WO2014129460A1 - インドキシル硫酸の測定方法 - Google Patents
インドキシル硫酸の測定方法 Download PDFInfo
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- WO2014129460A1 WO2014129460A1 PCT/JP2014/053777 JP2014053777W WO2014129460A1 WO 2014129460 A1 WO2014129460 A1 WO 2014129460A1 JP 2014053777 W JP2014053777 W JP 2014053777W WO 2014129460 A1 WO2014129460 A1 WO 2014129460A1
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- indoxyl sulfate
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- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Definitions
- the present invention relates to a measurement technique capable of detecting indoxyl sulfate in a sample easily and with high sensitivity.
- Indoxyl sulfate is a tryptophan metabolite derived from dietary protein and is known as a uremic substance. Uremic substances accumulate in the body as kidney dysfunction progresses, causing uremia symptoms such as decreased appetite, nausea and vomiting. In fact, in chronic renal failure patients, it has been reported that the concentration of indoxyl sulfate in serum is significantly increased to about 60 times that of normal subjects (see, for example, Non-Patent Document 1). Therefore, measuring the concentration of indoxyl sulfate in body fluids, especially serum, is extremely useful for diagnosis and testing of diseases, and the measurement methods include determination of therapeutic effects, prognosis estimation, dietary prescription diet It can be used for selection.
- indoxyl sulfate has been mainly measured using gas chromatography or high performance liquid chromatography.
- these methods are excellent in terms of sensitivity and accuracy, there is a problem in that they require skill in analysis and require high costs for measuring devices and equipment.
- an EIA method enzyme immunoassay
- an antibody specific for indoxyl sulfate has been reported (for example, see Patent Document 1). .
- the measurement method using the antigen-antibody reaction has a problem that the measurement time is long and the operation for measurement is complicated.
- An object of the present invention is to provide a simple measurement method capable of detecting indoxyl sulfate in a specimen quickly and with high sensitivity. Furthermore, an object of the present invention is to provide a kit for measuring indoxyl sulfate and a method for examining renal function using the measurement method.
- the present inventors produce a formazan dye by allowing sulfatase and a tetrazolium salt to act on indoxyl sulfate in a sample, and determine the amount of the formazan dye produced.
- indoxyl sulfate in a sample can be measured more easily, quickly and with higher sensitivity than in the past.
- the present inventors have found that the sulfatase derived from the genus Pseudomonas has a high activity of hydrolyzing the sulfuric acid site of indoxyl sulfate, and by using this, indoxyl sulfate can be more rapidly and sensitively used. It was found that it can be measured. Furthermore, it has been found that when sulfatase and tetrazolium salt are allowed to act on indoxyl sulfate in a specimen, the presence of albumin improves the dilution linearity of the specimen.
- the present invention provides a method for measuring indoxyl sulfate, a kit for measuring indoxyl sulfate, and a method for detecting renal function according to the following aspects.
- Item 1 A method for measuring indoxyl sulfate contained in a specimen, Characterized in that it comprises a step of allowing a sulfatase and a tetrazolium salt to act on a specimen and measuring the produced formazan dye. Method for measuring indoxyl sulfate.
- Item 2. The measurement method according to Item 1, wherein the sulfatase is an arylsulfatase.
- Item 3. Item 3.
- the measurement method according to Item 1 or 2 wherein the sulfatase is derived from at least one microorganism selected from the group consisting of Pseudomonas, Mycobacterium, Acinetobacter, Streptomyces, and Aspergillus.
- Item 4. Item 4. The measuring method according to any one of Items 1 to 3, wherein the sulfatase is derived from a bacterium belonging to the genus Pseudomonas.
- Item 6. Item 6.
- the sulfatase is an arylsulfatase comprising the polypeptide shown in any of the following (i) to (iv): (i) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, (ii) an indoxyl consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 1 and which is equal to or more than the polypeptide of (i) A polypeptide having an activity of catalyzing a hydrolysis reaction of sulfuric acid, (iii) an activity that catalyzes the hydrolysis reaction of indoxyl sulfate having an amino acid sequence having an identity of 60% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and at least equivalent to the polypeptide of (i) A polypeptide having (iv) consisting of an amino acid sequence encoded by a base sequence capable of hybrid
- Item 7. The measurement method according to any one of Items 1 to 6, wherein sulfatase and tetrazolium salt are allowed to act on the specimen in the presence of albumin.
- Item 8. The measurement method according to any one of Items 1 to 7, wherein a sulfatase and a tetrazolium salt are allowed to act on a specimen in the presence of at least one selected from the group consisting of an anionic surfactant and a thiol compound.
- Item 9. A kit for measuring indoxyl sulfate comprising sulfatase and a tetrazolium salt.
- the measurement kit according to Item 9 comprising a first reagent containing a tetrazolium salt and a second reagent containing sulfatase.
- Item 11. Item 11.
- the measurement kit according to Item 9 or 10 further comprising albumin.
- Item 12. Item 12.
- Item 13 Item 13.
- a method for examining renal function comprising a step of allowing a sulfatase and a tetrazolium salt to act on a specimen collected from a living body and measuring the produced formazan dye.
- Item 16. The test method according to Item 15, wherein the specimen is blood, serum, plasma, or urine.
- Item 17. The test method according to Item 15 or 16, which is used for testing renal failure.
- the measurement method of the present invention it is possible to measure indoxyl sulfate in a sample quickly and with high sensitivity by a simple technique. Therefore, by using the measurement method of the present invention, it is possible to easily detect renal dysfunction that is apparent from the amount of indoxyl sulfate, determination of renal function, examination and diagnosis of diseases accompanied by renal dysfunction. Etc. can be performed easily.
- Example 1 it is the figure which showed the relationship between the measured absorbance (vertical axis) and the known indoxyl sulfate concentration (horizontal axis) in the specimen.
- Example 9 it is the figure which showed the relationship between the measured light absorbency (vertical axis) and the dilution rate (horizontal axis) of the serum specimen which added indoxyl sulfate.
- the measurement method of the present invention is a method for measuring indoxyl sulfate contained in a sample, and includes a step of measuring the produced formazan dye by allowing a sulfatase and a tetrazolium salt to act on the sample. It is characterized by.
- the measuring method of the present invention will be described in detail.
- indoxyl Sulfate Measurement Although it is not desired to limit the interpretation of the present invention, it is thought that the measurement of the present invention enables the measurement of indoxyl sulfate by the mechanism shown in FIG. That is, indoxyl sulfate in the sample is hydrolyzed by the action of sulfatase to produce indoxyl. The produced indoxyl is easily oxidized to indigo, but when the tetrazolium salt coexists, the tetrazolium salt is reduced and a formazan dye is produced. Since this formazan dye reflects the concentration of indoxyl sulfate in the sample, the indoxyl sulfate in the sample can be determined by colorimetric determination of the produced formazan dye.
- the sample to be measured for indoxyl sulfate is not particularly limited as long as it is required to measure the presence or concentration of indoxyl sulfate.
- biological samples for example, biological samples, experimental samples Etc.
- a preferable example of a specimen is a biological sample collected for examination of renal function.
- biological samples include blood, serum, plasma, urine and the like.
- serum and plasma are more preferable.
- a mammal is mentioned, Preferably it is a human.
- the sample may be subjected to pretreatment such as removal of impurities as necessary when it is subjected to the measurement method of the present invention.
- Sulfatase used in the present invention is not particularly limited as long as it has an activity of catalyzing a reaction of hydrolyzing indoxyl sulfate to liberate a sulfate group, but preferably arylsulfatase (EC 3.1.6). .1).
- the sulfatase used in the present invention preferably has the characteristics that the Km value is small and the working pH is neutral to weakly alkaline from the viewpoint of increasing the measurement accuracy of indoxyl sulfate. Since the indoxyl sulfate concentration in the biological sample is very small and is required to have high sensitivity, the smaller the Km value, the higher the measurement accuracy. In the measurement method of the present invention, since the reaction pH of the tetrazolium salt is neutral to weakly alkaline, the formazan dye is efficiently generated when the action pH of the sulfatase is approximately the same as the reaction pH of the tetrazolium salt. It becomes possible.
- the origin of the sulfatase used in the present invention is not particularly limited, and examples thereof include microorganisms and molluscs.
- microorganisms producing sulfatase used in the present invention include, for example, Pseudomonas, Mycobacterium, Acinetobacter, Streptomyces, Klebsiella, Enterobacter, Serratia, Cytobacter, Escherichia, Bacteria belonging to the genus Dietia, Gracicola, Sphingomonas, Wickermomyces, Pseudoarteromonas, Sphingobium, Methylibium, etc .; Aspergillus, Candida, Arthroderma, Trichophyton, Penicillium, Unkinocalps, Schizophyllum, Exophia, Gentria, Magnaporte, Talaromyces, Schefelsomyces, Macrofomina, Fusarium, Kluywellomyces, Lachanthea, Jigo Examples include fungi of the genus Kalomyces, Trichoderma, Rodermyces, Meeologyma, Botryo
- fungi belonging to the genus Aspergillus include Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus fumigatus, Aspergillus fumigatus, Aspergillus fumigatus and Aspergillus fumigatus. terreus), Aspergillus flavus, Aspergillus clavatus, Aspergillus kawachii, and the like.
- fungi belonging to the genus Candida include Candida albicans, Candida tropicalis, Candida tenuis, Candida dubliniensis, and Candida olthopsis (Candida orthopsilosis), Candida parapsilosis and the like.
- fungi belonging to the genus Arthroderma include Arthroderma benhamiae, Arthroderma gypseum, Arthroderma otae, and the like.
- fungi belonging to the genus Trichophyton include Trichophyton tonsurans, Trichophyton equinum, Trichophyton rubrum, and Trichophyton verrucosum. Is mentioned.
- fungi belonging to the genus Penicillium include Penicillium chrysogenum.
- fungi belonging to the genus Unkinocarps include Uncinocarpus reesii.
- fungi belonging to the genus Ogataea include Ogataea parapolymorpha.
- fungi belonging to the genus Schizophyllum include Schizophyllum commune.
- Exophiala include Exophiala dermatitidis.
- fungi belonging to the genus Nectria include Nectria haematococca and the like.
- fungus belonging to the genus Magnaporte include Magnaporthe oryzae.
- fungi belonging to the genus Talaromyces include Talaromyces marneffei and Talaromyces stipitatus.
- Specific examples of the fungus belonging to the genus Scheffelomyces include Scheffersomyces stipitis.
- Specific examples of the fungus belonging to the genus Macrophomina include Macrophomina phaseolina.
- fungi belonging to the genus Fusarium include Fusarium pseudograminearum, Fusarium oxysporum, and the like.
- fungus belonging to the genus Kluyveromyces include Kluyveromyces lactis.
- fungi belonging to the genus Lachanthea include Lachancesa thermotolerans.
- fungi belonging to the genus Digosaccharomyces include Zygosaccharomyces rouxii.
- Specific examples of the fungus belonging to the genus Schizosaccharomyces include Schizosaccharomyces pombe.
- Trichoderma include Trichoderma virens, Trichoderma atroviride, Trichoderma reesei and the like.
- fungi belonging to the genus Clavispora include Clavispora lusitaniae.
- fungi belonging to the genus Rhodermyces include Lodderomyces elongisporus.
- Specific examples of the fungus belonging to the genus Meeologyma include Meyerozyma guilliermondii and Millerozyma farinosa.
- Specific examples of the fungus belonging to the genus Beauveria include Beauveria basiana.
- fungi belonging to the genus Botriotinia include Botryotinia fuckeliana.
- Specific examples of fungi belonging to the genus Wallemia include Wallemia sebi.
- Specific examples of the fungus belonging to the genus Coretricum include Colletotrichum gloeosporioides.
- Specific examples of the fungus belonging to the genus Spasaspora include Spasaspora passalidarum.
- Specific examples of the fungi belonging to the genus Cordyceps include Cordyceps militaris.
- Specific examples of fungi belonging to the genus Neosartoria include Neosartorya fischeri.
- Specific examples of fungi belonging to the genus Debaryomykes include Debaryomyces hansenii.
- Specific examples of fungi belonging to the genus Verticillium include Verticillium albo-atrum.
- Specific examples of fungi belonging to the genus Yarrowia include Yarrowia lipolytica.
- Specific examples of fungi belonging to the genus Astilago include Astilago maydis.
- mollusks producing sulfatase used in the present invention include snails (Helix pomatia), abalone (Abalone entrails), limpet (Patella vulgate) and the like.
- the sulfatase used in the present invention is preferably derived from a microorganism, more preferably Pseudomonas, Mycobacterium, Acinetobacter, Streptomyces. Genus, Aspergillus genus; more preferably Pseudomonas genus, particularly preferably Pseudomonas aeruginosa.
- a preferred example of the sulfatase used in the present invention is an arylsulfatase comprising the polypeptide shown in any of the following (i) to (iv): (i) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1. (ii) an indoxyl consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, added or inserted in the amino acid sequence represented by SEQ ID NO: 1 and which is equal to or more than the polypeptide of (i) A polypeptide having an activity of catalyzing a hydrolysis reaction of sulfuric acid.
- an activity that catalyzes the hydrolysis reaction of indoxyl sulfate having an amino acid sequence having an identity of 60% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and at least equivalent to the polypeptide of (i)
- the polypeptide (i) is an arylsulfatase derived from Pseudomonas aeruginosa, encoded by the base sequence represented by SEQ ID NO: 2, and can be obtained as a gene product of a gene having the base sequence.
- the number of amino acid residues to be substituted, deleted, added or inserted is an activity that catalyzes the hydrolysis reaction of indoxyl sulfate equal to or greater than that of the polypeptide of (i).
- an activity that catalyzes the hydrolysis reaction of indoxyl sulfate equal to or greater than that of the polypeptide of (i).
- 1 to 20 preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, particularly preferably 1 to 5, most preferably.
- Preferably 1 to 3 are mentioned.
- the substitution of amino acid residues is preferably conservative substitution based on the properties of the side chain functional group, but indoxyl equivalent to or higher than that of the polypeptide of (i). As long as it has an activity of catalyzing the hydrolysis reaction of sulfuric acid, it may be a non-conservative substitution in which the nature of the amino acid residue before substitution and the nature of the amino acid residue after substitution are different. Natural amino acids are classified into nonpolar amino acids, uncharged amino acids, acidic amino acids and basic amino acids according to the side chain functional groups. A conservative substitution is a substitution using an amino acid residue that falls into the same category as the amino acid residue before substitution.
- nonpolar amino acids include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan
- uncharged amino acids include glycine, serine, threonine, Cysteine, tyrosine, asparagine, and glutamine
- acidic amino acids include aspartic acid and glutamic acid
- basic amino acids include lysine, arginine, and histidine.
- substitution, deletion, or addition of amino acid residues is preferably introduced to the polypeptide (i) other than the site involved in sulfatase activity.
- the sites involved in the sulfatase activity include aspartic acid at position 13 in the sequence number 1, aspartic acid at position 14, cysteine at position 51, arginine at position 55, Examples include lysine at position 113, histidine at position 115, histidine at position 211, aspartic acid at position 317, aspartic acid at position 318, and lysine at position 375.
- the sites involved in sulfatase activity in the polypeptide (i) are disclosed in Structure, Vol. 9, 483-491, June, 2001. The amino acid residue to be added or inserted can be appropriately set.
- introducing mutations such as substitution, deletion, addition and insertion into an amino acid sequence
- it can be performed according to a conventionally known method.
- the method for introducing amino acid mutation include site-specific mutagenesis, which can be carried out by using a method based on Inverse PCR or a commercial kit of QuikChange II Kit (manufactured by Stratagene).
- a polynucleotide (DNA) encoding a sulfatase having a desired mutation can be obtained based on the base sequence represented by SEQ ID NO: 1.
- a sulfatase comprising the polypeptide (ii) can be obtained as a recombinant protein according to a known genetic engineering technique described later.
- the sequence identity to the amino acid sequence represented by SEQ ID NO: 1 is 60% or more, and catalyzes the hydrolysis reaction of indoxyl sulfate equal to or higher than that of the polypeptide (i). However, it is preferably 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more, Preferably, it is 96% or more, 97% or more, 98% or more, or 99% or more.
- the polypeptide of (iii) for the site where the amino acid sequence of the polypeptide of (i) is different from the amino acid residue, the polypeptide of (i) is the same as in the case of the polypeptide of (ii) above. In the above, it is desirable that the site is other than the site involved in the sulfatase activity.
- sequence identity For polypeptide sequence identity, the two polypeptides to be compared are optimally aligned, the number of positions where the amino acid matches in both sequences is divided by the total number of comparison amino acids, and the result multiplied by 100. Represented.
- sequence identity can be determined, for example, using a known algorithm such as BLAST, FASTA. Specific operation methods for determining sequence identity are described in, for example, Takahisa Toshihisa and Kanahisa Kei, “Genome Net Database Usage” Second Edition (1998), Kyoritsu Publishing (Tokyo, Japan). .
- the stringent condition is specifically 6 ⁇ SSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10 ⁇ SSC), 50% Hybrids were formed at 45 ° C. in a solution containing formamide, followed by washing at 50 ° C. with 2 ⁇ SSC (Molecular Biology, John John Wiley and Sons, N N Y Y (1989), 6.3.1-6.3 .6).
- the polypeptide of (iii) and (iv) is obtained by searching and obtaining a gene encoding the polypeptide from a genome base sequence of a living organism such as a microorganism or a mollusc using a known program and genetic engineering technology. Then, it can be obtained by introducing a mutation into the gene as necessary to obtain a gene product.
- the polypeptide of (iii) is obtained by introducing mutations such as substitution, deletion, addition, insertion, etc. into the amino acid sequence represented by SEQ ID NO: 1, similarly to the polypeptide of (ii). You can also.
- the sulfatase used in the present invention is prepared by introducing a gene encoding the sulfatase into an appropriate host cell based on a known genetic engineering technique, obtaining a transformant, and culturing the resulting transformant. It is preferably produced as a replacement protein, but may be obtained from culture of non-recombinant microorganisms that produce sulfatase, or may be obtained by extraction from non-recombinant microorganisms or mollusks that produce sulfatase. Good.
- sulfatase As a recombinant protein, see J. et al. Biol. Chem. 1998, 273, 25560-25564, and sulfatase can be obtained as a recombinant protein according to the technique described in this document.
- sulfatase when sulfatase is obtained as a recombinant protein, sulfatase has a protein such as a His tag, if necessary, at the C-terminus of the amino acid sequence, as long as the activity of catalyzing the hydrolysis of indoxyl sulfate is not inhibited.
- a sequence for purification may be added, and a sequence derived from an organism used as a transformant such as Escherichia coli (for example, a poly A addition signal) may be included.
- sulfatase may be used alone or in combination of two or more.
- Tetrazolium salt used in the present invention is a salt of a compound having a tetrazole ring and is not particularly limited as long as it can be reduced to produce a formazan dye.
- the tetrazolium salt used in the present invention is preferably a tetrazolium salt that forms a water-soluble formazan dye when reduced, and more preferably WST-1, WST-3, or WST-8.
- WST-8 has high solubility in water, high sensitivity, and excellent storage stability in an aqueous solution, and is particularly preferably used in the present invention.
- the tetrazolium salt may be used alone or in combination of two or more.
- albumin may coexist when sulfatase and tetrazolium salt are allowed to act on a specimen.
- albumin By coexisting albumin in this way, the dilution linearity of the specimen can be improved, and the measurement accuracy can be further enhanced.
- the albumin used in the present invention is not particularly limited, and examples thereof include human serum albumin, bovine serum albumin, ovalbumin and the like. These albumins may be used individually by 1 type, and may be used in combination of 2 or more type. These albumins may be purified from animals or egg whites, or may be produced as recombinant albumin by genetic engineering techniques.
- human serum albumin is preferable.
- the recombinant human serum albumin is particularly preferably used in the present invention because it does not contain human-derived viruses.
- an anionic surfactant and / or a thiol compound may coexist when a sulfatase and a tetrazolium salt are allowed to act on a specimen.
- the anionic surfactant used in the present invention is not particularly limited, and may be any of carboxylic acid type, sulfuric acid ester type, sulfonic acid type, and the like. Further, the carbon number of the anionic surfactant used in the present invention is not particularly limited, and examples thereof include 6 to 30, preferably 6 to 20, and more preferably 6 to 15.
- anionic surfactant used in the present invention include carboxylic acid type anionic surfactants such as sodium octoate, sodium N-lauroylsarcosine, sodium cholate, sodium deoxycholate; Examples thereof include sulfate type anionic surfactants such as sodium sulfate and lithium dodecyl sulfate; sulfonic acid type anionic surfactants such as sodium dodecylbenzenesulfonate. These anionic surfactants may be used individually by 1 type, and may be used in combination of 2 or more type.
- anionic surfactants from the viewpoint of more effectively improving the dilution linearity and measurement sensitivity of the sample, preferably a carboxylic acid type anionic surfactant, more preferably sodium octoate, N -Lauroyl sarcosine sodium.
- the thiol compound used in the present invention is a compound having hydrogenated sulfur at the end and a structure represented by R-SH (R ⁇ ⁇ is an organic group).
- the thiol compound used in the present invention is not particularly limited, and examples thereof include cysteine; cysteine derivatives such as homocysteine and N-acetylcysteine; and hydroxyl group-containing thiol compounds such as thioglycerol, 2-mercaptoethanol, and dithiothreitol. Is mentioned. These thiol compounds may be used individually by 1 type, and may be used in combination of 2 or more type.
- cysteine and cysteine derivatives are preferable, and N-acetylcysteine is more preferable, from the viewpoint of further effectively improving the dilution linearity and measurement sensitivity of the specimen.
- the anionic surfactant and / or thiol compound when used, only one of the anionic surfactant and the thiol compound may be used. It is preferable to use both an anionic surfactant and a thiol compound from the viewpoint of further improving the performance and measurement sensitivity more effectively.
- the sample, sulfatase, tetrazolium salt and, if necessary, albumin, anionic surfactant and / or thiol compound May be prepared and incubated under temperature conditions that allow the reaction shown in FIG. 1 to proceed.
- the concentration of the sample in the reaction solution is appropriately set according to the type of sample to be used, the amount of indoxyl sulfate contained in the sample, etc., but in the measurement method of the present invention, 0% of indoxyl sulfate is contained in the reaction solution. If the concentration is about 0.01 to 5 ⁇ g / ml, preferably about 0.03 to 2 ⁇ g / ml, it is possible to measure with high accuracy. Therefore, considering the measurable concentration range, the type of specimen, etc.
- the analyte concentration in the reaction solution may be set as appropriate.
- the concentration of sulfatase in the reaction solution is appropriately set according to the reaction time, the type of specimen, etc., and is, for example, 0.1 to 20 U / ml, preferably 0.2 to 10 U / ml.
- sulfatase 1U is an amount that hydrolyzes 1 ⁇ mol of p-nitrophenyl sulfate per minute.
- the concentration of the tetrazolium salt in the reaction solution is appropriately set according to the reaction time, the type of specimen, etc., and is, for example, 0.01 to 50 mmol / l, preferably 0.05 to 10 mmol / l.
- the concentration of albumin in the reaction solution is appropriately set according to the reaction time, the type of sample, etc., for example, 0.01 to 5% by weight, preferably 0. 04 to 1% by weight.
- the concentration of the anionic surfactant in the reaction solution is appropriately set according to the reaction time, the type of specimen, etc. 1 to 50 mmol / l, preferably 0.2 to 10 mmol / l.
- the concentration of the thiol compound in the reaction solution is appropriately set according to the reaction time, the type of sample, etc., for example, 0.01 to 5 mmol / l, preferably 0.02 to 1 mmol / l.
- the pH of the reaction solution may be set in a range that does not interfere with the reaction with the sulfatase and tetrazolium salt to be used, and examples thereof include 5 to 12, preferably 7 to 9.
- an electron carrier may be added to the reaction solution as necessary in order to promote the reduction of the tetrazolium salt.
- the electron carrier include phenanthine methosulfate (PMS), 1-methoxyphenandinium methyl sulfate (1-mPMS), 9-dimethylaminobenzo- ⁇ -phenazoxonium chloride (Meldra Blue), etc. Is mentioned. These electron carriers may be used individually by 1 type, and may be used in combination of 2 or more type.
- ascorbate oxidase ASOD
- other surfactants EDTA
- chelating agents for example, ethylenediaminetetraacetic acid (EDTA), etc.
- Etc. may be added.
- other additives such as a pH adjuster, a stabilizer, and a preservative (for example, sodium azide) may be added to the reaction solution as long as the effects of the present invention are not hindered.
- the order of adding the sample, sulfatase, and tetrazolium salt is not particularly limited, and (i) the sample, sulfatase, and tetrazolium salt are added simultaneously, (ii) Add sulfatase and tetrazolium salt simultaneously to the solution containing the sample, (iii) add the tetrazolium salt to the solution containing the sample and then add sulfatase, (iv) add the sulfatase to the solution containing the sample and then add the tetrazolium salt. Any of addition etc. may be sufficient.
- a tetrazolium salt is added to a solution containing a specimen to form a formazan dye at a temperature equal to 1 at the same heating temperature.
- a method of preparing a reaction solution by adding sulfatase after heating the solution for about 10 minutes to bring the solution to a constant temperature can be mentioned.
- albumin or an anionic surfactant and / or thiol compound is added to the reaction solution, the order of addition is also sulfatase and tetrazolium in the presence of these added components.
- the salt can act on the specimen.
- the temperature condition for incubating the reaction solution is appropriately set within the range where the working temperature of sulfatase and the reduction of the tetrazolium salt are possible.
- the temperature is 10 to 50 ° C., preferably 20 to 40 ° C. It is done.
- the incubation time of the reaction solution is also appropriately set according to the type of specimen used, the concentration of sulfatase and tetrazolium salt, etc., for example, 1 to 120 minutes, preferably 1 to 30 minutes, more preferably 1 to 10 minutes is mentioned.
- the “incubation time” is the time measured from the time when the specimen, sulfatase, and tetrazolium salt coexist.
- a formazan dye is produced by incubating a reaction solution containing a specimen, a sulfatase, and a tetrazolium salt. Since the concentration of the produced formazan dye reflects the concentration of indoxyl sulfate in the sample, the concentration of indoxyl sulfate in the sample can be measured by determining the concentration of the generated formazan dye.
- the method for measuring the concentration of formazan dye is not particularly limited, and a conventionally known method may be adopted, and a method for measuring the absorbance of formazan dye by an absorptiometric analysis method is preferable.
- the wavelength at which the absorbance of the formazan dye is measured is appropriately set according to the tetrazolium salt used and the type of formazan dye produced, and is not limited as long as it is a wavelength that can specifically absorb the formazan dye. It is set in a range of ⁇ 700 nm. More specifically, when WST-8 is used as the tetrazolium salt, the absorbance at 450 nm may be measured.
- a formazan dye that is insoluble or hardly soluble in water is produced.
- the absorbance may be measured by dissolving the formazan dye in an organic solvent such as isopropanol.
- the absorbance of the formazan dye before the start of incubation (absorbance A) and the absorbance of the formazan dye at the end of incubation (absorbance B) are measured, and the value obtained by subtracting absorbance A from absorbance B is The absorbance corresponds to the generated formazan dye concentration.
- the absorbance (absorbance A) of the formazan dye is measured after the tetrazolium salt is added to the solution containing the sample, and then sulfatase.
- the absorbance (absorbance B) of the formazan dye is measured, and the value obtained by subtracting the absorbance A from the absorbance B is calculated as the absorbance corresponding to the generated formazan dye concentration.
- the concentration of formazan dye produced is measured in advance using a known concentration of indoxyl sulfate and a calibration curve is prepared, the concentration of indoxyl sulfate in the sample can be measured quantitatively. .
- Kit for Measuring Indoxyl Sulfate The present invention further provides a kit for measuring indoxyl sulfate containing sulfatase and a tetrazolium salt. The kit of the present invention is used for carrying out the method for measuring indoxyl sulfate.
- the sulfatase and the tetrazolium salt may have a one-agent structure or a two-agent structure. That is, the kit of the present invention may be provided with a reagent containing sulfatase and a tetrazolium salt (hereinafter sometimes referred to as “one-agent constituent reagent”), but preferably contains a tetrazolium salt.
- a first reagent hereinafter also referred to as “two-agent-structured first reagent”
- a second reagent containing sulfatase hereinafter also referred to as “two-agent-structured second reagent”.
- the one-agent-constituting reagent, two-agent-constituting first reagent, and two-agent-constituting second reagent may be liquid, or may be solid such as a lyophilized state.
- the one-agent constituent reagent, the two-agent constituent first reagent, and the two-agent constituent second reagent include, as necessary, a buffer, an electron carrier, ascorbate oxidase (ASOD), a surfactant, a chelating agent (
- ASOD ascorbate oxidase
- EDTA ethylenediaminetetraacetic acid
- pH adjuster e.g., sodium azide, etc.
- a preservative for example, sodium azide, etc.
- various additives used for specimen pretreatment, reaction acceleration, etc. in addition to the one-agent constituent reagent, two-agent constituent first reagent, and two-agent constituent second reagent.
- the 3rd reagent containing may be contained.
- albumin may be contained in the kit of the present invention.
- the kit of the present invention has a two-agent constitution first reagent and a two-agent constitution second reagent and contains albumin
- the albumin is composed of the two-agent constitution first reagent and the two-agent constitution second reagent.
- it may be contained in any, it is preferable that it is contained in the 2 agent 1st reagent.
- the kit of the present invention may contain an anionic surfactant and / or a thiol compound.
- an anionic surfactant and The thiol compound may be contained in either the two-agent first reagent or the two-agent second reagent.
- the anionic surfactant is preferably contained in the two-agent first reagent.
- the kit of the present invention may contain indoxyl sulfate having a known concentration used for preparing a calibration curve, a diluent used for diluting a specimen, and the like.
- the kit of the present invention may include a measurement procedure manual showing the measurement conditions for the indoxyl sulfate.
- the kit of the present invention can be used as a kit for diagnosing renal function. It is suitable as a kit for diagnosis of a disease accompanied by a decrease in function. Specific examples of the disease accompanied by a decrease in renal function include renal failure (acute renal failure, chronic renal failure), uremia and the like. Among them, renal failure, particularly chronic renal failure, has a high blood indoxyl sulfate concentration, and a high correlation is observed between the blood indoxyl sulfate concentration and the degree of symptoms. It is highly useful for diagnosis of renal failure, particularly chronic renal failure.
- the present invention further provides a method for examining renal function using the method for measuring indoxyl sulfate.
- indoxyl sulfate concentration in biological samples such as blood, serum, plasma, urine, etc. reflects kidney function and can be an index for examining diseases associated with decreased kidney function.
- the method for measuring indoxyl sulfate can be used for examination of renal function.
- the method for examining renal function of the present invention is characterized by comprising a step of measuring a produced formazan dye by allowing a sulfatase and a tetrazolium salt to act on a specimen collected from a living body.
- the sample used may be one collected from a human who needs to be tested for renal function, such as blood, serum, plasma, urine, etc.
- a human who needs to be tested for renal function such as blood, serum, plasma, urine, etc.
- blood, serum and plasma are preferable, and serum and plasma are more preferable.
- the concentration of indoxyl sulfate contained in the sample is determined from the generated formazan dye, and compared with the concentration of indoxyl sulfate in the sample collected from a healthy person. Can be inspected. Specifically, the higher the indoxyl sulfate concentration in the sample is, the higher the level of healthy people, the more the kidney function is judged to be reduced. It is determined.
- the method for examining renal function of the present invention is particularly useful as a method for examining diseases associated with decreased renal function.
- diseases associated with a decrease in renal function are as described above.
- the results obtained by the method for examining renal function of the present invention include the determination of the presence or absence of a disease accompanied by a decrease in renal function, the determination of the therapeutic effect of the disease, the prognosis of the patient of the disease, and the diet therapy in the patient of the disease It can serve as a guideline for selection of prescription foods.
- Production Example 1 Preparation of sulfatase derived from Pseudomonas aeruginosa (1) Cloning of sulfatase gene The sulfatase gene was amplified by polymerase chain reaction (PCR) using genomic DNA derived from Pseudomonas aeruginosa (NBRC 106052G) as a template. PCR primers were designed based on the sulfatase gene sequence information in the database. The primer sequences are shown in SEQ ID NO: 1 (Forward Primer) and SEQ ID NO: 2 (Reverse Primer), respectively. A restriction enzyme EcoRI recognition sequence was introduced into the primer of SEQ ID NO: 1, and a SalI recognition sequence was introduced into the primer of SEQ ID NO: 2.
- the PCR reaction was performed using PrimeSTAR DNA polymerase (Takara Shuzo) with iCycler (BioRAD) for 30 cycles of heat denaturation at 98 ° C. for 10 seconds, annealing at 53 ° C. for 15 seconds, and extension reaction at 72 ° C. for 50 seconds. It was. As a result, a fragment of about 1620 bp which is the target size was amplified. When the amplified fragment was sequenced, it was confirmed that the base sequence of this PCR product was the same as the Pseudomonas aeruginosa sulfatase gene sequence in the database.
- the recombinant vector was introduced into Escherichia coli DH5 ⁇ competent cell (manufactured by Toyobo), applied to an LB agar medium containing 100 ⁇ g / ml ampicillin, and cultured overnight at 37 ° C. to obtain a transformant. Plasmid extraction was performed from this transformant and purified to obtain a recombinant vector.
- the obtained recombinant vector has a start codon before the start codon of the sulfatase gene, an extra amino acid sequence is added to the N-terminus of the sulfatase gene. Therefore, the excess sequence was deleted by performing inverse PCR using the obtained recombinant vector as a template.
- the base sequences of the primers used for the inverse PCR are represented by SEQ ID NO: 3 (Forward Primer) and SEQ ID NO: 4 (Reverse Primer) in the sequence listing.
- the PCR reaction was 12 cycles of heat denaturation at 98 ° C. for 10 seconds, annealing at 53 ° C. for 15 seconds, and extension reaction at 72 ° C. for 4 minutes.
- the obtained PCR product was digested with DpnI to cleave the template DNA, and the 5 ′ end of the amplified DNA fragment was phosphorylated with T4 polynucleotide kinase.
- this DNA fragment was ligated by reacting at 16 ° C. for 15 minutes using a Ligationhigh kit (Toyobo), then introduced into Escherichia coli DH5 ⁇ competent cell (Toyobo), and transformed by the same method as described above. Acquired a converter. Plasmid extraction was performed from this transformant and purified to obtain a recombinant vector.
- the obtained recombinant vector was named pTrc-SFT.
- pTrc-SFT was introduced into competent cells of Escherichia coli BL21 to obtain transformants.
- the sulfatase activity of the culture broth at the end of the culture was about 6.0 U / ml.
- the sulfatase activity is shown as 1 U in the amount of hydrolyzing 1 micromole of p-nitrophenyl sulfate per minute at 37 ° C. in Tris-HCl buffer (pH 8.0).
- the cells were collected by centrifugation, suspended in 20 mM Tris-HCl buffer (pH 8.0), and the cells were crushed with an ultrasonic crusher to obtain a crude enzyme solution.
- the crude enzyme solution was applied to a DEAE-Sepharose column equilibrated with 20 mM Tris-HCl buffer (pH 8.0) and eluted with a potassium chloride concentration gradient of 0 to 80% to recover a sulfatase enzyme activity fraction.
- the sulfatase separated and purified by this method was used in the following Test Examples 1 and 2 as a purified enzyme preparation.
- Example 1 Preparation of kit for measuring indoxyl sulfate-1 As a kit for measuring indoxyl sulfate, a first reagent and a second reagent having the following composition were prepared.
- Example 2 Measurement using physiological saline containing indoxyl sulfate of known concentration as a sample Using the indoxyl sulfate measurement kit of Example 1, the concentration of indoxyl sulfate in the sample was measured using an H7180 automatic analyzer ( Hitachi High-Technologies Corporation). As a specimen, physiological saline containing 4% HSA (human serum albumin) adjusted to have an indoxyl sulfate concentration of 0.1 to 5.0 mg / dl was used. Specific measurement conditions are as shown below.
- the first reagent 160 ⁇ l of the first reagent was mixed with 8 ⁇ l of the sample and heated at 37 ° C. for 5 minutes, and then the absorbance at 450 nm (absorbance A) was measured.
- 40 ⁇ l of the second reagent was added, and the mixture was heated at 37 ° C. for 5 minutes, and the absorbance at 450 nm (absorbance B) was measured.
- the value obtained by subtracting the absorbance A from the absorbance B was taken as the measured value.
- FIG. 2 the vertical axis is plotted as the actual measurement value (absorbance), and the horizontal axis is plotted as the known indoxyl sulfate concentration in the sample.
- Example 3 Serum sample obtained by adding a known concentration of indoxyl sulfate to commercially available control serum QAP Troll 1 ⁇ or QAP Troll 2 ⁇ (Sysmex Corporation) as a measurement sample using a serum sample containing indoxyl sulfate at a known concentration The absorbance of the formazan dye concentration produced was measured in the same manner as in Example 2 except that was used.
- the absorbance of the formazan dye concentration produced by the same method as described above was measured using a physiological saline containing 4% HSA adjusted to have an indoxyl sulfate concentration of 1 mg / dl, and the indoxyl sulfate concentration was measured.
- the absorbance of the formazan dye produced when 1 mg / dl was determined. Based on the relationship between the indoxyl sulfate concentration and the absorbance of the formazan dye, the indoxyl sulfate concentration contained in the serum sample was calculated.
- the addition recovery rate is the ratio (%) of the concentration (measurement concentration) of indoxyl sulfate in the serum sample obtained by measurement to the concentration (addition concentration) of indoxyl sulfate added to the serum sample. is there.
- the recovery rate of indoxyl sulfate was almost 100%, and the concentration of indoxyl sulfate added to the serum specimen and the actually measured concentration of indoxyl sulfate were almost the same value. From the above results, it was demonstrated that the concentration of indoxyl sulfate in serum can be accurately measured by using the first reagent and the second reagent of Example 1.
- Example 4 Preparation of indoxyl sulfate measurement kit-2 As a kit for measuring indoxyl sulfate, a first reagent and a second reagent having the following composition were prepared.
- Example 5 Preparation of kit for measuring indoxyl sulfate-3 As a kit for measuring indoxyl sulfate, a first reagent and a second reagent having the following composition were prepared.
- Commercially available snail (Helix pomatia) sulfatase manufactured by SIGMA-ALDRICH, product number S9626: 10 KU / L
- Example 6 Measurement using physiological saline containing indoxyl sulfate of known concentration as a sample Using the indoxyl sulfate measurement kits of Examples 4 and 5, the concentration of indoxyl sulfate in the sample was measured using a spectrophotometer UV. -265 (Shimadzu Corporation). As the specimen, physiological saline adjusted so that the indoxyl sulfate concentration was 10 mg / dl was used. Specific measurement conditions are as shown below.
- the absorbance at 450 nm was measured.
- 200 ⁇ l of the second reagent was added and heated at 37 ° C., and the absorbance (absorbance B) at 450 nm was measured 5 minutes and 90 minutes after the addition of the second reagent.
- the value obtained by subtracting the absorbance A from the absorbance B was taken as the measured value.
- Table 2 shows the results obtained.
- Pseudomonas aeruginosa-derived sulfatase showed an increase in absorbance at 450 nm due to formazan dye formation when 10 mg / dl indoxyl sulfate was used as a sample, confirming sulfatase activity against indoxyl sulfate.
- snail-derived sulfatase also showed an increase in absorbance at 450 nm due to formazan dye formation when 10 mg / dl indoxyl sulfate was used as a sample, but was smaller than that of Pseudomonas aeruginosa-derived sulfatase.
- Example 7 Preparation of kit for measuring indoxyl sulfate-4 As a kit for measuring indoxyl sulfate, a first reagent and a second reagent having the following composition were prepared.
- First reagent PIPES buffer pH 7.0: 50 mM 1-mPMS (manufactured by Dojindo Laboratories, product number M003): 0.05 mM Recombinant human serum albumin (HSA) (manufactured by Wako Pure Chemicals, product number 014-21543): 0.5% by weight
- Second reagent Tris-HCl buffer pH 8.2: 200 mM WST-8: 1 mM Pseudomonas aeruginosa-derived sulfatase obtained in Production Example 1: 10 KU / L
- Example 8 Preparation of kit for measuring indoxyl sulfate-5 As a kit for measuring indoxyl sulfate, a first reagent and a second reagent having the following composition were prepared.
- First reagent HEPES buffer (pH 7.8): 100 mM WST-8: 0.25 mM Sodium octanoate (manufactured by Wako Pure Chemicals, product number 196-08192): 5 mM Second reagent HEPES buffer (pH 7.8): 100 mM N-acetylcysteine (Roche, product number 068365): 2.5 mM Pseudomonas aeruginosa-derived sulfatase obtained in Production Example 1: 15 KU / L
- Example 9 Measurement of dilution linearity using a serum sample containing known concentrations of indoxyl sulfate
- a serum sample in which 4 to 6 mg / dl of indoxyl sulfate was added to commercially available control serum was 4 times or 16 times in physiological saline.
- the absorbance of the produced formazan dye concentration was measured for the specimen diluted 64 times.
- the absorbance of the formazan dye concentration produced by the same method as described above was measured using a physiological saline containing 4 wt% HSA adjusted to have an indoxyl sulfate concentration of 1 mg / dl, and indoxyl sulfate was measured.
- the absorbance of the formazan dye produced when the concentration was 1 mg / dl was determined. Based on the relationship between the indoxyl sulfate concentration and the absorbance of the formazan dye, the indoxyl sulfate concentration contained in the sample was calculated.
- the vertical axis represents the measured value and the horizontal axis represents the dilution factor.
- the indoxyl sulfate measurement kits of Examples 7 and 8 were superior to the indoxyl sulfate measurement kit of Example 1 in dilution linearity of serum samples. That is, from this test result, the dilution linearity of the sample containing indoxyl sulfate is improved when sulfatase and tetrazolium salt are allowed to act in the presence of albumin or in the presence of an anionic surfactant and a thiol compound. Became clear.
- Table 3 shows the absorbance of the formazan dye produced when the indoxyl sulfate concentration is 1 mg / dl.
- SEQ ID NO: 3 shows the base sequence of Forward Primer used for amplification of sulfatase gene.
- SEQ ID NO: 4 shows the base sequence of Reverse Primer used for amplification of the sulfatase gene.
- SEQ ID NO: 5 shows the base sequence of Forward Primer used for inverse PCR.
- SEQ ID NO: 6 shows the base sequence of Reverse Primer used for inverse PCR.
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Abstract
Description
項1. 検体に含まれるインドキシル硫酸を測定する方法であって、
検体にスルファターゼ及びテトラゾリウム塩を作用させ、生成されたホルマザン色素を測定する工程を含むことを特徴とする、
インドキシル硫酸の測定方法。
項2. 前記スルファターゼがアリールスルファターゼである、項1に記載の測定方法。
項3. 前記スルファターゼが、シュードモナス属、マイコバクテリウム属、アシネトバクター属、ストレプトマイセス属及びアスペルギルス属からなる群より選択される少なくとも1種の微生物由来である、項1又は2に記載の測定方法。
項4. 前記スルファターゼが、シュードモナス属に属する細菌由来である、項1~3のいずれかに記載の測定方法。
項5. 前記スルファターゼが、シュードモナス・アエルギノーザ由来である、項1~4のいずれかに記載の測定方法。
項6. 前記スルファターゼが、下記(i)~(iv)のいずれかに示すポリペプチドからなるアリールスルファターゼである、項1~5のいずれかに記載の測定方法:
(i)配列番号1で示されるアミノ酸配列からなるポリペプチド、
(ii)配列番号1で示されるアミノ酸配列において1又は数個のアミノ酸残基が置換、欠失、付加又は挿入されたアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド、
(iii)配列番号1で示されるアミノ酸配列に対して配列同一性が60%以上のアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド、
(iv)配列番号2で示される塩基配列の相補配列に対してストリンジェントな条件下でハイブリダイズし得る塩基配列によってコードされるアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド。
項7. アルブミン存在下で、検体にスルファターゼ及びテトラゾリウム塩を作用させる、項1~6のいずれかに記載の測定方法。
項8. 陰イオン性界面活性剤及びチオール化合物よりなる群から選択される少なくとも1種の存在下で、検体にスルファターゼ及びテトラゾリウム塩を作用させる、項1~7のいずれかに記載の測定方法。
項9. スルファターゼ及びテトラゾリウム塩を含むことを特徴とする、インドキシル硫酸の測定用キット。
項10. テトラゾリウム塩を含有する第1試薬と、スルファターゼを含む第2試薬を含む、項9に記載の測定用キット。
項11. 更にアルブミンを含む、項9又は10に記載の測定キット。
項12. 更に陰イオン性界面活性剤及びチオール化合物よりなる群から選択される少なくとも1種を含む、項9~11のいずれかに記載の測定キット。
項13. 腎機能の診断に使用される、項9~12のいずれかに記載の測定用キット。
項14. 腎不全の診断に使用される、項9~13のいずれかにのいずれかに記載の測定用キット。
項15. 生体から採取された検体に、スルファターゼ及びテトラゾリウム塩を作用させ、生成されたホルマザン色素を測定する工程を含むことを特徴とする、腎機能の検査方法。
項16. 前記検体が、血液、血清、血漿、又は尿である、項15に記載の検査方法。
項17. 腎不全の検査に使用される、項15又は16に記載の検査方法。
本発明の測定方法は、検体に含まれるインドキシル硫酸を測定する方法であって、検体にスルファターゼ及びテトラゾリウム塩を作用させ、生成されたホルマザン色素を測定する工程を含むことを特徴とする。以下、本発明の測定方法について詳述する。
本発明の限定的解釈を望むものではないが、本発明の測定は、図1に示される機序により、インドキシル硫酸の測定が可能になると考えられる。即ち、検体中のインドキシル硫酸はスルファターゼを作用させることにより加水分解され、インドキシルを生成する。生成したインドキシルは容易に酸化されインジゴとなるが、その際テトラゾリウム塩が共存するとテトラゾリウム塩が還元され、ホルマザン色素が生成される。このホルマザン色素は検体中のインドキシル硫酸濃度を反映することから、生成したホルマザン色素を比色定量することにより検体中のインドキシル硫酸の定量が可能となる。
本発明の測定方法において、インドキシル硫酸の測定対象となる検体は、インドキシル硫酸の有無や濃度の測定が必要とされるものであれば特に制限されないが、例えば、生体由来試料、実験サンプル等が挙げられる。特に、生体内のインドキシル硫酸量は腎機能との相関性が知られているため、検体の好適な例として、腎機能の検査のために採取された生体由来試料が挙げられる。このような生体由来試料としては、具体的には、血液、血清、血漿、尿等が挙げられる。これらの中でも、腎機能の状態がより正確に反映されるという観点から、好ましくは血液、血清、血漿、更に好ましくは血清、血漿が挙げられる。当該生体由来試料の由来については、特に制限されないが、例えば、哺乳動物が挙げられ、好ましくはヒトである。
本発明で使用されるスルファターゼとしては、インドキシル硫酸を加水分解して硫酸基を遊離させる反応を触媒する活性を有するものであれば特に限定されないが、好ましくはアリールスルファターゼ(EC3.1.6.1)が挙げられる。
(i)配列番号1で示されるアミノ酸配列からなるポリペプチド。
(ii)配列番号1で示されるアミノ酸配列において1又は数個のアミノ酸残基が置換、欠失、付加又は挿入されたアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド。
(iii)配列番号1で示されるアミノ酸配列に対して配列同一性が60%以上のアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド。
(iv)配列番号2で示される塩基配列の相補配列に対してストリンジェントな条件下でハイブリダイズし得る塩基配列によってコードされるアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド。
本発明で使用されるテトラゾリウム塩としては、テトラゾール環を有する化合物の塩であり、還元されてホルマザン色素を生成し得るものであれば特に限定されないが、例えば、インドテトラゾリウム(INT)、テトラゾリウムバイオレット(TV)、3-(4,5-ジメチル-2-チアゾリル)-2,5-ジフェニル-2H-テトラゾリウム(MTT)、2-(4-ヨードフェニル)-3-(4-ニトロフェニル)-5-(2,4-ジスルホフェニル)-2H-テトラゾリウム一ナトリウム塩(WST-1)、2-(4-ヨードフェニル)-3-(2,4-ジニトロフェニル)-5-(2,4-ジスルホフェニル)-2H-テトラゾリウム一ナトリウム塩(WST-3)、2-(2-メトキシ-4-ニトロフェニル)-3-(4-ニトロフェニル)-5-(2,4-ジスルホフェニル)-2H-テトラゾリウム一ナトリウム塩(WST-8)、ニトロテトラゾリウムブルー(NTB)等が挙げられる。
本発明の測定方法において、検体にスルファターゼ及びテトラゾリウム塩を作用させる際に、アルブミンを共存させてもよい。このようにアルブミンを共存させることによって、検体の希釈直線性を向上させることができ、測定精度をより一層高めることが可能になる。
本発明の測定方法において、検体にスルファターゼ及びテトラゾリウム塩を作用させる際に、陰イオン性界面活性剤及び/又はチオール化合物を共存させてもよい。このように陰イオン界面活性剤及び/又はチオール化合物を共存させることによって、検体の希釈直線性を向上させつつ、測定感度も高めることができ、測定精度をより一層高めることが可能になる。
本発明の測定方法において、前記検体に対してスルファターゼ及びテトラゾリウム塩を作用させるには、検体、スルファターゼ、テトラゾリウム塩、並びに必要に応じて、アルブミン、陰イオン性界面活性剤及び/又はチオール化合物を含む反応液を調製し、これを図1に示す反応が進行可能な温度条件下でインキュベートすればよい。
前述するように、検体、スルファターゼ及びテトラゾリウム塩を含む反応液をインキュベートすることによりホルマザン色素が生成する。生成したホルマザン色素の濃度は、検体中のインドキシル硫酸の濃度を反映しているので、生成したホルマザン色素の濃度を求めることによって、検体中のインドキシル硫酸の濃度を測定することができる。
本発明は、更に、スルファターゼ及びテトラゾリウム塩を含む、インドキシル硫酸の測定用キットを提供する。本発明のキットは、前記インドキシル硫酸の測定方法を実施するために使用される。
本発明は、更に、前記インドキシル硫酸の測定方法を利用した腎機能の検査方法をも提供する。
(1)スルファターゼ遺伝子のクローニング
シュードモナス・アエルギノーザ(Pseudomonas aeruginosa)由来のゲノムDNA(NBRC 106052G)を鋳型としたポリメラーゼチェーンリアクション(PCR)によりスルファターゼ遺伝子を増幅した。PCR用プライマーを、データベースのスルファターゼ遺伝子配列情報に基づいて設計した。プライマーの配列は配列表の配列番号1(Forward Primer)及び配列番号2(Reverse Primer)にそれぞれ示される。なお、配列番号1のプライマーには制限酵素EcoRI認識配列、配列番号2のプライマーにはSalIの認識配列を導入した。
スルファターゼ遺伝子をコードする遺伝子を含有する組換えベクターを作製した。前記(1)で取得したPCR産物のDNA断片を、制限酵素EcoRI(宝酒造社製)及び制限酵素SalI(宝酒造社製)にて切断した。一方、プラスミドベクターpTrc99aを同じ制限酵素にて切断し、アガロースゲル電気泳動により分離し、分子量が大きい方のDNA断片をGenElute Gel Extraction Kit(SIGMA-ALDRICH社製)を用いて回収した。次いでこれらのDNA断片をLigationhighキット(東洋紡績製)にて16℃で15分間反応させて連結し、シュードモナス・アエルギノーザ由来のスルファターゼ遺伝子を含む組換えベクターを得た。
得られた形質転換体BL21(pTrc-SFT)を30mlのLB液体培地(1.0%ポリペプトン、0.5%酵母エキス、0.5%NaCl、100μg/mlアンピシリン)に植菌し37℃、16時間培養し種培養液とした。この培養液を1.5Lの4×YT(3Lジャーファーメンター;3.2%ポリペプトン、2.0%酵母エキス、0.5%NaCl、1.0%グリセロール、pH7.5)に全量植菌し、30℃で68時間通気攪拌培養を行った。培養終了時の培養液のスルファターゼ活性は約6.0U/mlであった。なお、スルファターゼ活性は、トリス塩酸緩衝液(pH8.0)中で37℃、1分間に1マイクロモルのp-ニトロフェニル硫酸を加水分解する量を1Uとして示される。
インドキシル硫酸測定用キットとして、下記組成の第1試薬及び第2試薬を調製した。
トリス塩酸緩衝液(pH8.0):100mM
WST-8:0.5mM
EDTA:1mM
アジ化ナトリウム:0.1%
第2試薬
トリス塩酸緩衝液(pH8.0):100mM
製造例1で得られたシュードモナス・アエルギノーザ由来スルファターゼ:10KU/L
EDTA:1mM
アジ化ナトリウム:0.1%
実施例1のインドキシル硫酸測定用キットを用いて、検体中のインドキシル硫酸濃度を、H7180型自動分析装置(株式会社日立ハイテクノロジーズ)にて測定した。検体としては、インドキシル硫酸濃度が0.1~5.0mg/dlとなるよう調整した4%HSA(ヒト血清アルブミン)を含む生理食塩水を用いた。具体的測定条件は、以下に示す通りである。
検体として、市販管理血清QAPトロール1×又はQAPトロール2×(シスメックス株式会社)に既知濃度のインドキシル硫酸を添加した血清検体を使用したこと以外は、前記実施例2と同様の方法で、生成したホルマザン色素濃度の吸光度を測定した。
インドキシル硫酸測定用キットとして、下記組成の第1試薬及び第2試薬を調製した。
トリス塩酸緩衝液(pH8.0):100mM
WST-8:0.1mM
第2試薬
トリス塩酸緩衝液(pH8.0):100mM
製造例1で得られたシュードモナス・アエルギノーザ由来スルファターゼ:10KU/L
インドキシル硫酸測定用キットとして、下記組成の第1試薬及び第2試薬を調製した。
トリス塩酸緩衝液(pH8.0):100mM
WST-8:0.1mM
第2試薬
トリス塩酸緩衝液(pH8.0):100mM
市販のカタツムリ(Helix pomatia)由来スルファターゼ(SIGMA-ALDRICH社製、品番S9626):10KU/L
実施例4及び5のインドキシル硫酸測定用キットを用いて、検体中のインドキシル硫酸濃度を、分光光度計UV-265(株式会社島津製作所)にて測定した。検体としては、インドキシル硫酸濃度が10mg/dlとなるよう調整した生理食塩水を用いた。具体的測定条件は、以下に示す通りである。
インドキシル硫酸測定用キットとして、下記組成の第1試薬及び第2試薬を調製した。
PIPES緩衝液(pH7.0):50mM
1-mPMS(同仁化学研究所製、品番M003):0.05mM
遺伝子組換ヒト血清アルブミン(HSA)(和光純薬製、品番014-21543):0.5重量%
第2試薬
トリス塩酸緩衝液(pH8.2):200mM
WST-8:1mM
製造例1で得られたシュードモナス・アエルギノーザ由来スルファターゼ:10KU/L
インドキシル硫酸測定用キットとして、下記組成の第1試薬及び第2試薬を調製した。
HEPES緩衝液(pH7.8):100mM
WST-8:0.25mM
オクタン酸ナトリウム(和光純薬製、品番196-08192):5mM
第2試薬
HEPES緩衝液(pH7.8):100mM
N-アセチルシステイン(ロシュ製、品番068365):2.5mM
製造例1で得られたシュードモナス・アエルギノーザ由来スルファターゼ:15KU/L
市販管理血清に4~6mg/dlのインドキシル硫酸を添加した血清検体を生理食塩水で4倍、16倍、64倍希釈した検体について、実施例1、7及び8のインドキシル硫酸測定用キットを用いて、前記実施例2と同様の方法で、生成したホルマザン色素濃度の吸光度を測定した。
配列番号4は、スルファターゼ遺伝子の増幅に使用したReverse Primerの塩基配列を示す。
配列番号5は、インバースPCRに使用したForward Primerの塩基配列を示す。
配列番号6は、インバースPCRに使用したReverse Primerの塩基配列を示す。
Claims (17)
- 検体に含まれるインドキシル硫酸を測定する方法であって、
検体にスルファターゼ及びテトラゾリウム塩を作用させ、生成されたホルマザン色素を測定する工程を含むことを特徴とする、
インドキシル硫酸の測定方法。 - 前記スルファターゼがアリールスルファターゼである、請求項1に記載の測定方法。
- 前記スルファターゼが、シュードモナス属、マイコバクテリウム属、アシネトバクター属、ストレプトマイセス属及びアスペルギルス属からなる群より選択される少なくとも1種の微生物由来である、請求項1又は2に記載の測定方法。
- 前記スルファターゼが、シュードモナス属に属する細菌由来である、請求項1~3のいずれかに記載の測定方法。
- 前記スルファターゼが、シュードモナス・アエルギノーザ由来である、請求項1~4のいずれかに記載の測定方法。
- 前記スルファターゼが、下記(i)~(iv)のいずれかに示すポリペプチドからなるアリールスルファターゼである、請求項1~5のいずれかに記載の測定方法:
(i)配列番号1で示されるアミノ酸配列からなるポリペプチド、
(ii)配列番号1で示されるアミノ酸配列において1又は数個のアミノ酸残基が置換、欠失、付加又は挿入されたアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド、
(iii)配列番号1で示されるアミノ酸配列に対して配列同一性が60%以上のアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド、
(iv)配列番号2で示される塩基配列の相補配列に対してストリンジェントな条件下でハイブリダイズし得る塩基配列によってコードされるアミノ酸配列からなり、且つ前記(i)のポリペプチドと同等以上のインドキシル硫酸の加水分解反応を触媒する活性を有するポリペプチド。 - アルブミン存在下で、検体にスルファターゼ及びテトラゾリウム塩を作用させる、請求項1~6のいずれかに記載の測定方法。
- 陰イオン性界面活性剤及びチオール化合物よりなる群から選択される少なくとも1種の存在下で、検体にスルファターゼ及びテトラゾリウム塩を作用させる、請求項1~7のいずれかに記載の測定方法。
- スルファターゼ及びテトラゾリウム塩を含むことを特徴とする、インドキシル硫酸の測定用キット。
- テトラゾリウム塩を含有する第1試薬と、スルファターゼを含む第2試薬を含む、請求項9に記載の測定用キット。
- 更にアルブミンを含む、請求項9又は10に記載の測定キット。
- 更に陰イオン性界面活性剤及びチオール化合物よりなる群から選択される少なくとも1種を含む、請求項9~11のいずれかに記載の測定キット。
- 腎機能の診断に使用される、請求項9~12のいずれかに記載の測定用キット。
- 腎不全の診断に使用される、請求項9~13のいずれかにのいずれかに記載の測定用キット。
- 生体から採取された検体に、スルファターゼ及びテトラゾリウム塩を作用させ、生成されたホルマザン色素を測定する工程を含むことを特徴とする、腎機能の検査方法。
- 前記検体が、血液、血清、血漿、又は尿である、請求項15に記載の検査方法。
- 腎不全の検査に使用される、請求項15又は16に記載の検査方法。
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