serum can be obtained, for example, by standing or centrifuging blood collected from an animal and collecting the supernatant.
- Plasma can be obtained, for example, by mixing blood collected from an animal with an anticoagulant and then allowing to stand or centrifuge, and recovering the supernatant.
- the form of the sample is not particularly limited, and may be liquid or solid.
- liquid samples include liquid components such as sputum, cerebrospinal fluid, nasal wiping fluid, pharyngeal wiping fluid, nasal aspirate, ascites, bronchoalveolar lavage fluid (BALF), and blood-derived samples.
- suspensions of specimens examples include suspensions of specimens.
- the solid sample include, in addition to the above-described biopsy specimens, liquid components (spider, cerebrospinal fluid, nasal wipe, pharyngeal wipe, nasal aspirate, ascites, bronchoalveolar lavage fluid (BALF), blood, and the like.
- the sample may be a liquid sample itself, may be appropriately concentrated or diluted, or may be a solid sample dissolved appropriately.
- acid treatment is treatment for acidifying a sample, and can be performed by subjecting the sample to acidity.
- the pH when the sample is subjected to acid treatment (hereinafter referred to as “acid treatment”) is not particularly limited as long as it is acidic at 6 or less, and any pH can be selected.
- the pH during the acid treatment can be appropriately set according to, for example, the type of sample and the mode of measurement of zygomycetes.
- the pH including the lower limit may be, for example, pH 0 or more, pH 0.5 or more, pH 1 or more, pH 2 or more, pH 3 or more, pH 3.5 or more.
- the pH including the upper limit may be, for example, pH 6 or less, pH 5.5 or less, pH 5 or less, pH 4.5 or less, and the like. More specifically, pH 0 to 6, pH 0.5 to 5.5, pH 0 to 5, pH 0.5 to 5, pH 1 to 5, pH 2 to 5, pH 3 to 5, pH 3.5 to 4.5, etc. are exemplified. Is done.
- the concentration of the acidic component during the acid treatment is not particularly limited and can be appropriately set according to various conditions such as the kind of the acidic component and the desired pH.
- the concentration including the lower limit may be, for example, 0.001 mM or more, 0.01 mM or more, 0.1 mM or more, 1 mM or more, 10 mM or more, 50 mM or more, 100 mM or more.
- concentration including an upper limit may be 1 M or less, 500 mM or less, 250 mM or less, etc., for example.
- concentration in the case of using 2 or more types of acidic components is the value of the sum total of the density
- the concentration may mean the final concentration of the acidic component added to the sample.
- the temperature during acid treatment is not particularly limited, and any temperature can be selected.
- the acid treatment may be performed at a room temperature of 15 to 25 ° C., for example, or may be performed at a lower temperature or a higher temperature.
- the said temperature including the minimum may be 0 degreeC or more, 4 degreeC or more, 10 degreeC or more, 20 degreeC or more, etc., for example.
- the temperature including the upper limit may be, for example, 125 ° C. or lower, 100 ° C. or lower, 75 ° C. or lower, 50 ° C. or lower, 37 ° C. or lower. More specific examples of the temperature include 0 ° C. to 125 ° C., 4 ° C. to 100 ° C., 10 ° C. to 75 ° C., 20 ° C. to 75 ° C., etc. in addition to the normal temperature described above.
- the acid treatment can be performed at room temperature (24 to 26 ° C.), or can be performed by cooling or heating the sample.
- the sample can be cooled and heated using, for example, a device such as a heat block, a water bath, an oil bath, and an air bath.
- the time for performing the acid treatment is not particularly limited.
- the processing time including the lower limit may be, for example, 5 seconds or longer, 30 seconds or longer, 1 minute or longer, 5 minutes or longer, or the like.
- the processing time including the upper limit may be, for example, 24 hours or less, 2 hours or less, 1 hour or less, or 30 minutes or less. More specific examples of the treatment time include 5 seconds to 24 hours, 30 seconds to 2 hours, 1 minute to 1 hour, and 5 minutes to 30 minutes.
- the acid-treated sample can be used as it is for measurement of zygomycetes, but if the measurement is performed using a component that is not suitable for acidic use (for example, a protein such as an antibody), It is preferable to use a sample for which acid treatment has been completed for measurement of zygomycetes.
- a component that is not suitable for acidic use for example, a protein such as an antibody
- the acid treatment can be terminated, for example, by setting the sample to a pH higher than that suitable for acid treatment, preferably by making the sample neutral or basic.
- the acid treatment can be terminated by, for example, adding a neutralizing component or a basic component to the sample, or removing a component added to make the sample such as an acidic component acidic from the sample. .
- the acid treatment can be terminated, for example, by setting the sample to pH 6.5 or higher, preferably pH 7 or higher.
- Strong basicity is pH12 or more, pH12.5 or more, pH13 or more, pH13.5 or more, pH14 or more, for example.
- the acid treatment termination operation may be an operation of adjusting the sample to pH 6.5 to pH 12, more preferably pH 7 to pH 12, more preferably pH 7 to 10, and still more preferably pH 7 to pH 8.
- the sample that has been subjected to acid treatment and neutralized can be used as it is (without further treatment) as a sample to be measured for measurement of zygomycetes.
- the sample on which the zygote is measured is preferably a sample adjusted to the pH exemplified above after the acid treatment.
- the sample may be diluted or dissolved.
- the dilution ratio when the sample is diluted is not particularly limited, and the dilution ratio including the lower limit is, for example, more than 1 time, 1.1 times or more, 2 times or more, 5 times or more, 10 times or more, etc. Good.
- the dilution factor including the upper limit may be, for example, 500 times or less, 200 times or less, 100 times or less, and the like. More specifically, examples include more than 1 to 500 times, 1.1 to 500 times, 2 to 200 times, 5 to 200 times, 10 to 100 times, and the like.
- the acid treatment may be performed only once, or may be performed two or more times.
- the acid treatment may be performed by, for example, subjecting the sample to acidity, neutralizing, and then subjecting the sample to acidity again.
- various conditions for the acid treatment pH, type of acidic component, concentration of acidic component, temperature, time, etc. may be the same or different.
- the sample preparation agent of the present invention is an agent containing the acidic component exemplified above, and is an agent used for acid-treating a sample.
- the sample preparation agent of the present invention may be a liquid agent or a solid agent for dissolution at the time of use, such as powders, granules, tablets and the like.
- the sample preparation agent of the present invention may be the acidic component itself, or may be one obtained by diluting or dissolving the acidic component in a solvent such as water.
- the sample preparation agent of the present invention may contain other components other than the acidic component and the solvent for reasons of formulation.
- the other components are not particularly limited as long as they are pharmaceutically acceptable components for reagents and diagnostics.
- alkali metal salts examples thereof include alkali metal salts, alkaline earth metal salts, and various additives.
- the acidic component may exist in a non-ionized state, may exist in an ionized state, or may exist in a state in which they coexist.
- the weight percent concentration (w / w) occupied by the acidic component in the sample preparation of the present invention is not particularly limited, and any concentration can be selected.
- the concentration (w / w) including the lower limit is, for example, 0.001% or more, 0.01% or more, 0.1% or more, 1% or more, 5% or more, 10% or more, 25% or more, 50 % Or more.
- the concentration (w / w) including the upper limit may be, for example, 100% or less, 75% or less, 50% or less, 25% or less, 10% or less, 5% or less, 1% or less, and the like.
- the amount of the sample preparation of the present invention used is not particularly limited, and can be appropriately set according to various conditions such as the type and concentration of the acidic component contained in the sample preparation of the present invention.
- the sample preparation agent of the present invention can be used, for example, so that the amount of the acidic component when coexisting with the sample is an amount suitable for the acid treatment described above.
- the sample preparation agent of the present invention can be produced by a method usually used in the preparation of reagents and diagnostic agents.
- the sample By applying acid treatment to a sample for measuring zygomycetes using the sample preparation agent of the present invention, the sample can be brought into a state suitable for the measurement of zygomycetes, and the measurement of the desired zygomycetes can be performed with high sensitivity. It can be carried out.
- sample preparation method of the present invention a sample (sample for measuring zygomycetes) is converted into a sample for measurement of zygomycetes (sample for measuring zygomycetes) by acid treatment. It is a method to do.
- the sample preparation method of the present invention may further include other steps before, after, or simultaneously with the step.
- the sample preparation method of the present invention may include, for example, a step of terminating acid treatment, a step of dissolving a solid sample, a step of diluting or concentrating a liquid sample, and the like.
- the sample preparation method of the present invention may include, for example, a step of heating or cooling the sample so that the prepared sample has a temperature suitable for measurement of zygomycetes. Furthermore, the sample preparation method of the present invention may include, for example, a step of adjusting the pH of the sample so that the prepared sample has a pH suitable for measurement of zygomycetes.
- the sample can be brought into a state suitable for measurement of zygomycetes, and a desired zygote measurement can be performed with high sensitivity.
- the measurement method of the present invention is a method for measuring zygomycetes as described above.
- the zygomycetes to be measured in the present invention are acid-treated zygomycetes, and in one aspect, are components derived from zygomycetes.
- the zygote to be measured may be the microbial cell itself, may be a fragmented microbial cell, or may be a part or one component of the microbial cell.
- the zygomycetes to be measured in the present invention may be live cells or dead cells such as inactivated cells.
- the measurement of the zygomycete can be performed, for example, using a detection reagent for the zygomycete component (a reagent for detecting a component of the zygomycete).
- the component of the zygote to be measured is not particularly limited. Examples of the component include nucleic acids, proteins, lipids, carbohydrates and the like.
- the component is preferably a component having the same structure that is present in common in various zygomycete species.
- the zygote component to be measured is preferably a lipid or a carbohydrate, and more preferably a carbohydrate.
- the carbohydrate is preferably a carbohydrate, and more preferably chitosan.
- the measurement of the zygomycete is particularly preferably performed using chitosan as a component to be measured.
- measurement is a concept including detection, detection, and quantification. That is, the measurement of zygomycetes includes detection of zygomycetes, detection of zygomycetes, and determination of zygomycetes as concepts.
- the detection reagent means a reagent that is brought into contact with a zygote component and causes a physical or chemical change in the zygote component or the reagent itself. Such changes include binding, transition, rearrangement, addition, elimination, decomposition, oxidation, reduction, labeling, color development, luminescence, and the like.
- the change can be measured, for example, as a peak of a chromatogram, a contrast, a change in a measurement value of an analytical instrument, a signal detected with the change, or the like.
- the change can also be measured by further using, for example, a reagent for labeling or measuring the detection reagent or a reaction product generated upon contact with the detection reagent.
- Such a reagent may be a reagent that causes a physical or chemical change in any of the detection reagent, a reaction product that is brought into contact with the detection reagent, or the reagent itself.
- Examples of the reagent for labeling or measuring the detection reagent or the reaction product produced upon contact with the detection reagent include antibodies that bind to them.
- the detection reagent is preferably a reagent that enables specific detection of the measurement target.
- An example of such a reagent is an antibody.
- the measurement of zygomycetes is preferably performed by an immunoassay. As described above, it is particularly preferable that the measurement of the zygote is performed using chitosan as a component to be measured, and thus the detection reagent is particularly preferably an antibody that binds to chitosan (anti-chitosan antibody).
- the antibody used in the immunoassay may be a polyclonal antibody, a monoclonal antibody, or an antibody in which both are mixed.
- the antibody may be an antibody itself or an antibody labeled with another substance (labeled antibody).
- labeled antibodies include enzyme labeled antibodies, fluorescent labeled antibodies, biotin labeled antibodies, and the like.
- the type of immunoglobulin of the antibody is not particularly limited, but when the antibody is an anti-chitosan antibody, it is preferably prepared by immunizing birds for the reasons described later, and is preferably an IgY antibody. Since IgY is an immunoglobulin that does not exist in a mammal, the IgY antibody can be particularly preferably used when the sample is a sample derived from a mammal.
- the antibody may be an antibody in which two or more antibodies having the same epitope in the component to be measured are mixed, or may be an antibody in which two or more antibodies having different epitopes are mixed. .
- the usage mode of the antibody in the present invention may be the use of an antibody alone or may be used as an antibody preparation containing other components than the antibody.
- other components herein include alkali metal salts, alkaline earth metal salts, surfactants, buffers, and the like, as well as components other than antibodies derived from animals immunized with antigens.
- the weight volume percent concentration (w / v) occupied by the antibody itself is not particularly limited, and may be any concentration.
- the concentration (w / v) including the lower limit is, for example, 0.001% or more, 0.01% or more, 0.1% or more, 1% or more, 5% or more, 10% or more, 25% or more, 50 % Or more.
- the concentration (w / v) including the upper limit may be, for example, 100% or less, 75% or less, 50% or less, 25% or less, 10% or less, 5% or less, 1% or less, and the like.
- the immunoassay can be performed by a known method used for measuring an antigen by utilizing an antigen-antibody reaction, and its mode is not particularly limited.
- immunoassay include enzyme immunoassay (EIA or ELISA), immunochromatography, immunoblot (dot blot, slot blot, spot blot, etc.), agglutination, turbidimetry, and nephelometry.
- EIA or ELISA enzyme immunoassay
- immunochromatography immunoblot (dot blot, slot blot, spot blot, etc.), agglutination, turbidimetry, and nephelometry.
- a latex aggregation method is preferably exemplified.
- the ELISA method As the ELISA method, (a) a method in which an antibody (detection antibody) is added to a sample immobilized on a plate and the antigen contained in the sample is measured (binding method), and (b) the sample is immobilized on a plate.
- (c) a sample (anti-solid phase on the plate) Examples thereof include a method (competition method, inhibition method), etc., in which an antigen contained in a sample is measured by adding a competitive substance) and an antibody (detection antibody).
- the ELISA method can prepare a large number of plates in advance and stock them, so that the competitive method (hereinafter referred to as “competitive ELISA method”) or the sandwich method (hereinafter referred to as “sandwich ELISA method”). And the sandwich ELISA method is more preferable.
- the acid-treated sample and the detection antibody may be separately added to the plate in any order, or may be added to the plate after they coexist in advance.
- the time for allowing the sample and the detection antibody to coexist in advance is preferably several hours to about one day, and specific examples include 1 to 24 hours, 12 to 24 hours, and 18 to 24 hours. .
- the two types of antibodies (solid-phased antibody and detection antibody) used in the sandwich ELISA method are not limited as long as they are antibodies that bind to components of the conjugated bacteria, and may be antibodies that recognize different epitopes. May be the same antibody.
- the two types of antibodies may be the same antibody or different antibodies.
- the antibody used in the present invention can be produced by a known method used for producing an antibody, and the method is not particularly limited.
- a typical example of a method for producing the antibody is a method of immunizing an animal with an antigen.
- the antigen may be a zygote itself, a fragmented zygote, or a component of the zygote, and these may be fused with other substances to increase immunogenicity. It may be a complex. Examples of other substances used for forming the complex include proteins, and specifically bovine serum albumin (BSA).
- BSA bovine serum albumin
- the antigen may be used for animal immunization in the state where it coexists with an antigenic reinforcing agent (adjuvant).
- the zygote component used as an antigen is preferably chitosan as described above.
- the origin of chitosan used as an antigen is not particularly limited, and may be chitosan other than chitosan obtained by extraction from zygomycetes, for example, commercially available chitosan.
- Examples of chitosan used as an antigen include chitosan obtained by N-deacetylation of chitin.
- chitin is derived from crustaceans such as crabs and shrimps.
- the degree of polymerization of the chitosan that is, the molecular weight of the chitosan is not particularly limited, and may be a polymer or an oligomer (oligosaccharide), but when preparing a complex fused with another substance. From the viewpoint of the reaction yield, an oligomer is preferable. Examples of such oligomers include hexamers.
- the chitosan must be chitosan having an epitope sequence (sequence formed by repeated polymerization of GlcN residues) to which an anti-chitosan antibody binds in the molecule, and as long as it is chitosan containing only GlcN residues.
- the degree of deacetylation of the chitosan (hereinafter referred to as “degree of deacetylation”) is not particularly limited, but specifically, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98 % Or more, 99% or more, 100% or the like.
- degree of deacetylation is preferably high.
- the degree of deacetylation of chitosan means the ratio of the total number of GlcN residues to the total number of GlcN residues and GlcNAc residues constituting the chitosan skeleton. In other words, the total number of GlcN residues is expressed as GlcN residues. And the percentage of GlcNAc residues divided by the total number.
- the degree of deacetylation can be measured by a known method, specifically, for example, a nuclear magnetic resonance (NMR) method, an infrared absorption spectrum (IR) method, or a colloid titration method.
- Animals that are immunized in the production of anti-chitosan antibodies are immunized with the above chitosan.
- the animal is not particularly limited as long as it is an animal capable of producing an anti-chitosan antibody, but is preferably an animal that does not inherently have chitosan. Examples of such animals include mammals and birds, but birds are preferred, and chickens (Gallus gallus domesticus) are particularly preferred.
- the antibody may be a polyclonal antibody, a monoclonal antibody, or an antibody in which both are mixed.
- the anti-chitosan antibody obtained as a polyclonal antibody can be obtained from, for example, animal-derived components or animal cells, and specifically from body fluids or eggs of animals immunized with chitosan. Examples of animal body fluids herein include blood, serum, plasma, and ascites.
- the anti-chitosan antibody obtained as a monoclonal antibody can be obtained from, for example, a hybridoma of antibody-producing cells and myeloma cells of an animal immunized with chitosan, or a culture solution of the hybridoma.
- the anti-chitosan antibody obtained as a monoclonal antibody can be obtained, for example, from cells transformed with an anti-chitosan antibody expression vector or a culture solution of the cells.
- the cells can be produced, for example, by a method described in a document (WO2011 / 049082) or a method using a commercially available kit such as Mammalian PowerExpress System (manufactured by Toyobo Co., Ltd.).
- the cell may be a transient expression cell or a stable expression cell.
- the antibody may be a component derived from an animal, an animal cell, a hybridoma cell, or a culture solution of a hybridoma cell as it is, and an extract or a cell residue obtained by disrupting an animal cell or a hybridoma cell may be used as an antibody. Or a fraction obtained by purifying them may be used as an antibody.
- the disruption of cells can be performed by a method appropriately selected according to the type of cells. Examples of the method for disrupting cells include a method for homogenization, a method for ultrasonic treatment, a method for freeze-thawing, and a method for adding a surfactant.
- Antibody purification can be performed by a known technique used for protein separation and purification.
- the antibody can be purified by, for example, ammonium sulfate precipitation or various chromatographies.
- Antibody purification can also be performed using components that bind to antibodies such as protein A, protein G, and protein L, for example. Antibody purification may be performed to a desired degree.
- a zygomycete is defined as a fungus belonging to the Zygomycota.
- the fungi include fungi belonging to Zygomycetes and Trichomycetes.
- the fungi belonging to the zygomycetes are Dimargaritales, Endogonales, Entomophthorales, Glomales, Kickxellales, Mucorales, and Zoopagales.
- fungi belonging to Examples of the fungi belonging to the Trichomyces class include fungi belonging to the order of Amoebidiales, Asellariales, Eccrinales, and Harperellales.
- zygomycetes are also defined as fungi belonging to Glomeromycota, Entomophthoromycotina, Kickxellomycotina, Mucoromycotina, and Zoopagomycotina.
- Examples of fungi belonging to the Glomus gate include fungi belonging to the Glomeromycetes class.
- examples of fungi belonging to the Glomus class include fungi belonging to the order of Archaeoporales, Diversisporales, Glomerales, and Paraglomerales.
- Examples of the fungus belonging to the subfamily Hymenoptera include fungi belonging to the order of Entomophthorales.
- Examples of fungi belonging to the Kick sella subfamily include fungi belonging to the order of Asellariales, Dimargaritales, Harperellas, and Kickxellales.
- Examples of the fungus belonging to the subspecies Amanita include those belonging to the order of Endogonales, Mortierellales, and Mucorales.
- Examples of the fungus belonging to the subtropy submycosis include fungi belonging to the order of Zoopagales.
- Examples of the fungi belonging to the order of the fungi are those belonging to the family Mucoraceae, Cunninghamellaceae, Saksenaeaceae, and Thamnidiaceae.
- As the fungi belonging to the family Aceraceae fungi belonging to the genus Absidia, Apophysomyces, Lichtheimia, Mucor, Rhizomucor, Rhizopus Is mentioned.
- Examples of the fungus belonging to the family Cusumidae include fungi belonging to the genus Cunninghamella.
- Examples of fungi belonging to the family Saxenaea include fungi belonging to the genus Saxenaea.
- Examples of the fungus belonging to the family Eridaceae include fungi belonging to the genus Cokeromyces.
- the fungi belonging to the order of the fungus are Absidia butleri, Absidia mbcorymbifera, Apophysomyces elegans, and Apofisomyces trapezioformis.
- Amophysomyces trapeziformis Lichtheimia corymbifera, Mucor circinelloides, Mucor plumbeus, Mucor racemosus, Mucor ram rouxii), Rhizomucor pusillus, Rhizopus arrhizus, Rhizopus microspores, Rhizopus oryzae, Rhizopus oryzae, Rhizopus oryzae Chromatography (Rhizopus stolonifer), Cunninghamella-Berutore Tie (Cunninghamella bertholletiae), Cunninghamella-Ekinurata (Cunninghamella echinulata), Sakusenea-Bashiforumisu (Saksenaea vasiformis), Kokeromaisesu-
- Examples of the fungi belonging to the order of the fly fungus include fungi belonging to the family Entomophthoraceae and Basidiobolaceae.
- Examples of the fungus belonging to the family Fliesida include fungi belonging to the genus Conidiobolus.
- Examples of fungi belonging to the family Basidiobolus include fungi belonging to the genus Basidiobolus.
- the fungi belonging to the order of the flies are Conidiobolus brefeldianus, Conidiobolus coronatus, Conidiobolus incongruus, Conidiobolus onglangonges (Conidiobolus incongruus) Examples include Conidiobolus lamprauges), Basidiobolus meristosporus, Basidiobolus ⁇ ranarum, and the like.
- the measurement method of the present invention includes a step of acid treatment of the above-described sample and a step of detecting zygote by bringing the zygote component detection reagent into contact with the zygote (hereinafter referred to as zygote).
- zygote a step of detecting zygote by bringing the zygote component detection reagent into contact with the zygote
- other steps may be included as necessary.
- the other step includes a step (neutralization step) of making the acid-treated sample neutral or basic in order to end the acid treatment.
- the step of terminating the acid treatment is suitably performed, for example, when the detection reagent for the zygote component is a reagent that is not suitable for use under acidic conditions.
- the detection reagent for the conjugated bacteria component is a protein such as an antibody
- the use under acidic conditions may cause denaturation of the protein or inhibition of binding to the conjugated bacteria component.
- Prior to the neutralization step may be suitably
- the other process there is a process of converting the measurement value obtained in the zygote detection process into another numerical value.
- the other steps include, for example, detection of conjugated bacteria based on the relationship (standard curve) between the measured value obtained by bringing a standard substance with a known concentration into contact with the detection reagent for the zygote component and the concentration of the standard substance.
- This is a step of converting the measurement value obtained in the step into the amount or number of zygomycetes contained in the sample.
- the step may be a step of converting the measurement value obtained in the detection step of the zygomycete into the progress degree of the zygomycosis.
- the “standard curve” referred to here can be obtained, for example, by replacing the sample in which the zygote is measured with a standard substance and performing a zygote detection step.
- the zygote itself can be used as the standard substance, it is practically simple to use the zygote component to be measured.
- chitosan can be used as a zygote component to be measured.
- the measurement value obtained in the zygote detection step include a measurement value (fluorescence intensity) of a fluorometer and a measurement value (absorbance) of a spectrophotometer.
- the measuring method of the present invention it is possible to measure zygomycetes contained in a sample with high sensitivity.
- the measurement method of the present invention for example, it can be determined whether or not the sample contains a zygomycete, the fungus contained in the sample can be identified as a zygomycete, and further, The number can be quantified and the degree of progression of zygomycosis can be quantified. That is, in one aspect, the measurement method of the present invention is a method for determining whether a sample contains a zygomycete, a method for determining whether a fungus is a zygomycete, or a causative bacterium for mycosis.
- a method for determining whether the mycosis is zygomycosis a method for determining whether the mycosis is mucolosis, whether the mycosis is entomoftorosis It may be a method for determining whether or not, a method for quantifying the number of zygomycetes, or a method for quantifying the degree of progression of zygomycosis.
- the reagent kit of the present invention is a reagent kit for measuring conjugated bacteria, comprising at least one of a reagent containing an acidic component and a detection reagent for conjugated bacteria components as a component.
- the reagent kit of the present invention may be a zygomycosis diagnosis kit, a mucormycosis diagnosis kit, or an entomophthalosis diagnosis kit.
- the reagent kit of the present invention may further contain other components in addition to the above components.
- other components can be appropriately selected according to the measurement mode of the conjugated bacteria.
- reagents, buffers, neutralizing agents, standard substances for labeling or measuring the detection reagent of the conjugated bacteria component examples include microplates, antigens immobilized on plates, blocking agents, blood collection tubes, package inserts describing product information, and the like.
- Anti-chitosan antibody is exemplified as a detection reagent for zygote components, and chitosan is exemplified as an antigen or standard substance immobilized on a plate.
- each component may be included separately, or may be included in a coexisting state in any combination.
- an acidic component and a reagent containing the acidic component may be included in the reagent kit of the present invention in a form enclosed in a blood collection tube.
- the detection reagent for the conjugated bacterial component and the antigen to be immobilized on the plate may be included in the reagent kit of the present invention as a mixture with a buffer, and the solid phase immobilized on the microplate may be included. It may be included as a phased plate.
- the measurement of the zygomycete contained in the sample can be performed with high sensitivity and simplicity.
- measurement by the ELISA method was performed using a 96-well microplate (hereinafter simply referred to as “plate”).
- plate 96-well microplate
- F96 MaxiSorp Nunc-Immuno Plate manufactured by Nunk was used for the plate.
- CTS6-BSA chitosan hexamer
- SPDP solution N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP, Pierce) was dissolved in 5 mL of 0.1 M PBS (pH 6.0) so that BSA (made by Armor Pharmaceuticals) was 10 mg / mL.
- BSA-SH BSA having a thiol group
- BSA-SH total amount: 6 mL was obtained.
- CTSpAb Anti-chitosan polyclonal antibody
- Reference Example 2 Measurement of chitosan by ELISA using anti-chitosan polyclonal antibody (1) Measurement by binding method The binding specificity of CSpAb to CTS was confirmed by the binding method.
- Antigens immobilized on the plate include chitosan (CTS, manufactured by Seikagaku Corporation, degree of deacetylation: 80% or more), glycol chitosan (GCTS, manufactured by MP Biomedicals, degree of deacetylation: 76.2%) Alternatively, ethylene glycol chitin (EGCTN, manufactured by Seikagaku Corporation) was used.
- a solution prepared by adding 1% (v / v) acetic acid to each of the above antigens to give 2 ⁇ g / mL was added to the plate at 50 ⁇ L / well and allowed to stand at 4 ° C. overnight.
- the operation of adding 9.57 mM PBS (pH 7.5) at 300 ⁇ L / well and washing twice was repeated twice, and then 25% Block Ace (BA, manufactured by DS Pharma Biomedical) /9.57 mM PBS (pH 7.5). ) was added at 200 ⁇ L / well and allowed to stand for 1 hour to prepare solid-phase plates for the various antigens.
- PBS-T peroxidase-labeled anti-chicken IgY (IgG) (H + L) antibody (manufactured by KPL) diluted 5000 times with 10% BA / PBS-T.
- TMB reagent SureBlue Reserve TMB Microwell Peroxidase Substrate (hereinafter referred to as “TMB reagent”, manufactured by KPL) was added at 100 ⁇ L / well and allowed to stand for 30 minutes. And then 1N HCl was added at 100 ⁇ L / well. Absorbance was measured with Well Reader MP-96 (manufactured by Seikagaku Corporation), and the measurement wavelength was set to 450 nm and the control wavelength was set to 630 nm. The results are shown in Table 1.
- CTSpAb chicken serum immunized with chitosan showed the strongest response to CTS.
- CTSpAb showed a response to GCTS but no response to EGCTN. From the above, it was confirmed that the anti-chitosan polyclonal antibody (CTSpAb) did not react with chitin and was highly specific for chitosan.
- a solution prepared by adding 1% (v / v) acetic acid to CTS to 2 ⁇ g / mL was added to the plate at 50 ⁇ L / well and allowed to stand at 4 ° C. overnight. After repeating the operation of adding 9.57 mM PBS (pH 7.5) at 300 ⁇ L / well and washing twice, 25% BA / 9.57 mM PBS (pH 7.5) was added at 200 ⁇ L / well for 1 hour. The plate was allowed to stand to prepare a CTS solid-phase plate.
- Table 2 shows the results of percentage conversion of the rate of decrease in absorbance relative to the absorbance when no competing substance was added as an inhibition rate.
- the binding of CSpAb to CTS on the plate was inhibited by CTS or GCTS, but was not inhibited by EGCTN. From the above, it was confirmed by a competition method that the anti-chitosan polyclonal antibody (CTSpAb) does not react with chitin and has high specificity for chitosan.
- CTSpAb anti-chitosan polyclonal antibody
- a solution prepared by adding 1% (v / v) acetic acid to CTS to 2 ⁇ g / mL was added to the plate at 50 ⁇ L / well and allowed to stand at 4 ° C. overnight. After repeating the operation of adding 9.57 mM PBS (pH 7.5) at 300 ⁇ L / well and washing twice, 25% BA / 9.57 mM PBS (pH 7.5) was added at 200 ⁇ L / well for 1 hour. The plate was allowed to stand to prepare a CTS solid-phase plate.
- Table 3 shows the results of percentage conversion of the rate of decrease in absorbance relative to the absorbance when no CTS oligosaccharide was added as an inhibition rate.
- CSpAb was inhibited from binding to CTS immobilized on the plate by 3 to 6-mer CTS oligosaccharide. From the above, it was confirmed that the ELISA method using an anti-chitosan polyclonal antibody (CTSpAb) can measure chitosan having a molecular size of trimer or more.
- CTSpAb anti-chitosan polyclonal antibody
- CTS mAb Anti-chitosan monoclonal antibody
- RNA from lymphocytes isolated from the spleen of the chicken from which all blood was collected in ⁇ Reference Example 1> cDNA was synthesized, and then the gene sequence encoding the variable regions of the antibody heavy and light chains by PCR. Each amplification was performed.
- a single chain antibody single chain variable fragment, scFv expression vector
- the vector was packaged into phage particles. The phage particles were then infected with E. coli to amplify the phage. In this way, a phage antibody library was prepared.
- the phage antibody library obtained in (1) above was added to a plate on which CTS was immobilized. After washing the plate, the phage was recovered from E. coli bound to CTS. Thereafter, this phage was again infected with E. coli to amplify the phage.
- the thus obtained panned phage antibody library was subjected to a binding ELISA method using CTS as a solid phase sample, and a peroxidase-labeled anti-mouse IgG (H + L) antibody (manufactured by KPL) was used as a secondary antibody. The absorbance at the time was measured.
- Each of the gene sequences encoding the variable regions of the heavy chain and the light chain was amplified by PCR using the phage clone encoding the antibody that specifically binds to CTS obtained in (2) above as a template. These genes were then cloned into an IgY expression vector. Then, HEK293 cells transformed with this expression vector are cultured, and the culture solution is purified to obtain a monoclonal antibody (CTS mAb) that recognizes the same epitope as the polyclonal antibody (CTSpAb) obtained in ⁇ Reference Example 1>. It was.
- CTS mAb monoclonal antibody
- CTSpAb polyclonal antibody
- HRP-labeled anti-chitosan monoclonal antibody CTS mAb HRP-labeled CTS mAb labeled with horseradish peroxidase (HRP) was used as a detection antibody used in the ELISA method.
- HRP-labeled CTS mAb was prepared using Peroxidase Labeling Kit-NH 2 (manufactured by Dojindo). The preparation procedure of HRP-labeled CTS mAb is shown below.
- an HRP labeling solution (prepared by dissolving NH 2 -Reactive Peroxidase in 10 ⁇ L of Reaction Buffer) was added to the Filtration Tube and mixed, and then allowed to stand at 37 ° C. for 2 hours. Then, 100 ⁇ L of Washing Buffer was added to Filtration Tube and centrifuged at 8000 ⁇ g for 15 minutes. Subsequently, 200 ⁇ L of a stock solution (5% BA / 9.57 mM PBS (pH 7.5) /0.1% ProClin 950) was added to the Filtration Tube and mixed to recover the total amount. In this way, HRP-labeled CTS mAb was obtained.
- zygomycetes were measured using a sample containing cells prepared by culturing (hereinafter referred to as “cultured cells”).
- cultured cells a sample containing cells prepared by culturing
- the term “bacteria” is used as a general term for microorganisms including fungi and bacteria.
- Table 4 shows the cells used for the preparation of the cultured cells.
- Escherichia coli DH5 ⁇ is from Takara Bio Inc.
- other microbial cells are from American Cultured Cell Line Preservation Organization (American Type Culture Collection, ATCC), RIKEN Microbial Materials Development Office (Japan Collection , JCM), Teikyo University Institute of Medical Mycology, TIMM, or National Institute of Technology and Technology Evaluation (NITE) Biological Resource Center, NBRC.
- Various bacterial cells shown in Table 4 were inoculated into 10 mL of RPMI 1640 medium (Gibco) and cultured with shaking for 20 to 44 hours.
- the culture temperature was 37 ° C. for eubacteria and room temperature for fungi.
- the culture was terminated after confirming that the growth curve reached the stationary phase (stationary phase).
- the medium was removed from the culture solution thus obtained, and the cells were collected.
- the cells collected above were suspended in a washing solution (physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.)), the washing solution was removed, and the cells were collected again. The washing operation was repeated twice.
- a washing solution physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.)
- eubacteria Gram-negative and Gram-positive
- distilled water manufactured by Otsuka Pharmaceutical Co., Ltd.
- the bacterial cell suspension thus obtained was used as a “cultured bacterial cell suspension” in subsequent tests.
- fungi including zygomycetes
- inactivated cells cultured cells inactivated by high-pressure steam sterilization (hereinafter referred to as “inactivated cells”) for reasons of safety in work. )It was used.
- the procedure for preparing inactivated cells is shown below.
- eubacteria are subjected to cultured cell suspension, and fungi are subjected to high-pressure steam sterilization (121 ° C., 15 minutes), respectively. Activated.
- the agglomerates were precipitated by low-speed centrifugation, and then the cell suspension was recovered from the supernatant. Since the aggregate may physically inhibit the antigen-antibody reaction in ELISA, the solution from which the aggregate was removed (supernatant cell suspension) was used in subsequent tests.
- the inactivated cell suspension obtained in this way was used as a “inactivated cell suspension” in subsequent tests.
- CTS was measured by sandwich ELISA. The measurement procedure is shown below.
- a solution prepared by diluting CTS mAb with 9.57 mM PBS (pH 7.5) to 200 ng / mL was added to the plate at 100 ⁇ L / well and allowed to stand at 4 ° C. overnight. After repeating the operation of adding 9.57 mM PBS (pH 7.5) at 300 ⁇ L / well and washing twice, 25% BA / 9.57 mM PBS (pH 7.5) was added at 300 ⁇ L / well for 2 hours. The plate was allowed to stand to prepare a CTS mAb-immobilized plate.
- Table 5 shows the results of the above measurement.
- Example 1 Measurement of zygomycetes using human serum suffering from infection such as mycosis as a sample
- human serum suffering from infection such as mycosis is used as a sample.
- the fungus was measured.
- the sample was obtained from Teikyo University Institute of Medical Mycology (TIMM).
- the sera of humans suffering from infectious diseases such as mycosis were used as samples to measure zygomycetes.
- the measurement of zygomycetes was performed by competitive ELISA using CTSpAb, and the difference in the measurement results depending on the presence or absence of acid treatment was compared.
- Competitor substance (sample to be measured) was prepared as follows.
- Human serum (no acid treatment): Prepared by adding 0.225 mL of 10% BA / PBS-T to 0.025 mL of human serum.
- a solution prepared by adding 1% (v / v) acetic acid to CTS to 2 ⁇ g / mL was added to the plate at 50 ⁇ L / well and allowed to stand at 4 ° C. overnight. After repeating the operation of adding 9.57 mM PBS (pH 7.5) at 300 ⁇ L / well and washing twice, 25% BA / 9.57 mM PBS (pH 7.5) was added at 200 ⁇ L / well for 1 hour. The plate was allowed to stand to prepare a CTS solid-phase plate.
- Table 6 shows the results of percentage conversion of the rate of decrease in absorbance relative to absorbance when human serum without acid treatment (manufactured by Kojin Bio Inc.) was added as a competitor.
- Table 6 shows the results of percentage conversion of the rate of decrease in absorbance relative to absorbance when human serum without acid treatment (manufactured by Kojin Bio Inc.) was added as a competitor.
- Serum from humans with zygomycosis (Sample No .: specimen number: 5-9, including human serum from healthy individuals) was 10% or less. : 1 to 4, specimens were derived from different humans) showed an inhibition rate of 30% or more, and markedly inhibited the binding between CSpAb and CTS. From the above, it was confirmed in clinical specimens that by performing acid treatment on serum, it becomes possible to measure zygomycetes contained in the sample, and that only zygote can be specifically detected as bacterial cells.
- Example 2 Measurement of mating bacteria using human serum as a sample (1)
- a sample containing bacterial cells no acid treatment, with acid treatment
- a sample not containing bacterial cells blank sample
- a CTS standard solution used for creating a standard curve were prepared.
- Bacteria-containing sample (without acid treatment): A sample prepared by mixing 25 ⁇ L of the inactivated cell sample and 225 ⁇ L of human serum was mixed with 250 ⁇ L of 2M Tris-HCl (pH 8.0).
- (D) CTS standard solution The procedure of adding 1% (v / v) acetic acid to CTS and mixing an equal amount of 1% (v / v) acetic acid to a solution prepared to 200 ng / mL was repeated until 200 to 25 ng / mL. CTS dilutions were prepared. A standard sample containing 20 to 2.5 ng / mL of CTS was prepared by mixing 225 ⁇ L of rabbit serum with 25 ⁇ L of these CTS dilutions. This sample was mixed with 250 ⁇ L of 2M Tris-HCl (pH 8.0) and used for subsequent tests.
- Measurement of zygomycetes was performed by sandwich ELISA. The measurement procedure is shown below.
- a solution prepared by mixing 9.57 mM PBS (pH 7.5) with CTS mAb to a concentration of 0.2 ⁇ g / mL was added to the plate at 100 ⁇ L / well and allowed to stand at 4 ° C. overnight. After repeating the operation of adding 9.57 mM PBS (pH 7.5) at 300 ⁇ L / well and washing twice, 25% BA / 9.57 mM PBS (pH 7.5) was added at 300 ⁇ L / well for 2 hours. The plate was allowed to stand to prepare a CTS mAb-immobilized plate.
- the CTS concentration is a conversion value into a CTS concentration calculated from a measured value (average value) of absorbance using a standard curve.
- the measured value of the sample containing Escherichia coli which is an eubacteria (gram-negative bacterium) was below the blank value regardless of the presence or absence of acid treatment.
- the measured value of the sample containing the zygomycete was not more than the blank value when acid treatment was not performed, but showed a significant value above the blank value when acid treatment was performed.
- the presence of chitosan that is, the presence of zygomycetes was confirmed. Based on the above, it was confirmed in samples other than clinical specimens that the zygote contained in the sample can be measured by treating the serum with acid, and that only the zygote can be specifically detected as bacterial cells. did.
- Example 3 Measurement of mating bacteria using human serum as a sample (2) In accordance with the following procedure, a sample containing microbial cells, a sample not containing microbial cells, and a CTS standard solution used for preparing a standard curve were prepared.
- (C) CTS standard solution The procedure of adding 1% (v / v) acetic acid to CTS and mixing an equal amount of 1% (v / v) acetic acid to a solution prepared to 100 ng / mL was repeated until 25 to 1.56 ng. A / mL CTS dilution was prepared. A standard sample containing 2.5 to 0.156 ng / mL of CTS was prepared by mixing 12.5 ⁇ L of these CTS dilutions with 112.5 ⁇ L of rabbit serum. This sample was mixed with 250 ⁇ L of 0.8 M Tris-HCl (pH 8.0) and used for subsequent tests.
- the measured values of the samples containing Staphylococcus aureus which are eubacteria (gram-positive bacteria) or Candida albicans or Aspergillus fumigatus which are fungi other than the zygomycete were all below the blank value.
- the measured value of the sample containing the zygomycete showed a significant value equal to or greater than the blank value, and it was confirmed that chitosan was present in the sample, that is, the zygote was present.
- Example 2 and Example 3 it was found that only the zygote can be specifically detected from various cells by acid treatment.
- the measured values of the samples containing mucosal zygomycetes are all higher than the measured values of samples containing other zygomycetes (values of 1 ng / mL or more in terms of CTS concentration). showed that. Therefore, it was shown that the fungus belonging to the genus Mucor is particularly suitable as the zygote to be measured in the present invention.
- Example 2 and Example 3 when the sandwich ELISA method was used as a measurement method, the measured values of the samples containing cells other than the zygomycete were all blank (samples containing no cells). ) Showed no significant difference from the measured value, and it was found that the value was zero when converted to chitosan concentration. On the other hand, as shown in Example 1, when the competitive ELISA method was used as the measurement method, the measured value of the sample containing cells other than the zygomycete was also larger than the measured value of the blank (sample not containing cells).
- Example 4 Examination of the kind of acid According to the following procedures, the sample containing a microbial cell and the sample which does not contain a microbial cell were prepared.
- the acid treatment (v) was mixed with 25 ⁇ L of acid (citric acid) and left at room temperature for 1 minute for acid treatment. Thereafter, 200 ⁇ L of 0.4 M Tris-HCl (pH 8.0) was mixed and neutralized to finish the acid treatment.
- the acid concentration during acid treatment that is, the molar concentration of the acid when the acid was mixed with the sample (before neutralization treatment) Is 180 mM, 120 mM in hydrochloric acid, 260 mM in formic acid, 52 mM in citric acid, and 146 mM in phosphoric acid.
- Bacteria-containing sample (with acid treatment): 50 ⁇ L of 2% (v / v) acetic acid is mixed with a sample prepared by mixing 5 ⁇ L of inactivated cell sample and 45 ⁇ L of human serum, and left at room temperature for a predetermined time (10 minutes, 1 hour, or 2 hours). Then, acid treatment was performed. Thereafter, 200 ⁇ L of 0.4 M Tris-HCl (pH 8.0) was mixed and neutralized to finish the acid treatment.
- Tris-HCl pH 8.0
- the zygote contained in the sample could be measured without depending on the acid treatment time.
- Example 6 Examination of temperature for acid treatment According to the following procedure, a sample containing bacterial cells and a sample not containing bacterial cells were prepared.
- Bacteria-containing sample (with acid treatment): 50 ⁇ L of 2% (v / v) acetic acid is mixed with a sample prepared by mixing 5 ⁇ L of inactivated bacterial cell sample and 45 ⁇ L of human serum, and a predetermined temperature (room temperature (25 ° C.), 37 ° C., 50 ° C., or (70 ° C.) for 10 minutes, and the sample itself was subjected to acid treatment at each set temperature. Thereafter, 400 ⁇ L of 0.4 M Tris-HCl (pH 8.0) was mixed and neutralized to finish the acid treatment.
- a predetermined temperature room temperature (25 ° C.), 37 ° C., 50 ° C., or (70 ° C.
- the zygote contained in the sample could be measured without depending on the temperature during the acid treatment.
- Example 7 Examination of pH during acid treatment (1) According to the following procedures, a sample containing bacterial cells and a CTS standard solution used for preparing a standard curve were prepared.
- Bacteria-containing sample (with acid treatment): After adding 0.5 mL of human serum and 0.5 mL of distilled water to a 1 mm square cultured cell sample (Cunninghamella bertholletiae) and stirring for 30 seconds, 0.9 mL was removed from the supernatant after centrifugation. Next, after adding 0.45 mL of human serum and 0.45 mL of distilled water and stirring for 30 seconds, the operation of removing 0.9 mL from the supernatant after centrifugation was repeated twice.
- Bacteria-containing sample (without acid treatment) The same operation as the above (a) was performed using distilled water instead of acetic acid.
- (C) CTS standard solution The procedure of adding 1% (v / v) acetic acid to CTS and mixing an equal amount of 1% (v / v) acetic acid to a solution prepared to 1000 ng / mL was repeated until 1000 to 15.625 ng. A / mL CTS dilution was prepared. A standard sample containing 100 to 1.5625 ng / mL of CTS was prepared by mixing 0.9 mL of human serum with 0.1 mL of these CTS dilutions. This sample was mixed with 0.4 mL of 0.4 M Tris-HCl (pH 8.0) and used for subsequent tests.
- Measurement of zygomycetes was performed by sandwich ELISA. The measurement procedure is shown below.
- a solution prepared by mixing 9.57 mM PBS (pH 7.5) with CTS mAb to 1 ⁇ g / mL was added to the plate at 100 ⁇ L / well, and allowed to stand at 4 ° C. overnight. After repeating the operation of adding 9.57 mM PBS (pH 7.5) at 300 ⁇ L / well and washing twice, 25% BA / 9.57 mM PBS (pH 7.5) was added at 300 ⁇ L / well for 2 hours. The plate was allowed to stand to prepare a CTS mAb-immobilized plate.
- Example 8 Examination of pH during acid treatment (2) According to the following procedures, a sample containing bacterial cells and a CTS standard solution used for preparing a standard curve were prepared.
- Bacteria-containing sample (with acid treatment): After adding 0.5 mL of human serum and 0.5 mL of distilled water to a 1 mm square cultured cell sample (Rhizopus oryzae) and stirring for 30 seconds, 0.9 mL was removed from the supernatant after centrifugation. Next, after adding 0.45 mL of human serum and 0.45 mL of distilled water and stirring for 30 seconds, the operation of removing 0.9 mL from the supernatant after centrifugation was repeated twice.
- hydrochloric acid concentration indicates the final concentration of hydrochloric acid when the sample and hydrochloric acid are mixed (during acid treatment)
- pH during acid treatment indicates the pH of the sample solution during acid treatment
- the lower limit of the pH during acid treatment is not particularly limited, suggesting that it is possible to increase the sensitivity of measurement of zygomycetes by acid treatment at any pH of 0 or more. Specifically, it has been confirmed that it is possible to increase the sensitivity of measurement of zygomycetes by acid treatment at a pH of 0.7 or higher. In addition, it was confirmed that it is possible to increase the sensitivity of measurement of zygomycetes by acid treatment at a pH of 5.2 or lower.
- the pH range during acid treatment in which the measurement of zygomycetes is highly sensitive is 0 to 6, more preferably 0.5 to 6 or 0 to 5.5. More preferably, it was 0.5 to 5.5.
- Example 9 Measurement of mating bacteria using human serum as a sample (3) According to the following procedures, a sample containing bacterial cells and a CTS standard solution used for preparing a standard curve were prepared.
- Bacteria-containing sample (with acid treatment): Human serum (0.5 mL) and distilled water (0.5 mL) were added to 1 mm square cultured cell samples (Cunninghamella bertholletiae and Basidiobolus ranarum) and stirred for 30 seconds, and then 0.9 mL was removed from the supernatant after centrifugation. Next, after adding 0.45 mL of human serum and 0.45 mL of distilled water and stirring for 30 seconds, the operation of removing 0.9 mL from the supernatant after centrifugation was repeated twice.
- the acid treatment was effective not only for mucole zygomycetes (Cunninghamella bertholletiae), but also for entomophthala zygotes (Basidiobolus ranarum).
- Example 10 Measurement of mating bacteria using human serum as a sample (4) According to the following procedures, a sample containing bacterial cells and a CTS standard solution used for preparing a standard curve were prepared.
- Bacteria-containing sample (with acid treatment): After adding 0.5 mL of human serum and 0.5 mL of distilled water to a 1 mm square cultured cell sample (Cunninghamella bertholletiae) and stirring for 30 seconds, 0.9 mL was removed from the supernatant after centrifugation. Next, after adding 0.45 mL of human serum and 0.45 mL of distilled water and stirring for 30 seconds, the operation of removing 0.9 mL from the supernatant after centrifugation was repeated twice.
- “Acetic acid concentration” is the concentration of acetic acid added to the sample for acid treatment
- “Addition amount” is the amount of acetic acid added to the sample for acid treatment
- “Serum concentration” is during acid treatment.
- the concentration of human serum in, “pH during acid treatment” indicates the pH of the solution during acid treatment, respectively.
- Example 11 Measurement of zygomycetes using human serum as sample (5) According to the following procedure, a sample containing bacterial cells and a sample not containing bacterial cells were prepared.
- Bacteria-containing sample (with acid treatment): A sample prepared by mixing 8 ⁇ L of inactivated cell sample (Mucor racemosus) and 72 ⁇ L of human serum was mixed with 20 ⁇ L of 5% (v / v) acetic acid, and left at room temperature for 5 minutes for acid treatment. . Thereafter, 60 ⁇ L of 0.8 M Tris-HCl (pH 8.0) was mixed and neutralized to finish the acid treatment.
- Bacteria-containing sample (without acid treatment) The same operation as the above (a) was performed using distilled water instead of acetic acid.
- CTS mAb-sensitized latex solution A solution of latex particles bound with CTS mAb (CTS mAb-sensitized latex solution) was prepared using a commercially available kit (IMMUTEX P0307 (manufactured by JSR)) according to the method described in the package insert. The preparation procedure is shown below.
- 12.5 ⁇ L of 1% EDC ⁇ HCl (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) solution was mixed with this mixed solution and allowed to stand at room temperature for 1 hour. This mixture was centrifuged at 15,000 ⁇ g for 30 minutes, and the supernatant was removed to recover polymer particles. The particles were mixed with 0.5 mL of 25% Block Ace and then sonicated.
- the mixture was centrifuged at 15,000 ⁇ g for 30 minutes, the supernatant was removed, and the polymer particles were collected again.
- the particles were mixed with 0.5 mL of R2 Buffer and then sonicated.
- the mixture was allowed to stand at room temperature for 1 hour, and then subjected to ultrasonic treatment again to disperse the particles. Then, this liquid mixture was left still at 37 degreeC for 20 hours.
- the mixed solution thus obtained was used as a “CTS mAb-sensitized latex solution” in subsequent tests.
- Measurement of zygomycetes was performed by latex agglutination method. The measurement procedure is shown below.
- reaction plate Cho Direct “Eiken” Cryptocox, manufactured by Eiken Chemical Co., Ltd.
- 25 ⁇ L of a CTS mAb-sensitized latex solution was dropped.
- the reaction plate was shaken with a horizontal rotary shaker for 10 minutes to mix the sample and the latex liquid, and then visually checked for the presence of aggregates.
- the acid-treated sample produced an agglomerate, but the sample not subjected to acid treatment did not produce an agglomerate. That is, it was confirmed that the measurement of zygomycete is possible by the agglutination method. Therefore, it is easily understood by those skilled in the art that the zygote can be measured by other measurement methods (such as turbidimetric method and nephelometric method) using particles such as latex bound with an antibody. Furthermore, it is also easy for those skilled in the art to measure zygomycetes by using an antibody immunoassay method or an assay method other than an agglutination method (such as an immunochromatography method or an immunoblot method). Understood.
- Example 12 Measurement of zygomycetes using samples other than human serum According to the following procedure, a sample containing bacterial cells and a CTS standard solution used for preparing a standard curve were prepared.
- Bacteria-containing sample (with acid treatment): 1 mm square cultured cell sample (Rhizopus oryzae or Cunninghamella bertholletiae), plasma, etc. (human plasma (Cosmo Bio), horse serum (Cosmo Bio), bovine serum (Japan Biotest Laboratories), rabbit After adding 0.5 mL of serum (manufactured by Kojin Bio) or physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) and 0.5 mL of distilled water and stirring for 30 seconds, 0.9 mL was removed from the supernatant after centrifugation. .
- the sample for measuring zygote in the present invention may be bronchoalveolar lavage fluid (BALF). It was. Therefore, it was shown that the sample to be measured in the present invention is not particularly limited as long as it is a substance that can contain zygomycetes.
- BALF bronchoalveolar lavage fluid
- the present invention it is possible to measure zygomycetes contained in a sample with high sensitivity. Therefore, the present invention can be utilized as a means for realizing serological diagnosis useful for early diagnosis of zygomycosis such as mucormycosis and entomoftorosis.
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Abstract
Description
[1]
酸処理した試料を測定対象として接合菌を測定する、接合菌の測定方法。
[2]
酸処理が、酸性成分と試料を共存させる処理である、前記[1]に記載の接合菌の測定方法。
[3]
酸性成分が、オキソ酸、有機酸、無機酸からなる群より選択される1又は2以上の酸である、前記[2]に記載の接合菌の測定方法。
[4]
オキソ酸が、カルボン酸、スルホン酸、硫酸、リン酸、硝酸、ハロゲンオキソ酸からなる群より選択される1又は2以上の酸である、前記[3]に記載の接合菌の測定方法。
[5]
有機酸が、カルボン酸及び/又はスルホン酸である、前記[3]に記載の接合菌の測定方法。
[6]
カルボン酸が、酢酸、ギ酸、クエン酸、シュウ酸からなる群より選択される1又は2以上の酸である、前記[4]又は[5]に記載の接合菌の測定方法。
[7]
無機酸が、ハロゲン化水素、硫酸、リン酸、硝酸、ハロゲンオキソ酸からなる群より選択される1又は2以上の酸である、前記[3]に記載の接合菌の測定方法。
[8]
ハロゲン化水素が、塩酸である、前記[7]に記載の接合菌の測定方法。
[9]
ハロゲンオキソ酸が、次亜ハロゲン酸及び/又は過ハロゲン酸である、前記[4]又は[7]に記載の接合菌の測定方法。
[10]
次亜ハロゲン酸が、次亜塩素酸、次亜臭素酸、次亜ヨウ素酸からなる群より選択される1又は2以上の酸である、前記[9]に記載の接合菌の測定方法。
[11]
過ハロゲン酸が、過塩素酸、過臭素酸、過ヨウ素酸からなる群より選択される1又は2以上の酸である、前記[9]に記載の接合菌の測定方法。
[12]
接合菌の測定が、接合菌成分の検出試薬を用いた接合菌の測定である、前記[1]~[11]のいずれかに記載の接合菌の測定方法。
[13]
接合菌成分の検出試薬が、キトサンに結合する抗体である、前記[12]に記載の接合菌の測定方法。
[14]
接合菌の測定が、免疫測定法による接合菌の測定である、前記[1]~[13]のいずれかに記載の接合菌の測定方法。
[15]
免疫測定法が、キトサンに結合する抗体を用いた免疫測定法である、前記[14]に記載の接合菌の測定方法。
[16]
免疫測定法が、酵素免疫測定法、イムノクロマト法、イムノブロット法、凝集法、比濁法、又は比ろう法である、前記[14]又は[15]に記載の接合菌の測定方法。
[17]
凝集法が、ラテックス凝集法である、前記[16]に記載の接合菌の測定方法。
[18]
試料が、動物に由来する試料である、前記[1]~[17]のいずれかに記載の接合菌の測定方法。
[19]
動物に由来する試料が、血液由来試料、気管支肺胞洗浄液(BALF)、生検検体、喀痰、脳脊髄液、鼻腔拭い液、咽頭拭い液、鼻腔吸引液、又は腹水である、前記[18]に記載の接合菌の測定方法。
[20]
血液由来試料が、血清又は血漿である、前記[19]に記載の接合菌の測定方法。
[21]
接合菌の測定方法が、接合菌症の検知方法である、前記[1]~[20]のいずれかに記載の接合菌の測定方法。
[22]
接合菌症が、ムーコル症又はエントモフトラ症である、前記[21]に記載の接合菌の測定方法。
[23]
酸性成分を含有する、接合菌測定用試料の調製剤。
[24]
調製剤が、前記[1]~[22]のいずれかに記載の接合菌の測定方法を行う試料の酸処理に用いられる調製剤である、前記[23]に記載の調製剤。
[25]
接合菌の測定を行う試料の酸処理に使用するための酸性成分。
[26]
酸性成分が、前記[1]~[22]のいずれかに記載の接合菌の測定方法を行う試料の酸処理に用いられる酸性成分である、前記[25]に記載の酸性成分。
[27]
酸処理することにより、試料を接合菌測定用試料にする、接合菌測定用試料の調製方法。
[28]
酸処理が、試料を前記[1]~[22]のいずれかに記載の接合菌の測定方法を行うための試料とするための処理である、前記[27]に記載の調製方法。
[29]
酸処理が、酸性成分と試料を共存させる処理である、前記[27]又は[28]に記載の調製方法。
[30]
酸性成分と試料を共存させる処理が、前記[23]又は[24]に記載の調製剤と試料を共存させる処理である、前記[29]に記載の調製方法。
[31]
酸性成分を含有する試薬及び/又は接合菌成分の検出試薬を構成品として含む、接合菌の測定を行うための試薬キット。
[32]
酸性成分を含有する試薬及び/又は接合菌成分の検出試薬を構成品として含む、前記[1]~[22]のいずれかに記載の接合菌の測定方法を行うための試薬キット。
[33]
酸性成分を含有する試薬が、前記[23]又は[24]に記載の調製剤である、前記[31]又は[32]に記載の試薬キット。
[34]
接合菌成分の検出試薬が、キトサンに結合する抗体である、前記[31]~[33]のいずれかに記載の試薬キット。
[35]
試薬キットが、接合菌症の診断キットである、前記[31]~[34]のいずれかに記載の試薬キット。
[36]
試薬キットが、ムーコル症又はエントモフトラ症の診断キットである、前記[31]~[34]のいずれかに記載の試薬キット。
本発明により、酸処理した試料を測定対象として接合菌を測定することを特徴とする、接合菌の測定方法(以下「本発明の測定方法」という。)が提供される。本発明の測定方法は、例えば、接合菌の検知方法、接合菌の検出方法、接合菌の定量方法、接合菌症の検知方法、接合菌症の検出方法、又は接合菌症の定量化方法として用いることができる。また、本発明の測定方法は、例えば、酸処理した試料(検体)を測定対象として接合菌の測定(検体検査)を行い、動物が接合菌症に罹患しているか否かを診断するためのデータを取得することを特徴とする、接合菌症を診断するためのデータ取得方法として用いることもできる。
上記の通り、本発明において接合菌の測定が行われる試料は酸処理が施されている。当該酸処理は、試料に酸性成分を共存させて行うことが実用上簡便である。ここに酸性成分を含有することを特徴とする、接合菌測定用試料の調製剤(以下「本発明の試料調製剤」という。)が提供される。当該酸性成分を含む剤は、接合菌の測定を行う試料の酸処理剤として使用することができる。本発明の試料調製剤と当該酸処理剤は、どちらも酸性成分を剤に含有させる工程を含む任意の方法により製造することができる。また、本発明の試料調製剤と当該酸性成分は、どちらも本発明の測定方法を行う試料の酸処理に好適に用いることができる。
上記の通り、酸処理を行うことにより試料を接合菌測定用試料とすることができる。ここに酸処理することにより試料を接合菌測定用試料にすることを特徴とする、接合菌測定用試料の調製方法(以下「本発明の試料調製方法」という。)が提供される。本発明の試料調製方法は、本発明の測定方法を行う試料の酸処理に好適に用いることができる。本発明の試料調製方法において、酸処理は酸性成分と試料を共存させる処理であることが好適である。酸性成分と試料を共存させる処理は、本発明の試料調製剤と試料を共存させる処理であることが好適である。
本発明において接合菌の測定は、酸性成分を含有する試薬と接合菌成分の検出試薬のうち少なくとも一方を構成品として含む試薬キット(以下「本発明の試薬キット」という。)を用いて行うことが実用上簡便である。本発明の試薬キットは、本発明の測定方法を行うために好適に用いることができる。酸性成分を含有する試薬は、本発明の試料調製剤であることが好適である。接合菌成分の検出試薬は、キトサンに結合する抗体であることが好適である。本発明の試薬キットは、前記構成品以外に、さらに他の構成品を含んでいてもよい。当該他の構成品は、接合菌の測定の態様に応じて適宜選択できる。本発明の試薬キットは、例えば、ムーコル症やエントモフトラ症等の接合菌症の診断キットとしても用いることができる。
本発明において酸処理は、接合菌の測定を行う試料の前処理として行われる。当該酸処理は、試料を酸性にする処理であり、試料を酸性下に供することで行うことができる。当該酸処理は、試料に酸性成分を共存させて行われることが実用上簡便である。酸処理した試料は、酸性成分と共存させる処理が行われた試料であってよい。
本発明において酸性成分は、有機酸であってもよく、無機酸であってもよい。有機酸はカルボン酸であることが好適である。カルボン酸は酢酸、ギ酸、又はクエン酸であることが好適である。また、無機酸はハロゲン化水素、硫酸、又はリン酸であることが好適である。ハロゲン化水素は塩化水素(塩酸)であることが好適である。このような酸性成分は、1種のみを用いてもよく、2種又はそれ以上を組み合わせて用いてもよい。
本発明において接合菌の測定は、例えば、接合菌成分の検出試薬を用いて行うことができる。接合菌成分の検出試薬は、キトサンに結合する抗体であることが好適である。また、本発明において接合菌の測定は、例えば、免疫測定法により行うことができる。免疫測定法は、キトサンに結合する抗体を用いた免疫測定法であることが好適である。免疫測定法としては、酵素免疫測定(Enzyme ImmunoAssay、EIA)法、イムノクロマト法、イムノブロット法、凝集法、比濁法、又は比ろう法が例示される。凝集法としては、ラテックス凝集法が例示される。酵素免疫測定法は、ELISA(Enzyme-Linked ImmunoSorbent Assay)法であってよい。
本発明において接合菌の測定を行う試料は、接合菌を含有し得る物質である限り特に制限されないが、ヒトを含む動物に由来する試料(検体)であることが好適であり、本発明は検体検査において好適に用いられる。動物に由来する試料としては、動物の体から得られる試料や動物の体と接触させた液体を回収して得られる試料が例示される。動物の体と接触させる液体は、生体に適合可能な液体である限り特に制限されない。そのような液体としては、生理食塩水が挙げられる。動物の体と接触し、回収された液体は、そのまま接合菌の測定を行う試料として酸処理に供することができる。動物の体から得られる試料は、血液由来試料であることが好適である。血液由来試料は、血清又は血漿であることが特に好適である。動物の体と接触させた液体を回収して得られる試料は、気管支肺胞洗浄液(BALF)であることが好適である。また、ここにいう動物は、接合菌症に罹患した動物又は接合菌症に罹患した可能性のある動物であることが好適である。接合菌症としては、ムーコル症やエントモフトラ症が例示される。
GlcN:グルコサミン
GlcNAc:N-アセチル-D-グルコサミン
CTS:キトサン
CTS6:キトサンヘキサマー
GCTS:グリコールキトサン
CTN:キチン
EGCTN:エチレングリコールキチン
CTSpAb:抗キトサンポリクローナル抗体
CTSmAb:抗キトサンモノクローナル抗体
PBS:リン酸緩衝生理食塩水
本発明において接合菌の測定が行われる試料は、上述した通りに酸処理が施されて調製される。本発明において酸処理は、試料を酸性下に供する処理(酸性の状態にする処理)である限り、その手段は特に制限されない。そのような手段としては、陽イオン交換体、活性炭(酸性炭)、又は酸性成分と試料を共存させる方法が例示される。酸処理は、酸性成分と試料を共存させる処理であることが簡便かつ安価であり好適である。共存とは、対象物同士が接触し得る状態にあることを意味する。例えば、酸性成分と試料の共存は、試料に対して酸性成分を添加することにより行うことも可能であり、酸性成分に対して試料を添加することにより行うことも可能である。酸処理は液体中において行われることが好適である。また、酸処理が行われる液体は、水又は水溶液であることが好適である。
酸性成分は、水と共存した時に酸性を示す物質である限り特に制限されない。酸性成分は、水と共存した時に水素イオンを遊離する化合物であってよく、イオン化した時に水素イオンを遊離する化合物であってよい。すなわち、酸性成分はアレニウス酸であってよい。酸性成分としては、オキソ酸、有機酸、無機酸等が例示される。オキソ酸としては、カルボン酸、スルホン酸、硫酸、リン酸、硝酸、ハロゲンオキソ酸等が例示される。有機酸としては、カルボン酸、スルホン酸等が例示される。カルボン酸としては、酢酸、ギ酸、クエン酸、シュウ酸等が例示される。無機酸としては、ハロゲン化水素、硫酸、リン酸、硝酸、ハロゲンオキソ酸等が例示される。ハロゲン化水素としては、塩化水素(塩酸)等が例示される。ハロゲンオキソ酸としては、次亜ハロゲン酸、過ハロゲン酸等が例示される。次亜ハロゲン酸としては、次亜塩素酸、次亜臭素酸、次亜ヨウ素酸等が例示される。過ハロゲン酸としては、過塩素酸、過臭素酸、過ヨウ素酸等が例示される。酸性成分が有機酸である場合は、酸性成分はカルボン酸であることが好ましく、酢酸、ギ酸、又はクエン酸であることが特に好ましい。また、酸性成分が無機酸である場合は、酸性成分はハロゲン化水素、硫酸、又はリン酸であることが好ましく、ハロゲン化水素であることがより好ましい。ハロゲン化水素は塩化水素(塩酸)であることが特に好ましい。
試料は、接合菌を含有し得る物質である限り特に制限されない。試料としては、動物に由来する試料が挙げられる。動物としては、具体的には、ヒトをはじめ、ムーコル症やエントモフトラ症等の接合菌症の罹患の対象となり得るその他の動物、例えば、サル、イヌ、ネコ、ウサギ、ウマ、ウシ等の哺乳動物、トリ等が挙げられる。接合菌症は、ペット由来の感染や、ペット自体の接合菌症の罹患も問題になっており、ヒト以外の動物も試料の由来として重要である。動物に由来する試料は、動物の体から得られる試料であってもよいし、動物の体と接触させた液体を回収して得られる試料であってもよい。
本発明において酸処理は、試料を酸性にする処理であり、試料を酸性下に供することで行うことができる。試料の酸処理が行われる時(以下「酸処理時」という。)のpHは6以下の酸性である限り特に制限されず、任意のpHを選択することができる。酸処理時のpHは、例えば、試料の種類や接合菌の測定の態様に応じて適宜設定することができる。下限を含んだ当該pHは、例えば、pH0以上、pH0.5以上、pH1以上、pH2以上、pH3以上、pH3.5以上等であってよい。また上限を含んだ当該pHは、例えば、pH6以下、pH5.5以下、pH5以下、pH4.5以下等であってよい。さらに具体的には、pH0~6、pH0.5~5.5、pH0~5、pH0.5~5、pH1~5、pH2~5、pH3~5、pH3.5~4.5等が例示される。
本発明の試料調製剤は、上記に例示される酸性成分を含有する剤であり、試料を酸処理するために用いられる剤である。本発明の試料調製剤は、液剤であってもよいし、粉末剤、顆粒剤、錠剤等の用時溶解用の固形剤であってもよい。本発明の試料調製剤は、酸性成分そのものであってもよく、酸性成分を水等の溶媒に希釈又は溶解したものであってもよい。また、本発明の試料調製剤は、製剤上の理由等により、酸性成分や溶媒以外の他の成分を含有するものであってもよい。当該他の成分は、試薬・診断薬学的に許容される成分である限り特に制限されず、例えば、アルカリ金属塩、アルカリ土類金属塩、各種添加剤等が例示される。各種添加剤としては、安定化剤、保存剤、防腐剤、乳化剤、界面活性剤、湿潤剤、懸濁化剤、分散剤、浸透圧調整剤、等張化剤、緩衝剤、pH調整剤、抗酸化剤、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤等が例示される。
本発明の試料調製方法は、酸処理することにより試料(接合菌の測定を行う試料)を接合菌測定用試料(接合菌の測定が行われる試料)にするための方法である。本発明の試料調製方法は、酸処理する工程以外に、当該工程の前後、又は、同時において、さらに他の工程を含んでいてもよい。本発明の試料調製方法は、例えば、酸処理を終了する工程、固形状の試料を溶解する工程、液状の試料を希釈又は濃縮する工程等を含んでいてもよい。また、本発明の試料調製方法は、例えば、調製された試料が接合菌の測定に適した温度となるように、当該試料を加温又は冷却する工程を含んでいてもよい。さらに、本発明の試料調製方法は、例えば、調製された試料が接合菌の測定に適したpHとなるように、当該試料のpHを調整する工程を含んでいてもよい。
2-1: 測定手法
本発明の測定方法は、上述の通りに接合菌を測定する方法である。本発明において測定の対象となる接合菌は、具体的には、酸処理された接合菌であり、一態様においては、接合菌に由来する成分である。本発明において測定の対象となる接合菌は、菌体そのものであってもよく、断片化された菌体であってもよく、菌体の一部又は一成分であってもよい。また、本発明において測定の対象となる接合菌は、生菌体であってもよく、不活化された菌体等の死菌体であってもよい。接合菌の測定は、例えば、接合菌成分の検出試薬(接合菌が有する成分を検出するための試薬)を用いて行うことができる。
本発明において用いられる抗体は、抗体を作製するために用いられる公知の方法により作製することができ、その方法は特に制限されない。当該抗体を作製する方法としては、動物を抗原で免疫する方法が典型例として挙げられる。抗原は、接合菌そのものであってもよく、断片化された接合菌であってもよく、接合菌の一成分であってもよく、免疫原性を高めるためにこれらを他の物質と融合させた複合体であってもよい。当該複合体の形成に用いられる他の物質としては、タンパク質が挙げられ、具体的には、ウシ血清アルブミン(BSA)が挙げられる。また、抗原は、抗原性補強剤(アジュバント)と共存させた状態で動物の免疫に用いられてもよい。
本発明において測定の対象となり得る接合菌の種類は特に限定されない。接合菌は、接合菌門(Zygomycota)に属する真菌として定義される。当該真菌としては、接合菌綱(Zygomycetes)、トリコミケス綱(Trichomycetes)に属する真菌が挙げられる。接合菌綱に属する真菌としては、ディマルガリス目(Dimargaritales)、アツギケカビ目(Endogonales)、ハエカビ目(Entomophthorales)、グロムス目(Glomales)、キックセラ目(Kickxellales)、ケカビ目(Mucorales)、トリモチカビ目(Zoopagales)に属する真菌が挙げられる。トリコミケス綱に属する真菌としては、アメビディウム目(Amoebidiales)、アセラリア目(Asellariales)、エクリナ目(Eccrinales)、ハルペラ目(Harpellales)に属する真菌が挙げられる。
本発明の測定方法は、上述した試料の酸処理をする工程や接合菌成分の検出試薬と接合菌を接触させて接合菌を検知する工程(以下「接合菌の検知工程」という。)以外に、必要に応じて他の工程を含んでいてもよい。当該他の工程としては、酸処理を終了させるために酸処理をした試料を中性又は塩基性にする工程(中和工程)が挙げられる。酸処理を終了させる工程は、例えば、接合菌成分の検出試薬が酸性条件下での使用に適さない試薬である場合に好適に行われる。例えば、接合菌成分の検出試薬が抗体等のタンパク質である場合は、酸性条件下での使用がタンパク質の変性や接合菌成分との結合阻害等を引き起こす可能性があるため、接合菌の検知工程の前に中和工程が好適に行われてよい。
本発明の試薬キットは、酸性成分を含有する試薬と接合菌成分の検出試薬のうち少なくとも一方を構成品として含む、接合菌測定用の試薬キットである。本発明の試薬キットは、一態様として、接合菌症の診断キット、ムーコル症の診断キット、又はエントモフトラ症の診断キットであってもよい。
(1)免疫抗原の調製(キトサン-ウシ血清アルブミン複合体の調製)
動物を免疫する抗原には、キトサンヘキサマー(CTS6)にウシ血清アルブミン(BSA)を共有結合した複合体(以下「CTS6-BSA」という。)を用いた。CTS6-BSAの調製手順を以下に示す。
抗キトサンポリクローナル抗体(CTSpAb)は、CTS6-BSAを用いてニワトリを免疫して調製した。CTSpAbの調製手順を以下に示す。
(1)結合法による測定
結合法により、CTSpAbのCTSに対する結合特異性を確認した。プレートに固相化する抗原には、キトサン(CTS、生化学工業社製、脱アセチル化度:80%以上)、グリコールキトサン(GCTS、MP Biomedicals社製、脱アセチル化度:76.2%)、又はエチレングリコールキチン(EGCTN、生化学工業社製)を用いた。
競合法により、CTSpAbのCTSに対する結合特異性を確認した。競合物質としてはCTS、GCTS、又はEGCTNを用いた。
3~6量体のCTSオリゴ糖(生化学工業社製)を競合物質とした競合ELISA法により、CTSpAbによる測定が可能なCTSの分子サイズを確認した。
抗キトサンモノクローナル抗体(CTSmAb)の作製は、文献(WO2011/049082)に記載された方法を参照して行った。CTSmAbの調製手順を以下に示す。
ファージ抗体ライブラリーの作製は、文献(nakamura et al., J Vet Med Sci. 2004 Jul; 66(7): 807-14.)に記載された方法を参照して行った。
CTSに結合するファージ抗体のスクリーニングは、CTSを固相化したプレートを用いたパニングにより行った。
抗キトサンモノクローナルIgY抗体の調製は、文献(Shimamoto et al., Biologicals. 2005 Sep; 33(3): 169-74.)に記載された手法を参照して行った。
ELISA法に用いる検出抗体には西洋わさびペルオキシダーゼ(Horseradish Peroxidase、HRP)で標識したCTSmAb(HRP標識CTSmAb)を用いた。HRP標識CTSmAbはPeroxidase Labeling Kit-NH2(同仁化学社製)を用いて調製した。HRP標識CTSmAbの調製手順を以下に示す。
本願実施例の一部においては、培養により調製した菌体(以下「培養菌体」という。)を含有する試料を測定対象として接合菌の測定を行った。本願の実施例において、「菌体」の語は、真菌(Fungi)と真正細菌(Bacteria)を含む微生物の総称として使用される。培養菌体の調製に使用した菌体を表4に示す。
本願の実施例の一部においては、作業上の安全確保の理由により、高圧蒸気滅菌により不活化した培養菌体(以下「不活化菌体」という。)を使用した。不活化菌体の調製手順を以下に示す。
以下の手順に従い、CTS含有ヒト血清、及びCTS含有ウサギ血清を調製した。
本願実施例の一部においては、真菌症等の感染症に罹患したヒトの血清を試料として接合菌の測定を行った。当該試料は帝京大学医真菌研究センター(Teikyo University Institute of Medical Mycology, TIMM)から入手した。
ヒト血清0.025mLに10%BA/PBS-Tを0.225mL添加して調製した。
ヒト血清0.022mLに終濃度で1%(175mM)となるように2%(v/v)酢酸を0.022mL添加して30秒間撹拌した後、10%BA/PBS-Tを0.176mL添加して調製した。この時、ヒト血清に酢酸を添加した時のpHは4.1であり、10%BA/PBS-Tを添加した後のpHは4.9であった。
以下の手順に従い、菌体を含有する試料(酸処理なし、酸処理あり)、菌体を含有しない試料(ブランク試料)、及び標準曲線の作成に用いるCTS標準液を調製した。
不活化菌体試料25μLとヒト血清225μLを混合して調製した試料に対して2M Tris-HCl(pH8.0)を250μL混合した。
不活化菌体試料25μLとヒト血清225μLを混合して調製した試料に対して5%(v/v)酢酸を62.5μL混合し、室温に1分間静置して酸処理を行った。その後、2M Tris-HCl(pH8.0)を187.5μL混合して中和し、酸処理を終了した。
不活化菌体試料に代えて蒸留水を用い、上記(b)と同じ操作を行った。
CTSに1%(v/v)酢酸を添加して200ng/mLとなるように調製した溶液に対して等量の1%(v/v)酢酸を混合する操作を繰り返し、200~25ng/mLのCTS希釈液を調製した。これらのCTS希釈液25μLにウサギ血清225μLを混合して20~2.5ng/mLのCTSを含有する標準試料を調製した。この試料を2M Tris-HCl(pH8.0)250μLと混合し、以後の試験に用いた。
以下の手順に従い、菌体を含有する試料、菌体を含有しない試料、及び標準曲線の作成に用いるCTS標準液を調製した。
不活化菌体試料12.5μLとヒト血清112.5μLを混合して調製した試料に対して5%(v/v)酢酸を31.3μL混合し、室温に1分間静置して酸処理を行った。その後、0.8M Tris-HCl(pH8.0)を93.7μL混合して中和し、酸処理を終了した。
不活化菌体試料に代えて蒸留水を用い、上記(a)と同じ操作を行った。
CTSに1%(v/v)酢酸を添加して100ng/mLとなるように調製した溶液に対して等量の1%(v/v)酢酸を混合する操作を繰り返し、25~1.56ng/mLのCTS希釈液を調製した。これらのCTS希釈液12.5μLにウサギ血清112.5μLを混合して2.5~0.156ng/mLのCTSを含有する標準試料を調製した。この試料を0.8M Tris-HCl(pH8.0)250μLと混合し、以後の試験に用いた。
以下の手順に従い、菌体を含有する試料、及び菌体を含有しない試料を調製した。
不活化菌体試料2.5μLとヒト血清22.5μLを混合して調製した試料に対して2%(v/v)の酸(硫酸、塩酸、ギ酸、又はリン酸)又は2%(w/v)の酸(クエン酸)を25μL混合し、室温に1分間静置して酸処理を行った。その後、0.4M Tris-HCl(pH8.0)を200μL混合して中和し、酸処理を終了した。
不活化菌体試料に代えて蒸留水を用い、上記(a)と同じ操作を行った。
以下の手順に従い、菌体を含有する試料、及び菌体を含有しない試料を調製した。
不活化菌体試料5μLとヒト血清45μLを混合して調製した試料に対して2%(v/v)酢酸を50μL混合し、室温に所定時間(10分間、1時間、又は2時間)静置して酸処理を行った。その後、0.4M Tris-HCl(pH8.0)を200μL混合して中和し、酸処理を終了した。
不活化菌体試料に代えて蒸留水を用い、上記(a)と同じ操作を行った。
実施例3に記載した手順に従い調製した。
以下の手順に従い、菌体を含有する試料、及び菌体を含有しない試料を調製した。
不活化菌体試料5μLとヒト血清45μLを混合して調製した試料に対して2%(v/v)酢酸を50μL混合し、所定の温度(室温(25℃)、37℃、50℃、又は70℃)に10分間静置して、試料自体を各設定温度にして酸処理を行った。その後、0.4M Tris-HCl(pH8.0)を400μL混合して中和し、酸処理を終了した。
不活化菌体試料に代えて蒸留水を用い、上記(a)と同じ操作を行った。
以下の手順に従い、菌体を含有する試料、及び標準曲線の作成に用いるCTS標準液を調製した。
1mm四方の培養菌体試料(Cunninghamella bertholletiae)にヒト血清0.5mLと蒸留水0.5mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去した。次に、ヒト血清0.45mLと蒸留水0.45mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去する操作を2回繰り返した。続いて、ヒト血清0.45mLと酢酸(2%、1%、0.4%、又は0.1%)0.45mLを添加して30秒間撹拌した後、室温に5分間静置して酸処理を行った。その後、遠心後の上清0.1mLに0.4M Tris-HCl(pH8.0)を0.4mL混合して中和し、酸処理を終了した。
酢酸に代えて蒸留水を用い、上記(a)と同じ操作を行った。
CTSに1%(v/v)酢酸を添加して1000ng/mLとなるように調製した溶液に対して等量の1%(v/v)酢酸を混合する操作を繰り返し、1000~15.625ng/mLのCTS希釈液を調製した。これらのCTS希釈液0.1mLにヒト血清0.9mLを混合して100~1.5625ng/mLのCTSを含有する標準試料を調製した。この試料を0.4M Tris-HCl(pH8.0)0.4mLと混合し、以後の試験に用いた。
以下の手順に従い、菌体を含有する試料、及び標準曲線の作成に用いるCTS標準液を調製した。
1mm四方の培養菌体試料(Rhizopus oryzae)にヒト血清0.5mLと蒸留水0.5mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去した。次に、ヒト血清0.45mLと蒸留水0.45mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去する操作を2回繰り返した。続いて、ヒト血清0.45mLと塩酸(0.5N、0.1N、又は0.05N)0.45mLを添加して30秒間撹拌した後、室温に5分間静置して酸処理を行った。その後、遠心後の上清0.1mLに0.4M Tris-HCl(pH8.0)を0.4mL混合して中和し、酸処理を終了した。
塩酸に代えて蒸留水を用い、上記(a)と同じ操作を行った。
実施例7に記載した手順に従い調製した。
以下の手順に従い、菌体を含有する試料、及び標準曲線の作成に用いるCTS標準液を調製した。
1mm四方の培養菌体試料(Cunninghamella bertholletiae、及びBasidiobolus ranarum)にヒト血清0.5mLと蒸留水0.5mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去した。次に、ヒト血清0.45mLと蒸留水0.45mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去する操作を2回繰り返した。続いて、ヒト血清0.45mLと2%(v/v)酢酸0.45mLを添加して30秒間撹拌した後、室温に5分間静置して酸処理を行った。その後、遠心後の上清0.1mLに0.4M Tris-HCl(pH8.0)を0.4mL混合して中和し、酸処理を終了した。
実施例7に記載した手順に従い調製した。
以下の手順に従い、菌体を含有する試料、及び標準曲線の作成に用いるCTS標準液を調製した。
1mm四方の培養菌体試料(Cunninghamella bertholletiae)にヒト血清0.5mLと蒸留水0.5mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去した。次に、ヒト血清0.45mLと蒸留水0.45mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去する操作を2回繰り返した。続いて、ヒト血清0.45mLと酢酸(100%(v/v)酢酸0.01mL、25%(v/v)酢酸0.04mL、10%(v/v)酢酸0.1mL、又は2%(v/v)酢酸0.45mL)を添加して30秒間撹拌した後、室温に5分間静置して酸処理を行った。全量1mLとなるように蒸留水を混合した後、遠心後の上清0.1mLに0.4M Tris-HCl(pH8.0)を0.4mL混合して中和し、酸処理を終了した。
実施例7に記載した手順に従い調製した。
以下の手順に従い、菌体を含有する試料、及び菌体を含有しない試料を調製した。
不活化菌体試料(Mucor racemosus)8μLとヒト血清72μLを混合して調製した試料に対して5%(v/v)酢酸を20μL混合し、室温に5分間静置して酸処理を行った。その後、0.8M Tris-HCl(pH8.0)を60μL混合して中和し、酸処理を終了した。
酢酸に代えて蒸留水を用い、上記(a)と同じ操作を行った。
以下の手順に従い、菌体を含有する試料、及び標準曲線の作成に用いるCTS標準液を調製した。
1mm四方の培養菌体試料(Rhizopus oryzae、又はCunninghamella bertholletiae)に血漿等(ヒト血漿(コスモバイオ社製)、ウマ血清(コスモバイオ社製)、ウシ血清(日本バイオテスト研究所社製)、ウサギ血清(コージンバイオ社製)、又は生理食塩水(大塚製薬社製))0.5mLと蒸留水0.5mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去した。次に、血漿等0.45mLと蒸留水0.45mLを添加して30秒間撹拌した後、遠心後の上清から0.9mLを除去する操作を2回繰り返した。続いて、血漿等0.45mLと2%(v/v)酢酸0.45mLを添加して30秒間撹拌した後、室温に5分間静置して酸処理を行った。その後、遠心後の上清0.1mLに0.4M Tris-HCl(pH8.0)を0.4mL混合して中和し、酸処理を終了した。
実施例7に記載した手順に従い調製した。
Claims (15)
- 酸処理した試料を測定対象として接合菌を測定する、接合菌の測定方法。
- 酸処理が、酸性成分と試料を共存させる処理である、請求項1に記載の接合菌の測定方法。
- 接合菌の測定が、接合菌成分の検出試薬を用いた接合菌の測定である、請求項1又は2に記載の接合菌の測定方法。
- 接合菌の測定が、免疫測定法による接合菌の測定である、請求項1~3のいずれか1項に記載の接合菌の測定方法。
- 免疫測定法が、キトサンに結合する抗体を用いた免疫測定法である、請求項4に記載の接合菌の測定方法。
- 試料が、動物に由来する試料である、請求項1~5のいずれか1項に記載の接合菌の測定方法。
- 動物に由来する試料が、血液由来試料又は気管支肺胞洗浄液(BALF)である、請求項6に記載の接合菌の測定方法。
- 血液由来試料が、血清又は血漿である、請求項7に記載の接合菌の測定方法。
- 接合菌の測定方法が、接合菌症の検知方法である、請求項1~8のいずれか1項に記載の接合菌の測定方法。
- 接合菌症が、ムーコル症又はエントモフトラ症である、請求項9に記載の接合菌の測定方法。
- 酸性成分を含有する、接合菌測定用試料の調製剤。
- 酸処理することにより、試料を接合菌測定用試料にする、接合菌測定用試料の調製方法。
- 酸性成分を含有する試薬及び/又は接合菌成分の検出試薬を構成品として含む、接合菌の測定を行うための試薬キット。
- 試薬キットが、接合菌症の診断キットである、請求項13に記載の試薬キット。
- 試薬キットが、ムーコル症又はエントモフトラ症の診断キットである、請求項13に記載の試薬キット。
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