Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • 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
• • 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
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

• the present invention relates to a dry analytical element for bile acid analysis using 3 ⁇ -hydroxysteroid dehydrogenase, thionicotinamide coenzyme, and reduced nicotinamide coenzyme, and a method for measuring bile acids.
• Bile acids are synthesized in the liver by the oxidation of cholesterol.
• the synthesized bile acids are conjugated with taurine, glycine, sulfuric acid, and glucuronic acid to form various structures.
• the bile acids synthesized in this way are stored and concentrated in the gallbladder. Bile acids act as surfactants, and therefore play a role in promoting fat absorption, killing bacteria, and regulating cholesterol metabolism.
• bile acids are generally measured in body fluids, such as blood (whole blood, plasma, serum), cerebrospinal fluid, lymph, saliva, and urine.
• body fluids such as blood (whole blood, plasma, serum), cerebrospinal fluid, lymph, saliva, and urine.
• measuring the bile acid concentration in the blood is useful for liver function testing.
• Patent Document 1 discloses a method in which 3 ⁇ -hydroxysteroid dehydrogenase (hereinafter also referred to as "3 ⁇ -HSD”) is reacted with reduced nicotinamide coenzymes (hereinafter referred to as "NADHs”) and thionicotinamide coenzyme (thio-NAD) (or thio-NADP) as coenzymes, and the amount of thio-NADHs produced is measured.
• Patent Document 2 describes a dry chemistry reagent for sulfate-conjugated bile acids.
• Patent Document 2 is limited to "sulfate-conjugated bile acids," which are synthesized in the liver and excreted mainly in urine. Measurement of sulfate-conjugated bile acids is useful for testing for diseases such as liver cirrhosis, but it has been pointed out that the proportion of sulfate-conjugated bile acids present in the blood is only a few percent of the total bile acids in the blood, and testing for bile acids other than sulfate-conjugated bile acids is important for liver function and intestinal absorption tests for acute hepatitis and chronic liver disease.
• Patent Document 2 is promising for the qualitative measurement of "sulfate-conjugated bile acids," it has poor linearity (linearity) of the detected signal concentration relative to the amount of "sulfate-conjugated bile acids,” making it difficult to use it as a quantitative reagent.
• the problem to be solved by the present invention is to provide a dry analytical element for analyzing bile acids that is simple and inexpensive and can be used as a quantitative reagent for the amount of bile acids, not limited to sulfate conjugated types, and a method for measuring bile acids using the dry analytical element for analyzing bile acids.
• a dry chemistry reagent can be provided that does not require water supply and drainage equipment and can be used as a quantitative reagent for the amount of bile acids by using a dry analytical element for bile acid analysis in which at least one water-soluble polymer layer and at least one spreading layer are provided in this order on a support, and at least one of the water-soluble polymer layer and spreading layer contains 3 ⁇ -hydroxysteroid dehydrogenase, thionicotinamide coenzyme (thio-NAD), reduced nicotinamide coenzyme (NADH) and a buffer.
• thio-NAD thionicotinamide coenzyme
• NADH reduced nicotinamide coenzyme
• a dry analytical element comprising at least one water-soluble polymer layer and at least one spreading layer provided in this order on a support, At least one of the water-soluble polymer layer and the spreading layer contains 3 ⁇ -hydroxysteroid dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme and a buffer.
• the buffer has a buffering capacity in the pH range of 6.0 to 10.0.
• ⁇ 4> The dry analytical element for analyzing bile acids according to any one of ⁇ 1> to ⁇ 3>, wherein the buffer is trishydroxyaminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, or glycine.
• ⁇ 5> The dry analytical element for analyzing bile acid according to any one of ⁇ 1> to ⁇ 4>, wherein the thionicotinamide coenzyme is thio-NAD and the reduced nicotinamide coenzyme is NADH.
• ⁇ 6> The dry analytical element for analyzing bile acids according to any one of ⁇ 1> to ⁇ 5>, wherein the content of the thionicotinamide coenzyme is 0.05 to 0.6 g/ m2 .
• ⁇ 7> The dry analytical element for analyzing bile acids according to any one of ⁇ 1> to ⁇ 6>, wherein the content of the reduced nicotinamide coenzyme is 0.05 to 0.6 g/ m2 .
• ⁇ 8> The dry analytical element for analyzing bile acids according to any one of ⁇ 1> to ⁇ 7>, wherein the content of 3 ⁇ -hydroxysteroid dehydrogenase is 3.0 to 12.0 KU/ m2 .
• ⁇ 9> A method for measuring a bile acid, comprising spotting a bile acid-containing sample on the dry analytical element for analyzing a bile acid according to any one of ⁇ 1> to ⁇ 8>, and measuring color development.
• the dry analytical element for bile acid analysis and the method for measuring bile acid of the present invention make it possible to provide a dry chemistry reagent that does not require water supply and drainage equipment and can be used as a quantitative reagent for the amount of bile acid.
• FIG. 1 shows the relationship between the bile acid concentration and the ⁇ OD/min value in Example 1.
• FIG. 2 shows the relationship between the bile acid concentration and the ⁇ OD/min value in Example 2.
• FIG. 3 shows the relationship between the ⁇ OD/min value and the bile acid concentration in Example 3.
• FIG. 4 shows the relationship between the ⁇ OD/min value and the bile acid concentration in Example 4.
• FIG. 5 shows the relationship between the ⁇ OD/min value and the bile acid concentration in Example 5.
• FIG. 6 shows the relationship between the ⁇ OD/min value and the bile acid concentration in Example 6.
• the numerical range indicated using “to” means a range that includes the numerical values before and after “to” as the minimum and maximum values, respectively.
• the present invention relates to a dry analytical element for analyzing bile acids, which comprises at least one water-soluble polymer layer and at least one spreading layer provided in this order on a support, and at least one of the water-soluble polymer layer and the spreading layer contains 3 ⁇ -hydroxysteroid dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffering agent.
• the method of the present invention utilizes a reaction system in which 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSD) acts as a catalyst.
• 3 ⁇ -HSD 3 ⁇ -hydroxysteroid dehydrogenase
• thio-NAD thionicotinamide coenzyme
• NADH reduced nicotinamide coenzyme
• the color development of thio-NADH correlates with the reaction amount of bile acids, so the amount of bile acids can be quantified by measuring the color development of thio-NADH.
• the reaction speed of the analyte amount varies depending on the amount of bile acids, which is the analyte, and this results in differences in the rate method or the amount of color development (OD) at a fixed time. In this way, it is possible to quantify bile acids.
• the water-soluble polymer layer used in the present invention refers to a layer containing a water-soluble polymer on a support.
• the water-soluble polymer layer draws in a sample containing bile acid spotted on a spreading layer described later, thereby spreading the sample substantially uniformly in the analysis element.
• a preferred water-soluble polymer contained in the water-soluble polymer layer is gelatin. That is, the water-soluble polymer layer is preferably a gelatin layer.
• the water-soluble polymer has a particularly swelling property.
• the amount of the water-soluble polymer used is not particularly limited, but is preferably 8.0 g/m2 or more and 40.0 g/m2 or less , and more preferably 15.0 g/m2 or more and 30.0 g/ m2 or less.
• the spreading layer used in the present invention is a layer that has the function of spreading the aqueous liquid sample that has been spot-applied onto the upper surface of the dry analytical element for bile acid analysis in the lateral direction without causing the components contained in the aqueous liquid sample to be distributed unevenly, and supplying the sample to the lower layer containing a water-absorbent water-soluble polymer at a nearly constant volume per unit area (metering function).
• the spreading layer used in the present invention may be, for example, a woven spreading layer (e.g., plain weave fabric such as broadcloth or poplin) described in JP-A-55-164356, JP-A-57-66359, etc., a knitted spreading layer (e.g., tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric, etc.) described in JP-A-60-222769, etc., a spreading layer made of paper containing organic polymer fiber pulp described in JP-A-57-148250, a spreading layer made of paper containing organic polymer fiber pulp described in JP-B-53-21677, U.S. Pat. No.
• a woven spreading layer e.g., plain weave fabric such as broadcloth or poplin
• a knitted spreading layer e.g., tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric, etc.
• a membrane filter brush polymer layer
• a non-fibrous isotropic porous spreading layer consisting of a porous layer containing continuous microvoids (three-dimensional lattice-like granular structure layer) in which polymer microbeads described in JP-A-55-90859 are bonded in a point-contact manner with a polymer adhesive that does not swell in water can be used.
• a knitted spreading layer for example, tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric, etc. is preferable.
• an intermediate layer such as an adhesive layer can be provided between the support and the layer provided thereon, and between each layer provided on the support.
• the support is preferably a water-impermeable support.
• the material of the water-impermeable support is preferably a polymer such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, or cellulose ester (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), with polyethylene terephthalate being particularly preferred.
• the support may be a smooth, flat support having a thickness of about 50 ⁇ m to about 1 mm, preferably about 80 ⁇ m to about 300 ⁇ m, that is transparent, for example, transmits electromagnetic radiation having at least a part of a wavelength range of about 200 nm to about 900 nm.
• a known undercoat layer or adhesive layer may be provided on the surface of the support to strengthen adhesion to the intermediate layer.
• At least one of the water-soluble polymer layer and the spreading layer contains 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSD).
• 3 ⁇ -hydroxysteroid dehydrogenase is an enzyme that oxidizes 3 ⁇ -hydroxysteroids and is a conjugated enzyme.
• the content of 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSD) is preferably 3.0 to 12.0 KU/ m2 , and more preferably 4.0 to 8.0 KU/ m2 .
• the dry analytical element for bile acid analysis of the present invention contains (oxidized) thionicotinamide coenzyme.
• the (oxidized) thionicotinamide coenzyme is a coenzyme that acts by binding with various oxidoreductases in the same manner as nicotinamide coenzyme and is involved in hydrogen transfer in the living body, specifically meaning thio-NAD or thio-NADP.
• the content of the (oxidized) thionicotinamide coenzyme is preferably 0.05 to 1.0 g/ m2 , more preferably 0.05 to 0.6 g/ m2 , and even more preferably 0.1 to 0.5 g/ m2, in order to ensure sensitivity.
• the content of the (oxidized) thionicotinamide coenzyme is preferably 0.6 g/ m2 or less, since the background is reduced.
• the dry analytical element for bile acid analysis of the present invention contains a reduced nicotinamide coenzyme.
• the reduced nicotinamide coenzyme is a coenzyme for various dehydrogenases, specifically NADH or NADPH.
• the amount of the reduced nicotinamide coenzyme used is preferably 0.05 to 1.0 g/ m2 , more preferably 0.05 to 0.6 g/ m2 , and even more preferably 0.1 to 0.5 g/ m2, in order to ensure sensitivity.
• the content of the reduced nicotinamide coenzyme is preferably 0.6 g/m2 or less , since the background is reduced.
• the dry analytical element for analyzing bile acids of the present invention contains a buffer.
• the buffering agent is preferably a buffering agent having a buffering capacity in the range of pH 6.0 to 10.0, more preferably a buffering agent having a buffering capacity in the range of pH 7.0 to 9.0.
• Types of buffers include known buffers such as trishydroxyaminomethane, glycine, phosphate, and Good's buffer.
• the buffer is preferably trishydroxyaminomethane (also called Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (also called HEPES), N-[tris(hydroxymethyl)methyl]glycine (also called Tricine), N,N-bis(2-hydroxyethyl)glycine (also called Bicine), or glycine.
• the buffer is more preferably trishydroxyaminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, or glycine.
• the content of the buffer is not particularly limited as long as it is not affected by the pH of the sample, but is preferably 1.0 to 10.0 g/ m2 , and more preferably 3.0 to 8.0 g/ m2 .
• the amount of bile acid can be quantified by including 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSD), thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer in at least one of the water-soluble polymer layer and the spreading layer. That is, 3 ⁇ -hydroxysteroid dehydrogenase, (oxidized) thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer may be included in the water-soluble polymer layer, the spreading layer, or both the water-soluble polymer layer and the spreading layer. It is more preferable to include these in the spreading layer.
• the dry analytical element for bile acid analysis of the present invention may further include a reagent layer, a reflective layer, a light-shielding layer, a filtration layer, an undercoat layer, and other layers.
• a reagent layer a reflective layer
• a light-shielding layer a filtration layer
• an undercoat layer an undercoat layer
• other layers such analytical elements are disclosed in U.S. Pat. Nos. 3,992,158 and 4,042,335, but the preferred configuration of the present invention is an integrated multi-layer analytical element produced by sequentially laminating and integrating a water-soluble polymer layer having water absorption properties and a spreading layer that spreads the sample laterally on a light-transmitting, water-impermeable support.
• the dry analytical element for bile acid analysis of the present invention can be prepared by a method known to those skilled in the art. For example, a coating liquid prepared as an intermediate layer coating liquid is applied to a support and dried to prepare a dry film of about 40 ⁇ m thickness, and then a woven fabric for the spreading layer is laminated to prepare the dry analytical element for bile acid analysis.
• the dry analytical element for bile acid analysis is then cut into square pieces with sides of about 15 mm to about 30 mm or pieces of similar size and shape, and is preferably used as a chemical analysis slide by placing it in a slide frame described in JP-B-57-28331, JP-A-56-142454, JP-A-57-63452, JP-A-58-32350, JP-T-58-501144, etc., from various viewpoints such as production, packaging, transportation, storage, and measurement operation. Depending on the intended use, it can be used in the form of a long tape stored in a cassette or magazine, or in small pieces attached or stored in a card with an opening.
• the present invention provides a method for measuring bile acids, which includes spotting a bile acid-containing sample on the dry analytical element for analyzing bile acids of the present invention and measuring the color development.
• aqueous liquid sample such as whole blood, plasma, serum, lymph, or urine
• a substantially constant temperature in the range of about 20° C. to about 40° C., preferably at a substantially constant temperature around 37° C., for about 1 to about 10 minutes, preferably about 2 to about 7 minutes, and detectable changes such as color change and color development in the dry analytical element for bile acid analysis are measured by reflection photometry from the support side, and the content of bile acid in the liquid sample can be determined by the principle of colorimetric measurement.
• the optical density of the spreading layer is measured by reflection photometry using light at or near the absorption maximum wavelength of bile acid, and the content of bile acid in the liquid sample can be determined by the principle of colorimetric measurement using a previously prepared calibration curve.
• quantitative analysis of bile acid can be performed with high accuracy. Measurement operations can be performed with extremely simple operations and highly accurate quantitative analysis using chemical analysis devices described in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867, etc.
• Example 1 Preparation of coated film and dried slide An aqueous solution of the following composition-1 was applied to a smooth, colorless, transparent, 180 ⁇ m polyethylene terephthalate (PET) film that had been gelatin-subbed, so that the thickness after drying was 40 ⁇ m, and then dried to provide a water-absorbing layer.
• PET polyethylene terephthalate
• composition-1 Gelatin 17g/ m2
• surfactant 0.2g/ m2 The surfactant used here was polyoxy(2-hydroxy)propylene nonylphenyl ether (Surfactant 10G, manufactured by Olin).
• water was supplied to the front surface of the film at a supply rate of approximately 30 g/ m2 to moisten it, and then a polyester spun yarn tricot knit fabric equivalent to 50 denier was attached to it using a wet lamination method while applying light pressure to form a spread layer.
• aqueous solution A having the following composition was applied onto the spreading layer so that the amounts of each component were as follows, and then dried to prepare a dry analytical element for bile acid analysis according to the present invention.
• the above dry analytical element for bile acid analysis was cut to a size of 12 mm x 13 mm, and a slide was prepared according to the method described in JP-A-57-63452, to produce a dry analytical slide for bile acid analysis (1).
• Table 1 shows the bile acid concentration and the change in reflection concentration per minute ( ⁇ OD/min) at the reflection concentration that increases between 60 and 180 seconds of measurement
• Figure 1 shows the relationship between the bile acid concentration and the ⁇ OD/min value, with the bile acid concentration on the horizontal axis and the ⁇ OD value on the vertical axis.
• Example 2 (1) Preparation of coated film and dried slide An aqueous solution of Composition-2 below was applied to a smooth, colorless, transparent PET film of 180 ⁇ m thick and undercoated with gelatin, so that the thickness after drying would be 40 ⁇ m, and then dried to provide a water-absorbing layer.
• composition-2 Polyvinyl alcohol 23g/ m2 Surfactant 0.2g/ m2
• the surfactant used here was polyoxy(2-hydroxy)propylene nonylphenyl ether (Surfactant 10G, manufactured by Olin).
• Example 2 a polyester spun yarn tricot knit fabric equivalent to about 50 denier was attached by wet lamination to provide a spreading layer.
• aqueous solution A as in Example 1 was applied onto the spreading layer and dried to prepare a dry analysis slide (2) for bile acid analysis according to the present invention.
• Table 2 summarizes the bile acid concentration and the change in reflection concentration per minute ( ⁇ OD/min) at the reflection concentration that increases between 60 and 180 seconds of measurement, and Figure 2 shows the relationship between the bile acid concentration and the ⁇ OD/min value, with the bile acid concentration on the horizontal axis and the ⁇ OD value on the vertical axis.
• Examples 1 and 2 show that both dry analytical elements for bile acid analysis have good linearity and exhibit sufficient performance as test agents capable of quantitatively measuring bile acids.
• the use of gelatin as the water-absorbing layer in the dry analytical element is more preferable because a larger signal-to-noise ratio (S/N) can be obtained by using gelatin.
• Example 3 The following levels of buffer were prepared and slides were prepared for confirmation.
• a second experiment (Example 3) was performed using the aqueous solution A of Example 1, and the trishydroxyaminomethane (Tris) in Example 3 was replaced with HEPES (Example 4), Tricine (Example 5), or Glycine (Example 6), and dry analysis slides were prepared in the same manner as in Example 1.
• human pool serum specimens were prepared so that the bile acid concentration (measured value by the solution method) was 0 ⁇ mol/L, 118 ⁇ mol/L, or 186 ⁇ mol/L, and 10 ⁇ L of each specimen was spotted on a dry analysis slide using the above-mentioned buffer solution.
• Table 3 shows the bile acid concentration and the change in reflection concentration per minute ( ⁇ OD/min) at the reflection concentration that increases between 60 and 180 seconds of measurement.
• Figures 3 to 6 show the relationship between the bile acid concentration and the ⁇ OD/min value, with the bile acid concentration on the horizontal axis and the ⁇ OD value on the vertical axis.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Genetics & Genomics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Medicinal Chemistry (AREA)
• Biomedical Technology (AREA)
• Analytical Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Sustainable Development (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Immunology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=94374477

Family Applications (1)

Country Status (2)

Citations (4)

• 2024
  • 2024-07-23 JP JP2025535833A patent/JPWO2025023240A1/ja active Pending
  • 2024-07-23 WO PCT/JP2024/026307 patent/WO2025023240A1/ja active Pending

Patent Citations (4)

Also Published As

Similar Documents

Legal Events