WO2019065936A1 - Procédé de mesure de la quantité de muc5ac dans les larmes - Google Patents

Procédé de mesure de la quantité de muc5ac dans les larmes Download PDF

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WO2019065936A1
WO2019065936A1 PCT/JP2018/036175 JP2018036175W WO2019065936A1 WO 2019065936 A1 WO2019065936 A1 WO 2019065936A1 JP 2018036175 W JP2018036175 W JP 2018036175W WO 2019065936 A1 WO2019065936 A1 WO 2019065936A1
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muc5ac
tear
amount
tears
dry eye
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PCT/JP2018/036175
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English (en)
Japanese (ja)
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直人 森
秀樹 三宅
健治 上田
英俊 真野
崇博 今中
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参天製薬株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4704—2-Quinolinones, e.g. carbostyril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a method of measuring the amount of MUC5AC in tear fluid.
  • Mucin is a type of glycoprotein, and is a main component of mucus such as tears, liquid drops, gastric juices and intestinal juices.
  • the basic structure of mucin is a glycoprotein in which an O-type sugar chain is bound to a core protein having a repeating structure of 10 to 80 amino acid residues.
  • Mucin has a molecular weight of 1,000,000 to 10,000,000, of which 50 to 80% is occupied by sugar chains.
  • Mucins in tears can be classified into secreted mucins and membrane mucins.
  • secreted mucins There are two types of secreted mucins: MUC5AC secreted from conjunctival goblet cells and MUC7 secreted from lacrimal gland, and membrane mucins As MUC1, MUC4, MUC16 etc. are known.
  • MUC5AC is considered to greatly affect the onset and / or deterioration of dry eye.
  • MUC5AC in tears of dry eye patients is lower than that of healthy people (Non-patent Document 1). Therefore, measurement of the amount of MUC5AC in tears has been attempted in various clinical studies including examination of dry eye.
  • the Schirmer test which measures tear volume, and the phenol red cotton thread method are known as a means to evaluate the test subject's tear secretion ability. These tests are intended to measure the tear fluid volume of the subject depending on how much the tear fluid is absorbed by the tear fluid collection member such as Silmer's test paper or cotton yarn stained with phenol red. It is used widely in the clinic for diagnosis of dry eye etc.
  • An object of the present invention is to provide a method for easily and accurately measuring the amount of MUC5AC in tears of a subject and a kit for measurement.
  • the present inventors collect tears of a subject using tear collection members such as Schirmel test paper used for measuring tear volume, and tears from the members.
  • the tear extract obtained by extracting the extract is subjected to a sugar chain cleaving treatment by a sugar chain cleaving enzyme treatment or a chemical treatment, and the immunological measurement is carried out to rapidly and promptly measure the amount of MUC5AC in the tears of the subject. It has been found that accurate measurement can be made and dry eye patients having a reduced amount of MUC5AC in tears can be efficiently sorted based on the measurement results, and the present invention has been completed.
  • a method of measuring the amount of MUC5AC in tears of a subject comprising the steps of: (A) absorbing tears by a water-absorbent tear collection member; (B) extracting tear from the tear collection member using an extraction solvent containing a surfactant to obtain a tear extract; (C) applying a glycolytic enzyme treatment to the lacrimal fluid extract; and (d) in a lacrimal fluid extract which has been glycolytic enzyme-treated by an immunological measurement method using an anti-MUC5AC antibody.
  • a method of measuring the amount of MUC5AC in tears of a subject comprising the steps of: (A) absorbing tears by a water-absorbent tear collection member; (B) extracting tear from the tear collection member using an extraction solvent containing a surfactant to obtain a tear extract; (C) applying a glycolytic enzyme treatment to the lacrimal fluid extract; and (d) in a lacrimal fluid extract which has been glycolytic enzyme-treated by an immunological measurement method using an anti-MUC5AC antibody.
  • the method according to (1) further comprising the step of measuring the total amount of protein in the tear solution extract and determining the ratio of the amount of MUC5AC in the tear solution extract to the total amount of protein.
  • the surfactant is a nonionic surfactant.
  • the water-absorbent tear fluid collecting member is a filter paper, a cotton thread, a cellulose membrane, a non-woven fabric, or a sponge.
  • the water-absorbing tear fluid collecting member is Schirmel's test paper.
  • the sugar chain cleaving enzyme is neuraminidase (sialidase), galactosidase, mannosidase, fucosidase, N-acetylglucosaminidase, N-acetylgalactosaminidase, glucosidase, xylosidase, peptide N-glycosidase F, N-acetylhexosaminidase F Or the method according to (1), which is at least one selected from glucuronidase.
  • the immunoassay is an ELISA method.
  • the method according to (1), wherein the subject is a dry eye patient.
  • the surfactant is a nonionic surfactant
  • the water-absorbent tear fluid collecting member is Schirmer's test paper
  • the sugar chain cleaving enzyme is neuraminidase (sialidase)
  • the immunological measurement The method according to (1), wherein the method is an ELISA method, and the subject is a dry eye patient.
  • a kit for measuring the amount of MUC5AC in tears comprising a water-absorbent tear fluid collecting member, an extraction solvent containing a surfactant, a glycolytic enzyme, and an anti-MUC5AC antibody.
  • a therapeutic agent for dry eye characterized in that it is administered to dry eye patients having a decreased amount of MUC5AC in tears, and is selected from the group consisting of diquafosol sodium, rebamipide, and salts thereof
  • An agent for treating dry eye which comprises at least one compound as an active ingredient.
  • a method of measuring the amount of MUC5AC in the tears of a subject comprising the steps of: (A) absorbing tears by a water-absorbent tear collection member; (B) extracting tear from the tear collection member using an extraction solvent containing a surfactant to obtain a tear extract; (C ') a step of applying a sugar chain cleavage treatment to the lacrimal fluid extract by a chemical treatment; and (d) a sugar chain cleavage treatment is applied by a chemical treatment by an immunological measurement method using an anti-MUC5AC antibody Measuring the amount of MUC5AC in the tear fluid extract.
  • the surfactant is a nonionic surfactant.
  • the water-absorbent tear fluid collecting member is a filter paper, a cotton thread, a cellulose membrane, a non-woven fabric, or a sponge.
  • the water-absorbing tear fluid collecting member is Schirmel's test paper.
  • the surfactant is a nonionic surfactant
  • the water-absorbent tear fluid collecting member is Schirmer's test paper
  • the immunological assay is an ELISA method
  • the subject is a patient with dry eye
  • the method according to (14) which is (23) A method for selecting a subject with dry eye having a decreased amount of MUC5AC in tears from a subject based on the amount of MUC5AC in tears measured by the method according to any one of (14) to (22).
  • a kit for measuring the amount of MUC5AC in tears comprising a water-absorbent tear fluid collecting member, an extraction solvent containing a surfactant, a sugar chain cleaving agent, and an anti-MUC5AC antibody.
  • the present invention there is provided a method and a kit for measuring the amount of MUC5AC in tear fluid rapidly, simply and accurately. Since the method of the present invention uses the Schirmel test or the like, which is widely used to measure tear volume at the clinical site, it measures both tear volume and MUC5AC volume in tear without putting a burden on the subject. can do. In addition, based on the measurement results, screening of dry eye patients with decreased amounts of MUC5AC in tear fluid with effective mucin secretion promoting action is effective, understanding of the condition and cause of dry eye, determination of treatment policy, treatment effect You can check the
  • FIG. 1 shows the extraction rate of MUC5AC and total protein with each concentration (0%, 0.05%, 0.2%, 0.5%) of polysorbate 20-containing extraction solvent.
  • FIG. 2 shows the results of measurement of MUC5AC by ELISA of tear extract with or without glycolytic enzyme treatment.
  • the present invention is a method of measuring the amount of MUC5AC in the tears of a subject, comprising the following steps. (A) absorbing tears by a water-absorbent tear collection member; (B) extracting tear from the tear collection member using an extraction solvent containing a surfactant to obtain a tear extract; (C) applying a glycolytic enzyme treatment to the lacrimal fluid extract; and (d) in a lacrimal fluid extract which has been glycolytic enzyme-treated by an immunological measurement method using an anti-MUC5AC antibody. To measure the amount of MUC5AC.
  • another aspect of the present invention is a method of measuring the amount of MUC5AC in tears of a subject, comprising the following steps.
  • a sugar chain cleavage treatment is applied by a chemical treatment by an immunological measurement method using an anti-MUC5AC antibody Measuring the amount of MUC5AC in the tear fluid extract.
  • MUC5AC mucin-forming secreted mucin, which has a repeating sequence rich in serine and threonine as a core protein and is present in normal gastric mucosa and bronchial mucosa, as described above, It is secreted from conjunctival goblet cells and plays an important role in tear stability.
  • the sequence of MUC5AC is well known. For example, as MUC5AC derived from human, the base sequence is registered as GenBank accession number: NM — 020134359.1, and the amino acid sequence based thereon is accession number: NP — 001291288.1.
  • the “subject” is typically a human but may be a mammal such as monkey, dog, cat, cow, horse, pig, rabbit, mouse, rat and the like.
  • subjects include those who are the target of the presence or absence of disease related to the decrease in the amount of MUC5AC in tears, determination of the degree of progression, confirmation of treatment effects, prognosis, and even if they are normal persons.
  • Diseases related to the decrease in the amount of MUC5AC in tears include dry eye (in particular, "BUT-shortened dry eye” in which tear eye volume is normal but various symptoms specific to dry eye appear), Sjogren's syndrome Be
  • the "water-absorbent tear fluid collecting member” used herein is not particularly limited in shape, size, etc., as long as it is used for collecting tear fluid and is water-absorbent.
  • filter paper, cotton yarn, cellulose membrane, non-woven fabric, sponge and the like can be mentioned.
  • the lacrimal fluid collecting member used in the present invention one capable of confirming the amount of lacrimal fluid absorbed by the length, area, etc. is preferable.
  • a paper tear collection member with a scale for measuring tears is particularly preferable, but among them, because of its simplicity and easiness of collection, it is commonly used for measurement of tear volume.
  • Production Measuring Strips (Scaled)) and phenol red cotton yarn are preferable, and commercially available products thereof can be used.
  • the tear fluid collecting member having absorbed the tear fluid can also be stored frozen.
  • the method for absorbing tear fluid into the tear fluid collection member may be performed according to a predetermined method of use of those commercially available products.
  • the amount of tear fluid to be absorbed by the tear fluid collecting member is not particularly limited as long as measurement of the amount of MUC5AC in the tear fluid is possible.
  • the "extraction solvent” used herein is not limited as long as it can extract sufficient tear fluid from the above tear fluid collecting member, but an aqueous solvent such as phosphate buffer, Tris buffer, sodium acetate buffer Solution, buffers such as borate buffer, glycine buffer, distilled water, and physiological saline.
  • the pH of the extraction solvent is not particularly limited as long as it does not affect the extraction and measurement of tear fluid, but can be set to, for example, about 6.0 to 8.0.
  • the salt concentration of the extraction solvent is not particularly limited as long as it does not affect the extraction and measurement of the tear fluid, but it may be adjusted to a concentration to be a physiologically isotonic solution.
  • the surfactant to be contained in the above-mentioned extraction solvent may be either a nonionic surfactant or an ionic surfactant, but a nonionic surfactant is preferred.
  • nonionic surfactants include polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene castor oil, polyoxyethylene polyoxypropylene glycol, sucrose fatty acid ester, etc. Be Among these, polyoxyethylene sorbitan fatty acid ester is preferable.
  • polyoxyethylene sorbitan fatty acid ester examples include, for example, polysorbate 20 (polyoxyethylene sorbitan monolaurate: Tween 20), polysorbate 40 (polyoxyethylene sorbitan monopalmitate: Tween 40), polysorbate 60 (polyoxyethylene monostearate) Sorbitan: Tween 60), polysorbate 65 (polyoxyethylene sorbitan tristearate: Tween 65), and the like.
  • the concentration of the surfactant in the extraction solvent is preferably 0.005% to 0.5%, more preferably 0.01% to 0.2%, and still more preferably 0.025% to 0.1%. 0.05% is most preferred.
  • the extraction method is also not particularly limited, but it is carried out by immersing the tear fluid-collecting member having absorbed tear fluid in the above-mentioned extraction solvent in a container such as a microtube and incubating it while shaking for a fixed time. Extraction may be performed, for example, at 25 to 40 ° C. for about 30 minutes to 1 hour.
  • the sugar chain cleaving enzyme is not particularly limited as long as it can cleave mucin-type sugar chains, and, for example, neuraminidase (sialidase), galactosidase, mannosidase, fucosidase, N-acetylglucosaminidase, N-acetylgalactosaminidase, glucosidase, xylosidase , Peptide N-glycosidase F (Peptide-N-glycosidase F), N-acetylhexosaminidase F (N-Acetyl hexosaminidase F) or glucuronidase etc.
  • glycoslation enzyme also called. These enzymes may be used alone or in combination of two or more.
  • the enzyme treatment can be carried out by mixing the tear solution extract and the glycolytic enzyme and reacting them, for example, at 25 to 40 ° C. for about 30 minutes to 1 hour.
  • the substance used for the chemical treatment is not particularly limited as long as it can cleave mucin-type sugar chains, and examples thereof include ammonia or ammonium salts such as ammonium carbonate, ammonium bicarbonate and ammonium carbamate (see These are collectively referred to as "a sugar chain cleaving agent").
  • the chemical treatment can be carried out by mixing the lacrimal fluid extract and the sugar chain cleaving agent and reacting at, for example, 40 to 80 ° C. for about 10 to 40 hours.
  • step (c) or step (c ') can be carried out integrally and continuously with the antigen-antibody reaction of step (d) described later.
  • Anti-MUC5AC antibodies used for immunological measurements can be obtained using methods well known to those skilled in the art.
  • the antibody may be either a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
  • An antibody can be produced according to a known method using MUC5AC of an animal species to be measured or a partial polypeptide thereof as an antigen.
  • MUC5AC or a partial polypeptide thereof used as an antigen is prepared, for example, by incorporating a polynucleotide consisting of a known base sequence of MUC5AC or a partial sequence thereof into an expression vector and introducing it into an appropriate host cell to prepare a transformant.
  • the transformant can be cultured to express a recombinant protein, and the expressed recombinant protein can be obtained by purification from a culture or a culture supernatant.
  • a polypeptide consisting of a known amino acid sequence of MUC5AC or a partial sequence thereof can be chemically synthesized and used as an immunogen.
  • antibody-producing cells spleen cells, lymph node cells, etc.
  • the hybridoma thus obtained can be cloned, and the antibody can be recovered from the culture to form a monoclonal antibody.
  • polyclonal antibodies can be prepared by collecting blood from an antigen-sensitized mammal (eg, rabbit, rat, mouse, etc.) and separating the serum from the blood by a known method.
  • the antibody can also be used as a fragment as long as it can specifically bind to MUC5AC.
  • active fragments of antibodies include Fab fragments, F (ab ') 2 fragments, single chain antibodies (scFv) and the like. Preparation of these active fragments can be carried out by applying known methods such as papain, pepsin and trypsin treatment.
  • the labeling substance is not particularly limited as long as it is a labeling substance that enables the immunological measurement of MUC5AC, and for example, enzymes (peroxidase, ⁇ -galactosidase, alkaline phosphatase, etc.), fluorescence Substance (FITC, RITC etc.), Chemiluminescent substance (Acridinium, Ruthenium, Loffin etc.), Radioisotope ( 3 H, 14 C, 35 S, 32 P, 125 I, 131 I etc.), Biotin, Digoxigenin, Tag sequence And polypeptides containing gold, colloidal gold particles, and the like.
  • the labeling substance may be directly bound to the antibody, or may be indirectly labeled using an antibody that recognizes the labeling substance, an avidin-biotin system, or the like.
  • the immunological assay is not particularly limited, and conventionally known methods such as enzyme-linked immunosorbent assay (EIA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), radioimmunoassay (RIA) ), Chemiluminescence immunoassay (CLIA), chemiluminescence enzyme immunoassay (CLEIA), electrochemiluminescence immunoassay (ECLIA), immunoblotting, western blotting, agglutination, luminescence immunoassay, spin immunoassay A method, a turbidimetric method which measures turbidity accompanying antigen-antibody complex formation, etc. are used.
  • EIA enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • FIA fluorescent immunoassay
  • RIA radioimmunoassay
  • CLIA Chemiluminescence immunoassay
  • CLIA chemiluminescence enzyme immunoassay
  • the immunoassay is performed by sandwich ELISA, for example, it can be performed as follows. First, the anti-MUC5AC antibody is immobilized on a carrier, and the sample is added and incubated.
  • a carrier for immobilizing the anti-MUC5AC antibody for example, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyester, fluorocarbon resin, glass, metal and the like can be used.
  • the form of the carrier includes plates, beads, cells, test tubes and the like.
  • commercially available solid phase carriers such as microtiter plates for ELISA (96-well microtiter plates) can be used.
  • the method for immobilizing the anti-MUC5AC antibody may be performed according to known methods.
  • an anti-MUC5AC antibody solution dissolved in a suitable buffer is brought into contact with a carrier, and left at 4-37 ° C. for 1-24 hours to immobilize the anti-MUC5AC antibody.
  • a buffer solution such as 1-10% albumin, gelatin, phosphate buffered saline (PBS) containing powdered milk or fetal bovine serum, or Tris buffer (TBS) is used. It is preferable to use as a blocking solution and to block the non-adsorbed portion. After immobilization, the carrier is washed with an appropriate buffer.
  • anti-MUC5AC antibodies that are already immobilized on a solid phase are also commercially available, and, for example, accessories of Cloud-Clone Corp.'s Enzyme-linked immunosorbent assay Kit For Mucin 5 subtype AC (MUC5AC) can be used. .
  • a sample diluted to an appropriate concentration (a tear extract subjected to a sugar chain cleavage treatment by a sugar chain cleaving enzyme treatment or a chemical treatment) is added to the carrier prepared as described above, and the sample is incubated.
  • the existing MUC5AC is reacted with the anti-MUC5AC antibody on a carrier.
  • the incubation is not particularly limited as long as the time is sufficient for the anti-MUC5AC antibody and the MUC5AC contained in the sample to bind to form a complex, but is usually several seconds to several tens of hours.
  • the temperature conditions for incubation are usually 4 ° C. to 50 ° C., preferably 4 ° C. to 45 ° C., more preferably room temperature of about 15 ° C. to 40 ° C., and most preferably 37 ° C.
  • the pH conditions under which the reaction is carried out are not particularly limited as long as it is suitable for the anti-MUC5AC antibody and MUC5AC contained in the sample to bind to form a complex, but, for example, from 6.0 to 8.0 It can be set in the sex area. After the reaction, the reaction solution is removed and the carrier is washed.
  • an enzyme-labeled anti-MUC5AC antibody is added.
  • Enzymes used for labeling include peroxidase, alkaline phosphatase, ⁇ -galactosidase and the like.
  • the enzyme-labeled antibody may be diluted about 100 to 10000 times before incubation. The reaction is carried out, for example, at 4 ° C to 37 ° C for about 1 to 2 hours. After completion of the reaction, the reaction solution is removed and the carrier is washed.
  • the measurement is carried out by adding known color formers (chromogens) according to the enzymes used for labeling.
  • color formers chromogens
  • TMB 3,3'5,5'-tetramethylbenzidine
  • OPD o-phenylenediamine
  • ABTS 2,2'-azino-bis- (3'-ethylbenzodiazo] Phosphorus sulfonic acid
  • DAB diaminobenzidine
  • alkaline phosphatase substrates such as p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, etc.
  • ⁇ -galactosidase Substrates such as o-nitrophenyl- ⁇ -D-galactoside and 4-methylumbelliferyl- ⁇ -D-galactoside may be used as a color former.
  • a biotin-avidin complex system may be used.
  • MUC5AC in a sample is captured with an anti-MUC5AC antibody immobilized on a carrier, and the captured MUC5AC is reacted with another anti-MUC5AC antibody labeled with biotin, and the resulting complex is enzyme-labeled
  • the avidin is added to carry out the biotin-avidin reaction. Then, the amount of MUC5AC is measured by measuring this enzyme activity.
  • enzymes used for labeling include peroxidase, alkaline phosphatase, ⁇ -galactosidase and the like.
  • a biotin-labeled anti-MUC5AC antibody can be produced by binding biotin and an anti-MUC5AC antibody by a method known per se. Such labeling can be performed, for example, using a commercially available biotin labeling kit. Commercially available enzyme-labeled avidin can also be used.
  • a complex is formed by reacting MUC5AC in a sample with another enzyme-labeled anti-MUC5AC antibody and another anti-MUC5AC antibody labeled with biotin, and the resulting complex is used as streptabindin It may be applied to the immobilized plate and the complex may be supplemented by the biotin-avidin reaction. Subsequently, the amount of MUC5AC can be measured by measuring this enzyme activity.
  • enzymes used for labeling include the peroxidase, alkaline phosphatase and ⁇ -galactosidase described above.
  • the enzyme activity is measured by measuring the absorbance of each well (the measurement wavelength differs depending on the substrate and 450 ⁇ 10 nm in the case of TMB) using a commercially available microplate reader.
  • a standard curve calibration curve
  • the MUC5AC standard prepared at a concentration of 5 to 10 was prepared and this standard was used as a sample to measure the absorbance and the concentration of the MUC5AC standard. If the absorbance measured using the subject's tear extract is compared with the standard curve, the MUC5AC concentration in the subject's tear extract can be known.
  • another preferable immunological assay includes electrochemiluminescence immunoassay (ECLIA), and when the immunoassay is performed by sandwich ECLIA, for example, it can be performed as follows.
  • ECLIA electrochemiluminescence immunoassay
  • the anti-MUC5AC antibody is immobilized on a carrier, and the sample is added and incubated.
  • the type and shape of the carrier for immobilizing the anti-MUC5AC antibody may be the same as the sandwich ELISA method described above, and the method for immobilizing the anti-MUC5AC antibody may be performed according to the aforementioned known method.
  • a sample diluted to an appropriate concentration (a tear extract subjected to a sugar chain cleavage treatment by a sugar chain cleaving enzyme treatment or a chemical treatment) is added to the carrier prepared as described above, and the sample is incubated.
  • the existing MUC5AC is reacted with the anti-MUC5AC antibody on a carrier.
  • the conditions for incubation time, temperature, pH
  • the reaction solution is removed and the carrier is washed.
  • a metal complex-labeled anti-MUC5AC antibody is added and reacted, and then washed. Electric energy is applied to the support after the washing on the electrode, and the excitation light emission is measured by the oxidation reaction due to the charge of the electrode and the reduction reaction with tripropylamine (TPA) or the like.
  • TPA tripropylamine
  • a ruthenium complex an osmium complex or the like is used.
  • a ruthenium complex for example, a ruthenium pyridine complex such as tris (2,2'-bipyridyl) ruthenium (II) is preferable.
  • the concentration of MUC5AC in the tear fluid extract can be measured, the amount of MUC5AC present on the eye surface is estimated can do.
  • a step of correcting the measured value can also be performed. The correction can be performed on the basis of the total protein concentration, for example, by dividing the MUC5AC concentration in the tear extract measured in step (d) by the total protein concentration of the tear extract, The relative amount of MUC5AC (the ratio of the amount of MUC5AC in tear fluid extract to the total amount of protein) per amount is calculated.
  • step (d) it is preferable to measure the total protein concentration in the sample as well.
  • the method for measuring the total protein concentration in a sample There are no particular limitations on the method for measuring the total protein concentration in a sample, and BCA method, Bradford method, Lowry method and the like can be mentioned.
  • the present invention also provides a kit for measuring the amount of MUC5AC for carrying out the above-mentioned measuring method.
  • the kit for measuring the amount of MUC5AC of the present invention contains, for example, a "water-absorbent tear fluid collecting member", an “extraction solvent containing a surfactant", a "glycosylation enzyme", and an "anti-MUC5AC antibody”.
  • anti-MUC5AC may be labeled.
  • the anti-MUC5AC antibody may be immobilized on a carrier (eg, a membrane, beads, etc.).
  • the kit for measuring the amount of MUC5AC of the present invention can further include any other reagent, material and the like necessary for carrying out the above-mentioned measuring method.
  • Examples thereof include immobilized carriers, labeled secondary antibodies, reagents for detection, reaction buffers, washing buffers, reaction stop solutions, color reagents, standard substances (positive control, negative control), It may also contain dilution reagents, instructions for use, and the like.
  • the “sugar chain cleaving agent” can be included instead of the “sugar chain cleaving enzyme” in the kit.
  • the amount of MUC5AC in tears is measured using the above-mentioned immunological measurement method
  • Diseases associated with decreased levels of MUC5AC in tears can be examined or diagnosed.
  • “examination” or “diagnosis” typically means determination of whether or not a subject suffers from a disease associated with a decrease in the amount of MUC5AC in tears, but is not limited thereto. It does not include the determination of the degree of progression of the disease, the determination of the therapeutic effect on the disease, and the determination of whether or not there is a risk of recurrence of the disease after treatment.
  • “examination” or “diagnosis” can be replaced with the term “examination of examination” or “diagnosis assistance” not including diagnosis by a doctor, and specifically, "examination” or “diagnosis” To get the data for.
  • Non-Sjogren's syndrome type dry eye is associated with lacrimal gland diseases such as congenital alacriminosis, sarcoidosis, graft versus host disease (GVHD) due to bone marrow transplantation; Pemphigus ophthalma, Stevens ⁇ Stevens ⁇ Associated with lacrimal obstruction caused by Johnson's syndrome, trachoma, etc .; Included are those associated with decreased reflex secretion caused by diabetes, corneal refractive surgery (LASIK: Laser (-assisted) in Situ Keratomileus), etc.
  • LASIK Laser (-assisted) in Situ Keratomileus
  • Be "Tear Evaporation Type Dry Eye” is associated with loss of oil layer caused by meibom's gland dysfunction, blepharitis, etc .; with blink failure or closure failure caused by ocular protrusion, eyelid etc .; With the fall of the tear fluid stability by contact lens wearing; With the mucin secretion from goblet cells; With the thing of VDT work etc. are mentioned.
  • the presence or absence of the onset of the disease and / or the progress of the disease can be determined by the amount of MUC5AC by comparing the amount of MUC5AC in the sample derived from the subject with the amount of MUC5AC in the control sample.
  • the control sample refers to a sample serving as a reference for comparison, which is a tear extract of a normal person (negative control), a tear extract of dry eye patients having a lower MUC5AC amount than a predetermined reference value (positive control) It may be any.
  • the measurement of the amount of MUC5AC in the control sample can be performed by the same procedure as the measurement of the amount of MUC5AC in the sample derived from the subject.
  • the amount of MUC5AC in the control sample may be measured each time the amount of MUC5AC in the subject sample is measured, or may be measured in advance. If the amount of MUC5AC in the subject-derived sample is significantly lower than the amount of MUC5AC in the negative control, then the subject is determined to suffer from a disease associated with a decrease in the amount of MUC5AC and is equivalent to the amount of MUC5AC in the negative control It is determined that the subject is not afflicted with a disease associated with a decrease in the amount of MUC5AC.
  • the amount of MUC5AC in the subject-derived sample is significantly higher than the amount of MUC5AC in the positive control, it is determined that the subject is not afflicted with a disease associated with a decrease in the amount of MUC5AC, or that the symptom has been improved by the treatment. it can.
  • the measurement of the amount of MUC5AC in the subject-derived sample may be performed by measuring the absolute amount by comparison with the amount of MUC5AC in the control sample, etc., it is not necessary to measure the absolute amount of MUC5AC. It is sufficient if the relative relationship with the amount of MUC5AC is revealed.
  • a method of selecting a patient having a decreased amount of MUC5AC based on the amount of MUC5AC in the subject's tear fluid measured by the above-mentioned measurement method By using this method, it is possible to select patients with a decreased amount of MUC5AC, but by correcting the MUC5AC concentration in the obtained tear solution extract with the total protein concentration in the tear solution extract, it is in the tears of the subject.
  • the amount of MUC5AC in the total protein of can be calculated with higher accuracy.
  • the cut-off value of the amount of MUC5AC in the tear fluid may be set in advance and compared with the measured amount of MUC5AC.
  • the reference MUC5AC amount (cutoff value) is an appropriate reference set according to the purpose.
  • the “cut-off value” is a value that can satisfy both high diagnostic sensitivity and high diagnostic specificity when determining the onset of a disease based on that value.
  • the amount of MUC5AC in the tear fluid can be set as a cutoff value, which shows a high positive rate in dry eye patients and a high negative rate in normal people.
  • cutoff values are well known in the art. For example, the amount of MUC5AC in tears of dry eye patients and normal persons is measured to determine the diagnostic sensitivity and diagnostic specificity at the measured values, and ROC (Receiver) is obtained using commercially available analysis software based on these values. Operating Characteristic) Create a curve. Then, a value when the diagnostic sensitivity and the diagnostic specificity are as close to 100% as possible is obtained, and the value can be used as a cutoff value.
  • Dry eye can be treated by administering a therapeutically effective amount of the dry eye therapeutic agent to a patient having a decreased amount of MUC5AC selected by the above method.
  • the dry eye therapeutic agent is not particularly limited as long as it is a mucin secretion production promoter, and examples thereof include diquafosol sodium, rebamipide, or salts thereof, and those containing diquafosol sodium as the only active ingredient Is preferred.
  • the preferred concentration of diquafosol sodium is 1 to 5% (w / v), more preferably 3% (w / v).
  • Test Example 1 Extraction of MUC 5 AC and Total Protein from Silmer Test Paper (Influence of Surfactant) (1) Test method 10 ⁇ L of commercially available normal human tear fluid (collecting manufacturer: Lee BioSolutions, Inc., import sales: Vidicom Japan Ltd.) was spotted with Schilmel test paper (Ayumi Pharmaceutical Co., Ltd.) with a pipette.
  • the tear-impregnated Schirmel test paper is placed in a cryotube (Corning Inc.), and as a pretreatment solution (extraction solvent), 0.05%, 0.2% or 0.5% polysorbate 20 (product name: polyoxyethylene ( 20)
  • a pretreatment solution extraction solvent
  • polysorbate 20 product name: polyoxyethylene ( 20)
  • the paper was immersed in each of these solutions and extracted by shaking at 25 ° C. for 1 hour with a plate mixer equipped with a thermostat function.
  • sample The concentration of MUC5AC in the tear solution extract (hereinafter referred to as "sample") obtained by the above extraction was measured using the following ELISA method, and the total protein concentration in the sample was measured using the BCA protein assay.
  • Reference sample 10 ⁇ L of normal human tear fluid and 590 ⁇ L of each PBS / 12 mM EDTA / polysorbate 20 solution different in content of polysorbate 20 mixed (hereinafter referred to as “ Reference sample was used.
  • ELISA method The amount of MUC5AC in the sample (as well as the reference sample) was measured according to the following procedure. The measurement was performed using a Cloud-Clone Corp. ELISA kit: Enzyme-linked immunosorbent Assay Kit For Mucin 5 Subtype AC (MUC5AC).
  • Detection Reagent A solution was prepared by diluting Detection Reagent A (the kit accessory) 100 times with Assay Diluent A (the kit accessory) and mixing. The contents of the plate were discarded and each well was washed 3 times with Wash solution.
  • the Wash solution was prepared by diluting Wash Buffer (30 ⁇ concentrate) (the above kit accessory) with ultrapure water (manufactured by an ultrapure water production apparatus) 30 times and mixing.
  • Detection Reagent B solution 100 ⁇ L was added to each well of the plate, and incubation (setting: 400 rpm) was carried out at 37 ° C. for 30 minutes in the dark with a plate mixer equipped with a thermostatic function.
  • the Detection Reagent B solution was prepared by diluting Detection Reagent B (the kit accessory) 100 times with Assay Diluent B (the kit accessory) and mixing. The contents of the plate were discarded and each well was washed 5 times with Wash solution.
  • TMB Substrate (the above kit accessory) was added, and incubation (setting: 400 rpm) was carried out at 37 ° C. for 10 to 20 minutes in the dark with a plate mixer equipped with a thermostatic function.
  • 50 ⁇ L of Stop Solution (kit of the above kit) was added and mixed using a plate mixer. The absorbance at 450 nm was immediately measured with a microplate reader.
  • BCA protein assay The total protein concentration in the samples was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Specifically, a standard sample solution for a calibration curve prepared by diluting 2 mg / mL of Albumin Standard Ampules (the above kit accessory) with PBS / 12 mM EDTA / 0.05% polysorbate 20 in a 96-well blank plate and tear fluid 10 ⁇ L of extract was added.
  • Test Example 2 Extraction of MUC 5 AC and Total Protein from Silmer Test Paper (Influence of Extraction Temperature)
  • Test method 10 ⁇ L of commercially available normal human tear fluid collecteding manufacturer: Lee BioSolutions, Inc., import sales: Vidicom Japan Ltd.
  • Schilmel test paper Auto-impregnated Schirmel test paper
  • the tear-impregnated Schirmel test paper is placed in a cryotube (Corning Inc.), to which 600 ⁇ L of PBS or PBS / 12 mM EDTA / 0.05% polysorbate 20 solution is added as a pretreatment solution (extraction solvent).
  • the Schirmel test paper was dipped in the solution and extracted by shaking for 1 hour at 25 ° C. or 37 ° C. using a plate mixer equipped with a thermostatic function.
  • the MUC5AC concentration and total protein concentration in the solution after extraction were measured in the same manner as in Test Example 1, and the extraction rate was calculated by the above (formula I).
  • a reference sample for calculating the extraction rate a mixture of 10 ⁇ L of human tears of the normal person and 590 ⁇ L of PBS or PBS / 12 mM EDTA / 0.05% polysorbate 20 solution was used.
  • Example with enzyme treatment 2000 units / mL of Neuraminidase A solution ( ⁇ 2-3, 6, 8, 9 Neuraminidase A (New England Biolabs Inc.) was added to 220 ⁇ L of the above tear solution extract before adding ELISA plate. 10 ⁇ L of the treatment solution (diluted 10-fold with extraction solvent) was added and mixed. As a sample without enzyme treatment, 10 ⁇ L of pretreatment solution (extraction solvent) was added instead of 2000 units / mL of Neuraminidase A solution.
  • the MUC5AC concentration in the obtained lacrimal fluid extract hereinafter, referred to as “sample with enzyme treatment”, “sample without enzyme treatment” was measured by the ELISA method in the same manner as in Test Example 1. The measurement was performed on 2 human tears (tears 1 and 2).
  • Test Example 4 Confirmation of Dilution Linearity (1) Test Method A commercially available normal human human tear fluid (collection maker: Lee BioSolutions, Inc., import and sale: Vidicom Japan Co., Ltd.) is pretreated (PBS / 12 mM EDTA) Dilute to 60, 120 times, 240 times, 480 times with 0.05% polysorbate 20 (also referred to as “extraction solvent”) and add 2000 units / mL of Neuraminidase A solution ( ⁇ After 3,6,8,9 Neuraminidase A (New England Biolabs Inc.) was diluted 10-fold with a pretreatment solution (extraction solvent), each MUC5AC concentration was measured by ELISA. The measurement was performed on 2 human tears (tears 3 and 4).
  • the ELISA method was carried out in the same manner as in Test Example 1 except that blocking treatment was carried out before sample addition.
  • blocking treatment add 1 bag of Block Ace powder (DS Pharma Biomedical Co., Ltd.) to 100 mL of blocking solution (Kyoei Pharmaceutical Co., Ltd.) and dissolve it, and then dilute this with 4-fold dilution with purified water. Prepared by adding 300 .mu.L to each well of the plate and leaving it for 30 minutes or more at room temperature. After blocking, the contents of the plate were discarded (not washed). The converted value was calculated for each sample by the following formula (II).
  • a dilution linearity test is one of methods for evaluating the measurement accuracy of the measurement object in a biological sample (tears etc.) by an immunological method. As shown in Table 4, it was confirmed that the method of the present invention was a highly accurate and reliable measurement system for the amount of MUC5AC because good dilution linearity was obtained.
  • Test Example 5 Measurement of MUC5AC amount in tears of dry eye patients (1) Test method From the dry eye patient group (25 eyes), normal human (non dry eye) group (26 eyes) Schirmer's test paper (Ayumi Pharmaceutical Co., Ltd.) Cryotube (Corning Inc.) containing 600 ⁇ L of pretreatment solution (PBS / 12 mM EDTA / 0.05% polysorbate 20, also referred to as “extraction solvent”) after collecting tears for 5 minutes using a corporation It was mixed and tumbled. Immediately thereafter, it was frozen with dry ice. Frozen samples were stored frozen at -80.degree. C. until measurement. At the time of measurement, it was thawed at room temperature, and after thawed, the plate mixer was put in an incubator at 37 ° C., shake extracted (setting: 700 rpm) for 1 hour with the mixer, tear fluid was extracted.
  • pretreatment solution PBS / 12 mM EDTA / 0.05% polysorbate 20
  • a solution of 2000 units / mL of Neuraminidase A solution ( ⁇ 2-3, 6, 8, 9 Neuraminidase A (New England Biolabs Inc.) was diluted 10-fold with 220 ⁇ L of the obtained tear solution extract with a pretreatment solution (extraction solvent) After addition of 10 ⁇ L, each MUC5AC concentration was measured by ELISA in the same manner as in Test Example 1.
  • the ELISA plate was subjected to blocking treatment in advance in the same manner as in Test Example 4. Further, the total protein concentration in the tear solution extract was measured by BCA protein assay in the same manner as in Test Example 1.
  • Test Example 6 Clinical Test Using Diquafosol Sodium Ophthalmic Solution The clinical application of the method for measuring the amount of MUC5AC in tears of the present invention will be described.
  • Schirmer test method I keratoconjunctive for about 2 weeks (14 ⁇ 3 days), eye drops of placebo eye drops six times a day for patients diagnosed as dry eye in both eyes Fluorescein staining and confirmation of the presence / degree of subjective symptoms.
  • a placebo a base of 3% (w / v) diquafosol sodium ophthalmic solution described later is used.
  • a solution of 2000 units / mL of Neuraminidase A solution ( ⁇ 2-3, 6, 8, 9 Neuraminidase A (New England Biolabs Inc.) was diluted 10-fold with 220 ⁇ L of the obtained tear solution extract with a pretreatment solution (extraction solvent) 1)
  • the concentration of each MUC5AC is measured by the ELISA method described in Test Example 1.
  • the ELISA plate is subjected to blocking treatment in advance in the same manner as in Test Example 4.
  • the total protein concentration in the tear fluid extract is measured by the BCA protein assay described in Test Example 1.
  • the present invention is available in the field of dry eye test drug production.

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Abstract

La présente invention aborde le problème de la fourniture d'un procédé et d'un kit pour mesurer de manière simple et précise une quantité de MUC5AC dans les larmes d'un sujet. Selon la présente invention, un procédé de mesure d'une quantité de MUC5AC dans les larmes d'un sujet comprend les étapes suivantes : (a) une étape consistant à absorber une larme dans un élément absorbant de collecte de larme ; (b) une étape consistant à extraire, à partir de l'élément de collecte de larme, une larme à l'aide d'un solvant d'extraction contenant un tensio-actif et obtenir un extrait de larme ; (c) une étape consistant à effectuer un traitement de clivage de chaîne glucidique sur l'extrait de larme par l'intermédiaire d'un traitement enzymatique ou d'un traitement chimique de clivage de chaîne glucidique ; et (d) une étape consistant à mesurer une quantité de MUC5AC dans l'extrait de larme soumis au traitement enzymatique de clivage de chaîne glucidique par un dosage immunologique à l'aide d'un anticorps anti-MUC5AC.
PCT/JP2018/036175 2017-09-29 2018-09-28 Procédé de mesure de la quantité de muc5ac dans les larmes WO2019065936A1 (fr)

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