WO2012077737A1 - Anti-psk polyclonal antibody, psk immunological analysis method using same, and psk immunological analysis kit - Google Patents

Anti-psk polyclonal antibody, psk immunological analysis method using same, and psk immunological analysis kit Download PDF

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Publication number
WO2012077737A1
WO2012077737A1 PCT/JP2011/078368 JP2011078368W WO2012077737A1 WO 2012077737 A1 WO2012077737 A1 WO 2012077737A1 JP 2011078368 W JP2011078368 W JP 2011078368W WO 2012077737 A1 WO2012077737 A1 WO 2012077737A1
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psk
antibody
glucan
polyclonal antibody
antigen
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PCT/JP2011/078368
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French (fr)
Japanese (ja)
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裕孝 星
光 齋藤
勉 和田
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株式会社クレハ
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present invention relates to an anti-PSK polyclonal antibody and an immunological analysis method for PSK using the same.
  • Protein polysaccharides extracted from Kawaratake show antitumor activity and the like, and antitumor agents containing the protein polysaccharide as an active ingredient are described in, for example, JP-A-60-45533 (Patent Document 1).
  • PSK registered trademark
  • Kirestin registered trademark
  • PSK is a protein polysaccharide containing about 18 to 38% protein, and has a molecular weight of 5000 or more (gel filtration method), for example, 5000 to 300,000 (gel filtration method).
  • the sugar part of the main fraction is ⁇ -D-glucan, and the structure of this glucan part is a branched structure containing 1 ⁇ 3, 1 ⁇ 4 and 1 ⁇ 6 bonds.
  • Non-Patent Document 1 discloses a rabbit polyclonal antibody against PSK, which describes that PSK can be detected by a fluorescent antibody method using this polyclonal antibody.
  • this polyclonal antibody uses PSK as an immunogen, it is an antibody that binds to polysaccharides other than PSK and is not an antibody specific to PSK.
  • PSK was obtained by extracting with hot water or an alkali solution (for example, an alkali metal hydroxide, particularly an aqueous solution of sodium hydroxide), followed by purification and drying as described below.
  • an alkali solution for example, an alkali metal hydroxide, particularly an aqueous solution of sodium hydroxide
  • PSK produced by such a method has a sufficient effect as a pharmaceutical, but when this PSK is used as an immunogen for obtaining an antibody, as described in Non-patent Document 1.
  • only polyclonal antibodies that bind to polysaccharides other than PSK were obtained, and PSK could not be measured specifically.
  • an object of the present invention is to provide a polyclonal antibody that reacts specifically with PSK, and to provide an immunological analysis method of PSK that can specifically detect PSK.
  • the present invention relates to [1] an immunological analysis method for PSK, which comprises using an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof.
  • the PSK immunological analysis method of the present invention enables specific detection of PSK.
  • the present invention also provides [2] the immunological analysis of PSK according to [1], wherein the anti-PSK polyclonal antibody is an anti-PSK polyclonal antibody that recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK. It relates to the analysis method.
  • the present invention also provides [3] that the anti-PSK polyclonal antibody reacts with PSK and does not react with the ⁇ 1,4 glucan glucan structure of cellulose and the ⁇ 1,3 glucan of laminarin and the glucan structure of ⁇ 1,6 glucan.
  • the present invention relates to an immunological analysis method for PSK [1] or [2], which is an anti-PSK polyclonal antibody.
  • the present invention also provides [4] a glucan structure of ⁇ 1,4 glucan of cellulose or ⁇ 1,3 glucan of laminarin and glucan of ⁇ 1,6 glucan when the binding force of the anti-PSK polyclonal antibody to PSK is 100.
  • the present invention relates to the immunological analysis method for PSK according to [3], wherein the binding force to the structure is 30% or less.
  • the present invention also provides [5] enzyme immunoassay, immunohistochemical staining, surface plasmon resonance, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation, western blot
  • the present invention relates to an immunological analysis method for PSK according to any one of [1] to [4], using a method, an immunochromatographic method, a magnetic bead aggregation method, or a magnetic bead enzyme immunization method. By using these analysis methods, various PSK measurements can be performed.
  • the present invention also uses [6] an anti-PSK polyclonal antibody or antigen-binding fragment thereof that specifically binds to PSK, and a second antibody or antigen-binding fragment thereof that reacts with PSK, [1 ] To an immunological analysis method for PSK according to any one of [5] to [5].
  • the present invention also provides [7] Immobilization wherein the anti-PSK polyclonal antibody or antigen-binding fragment thereof, or the second antibody or antigen-binding fragment thereof is immobilized, or an antigen-binding fragment thereof.
  • An insoluble carrier having an antibody, a test sample possibly containing PSK, the remaining antibody or a labeled antibody labeled with an antigen-binding fragment thereof is contacted, and the immobilized antibody and PSK are labeled on the insoluble carrier.
  • the present invention relates to the immunological analysis method for PSK of [6], comprising a complex formation step for forming a complex with an antibody, and an analysis step for analyzing a signal from a label in the complex.
  • the present invention also provides [8] an insoluble carrier in which the complex-forming step immobilizes an anti-PSK monoclonal antibody or an antigen-binding fragment thereof as a second antibody, and a test sample that may contain PSK.
  • One of the specific embodiments in this embodiment is a sandwich method. By using the sandwich method as the immunological analysis method for PSK of the present invention, it is possible to measure a wide dynamic range of PSK.
  • the present invention also relates to [9] the immunological analysis method for PSK according to any one of [6] to [8], wherein the second antibody is an anti-PSK monoclonal antibody.
  • the second antibody is an anti-PSK monoclonal antibody.
  • the present invention also relates to [10] an immunological analysis method for PSK according to any one of [1] to [9], which measures blood concentration. By measuring the blood concentration, it is possible to predict the efficacy of PSK and measure the optimal concentration of PSK.
  • the present invention also relates to [11] an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof.
  • an anti-PSK polyclonal antibody or antigen-binding fragment thereof of the present invention is used in the immunological analysis method for PSK, it can measure PSK specifically.
  • the present invention also provides [13]
  • the anti-PSK polyclonal antibody reacts with PSK and does not react with the ⁇ 1,4 glucan glucan structure of cellulose, and the ⁇ 1,3 glucan of laminarin and the glucan structure of ⁇ 1,6 glucan. [11] or [12], or an antigen-binding fragment thereof.
  • the present invention also provides [14] wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2 , Fv fragment, diabody, single chain antibody molecule, and multispecific antibody.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2 , Fv fragment, diabody, single chain antibody molecule, and multispecific antibody.
  • the present invention relates to a kit for immunological analysis of PSK, comprising [15] an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof.
  • a kit for immunological analysis of PSK of the present invention PSK-specific measurement can be performed more simply.
  • the present invention also relates to [16] the kit for immunological analysis of PSK according to [15], wherein the anti-PSK polyclonal antibody recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK. .
  • the present invention also provides [17]
  • the anti-PSK polyclonal antibody reacts with PSK and does not react with the ⁇ 1,4 glucan glucan structure of cellulose, and the ⁇ 1,3 glucan of laminarin and the glucan structure of ⁇ 1,6 glucan.
  • the kit for immunological analysis of PSK according to [15] or [16].
  • the present invention also relates to a kit for immunological analysis of PSK according to any one of [15] to [17], further comprising [18] an anti-PSK monoclonal antibody or an antigen-binding fragment thereof.
  • an anti-PSK polyclonal antibody specific for PSK is used.
  • an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK or an antigen-binding fragment thereof is used.
  • an anti-PSK polyclonal antibody that reacts with PSK and does not react with the ⁇ 1,4 glucan glucan structure of cellulose and the ⁇ 1,3 glucan and ⁇ 1,6 glucan glucan structures of laminarin can also be used.
  • the anti-PSK polyclonal antibody of the present invention since the anti-PSK polyclonal antibody of the present invention has high specificity for PSK, specific detection of PSK is possible by using the polyclonal antibody of the present invention. Furthermore, it is possible to increase the measurement sensitivity in the analytical method and reduce the amount of anti-PSK polyclonal antibody used. In addition, when an anti-PSK polyclonal antibody is used in the sandwich method, measurement with a wide dynamic range of PSK is possible.
  • the immunological analysis method for PSK according to the present invention has an effect that the amount of PSK contained in a medicine can be accurately measured and used for quality control of the medicine. Moreover, it is possible to measure PSK administered to a living body, and it is possible to examine the pharmacokinetics of PSK.
  • the immunological analysis method of PSK according to the present invention can examine the pharmacokinetics of PSK, it should also be used for data measurement for predicting the efficacy of PSK and grasping the optimal concentration of PSK. Can do.
  • the anti-PSK polyclonal antibody of the present invention is an anti-PSK polyclonal antibody that specifically binds to PSK. It may also be an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK, and reacts with PSK, and the glucan structure of ⁇ 1,4 glucan of cellulose, and laminarin. It may be an anti-PSK polyclonal antibody that does not react with the glucan structure of ⁇ 1,3 glucan and ⁇ 1,6 glucan.
  • PSK is obtained by extracting the mycelium of Kawaratake fungus CM101 strain [FERM-P2412 (ATCC20547)] with an aqueous solvent, for example, hot water or an alkaline solution (for example, an alkali metal hydroxide, particularly an aqueous solution of sodium hydroxide). It can be obtained after purification and drying.
  • the sugar part of the main fraction is ⁇ -D-glucan, and the structure of this glucan part is a branched structure containing ⁇ 1 ⁇ 3, ⁇ 1 ⁇ 4 and ⁇ 1 ⁇ 6 linkages, and the main constituent monosaccharides are glucose and mannose. Yes, containing about 18-38% protein.
  • the constituent amino acids of proteins are mostly acidic amino acids such as aspartic acid and glutamic acid, and neutral amino acids such as valine and leucine, and few basic amino acids such as lysine and arginine. Although soluble in water, it is hardly soluble in methanol, pyridine, chloroform, benzene or hexane.
  • the anti-PSK polyclonal antibody of the present invention does not recognize laminarin ⁇ 1,3 glucan.
  • ⁇ 1,3 glucan is a polysaccharide in which glucose is linked by ⁇ 1-3 type bonds. Widely distributed in nature such as plants, fungi and bacteria.
  • ⁇ 1,3 glucan is a glucan present in laminarin or yeast glucan, but the anti-PSK polyclonal antibody of the present invention is a polyclonal antibody that does not bind to laminarin and therefore does not recognize laminarin ⁇ 1,3 glucan. .
  • the anti-PSK polyclonal antibody of the present invention does not recognize the glucan structure of ⁇ 1,4 glucan of cellulose.
  • ⁇ 1,4 glucan is present in cellulose or glycogen.
  • Cellulose is a polysaccharide represented by the molecular formula (C 6 H 10 O 5 ) n and is the main component of plant cell walls and fibers.
  • the anti-PSK polyclonal antibody of the present invention is a polyclonal antibody that does not bind to cellulose and therefore does not recognize the glucan structure of ⁇ 1,4 glucan of cellulose.
  • the anti-PSK polyclonal antibody of the present invention does not recognize ⁇ 1,6 glucan of laminarin.
  • ⁇ 1,6 glucan is present in laminarin or dextran.
  • Dextran is a kind of polysaccharide consisting only of glucose.
  • the anti-PSK polyclonal antibody of the present invention is a polyclonal antibody that does not bind to laminarin and therefore does not recognize ⁇ 1,6 glucan of laminarin.
  • “specifically binds to PSK” means that it binds to PSK and does not substantially bind to at least laminarin and cellulose glucan.
  • the fact that it does not substantially bind to laminarin and cellulose glucan means that nonspecific reaction does not occur at a practical level when the antibody of the present invention is used in the PSK analysis method.
  • the determination can be made by examining the binding of the immobilized anti-PSK polyclonal antibody to PSK, laminarin or cellulose using Biacore.
  • the force is preferably 30% or less, more preferably 20% or less, and most preferably 10% or less.
  • the protein portion of PSK that may be recognized by the anti-polyclonal antibody of the present invention occupies about 18 to 38% of the protein of PSK, and the amino acids constituting the protein include acidic amino acids such as aspartic acid and glutamic acid, valine, leucine and the like. There are many neutral amino acids and few basic amino acids such as lysine and arginine.
  • the glucan structure specific to PSK that may be recognized by the anti-polyclonal antibody of the present invention is a branched structure containing ⁇ 1 ⁇ 3, ⁇ 1 ⁇ 4 and ⁇ 1 ⁇ 6 bonds, and the main constituent monosaccharides are glucose and mannose. is there.
  • the glucan structure specific to PSK is different from the glucan structure of ⁇ 1,4 glucan of cellulose or the glucan structure of ⁇ 1,3 glucan and ⁇ 1,6 glucan of laminarin.
  • the anti-PSK polyclonal antibody of the present invention does not substantially contain an antibody that binds to the laminarin ⁇ 1,3 glucan and ⁇ 1,6 glucan glucan structures and the cellulose ⁇ 1,4 glucan glucan structure.
  • the reason for this is that the hot water / alkaline extract of Kawaratake used for immunization substantially contains laminarin ⁇ 1,3 glucan and ⁇ 1,6 glucan, and cellulose ⁇ 1,4 glucan identical or similar in structure to glucan. This is thought to be because there is not.
  • the hot water / alkaline extract of Kawaratake used to obtain the anti-PSK polyclonal antibody of the present invention can be produced, for example, as follows.
  • the obtained fungus moss is homogenized to prepare the inoculum.
  • the inoculum is cultured according to a conventional method and dried after the culture to obtain a dried mycelium. Extraction is performed from the dried mycelium using hot water and an alkaline solution.
  • the obtained hot water extract and alkali extract are mixed, neutralized with dilute hydrochloric acid, and further concentrated. Salt out and collect the precipitate. From the obtained precipitate, a hot water / alkali extract can be obtained as a lyophilized product.
  • extraction with hot water and an alkaline solution extraction with hot water is first performed, and the aqueous solution is extracted from the residue obtained by centrifugation.
  • the obtained hot water extract and the aqueous alkaline solution are mixed and subjected to processes such as salting out and precipitation to obtain a hot water / alkaline extract.
  • the temperature of hot water is not particularly limited as long as it is 50 ° C or higher, but is preferably 70 ° C or higher, more preferably 80 ° C or higher, and most preferably 95 to 99 ° C.
  • the extraction time with hot water is not particularly limited, but is preferably 10 minutes to 6 hours, more preferably 1 hour to 3 hours, and most preferably 30 minutes to 90 minutes.
  • the alkali solution is not particularly limited.
  • an alkali metal hydroxide can be used, and a sodium hydroxide aqueous solution is preferable.
  • the concentration is not particularly limited as long as it is 0.2 M or higher, but is preferably 0.3 M to 3 M, more preferably 0.5 M to 1.5 M, and Most preferred is 7M to 1.3M.
  • the temperature of the alkaline solution is not particularly limited as long as it is 4 ° C. or higher, but is preferably 25 ° C. or higher, more preferably 50 ° C. or higher, and most preferably 95 to 99 ° C.
  • the extraction time with an alkaline solution is not particularly limited, but is preferably 10 minutes to 6 hours, more preferably 1 hour to 3 hours, and most preferably 30 minutes to 90 minutes.
  • a conventional method for extracting PSK is, for example, extraction with a 0.1 M aqueous sodium hydroxide solution as described in the Examples of Patent Document 2.
  • the PSK obtained by such an extraction method is considered to contain glucan having the same or similar structure to ⁇ 1,3 glucan and ⁇ 1,6 glucan of laminarin and ⁇ 1,4 glucan of cellulose.
  • ⁇ 1,3 glucan and ⁇ 1,6 glucan of laminarin, and ⁇ 1 of cellulose , 4 glucan is considered to be obtained that does not contain the same or similar structure glucan.
  • the antigen-binding fragment of the present invention is not particularly limited as long as it contains the antigen-binding site of the anti-PSK polyclonal antibody of the present invention and has binding ability to PSK.
  • These antigen-binding fragments can be obtained, for example, by digesting an antibody with a proteolytic enzyme (for example, pepsin or papain) by a conventional method, and subsequently purifying the protein by a conventional method for separating and purifying proteins. Can do.
  • a proteolytic enzyme for example, pepsin or papain
  • multispecific antibodies formed from diabodies, single chain antibody molecules, and antibody fragments can be prepared by genetic recombination.
  • the immunological analysis method of PSK of the present invention is characterized by using an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof.
  • the anti-PSK polyclonal antibody or antigen-binding fragment thereof that specifically binds to PSK the antibody described in the item “[1] Anti-PSK polyclonal antibody” can be used.
  • an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific for PSK may be used, reacts with PSK, and the glucan structure of ⁇ 1,4 glucan of cellulose, and of laminarin.
  • An anti-PSK polyclonal antibody that does not react with the glucan structure of ⁇ 1,3 glucan and ⁇ 1,6 glucan may be used.
  • PSK can be quantitatively or semi-quantitatively determined using the anti-PSK polyclonal antibody of the present invention or an antigen-binding fragment thereof, or the presence of PSK.
  • enzyme immunoassay, immunohistochemical staining, surface plasmon resonance (SPR method: Biacore method), latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation, western A blotting method, an immunochromatography method, a magnetic bead aggregation method, or a magnetic bead enzyme immunization method can be mentioned.
  • analysis means both “measurement” that quantitatively or semi-quantitatively determines the amount of an analyte, and “detection” that determines the presence or absence of the analyte. Is included.
  • test sample The test sample that can be used in the immunological analysis method of PSK is not particularly limited as long as it is a sample that may contain PSK, but in particular, it may contain PSK having physiological activity.
  • examples include biological samples or biological samples of patients who have sex medicine or food or drink, or PSK.
  • pharmaceuticals or foods and drinks pharmaceutical compositions, pharmaceutical preparations, hot water / alkaline extracts derived from fungi used as raw materials thereof, health foods, functional foods, or raw materials thereof And hot water / alkaline extract derived from fungi.
  • biological samples or biological samples include urine, blood, serum, plasma, feces, spinal fluid, saliva, cells, tissues, organs, or preparations thereof (for example, biopsy specimens). be able to.
  • a specific hot water / alkaline extract can be detected qualitatively and quantitatively from medicines, foods and drinks, blood and tissues after taking PSK. Therefore, it is very useful because the intake (dose) of physiologically active PSK can be grasped. For example, since the blood concentration after PSK administration and the degree of PSK reaching the tumor can be determined easily and with high accuracy, pharmacokinetics and drug efficacy can be evaluated easily and with high accuracy.
  • the blood concentration by the immunological analysis method of PSK of the present invention can be used for the measurement of data for predicting the drug effect of PSK and grasping the optimum concentration of PSK.
  • the blood concentration of PSK in a patient to whom PSK is administered is measured after a certain time after PSK administration. From the obtained blood concentration, if the blood concentration is above a certain level, it can be predicted that the PSK will have high efficacy, and if it is below a certain level, it can be predicted that the PSK will have low efficacy. It is.
  • the optimal concentration of PSK may vary depending on the patient.
  • grasping the optimal concentration of PSK is important in the treatment of patients.
  • the blood concentration of PSK in a patient to whom PSK is administered is measured after a certain time after the administration of PSK.
  • the dose of PSK can be increased.
  • the blood concentration is high, the dose of PSK can be decreased.
  • PSK can be analyzed using an anti-PSK polyclonal antibody that specifically binds to PSK or one of its antigen-binding fragments.
  • an anti-PSK polyclonal antibody that specifically binds to PSK or one of its antigen-binding fragments.
  • immunohistological staining method surface plasmon resonance method (SPR method: Biacore method), latex agglutination immunoassay method, fluorescent antibody method, radioimmunoassay method, immunoprecipitation method, or Western blot method may be used.
  • SPR method surface plasmon resonance method
  • latex agglutination immunoassay method fluorescent antibody method
  • radioimmunoassay method radioimmunoassay method
  • immunoprecipitation method or Western blot method.
  • an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof, and a second antibody that reacts with PSK or its PSK can be analyzed using antigen-binding fragments.
  • the anti-PSK polyclonal antibody used in this embodiment is the anti-PSK polyclonal antibody of the present invention.
  • Specific examples of this embodiment include a sandwich method using an anti-PSK polyclonal antibody and a second antibody.
  • immunohistological staining method using a mixture of anti-PSK polyclonal antibody and second antibody, surface plasmon resonance method (SPR method: Biacore method), latex agglutination immunoassay method, fluorescent antibody method, radioimmunoassay method, immunoprecipitation method Or Western blotting.
  • SPR method surface plasmon resonance method
  • latex agglutination immunoassay method fluorescent antibody method
  • radioimmunoassay method radioimmunoassay method
  • immunoprecipitation method Or Western blotting.
  • two or more types of second antibodies can also be used.
  • the second antibody is not particularly limited as long as it can bind to PSK.
  • it recognizes PSK and other polysaccharides ⁇ 1,3 glucan, ⁇ 1,4 glucan, and ⁇ 1,6
  • the polyclonal antibody described in Non-Patent Document 1 that recognizes glucan or the anti-PSK antibody that specifically binds to PSK can be mentioned, and a PSK-specific antibody is preferred.
  • PSK-specific antibody examples include an anti-PSK polyclonal antibody that specifically binds to the PSK of the present invention (for example, an anti-PSK polyclonal antibody that recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK).
  • an anti-PSK polyclonal antibody that reacts with PSK and does not react with the ⁇ 1,4 glucan glucan structure of cellulose and the ⁇ 1,3 glucan and ⁇ 1,6 glucan glucan structures of laminarin); and recognizes PSK and dextran
  • Examples include monoclonal antibodies that do not recognize ⁇ 1,3 glucan, ⁇ 1,4 glucan, and ⁇ 1,6 glucan of glycogen, laminarin, and cellulose; or monoclonal antibodies that recognize a physiologically active site of PSK, most preferably Physiological activity of PSK Place is a monoclonal antibody that recognizes. This is because PSK having activity can be accurately measured.
  • the second antibody can be prepared according to a conventional method except for the anti-PSK polyclonal antibody of the present invention.
  • monoclonal antibodies can be prepared according to the method of Koehler and Milstein (Nature 256: 495-497, 1975).
  • the immunizing antigen for obtaining the anti-PSK antibody is obtained by using a mycelium of Kawaratake fungus CM101 strain [FERM-P2412 (ATCC20547)] in an aqueous solvent such as hot water or an alkaline solution (for example, alkali metal hydroxide, particularly It can be used without particular limitation as long as it is obtained by extraction with an aqueous solution of sodium hydroxide), purification and drying, and it has antitumor activity.
  • monoclonal antibodies that recognize PSK bioactive sites include the following antibodies.
  • the monoclonal antibody (A) is typified by the 2G9 antibody described in Reference Examples described later.
  • the heavy chain variable region domain is preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 (SYGMS), and an H-CDR2 polymorphism consisting of the amino acid sequence represented by SEQ ID NO: 10 (TISSGGSYTYYPDSVKG).
  • the peptide includes an H-CDR3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 (RITTVVARSFYFDY).
  • the light chain variable region domain of this antibody is preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22 (RASKSVSSTSGYSYMH), and an L-sequence consisting of the amino acid sequence represented by SEQ ID NO: 26 (LVSNLES).
  • -A polypeptide of CDR2 an L-CDR3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 30 (QHIRELTRS).
  • the heavy chain variable region domain of the monoclonal antibody (A) is more preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and an H-- consisting of the amino acid sequence represented by SEQ ID NO: 10.
  • the light chain variable region domain of this antibody is more preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22, and an L-CDR2 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 26
  • An L-CDR3 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 30 and a light chain variable region framework polypeptide, most preferably a light chain variable comprising the amino acid sequence represented by SEQ ID NO: 18.
  • the monoclonal antibody (A) includes, for example, an antibody that competes with the 2G9 antibody for binding to the epitope, and particularly includes an antibody that binds to the same epitope as the PSK epitope to which the 2G9 antibody binds.
  • the PSK epitope to which the 2G9 antibody binds is likely to be an epitope present in the physiologically active site exhibiting the cytotoxic activity of PSK or an epitope in the vicinity thereof, and the 2G9 antibody binds to the epitope.
  • the PSK epitope to which the 2G9 antibody binds is likely to be an epitope present in the physiologically active site exhibiting TGF- ⁇ 1 inhibitory activity of PSK, or an epitope in the vicinity thereof, and the 2G9 antibody is considered to be the epitope.
  • it is an epitope that can suppress the activity of a physiologically active site showing TGF- ⁇ 1 inhibitory activity of PSK.
  • an antibody that competes for binding to an epitope includes all antibodies that have shown a competitive action in an epitope competition test using two antibodies. In an epitope competition test using two antibodies, the competition rate can be calculated. “Antibodies that compete for binding to the epitope” may exhibit a competition rate of 1% to 100%, specifically Includes antibodies exhibiting a competitive rate of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
  • binding to the same epitope means that the epitope to which the antigen-binding site of the antibody binds is the same, and this antibody exhibits a competitive action in an epitope competition test using two antibodies.
  • the competition rate in the epitope competition test of an antibody “binding to the same epitope” is not particularly limited. This is because the competition rate in the epitope competition test is determined by the titer, binding constant, dissociation constant, affinity constant, and the like of the two antibodies. Therefore, an antibody that “binds to the same epitope” may exhibit a competition rate of 1% to 100%, specifically 10% or more, 20% or more, 30% or more, 40% or more, 50% or more. , 60% or higher, 70% or higher, 80% or higher, or 90% or higher.
  • the epitope competition test can be performed by the following method.
  • PSK is coated at a concentration of 1 ⁇ g / well on a 96-well plate overnight at 4 ° C., and then blocked with 1% BSA to prepare a plate on which PSK is immobilized.
  • 0.1 ⁇ g / mL, 0.5 ⁇ g / mL, 1 ⁇ g / mL, or 5 ⁇ g / mL first antibody is added and incubated at 25 ° C. for 3 hours.
  • a second HRP-labeled antibody solution prepared to a concentration of 0.5 ⁇ g / mL is added and incubated at 25 ° C. for 1 hour.
  • the monoclonal antibody (B) is typified by the 5G5 antibody described in Reference Examples described later.
  • the heavy chain variable region domain is preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 38 (GYTMN), and an H-CDR2 polymorphism consisting of the amino acid sequence represented by SEQ ID NO: 42 (LINPYNGGTSYNQKFKG).
  • the peptide includes an H-CDR3 polypeptide consisting of the amino acid sequence (GGKFAGTTSY) represented by SEQ ID NO: 46.
  • the light chain variable region domain of this antibody is preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 54 (RSSTGAVTTSNYAN) and an L-sequence consisting of the amino acid sequence represented by SEQ ID NO: 58 (GTNNRAP).
  • -A polypeptide of CDR2 an L-CDR3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 62 (ALWYSNHWV).
  • the heavy chain variable region domain of the monoclonal antibody (B) is more preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 38, and an H-- consisting of the amino acid sequence represented by SEQ ID NO: 42.
  • a heavy chain variable region domain polypeptide is more preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 38, and an H-- consisting of the amino acid sequence represented by SEQ ID NO: 42.
  • the light chain variable region domain of this antibody is more preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 54 and an L-CDR2 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 58
  • Monoclonal antibody (B) includes, for example, an antibody that competes with the 5G5 antibody for binding to the epitope, and in particular, an antibody that binds to the same epitope as the epitope of PSK to which the 5G5 antibody binds.
  • the PSK epitope to which the 5G5 antibody binds is likely to be an epitope present in the physiologically active site exhibiting the cytotoxic activity of PSK or an epitope in the vicinity thereof, and the 5G5 antibody binds to the epitope.
  • the PSK epitope to which the 5G5 antibody binds is likely to be an epitope present in the physiologically active site exhibiting TGF- ⁇ 1 inhibitory activity of PSK, or an epitope in the vicinity thereof, and the 5G5 antibody is considered to be the epitope.
  • it is an epitope that can suppress the activity of a physiologically active site showing TGF- ⁇ 1 inhibitory activity of PSK.
  • H-CDR1 polypeptide, H-CDR2 polypeptide, H-CDR3 polypeptide, L-CDR1 polypeptide, L-CDR2 polypeptide, and L -CDR3 polypeptides may each have one or several amino acids deleted, substituted, inserted or added.
  • the epitope to which the antigen-binding site formed from the heavy chain variable region domain and the light chain variable region domain containing the polypeptide having the deletion, substitution, insertion or addition thereof binds to the epitope to which the 2G9 antibody or 5G5 antibody binds It is the same, and its binding can suppress the cytotoxic activity of PSK.
  • the heavy chain variable region domain or light chain variable region domain polypeptide of the monoclonal antibody (A) and the monoclonal antibody (B) may be deleted, substituted, inserted, or added, respectively.
  • the epitope to which the antigen-binding site formed from the heavy chain variable region domain and the light chain variable region domain containing the polypeptide having the deletion, substitution, insertion or addition thereof binds to the epitope to which the 2G9 antibody or 5G5 antibody binds It is the same, and its binding can suppress the cytotoxic activity of PSK.
  • amino acid deletion, substitution, insertion, or addition is preferably 3 or less, more preferably 2 or less, and most preferably 1 in each polypeptide.
  • the hydrophilic amino acid is a hydrophilic amino acid
  • the hydrophobic amino acid is a hydrophilic amino acid
  • the basic amino acid is a basic amino acid
  • an acidic amino acid is not limited.
  • the amino acid is preferably substituted with an acidic amino acid.
  • substitution of amino acids having similar properties the three-dimensional structure of the protein is often maintained, and thus the three-dimensional structure of the antigen-binding site of the anti-PSK antibody is also maintained, and the anti-PSK antibody binds to PSK. Can do.
  • the sandwich method is performed by using an anti-PSK polyclonal antibody or an antigen-binding fragment thereof or a second antibody or an antigen-binding fragment thereof.
  • An insoluble carrier on which any one of the antibodies or antigen-binding fragments thereof is immobilized, a test sample that may contain PSK, and the remaining one of the antibodies or antigen-binding fragments thereof are contacted with a labeled antibody.
  • a step of forming a complex of an immobilized antibody, PSK and a labeled antibody on an insoluble carrier (hereinafter referred to as a complex formation step), and a step of analyzing a signal from the label in the complex.
  • the order of contact of the insoluble carrier on which the antibody or the like is immobilized, the test sample that may contain PSK, and the labeled antibody is not particularly limited, and the insoluble carrier and the test are not limited.
  • the sample may be contacted first, and then the labeled antibody may be contacted, the labeled antibody and the test sample may be contacted, and then the insoluble carrier may be contacted, and the insoluble carrier, test sample, and labeled antibody may be contacted. They may be contacted together.
  • the “second antibody” in the present specification and the like is not particularly limited as long as it can bind to PSK.
  • it recognizes PSK and other polysaccharides ⁇ 1,3 glucan, ⁇ 1 , 4 glucan, and polyclonal antibody described in Non-Patent Document 1 that recognizes ⁇ 1,6 glucan, or anti-PSK antibody that specifically binds to PSK, but anti-PSK antibody that specifically binds to PSK Is preferred.
  • an anti-PSK polyclonal antibody that specifically binds to the PSK of the present invention for example, an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK, or PSK
  • a second antibody or an antigen-binding fragment thereof is immobilized on an insoluble carrier, and then contacted with a test sample that may contain PSK.
  • the anti-PSK polyclonal antibody or the antigen-binding fragment thereof is contacted with a labeled antibody to form a sandwich complex composed of an immobilized antibody, PSK and the labeled antibody on an insoluble carrier, and the anti-PSK polyclonal antibody
  • the signal from the label can be detected.
  • the anti-PSK polyclonal antibody or antigen-binding fragment thereof is immobilized on an insoluble carrier, the antibody-immobilized insoluble carrier is brought into contact with a test sample that may contain PSK, and then the second antibody or its The antigen-binding fragment is contacted with a labeled antibody, and a sandwich-like complex composed of an immobilized antibody, PSK, and a labeled antibody is formed on an insoluble carrier, and a signal for labeling the second antibody is detected. May be.
  • the complex formation step comprises contacting an insoluble carrier on which an anti-PSK monoclonal antibody or antigen-binding fragment thereof is immobilized with a test sample that may contain PSK, and then anti-PSK polyclonal antibody or antigen-binding fragment thereof.
  • the step is preferably a step of contacting a labeled antibody with a label.
  • the second antibody is an anti-PSK monoclonal antibody.
  • a sandwich complex composed of an anti-PSK monoclonal antibody, PSK and a labeled antibody (anti-PSK polyclonal antibody), and detect the signal of the anti-PSK polyclonal antibody label.
  • an unlabeled second antibody or anti-PSK polyclonal antibody may be used instead of the labeled second antibody or anti-PSK polyclonal antibody.
  • the signal is detected using a labeled antibody that binds to the unlabeled second antibody or anti-PSK polyclonal antibody.
  • a biotin-labeled second antibody or anti-PSK polyclonal antibody can be used, and a signal can be detected using labeled avidin.
  • the insoluble carrier that can be used in the immunological analysis method according to the sandwich method of the present invention is not particularly limited, and examples thereof include polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, and crosslinked dextran. And polymers such as polysaccharides, other nitrocellulose, paper, agarose, and combinations thereof.
  • an enzyme a fluorescent substance, or a luminescent substance
  • examples of enzymes include alkaline phosphatase, peroxidase, ⁇ -D-galactosidase, etc.
  • examples of fluorescent materials include fluorescein isothiocyanate
  • examples of luminescent materials include acridinium esters, luciferin, etc. can do.
  • kits for immunological analysis of PSK of the present invention comprises an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof.
  • the anti-PSK polyclonal antibody that specifically binds to PSK may be an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK, and reacts with PSK, and It may be an anti-PSK polyclonal antibody that does not react with the glucan structure of ⁇ 1,4 glucan of cellulose and the glucan structure of ⁇ 1,3 glucan and ⁇ 1,6 glucan of laminarin.
  • the PSK immunological analysis kit of the present invention may further contain a second antibody that reacts with PSK or an antigen-binding fragment thereof.
  • the PSK analysis kit of this embodiment is a kit for immunological analysis of PSK containing the anti-PSK polyclonal antibody of the present invention or an antigen-binding fragment of the antibody.
  • the kit for immunological analysis of PSK can be used for the immunological analysis method of PSK of the present invention.
  • the immunological analysis kit for PSK of the present invention comprises an enzyme immunoassay method, an immunohistochemical staining method, a surface plasmon resonance method (SPR method: Biacore method), a latex agglutination immunoassay method, a chemiluminescence immunoassay method, a fluorescence It includes kits used for antibody methods, radioimmunoassay methods, immunoprecipitation methods, Western blotting methods, immunochromatographic methods, magnetic bead agglutination methods, magnetic bead enzyme immunization methods, and the like.
  • kits for immunological analysis of PSK is an enzyme immunoassay, for example, a sandwich kit, a carrier having an anti-PSK polyclonal antibody or a second antibody, or an antigen-binding fragment thereof immobilized on its surface
  • a carrier having an anti-PSK polyclonal antibody or a second antibody, or an antigen-binding fragment thereof immobilized on its surface Polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharides and other polymers, nitrocellulose, paper, agarose and combinations thereof
  • labeled first anti-PSK polyclonal An antibody or a second antibody, or an antigen-binding fragment thereof, an enzyme, a chromogenic substrate thereof, and other ELISA reagents (for example, a washing solution) can be appropriately combined.
  • the PSK analysis kit is an immunohistochemical staining kit
  • biotinylated anti-PSK polyclonal antibody of the present invention HRP labeled streptavidin, DAB substrate, or unlabeled anti-PSK polyclonal antibody, HRP Labeled anti-mouse IgG antibodies, substrates, etc.
  • HRP Labeled anti-mouse IgG antibodies, substrates, etc. can be included.
  • the PSK analysis kit When the PSK analysis kit is an SPR analysis kit, it includes a sensor chip on which the anti-PSK polyclonal antibody of the present invention is immobilized.
  • the PSK analysis kit contains an anti-PSK polyclonal antibody or an antigen-binding fragment thereof in a desired form depending on the immunological technique used.
  • the labeling substance include peroxidase, alkaline phosphatase, ⁇ -D-galactosidase or glucose oxidase as an enzyme, fluorescein isothiocyanate or rare earth metal chelate as a fluorescent substance, 3 H as a radioisotope,
  • biotin, avidin, a chemiluminescent substance, and the like can be given.
  • an enzyme, a chemiluminescent substance, or the like it is not possible to provide a measurable signal by itself, and therefore it is preferable to select and include a corresponding appropriate substrate.
  • the kit for immunological analysis of PSK is capable of specifically analyzing PSK, and includes an instruction manual describing the fact. Moreover, it may be described that PSK can be specifically analyzed on the packaging of a kit or the like.
  • Ammonium sulfate was added to 1 liter of the concentrated solution to form a saturated solution, salted out, and the resulting precipitate was collected by centrifugation.
  • the fractionated precipitate was redissolved in distilled water and subjected to dialysis and ultrafiltration to remove low molecular weight substances having a molecular weight of 5000 or less. Subsequently, it was freeze-dried to obtain 15 g of hot water / alkali extract powder of Kawaratake.
  • Example 1 Preparation of anti-PSK rabbit polyclonal antibody Equivalent amounts of PBS solution of Kawaratake hot water / alkaline extract powder obtained in Production Example 1 and Freund's Complete Adjuvant (Sigma-Aldrich, Tokyo) were mixed, and high-viscosity was produced using an ultrasonic generator. An emulsion was prepared. The emulsion solution was subcutaneously injected into the back of female New Zealand white rabbits in a PSK amount of 0.5 mg / feather. Two weeks later, an emulsion of PSK solution and Freund's Incomplete Adjuvant (Sigma-Aldrich) was injected subcutaneously into the back of the rabbit so that the amount of PSK was 0.5 mg / feather.
  • the antibody titer of the immune serum was measured by ELISA. Specifically, a 96-well plate coated with PSK at a concentration of 50 ng / well was dispensed with a 100 to 2187,000-fold dilution of the obtained serum at 100 ⁇ g / L, reacted at room temperature for 3 hours, It was washed 3 times with% Tween 80-added TBS (hereinafter referred to as “TBS-T”). Next, an HRP-labeled anti-rabbit IgG antibody solution was added to each well of the plate, reacted at room temperature for 1 hour, and then washed 3 times with TBS-T. After adding ABTS substrate solution to develop color for 5-15 minutes, absorbance at 405-630 nm was measured using a plate reader. The antibody titer measurement results are shown in FIG. In the subsequent experiments, the individual No. Two antisera were used.
  • the anti-PSK polyclonal antibody was purified by affinity chromatography using a PSK solid phase column.
  • pepsin treatment and reduction treatment were performed, followed by horseradish peroxidase (HRP) labeling. It was confirmed by the aforementioned ELISA method that the obtained Fab′-HRP maintained the antigen recognition ability (Table 1).
  • the dry mycelium yield was 2.0 to 4.5 g per incubator.
  • To 100 g of this mycelium was added 3 liters of a 0.1N aqueous sodium hydroxide solution, and the mixture was extracted with stirring at 97 ° C. for 1 hour. After completion of extraction, the mixture was cooled to room temperature, and the extract was collected by filtration with filter paper. Next, the solution was neutralized with dilute hydrochloric acid and concentrated with an evaporator. Ammonium sulfate was added to 1 liter of the concentrated solution to form a saturated solution, salted out, and the resulting precipitate was collected by centrifugation.
  • the fractionated precipitate was redissolved in distilled water and subjected to dialysis and ultrafiltration to remove low molecular weight substances having a molecular weight of 5000 or less. Subsequently, it was freeze-dried to obtain 15 g of protein polysaccharide powder.
  • Comparative Example 1 Production of conventional anti-PSK rabbit polyclonal antibody >> The procedure of Example 1 was repeated except that the protein polysaccharide obtained in Comparative Production Example 1 was used instead of the hot water / alkaline extract powder obtained in Production Example 1. An anti-PSK rabbit polyclonal antibody was obtained.
  • Example 2 Specificity evaluation of anti-PSK rabbit polyclonal antibody >> The specificity of the anti-PSK rabbit polyclonal antibody for PSK was evaluated using Biacore. First, an anti-rabbit IgG antibody was immobilized on the surface of the Sensor Chip CM5 sensor by an amino coupling method. Next, the conventional anti-PSK rabbit polyclonal antibody prepared in Comparative Example 1 (old rabbit anti-PSK pAb) or the anti-PSK rabbit polyclonal antibody prepared in Example 1 (new rabbit anti-PSK pAb) is used against the sensor chip. A certain amount of was supplemented.
  • Binding analysis was performed on these sensor chips using PSK, ⁇ 1-4 glucan, or ⁇ 1-3, 1-6 glucan adjusted to 1 ⁇ g / mL as an analyte. Note that cellulose was used as ⁇ 1-4glucan, and laminarin was used as ⁇ 1-3,1-6glucan.
  • the conventional old rabbit anti-PSK pAb also binds to glucans other than PSK, whereas the new rabbit anti-PSK pAb of the present invention is not included in PSK. It was found to bind specifically to PSK without binding to the ⁇ 1,3 glucan, ⁇ 1,4 glucan, and ⁇ 1,6 glucan structures.
  • This emulsion solution was subcutaneously injected into 6-week-old female Balb / c mice (Oriental Yeast Co., Ltd.) so that the PSK amount was 0.1 mg / animal.
  • a second immunization was performed one week later.
  • PSK in PBS and Freund's Incomplete Adjuvant (Sigma-Aldrich) were mixed to prepare an emulsion.
  • the intraperitoneal injection was performed so that the PSK amount was 0.1 mg / animal.
  • Immunization was performed in the same procedure every week, and after the eighth immunization, blood was collected from the tail vein and the titer was measured. Individuals in which an increase in antibody titer was observed were boosted by intraperitoneal injection of PSK, and then cell fusion was performed to obtain a hybridoma.
  • FIG. 3 shows the results of antibody titer measurement by ELISA.
  • the horizontal axis of the graph represents the serum dilution ratio, and the vertical axis represents the absorbance (titer).
  • the IgM fraction was collected by a HiTrap IgM column (Amersham) or Sepharose HP column (Amersham) by a conventional method. Further, the IgM fraction was fractionated with a Sepharose 200 pg column to purify pentamer IgM. The titers of the obtained 2G9 antibody and 5G5 antibody are shown in FIG.
  • PSK has ⁇ 1,3 glucan, ⁇ 1,4 glucan, and ⁇ 1,6 glucan
  • cellulose has ⁇ 1,4 glucan
  • laminarin has ⁇ 1,3 glucan and ⁇ 1,6 glucan
  • Glycogen has ⁇ 1,4 glucan
  • dextran has ⁇ 1,6 glucan.
  • RNA was extracted from the hybridoma producing 2G9 antibody or 5G5 antibody by a conventional method, and reverse transcription was performed using an oligo dT primer to prepare cDNA. From the obtained cDNA, PCR was performed according to the attached protocol using a mouse Ig primer set (Novagen) to amplify the variable region gene. The obtained antibody variable region gene was TA cloned into the pCR2.1 vector and the sequence was determined.
  • FIGS. Shown in H-FR1, H-CDR1, H-FR2, H-CDR2, H-FR3, H-CDR3, and H-FR4, and L-FR1, L-CDR1, L-FR2, L-
  • the amino acid sequences of CDR2, L-FR3, L-CDR3, and L-FR4 are shown below.
  • H-FR1 of heavy chain variable region domain of 2G9 antibody GVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFS (SEQ ID NO: 4)
  • H-CDR1 SYGMS (SEQ ID NO: 6)
  • H-FR2 WVRQTPDKRLEWVA (SEQ ID NO: 8)
  • H-CDR2 TISSGGSYTYYPDSVKG (SEQ ID NO: 10)
  • H-FR3: RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR SEQ ID NO: 12
  • H-CDR3 RITTVVARSFYFDY (SEQ ID NO: 14)
  • H-FR4 WGQG (SEQ ID NO: 16)
  • L-FR1 of the light chain variable region domain of 2G9 antibody GSTGDIVLTQSPASLAVSLGQRATISY (SEQ ID NO: 20)
  • L-CDR1 RASKSVSTSGYSYMH (SEQ ID NO: 22)
  • L-FR2 WNQQKPGQPPRLLIY (SEQ ID NO: 24)
  • L-CDR2 LVSNLES (SEQ ID NO: 26)
  • L-FR3: GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC SEQ ID NO: 28
  • L-CDR3 QHIRELTRS (SEQ ID NO: 30)
  • L-FR4 EGGP (SEQ ID NO: 32)
  • H-FR1 of heavy chain variable region domain of 5G5 antibody GVHSEVQLQQSGPELVKPGASMKISCKASGYSFT (SEQ ID NO: 36)
  • H-CDR1 GYTMN (SEQ ID NO: 38)
  • H-FR2 WVKQSHGKNLEWIG (SEQ ID NO: 40)
  • H-CDR2 LINPYNGGTSYNQKFKG (SEQ ID NO: 42)
  • H-FR3: KATLTVDKSSSTAYMELLSLTSEDSAVYYCAR SEQ ID NO: 44
  • H-CDR3 GGKFATGTSY (SEQ ID NO: 46)
  • H-FR4 WGQG (SEQ ID NO: 48)
  • L-FR1 of the light chain variable region domain of the 5G5 antibody GAISQAVVTQESALTTSPGETVTLTC (SEQ ID NO: 52)
  • L-CDR1 RSSTGAVTTSNYAN (SEQ ID NO: 54)
  • L-FR2 WVQEKPDHLFTGLIG (SEQ ID NO: 56)
  • L-CDR2 GTNNRAP (SEQ ID NO: 58)
  • L-FR3 GVPARFSGSLIGDKAALTITGAQTEDEAIYFC (SEQ ID NO: 60)
  • L-FR4 FGGG (SEQ ID NO: 64)
  • Example 3 Construction of sandwich ELISA system The detection system for PSK was performed by sandwich ELISA.
  • Anti-PSK monoclonal antibodies (2G9 antibody and 5G5 antibody) dissolved in carbonate buffer were coated on a 96-well plate at 500 ng / well. After blocking with PBS containing 1% BSA, 62.5-500 ng / mL PSK solution was added and incubated at room temperature for 1 hour. After washing the plate with TBS-T, HRP-labeled anti-PSK Fab ′ antibody was added and incubated at room temperature for an additional 30 minutes.
  • the ELISA system constructed as shown in FIG. 6 was able to detect PSK with high sensitivity and higher specificity.
  • Example 4 Measurement of blood concentration of PSK in mouse by sandwich ELISA After a single intraperitoneal administration of PSK (50 mg / kg) to Balb / c mice (female, 6 weeks old), blood was collected 8 hours later, and the blood concentration was measured by the sandwich ELISA method. As a result, it was possible to detect PSK taken up in blood (Table 2).
  • the immunological analysis method of PSK of the present invention can be used for pharmaceutical quality control.
  • the immunological analysis method for PSK of the present invention can be used for examination of pharmacokinetics of PSK.

Abstract

The problem is to provide a polyclonal antibody that reacts specifically to PSK and a PSK immunological analysis method with which it is possible to specifically detect PSK. The problem can be solved by providing: a PSK immunological analysis method characterized by using an anti-PSK polyclonal antibody that binds specifically to PSK or an antigen-binding fragment thereof; and an anti-PSK polyclonal antibody that binds specifically to PSK or an antigen-binding fragment thereof.

Description

抗ＰＳＫポリクローナル抗体並びにそれを用いたＰＳＫの免疫学的分析方法およびＰＳＫの免疫学的分析用キットAnti-PSK polyclonal antibody, immunological analysis method of PSK using the same, and kit for immunological analysis of PSK
　本発明は、抗ＰＳＫポリクローナル抗体及びそれを用いたＰＳＫの免疫学的分析方法に関する。 The present invention relates to an anti-PSK polyclonal antibody and an immunological analysis method for PSK using the same.
　カワラタケから抽出される蛋白多糖体は、抗腫瘍活性などを示し、前記蛋白多糖体を有効成分とする抗腫瘍剤などが、例えば、特開昭６０－４５５３３号公報（特許文献１）などに記載されている。このような蛋白多糖体のなかで、カワラタケ由来の前記蛋白多糖体の一種であるＰＳＫ（登録商標）〔商品名「クレスチン」（登録商標）〕は、皮内投与や静脈内投与だけでなく、経口投与によっても抗腫瘍活性を示すことが特長であり、臨床的にも経口投与製剤として用いられている。 Protein polysaccharides extracted from Kawaratake show antitumor activity and the like, and antitumor agents containing the protein polysaccharide as an active ingredient are described in, for example, JP-A-60-45533 (Patent Document 1). Has been. Among such protein polysaccharides, PSK (registered trademark) [trade name “Krestin” (registered trademark)], which is a kind of the protein polysaccharide derived from Kawaratake, is not only administered intradermally or intravenously, It is characterized by exhibiting antitumor activity even by oral administration, and is clinically used as an oral administration preparation.
　ＰＳＫは、約１８～３８％の蛋白質を含む蛋白多糖体であり、５０００以上（ゲル濾過法）の分子量、例えば５０００～３０００００（ゲル濾過法）の分子量を有するものである。主要画分の糖部分はβ－Ｄ－グルカンで、このグルカン部分の構造は、１→３、１→４及び１→６結合を含む分枝構造である。 PSK is a protein polysaccharide containing about 18 to 38% protein, and has a molecular weight of 5000 or more (gel filtration method), for example, 5000 to 300,000 (gel filtration method). The sugar part of the main fraction is β-D-glucan, and the structure of this glucan part is a branched structure containing 1 → 3, 1 → 4 and 1 → 6 bonds.
　このＰＳＫに対する抗体としては、非特許文献１に、ＰＳＫに対するウサギポリクローナル抗体が開示されており、このポリクローナル抗体を用いた蛍光抗体法により、ＰＳＫを検出できることが記載されている。しかしながら、このポリクローナル抗体は、ＰＳＫを免疫原として用いたものであるが、ＰＳＫ以外の多糖類にも結合する抗体であり、ＰＳＫに特異的な抗体ではなかった。 As an antibody against this PSK, Non-Patent Document 1 discloses a rabbit polyclonal antibody against PSK, which describes that PSK can be detected by a fluorescent antibody method using this polyclonal antibody. However, although this polyclonal antibody uses PSK as an immunogen, it is an antibody that binds to polysaccharides other than PSK and is not an antibody specific to PSK.
特開昭６０－４５５３３号公報Japanese Patent Laid-Open No. 60-45533 特開平８－２０８７０４号公報JP-A-8-208704
　従来のＰＳＫは、後述のように熱水又はアルカリ溶液（例えば、アルカリ金属の水酸化物、特には水酸化ナトリウムの水溶液）で抽出し、精製した後に乾燥して得られていた。具体的には、例えば特許文献２の実施例２に記載のように、カワラタケ菌ＣＭ１０１株を培養し、得られた乾燥菌糸体から、０．１Ｍ水酸化ナトリウムで抽出することにより得られていた。このような方法で製造されたＰＳＫは、医薬品としては十分な効果を有するものであったが、このＰＳＫを、抗体を取得するための免疫原として用いた場合、非特許文献１に記載のように、ＰＳＫ以外の多糖類に結合するポリクローナル抗体しか得られず、ＰＳＫを特異的に測定することができなかった。 Conventional PSK was obtained by extracting with hot water or an alkali solution (for example, an alkali metal hydroxide, particularly an aqueous solution of sodium hydroxide), followed by purification and drying as described below. Specifically, for example, as described in Example 2 of Patent Document 2, it was obtained by culturing Kawaratake fungus CM101 strain and extracting the resulting dried mycelium with 0.1 M sodium hydroxide. . PSK produced by such a method has a sufficient effect as a pharmaceutical, but when this PSK is used as an immunogen for obtaining an antibody, as described in Non-patent Document 1. Furthermore, only polyclonal antibodies that bind to polysaccharides other than PSK were obtained, and PSK could not be measured specifically.
　従って、本発明の目的は、ＰＳＫに特異的に反応するポリクローナル抗体を提供すること、及びＰＳＫを特異的に検出することのできるＰＳＫの免疫学的分析方法を提供することである。 Therefore, an object of the present invention is to provide a polyclonal antibody that reacts specifically with PSK, and to provide an immunological analysis method of PSK that can specifically detect PSK.
　本発明者は、ＰＳＫに特異的に反応するポリクローナル抗体について、鋭意研究した結果、驚くべきことに、カワラタケの熱水・アルカリ抽出物を免疫することによって、セルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応せず、ＰＳＫに特異的に反応するポリクローナル抗体を得ることができることを見出した。 As a result of diligent research on polyclonal antibodies that specifically react with PSK, the present inventors have surprisingly immunized hot water / alkaline extract of Kawaratake to give a glucan structure of β1,4 glucan of cellulose, It was also found that a polyclonal antibody that specifically reacts with PSK without reacting with the glucan structures of laminarin β1,3 glucan and β1,6 glucan can be obtained.
　従って、本発明は、［１］ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片を用いることを特徴とするＰＳＫの免疫学的分析方法に関する。本発明のＰＳＫの免疫学的分析方法によって、ＰＳＫの特異的な検出が可能である。 Therefore, the present invention relates to [1] an immunological analysis method for PSK, which comprises using an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof. The PSK immunological analysis method of the present invention enables specific detection of PSK.
　また、本発明は［２］前記抗ＰＳＫポリクローナル抗体が、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体である、［１］のＰＳＫの免疫学的分析方法に関する。 The present invention also provides [2] the immunological analysis of PSK according to [1], wherein the anti-PSK polyclonal antibody is an anti-PSK polyclonal antibody that recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK. It relates to the analysis method.
　また、本発明は、［３］前記抗ＰＳＫポリクローナル抗体が、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体である、［１］又は［２］のＰＳＫの免疫学的分析方法に関する。また、本発明は［４］前記抗ＰＳＫポリクローナル抗体のＰＳＫに対する結合力を１００とした場合の、セルロースのβ１，４グルカンのグルカン構造、又はラミナリンのβ１，３グルカン、及びβ１，６グルカンのグルカン構造に対する結合力が３０％以下である、［３］のＰＳＫの免疫学的分析方法に関する。 The present invention also provides [3] that the anti-PSK polyclonal antibody reacts with PSK and does not react with the β1,4 glucan glucan structure of cellulose and the β1,3 glucan of laminarin and the glucan structure of β1,6 glucan. The present invention relates to an immunological analysis method for PSK [1] or [2], which is an anti-PSK polyclonal antibody. The present invention also provides [4] a glucan structure of β1,4 glucan of cellulose or β1,3 glucan of laminarin and glucan of β1,6 glucan when the binding force of the anti-PSK polyclonal antibody to PSK is 100. The present invention relates to the immunological analysis method for PSK according to [3], wherein the binding force to the structure is 30% or less.
　また、本発明は［５］酵素免疫測定法、免疫組織染色法、表面プラズモン共鳴法、ラテックス凝集免疫測定法、化学発光免疫測定法、蛍光抗体法、放射免疫測定法、免疫沈降法、ウエスタンブロット法、イムノクロマトグラフ法、磁気ビーズ凝集法、又は磁気ビーズ酵素免疫法を用いる［１］～［４］のいずれかのＰＳＫの免疫学的分析方法に関する。これらの分析方法を用いることにより、多様なＰＳＫの測定を行うことができる。 The present invention also provides [5] enzyme immunoassay, immunohistochemical staining, surface plasmon resonance, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation, western blot The present invention relates to an immunological analysis method for PSK according to any one of [1] to [4], using a method, an immunochromatographic method, a magnetic bead aggregation method, or a magnetic bead enzyme immunization method. By using these analysis methods, various PSK measurements can be performed.
　また、本発明は［６］前記ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片と、ＰＳＫと反応する第２の抗体又はその抗原結合性断片と、を使用する、［１］～［５］のいずれかのＰＳＫの免疫学的分析方法に関する。 The present invention also uses [6] an anti-PSK polyclonal antibody or antigen-binding fragment thereof that specifically binds to PSK, and a second antibody or antigen-binding fragment thereof that reacts with PSK, [1 ] To an immunological analysis method for PSK according to any one of [5] to [5].
　また、本発明は［７］前記抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片、又は前記第２の抗体若しくはその抗原結合性断片のいずれか一方の抗体若しくはその抗原結合性断片を固定化した固定化抗体を有する不溶性担体、ＰＳＫを含む可能性のある被検試料、残る一方の抗体若しくはその抗原結合性断片に標識を付した標識抗体を接触させ、前記不溶性担体上に固定化抗体とＰＳＫと標識抗体との複合体を形成する複合体形成工程と、前記複合体における標識からの信号を分析する分析工程と、を含む、［６］のＰＳＫの免疫学的分析方法に関する。 The present invention also provides [7] Immobilization wherein the anti-PSK polyclonal antibody or antigen-binding fragment thereof, or the second antibody or antigen-binding fragment thereof is immobilized, or an antigen-binding fragment thereof. An insoluble carrier having an antibody, a test sample possibly containing PSK, the remaining antibody or a labeled antibody labeled with an antigen-binding fragment thereof is contacted, and the immobilized antibody and PSK are labeled on the insoluble carrier. The present invention relates to the immunological analysis method for PSK of [6], comprising a complex formation step for forming a complex with an antibody, and an analysis step for analyzing a signal from a label in the complex.
　また、本発明は［８］前記複合体形成工程が、第２の抗体として抗ＰＳＫモノクローナル抗体若しくはその抗原結合性断片を固定化した不溶性担体と、ＰＳＫを含む可能性のある被検試料とを接触させ、そして前記抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片に標識を付した標識抗体を接触させる工程である、［７］のＰＳＫの免疫学的分析方法に関する。本態様における、具体的な態様の１つはサンドイッチ法である。本発明のＰＳＫの免疫学的分析方法として、サンドイッチ法を用いることにより、ＰＳＫのダイナミックレンジの広い測定が可能である。 The present invention also provides [8] an insoluble carrier in which the complex-forming step immobilizes an anti-PSK monoclonal antibody or an antigen-binding fragment thereof as a second antibody, and a test sample that may contain PSK. The method for immunological analysis of PSK according to [7], which is a step of contacting and contacting the labeled antibody labeled with the anti-PSK polyclonal antibody or antigen-binding fragment thereof. One of the specific embodiments in this embodiment is a sandwich method. By using the sandwich method as the immunological analysis method for PSK of the present invention, it is possible to measure a wide dynamic range of PSK.
　また、本発明は［９］前記第２の抗体が、抗ＰＳＫモノクローナル抗体である、［６］～［８］のいずれかのＰＳＫの免疫学的分析方法に関する。抗ＰＳＫモノクローナル抗体を用いることにより、更に特異性の高いＰＳＫの測定を行うことができる。 The present invention also relates to [9] the immunological analysis method for PSK according to any one of [6] to [8], wherein the second antibody is an anti-PSK monoclonal antibody. By using an anti-PSK monoclonal antibody, more specific PSK can be measured.
　また、本発明は［１０］血中濃度の測定を行うものである、［１］～［９］のいずれかのＰＳＫの免疫学的分析方法に関する。血中濃度の測定を行うことにより、ＰＳＫの薬効の予測およびＰＳＫの投与至適濃度の測定を行うことができる。 The present invention also relates to [10] an immunological analysis method for PSK according to any one of [1] to [9], which measures blood concentration. By measuring the blood concentration, it is possible to predict the efficacy of PSK and measure the optimal concentration of PSK.
　また、本発明は、［１１］ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片に関する。本発明の抗ＰＳＫポリクローナル抗体又はその抗原結合性断片は、前記ＰＳＫの免疫学的分析方法に用いた場合、ＰＳＫの特異的な測定をすることができる。 The present invention also relates to [11] an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof. When the anti-PSK polyclonal antibody or antigen-binding fragment thereof of the present invention is used in the immunological analysis method for PSK, it can measure PSK specifically.
　また、本発明は［１２］前記抗ＰＳＫポリクローナル抗体が、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する、［１１］に記載の抗ＰＳＫポリクローナル抗体又はその抗原結合性断片に関する。 [12] The anti-PSK polyclonal antibody according to [11], wherein the anti-PSK polyclonal antibody recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK, or an antigen binding property thereof. Regarding fragments.
　また、本発明は［１３］前記抗ＰＳＫポリクローナル抗体が、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン、及びβ１，６グルカンのグルカン構造に反応しない、［１１］又は［１２］の抗ＰＳＫポリクローナル抗体又はその抗原結合性断片に関する。 The present invention also provides [13] The anti-PSK polyclonal antibody reacts with PSK and does not react with the β1,4 glucan glucan structure of cellulose, and the β1,3 glucan of laminarin and the glucan structure of β1,6 glucan. [11] or [12], or an antigen-binding fragment thereof.
　また、本発明は［１４］前記抗原結合性断片が、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ断片、ディアボディー、単一鎖抗体分子、及びマルチ特異性抗体からなる群から選択される、［１１］～［１３］のいずれかに記載の抗原結合性断片に関する。このような抗原結合性断片をもちいることにより、Ｆｃ断片によって起こる非特異反応を除くことができる。 The present invention also provides [14] wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2 , Fv fragment, diabody, single chain antibody molecule, and multispecific antibody. This relates to the antigen-binding fragment according to any one of [11] to [13]. By using such an antigen-binding fragment, a non-specific reaction caused by the Fc fragment can be eliminated.
　更に、本発明は、［１５］ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片、を含むことを特徴とする、ＰＳＫの免疫学的分析用キットに関する。本発明のＰＳＫの免疫学的分析用キットによれば、ＰＳＫの特異的な測定を、より簡便に行うことが可能である。 Furthermore, the present invention relates to a kit for immunological analysis of PSK, comprising [15] an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof. According to the kit for immunological analysis of PSK of the present invention, PSK-specific measurement can be performed more simply.
　また、本発明は［１６］前記抗ＰＳＫポリクローナル抗体が、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する、［１５］に記載のＰＳＫの免疫学的分析用キットに関する。 The present invention also relates to [16] the kit for immunological analysis of PSK according to [15], wherein the anti-PSK polyclonal antibody recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK. .
　また、本発明は［１７］前記抗ＰＳＫポリクローナル抗体が、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン、及びβ１，６グルカンのグルカン構造に反応しない、［１５］又は［１６］に記載のＰＳＫの免疫学的分析用キットに関する。 [17] The present invention also provides [17] The anti-PSK polyclonal antibody reacts with PSK and does not react with the β1,4 glucan glucan structure of cellulose, and the β1,3 glucan of laminarin and the glucan structure of β1,6 glucan. The kit for immunological analysis of PSK according to [15] or [16].
　また、本発明は［１８］抗ＰＳＫモノクローナル抗体又はその抗原結合性断片を更に含む、［１５］～［１７］のいずれかのＰＳＫの免疫学的分析用キットに関する。 The present invention also relates to a kit for immunological analysis of PSK according to any one of [15] to [17], further comprising [18] an anti-PSK monoclonal antibody or an antigen-binding fragment thereof.
　本発明に係るＰＳＫの免疫学的分析方法では、ＰＳＫに特異的な抗ＰＳＫポリクローナル抗体を用いる。特には、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片を用いる。又は、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体を用いることもできる。また、本発明の抗ＰＳＫポリクローナル抗体はＰＳＫに対する特異性が高いため、本発明のポリクローナル抗体を用いることによって、ＰＳＫの特異的な検出が可能である。更には、分析方法における測定感度の上昇、及び使用する抗ＰＳＫポリクローナル抗体量を減らすことも可能である。また、抗ＰＳＫポリクローナル抗体をサンドイッチ法に用いた場合には、ＰＳＫのダイナミックレンジの広い測定が可能である。 In the immunological analysis method for PSK according to the present invention, an anti-PSK polyclonal antibody specific for PSK is used. In particular, an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK or an antigen-binding fragment thereof is used. Alternatively, an anti-PSK polyclonal antibody that reacts with PSK and does not react with the β1,4 glucan glucan structure of cellulose and the β1,3 glucan and β1,6 glucan glucan structures of laminarin can also be used. In addition, since the anti-PSK polyclonal antibody of the present invention has high specificity for PSK, specific detection of PSK is possible by using the polyclonal antibody of the present invention. Furthermore, it is possible to increase the measurement sensitivity in the analytical method and reduce the amount of anti-PSK polyclonal antibody used. In addition, when an anti-PSK polyclonal antibody is used in the sandwich method, measurement with a wide dynamic range of PSK is possible.
　すなわち、本発明に係るＰＳＫの免疫学的分析方法は、医薬に含まれるＰＳＫの量を正確に測定し、医薬の品質管理に用いることができる効果を奏する。また、生体に投与されたＰＳＫを測定することが可能であり、ＰＳＫの体内動態を調べることができる効果を奏する。 That is, the immunological analysis method for PSK according to the present invention has an effect that the amount of PSK contained in a medicine can be accurately measured and used for quality control of the medicine. Moreover, it is possible to measure PSK administered to a living body, and it is possible to examine the pharmacokinetics of PSK.
　なお、本発明に係るＰＳＫの免疫学的分析方法では、ＰＳＫの体内動態を調べることができるため、ＰＳＫの薬効の予測およびＰＳＫの投与至適濃度を把握するためのデータ測定にも利用することができる。 In addition, since the immunological analysis method of PSK according to the present invention can examine the pharmacokinetics of PSK, it should also be used for data measurement for predicting the efficacy of PSK and grasping the optimal concentration of PSK. Can do.
本発明の抗ＰＳＫポリクローナル抗体の力価を示したグラフである。It is the graph which showed the titer of the anti- PSK polyclonal antibody of this invention. 本発明の抗ＰＳＫウサギポリクローナル抗体の特異性をＢｉａｃｏｒｅにより評価した結果を示すグラフである。It is a graph which shows the result of having evaluated the specificity of the anti- PSK rabbit polyclonal antibody of this invention by Biacore. Ｂａｌｂ／ｃマウスにＰＳＫを免疫し、血清中の抗体価を測定したグラフである。It is the graph which immunized the Balb / c mouse | mouth with PSK and measured the antibody titer in serum. 精製された抗ＰＳＫモノクローナル抗体の抗体価を示すグラフである。It is a graph which shows the antibody titer of the refined anti- PSK monoclonal antibody. 抗ＰＳＫマウスモノクローナル抗体の特異性を競合ＥＬＩＳＡにより評価したグラフである。It is the graph which evaluated the specificity of the anti- PSK mouse monoclonal antibody by competition ELISA. 本発明の抗ＰＳＫモノクローナル抗体と、２Ｇ９抗体又は５Ｇ５抗体とを用いたサンドイッチＥＬＩＳＡによるＰＳＫの検出を示したグラフである。It is the graph which showed the detection of PSK by sandwich ELISA using the anti- PSK monoclonal antibody of this invention, and 2G9 antibody or 5G5 antibody. ２Ｇ９抗体の重鎖可変領域ドメインのヌクレオチドの塩基配列及びアミノ酸配列を示す図である。It is a figure which shows the base sequence and amino acid sequence of the nucleotide of the heavy chain variable region domain of 2G9 antibody. ２Ｇ９抗体の軽鎖可変領ドメインのヌクレオチドの塩基配列及びアミノ酸配列を示す図である。It is a figure which shows the base sequence and amino acid sequence of the light chain variable region domain of 2G9 antibody. ５Ｇ５抗体の重鎖可変領域ドメインのヌクレオチドの塩基配列及びアミノ酸配列を示す図である。It is a figure which shows the base sequence and amino acid sequence of the nucleotide of the heavy chain variable region domain of 5G5 antibody. ５Ｇ５抗体の軽鎖可変領ドメインのヌクレオチドの塩基配列及びアミノ酸配列を示す図である。It is a figure which shows the base sequence and amino acid sequence of the light chain variable region domain of 5G5 antibody.
［１］抗ＰＳＫポリクローナル抗体
　本発明の抗ＰＳＫポリクローナル抗体は、ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体である。また、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体であってもよく、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体であってもよい。 [1] Anti-PSK polyclonal antibody The anti-PSK polyclonal antibody of the present invention is an anti-PSK polyclonal antibody that specifically binds to PSK. It may also be an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK, and reacts with PSK, and the glucan structure of β1,4 glucan of cellulose, and laminarin. It may be an anti-PSK polyclonal antibody that does not react with the glucan structure of β1,3 glucan and β1,6 glucan.
　抗ＰＳＫポリクローナル抗体は、ＰＳＫを認識する。ＰＳＫは、カワラタケ菌ＣＭ１０１株〔ＦＥＲＭ－Ｐ２４１２（ＡＴＣＣ２０５４７）〕の菌糸体を水系溶媒、例えば、熱水又はアルカリ溶液（例えば、アルカリ金属の水酸化物、特には水酸化ナトリウムの水溶液）で抽出し、精製した後に乾燥して得ることができる。主要画分の糖部分はβ－Ｄ－グルカンで、このグルカン部分の構造はβ１→３、β１→４及びβ１→６結合を含む分枝構造であり、主な構成単糖はグルコースやマンノースであり、約１８～３８％のタンパク質を含む。タンパク質の構成アミノ酸は、アスパラギン酸やグルタミン酸等の酸性アミノ酸と、バリンやロイシン等の中性アミノ酸が多く、リジンやアルギニン等の塩基性アミノ酸は少ない。水に可溶であるが、メタノール、ピリジン、クロロホルム、ベンゼン又はヘキサンには殆ど溶けない。 Anti-PSK polyclonal antibody recognizes PSK. PSK is obtained by extracting the mycelium of Kawaratake fungus CM101 strain [FERM-P2412 (ATCC20547)] with an aqueous solvent, for example, hot water or an alkaline solution (for example, an alkali metal hydroxide, particularly an aqueous solution of sodium hydroxide). It can be obtained after purification and drying. The sugar part of the main fraction is β-D-glucan, and the structure of this glucan part is a branched structure containing β1 → 3, β1 → 4 and β1 → 6 linkages, and the main constituent monosaccharides are glucose and mannose. Yes, containing about 18-38% protein. The constituent amino acids of proteins are mostly acidic amino acids such as aspartic acid and glutamic acid, and neutral amino acids such as valine and leucine, and few basic amino acids such as lysine and arginine. Although soluble in water, it is hardly soluble in methanol, pyridine, chloroform, benzene or hexane.
　本発明の抗ＰＳＫポリクローナル抗体は、ラミナリンのβ１，３グルカンを認識しない。β１，３グルカンは、グルコースがβ１－３型の結合で連なった多糖である。植物や菌類、細菌など自然界に広く分布する。例えば、β１，３グルカンは、ラミナリン又はイーストグルカンなどに存在するグルカンであるが、本発明の抗ＰＳＫポリクローナル抗体は、ラミナリンに結合せず、従ってラミナリンのβ１，３グルカンを認識しないポリクローナル抗体である。 The anti-PSK polyclonal antibody of the present invention does not recognize laminarin β1,3 glucan. β1,3 glucan is a polysaccharide in which glucose is linked by β1-3 type bonds. Widely distributed in nature such as plants, fungi and bacteria. For example, β1,3 glucan is a glucan present in laminarin or yeast glucan, but the anti-PSK polyclonal antibody of the present invention is a polyclonal antibody that does not bind to laminarin and therefore does not recognize laminarin β1,3 glucan. .
　本発明の抗ＰＳＫポリクローナル抗体は、セルロースのβ１，４グルカンのグルカン構造を認識しない。β１，４グルカンは、セルロース、又はグリコーゲンに存在する。セルロースは、分子式（Ｃ６Ｈ１０Ｏ５）ｎで表される多糖類であり、植物細胞の細胞壁および繊維の主成分である。本発明の抗ＰＳＫポリクローナル抗体は、セルロースに結合せず、従ってセルロースのβ１，４グルカンのグルカン構造を認識しないポリクローナル抗体である。 The anti-PSK polyclonal antibody of the present invention does not recognize the glucan structure of β1,4 glucan of cellulose. β1,4 glucan is present in cellulose or glycogen. Cellulose is a polysaccharide represented by the molecular formula (C 6 H 10 O 5 ) n and is the main component of plant cell walls and fibers. The anti-PSK polyclonal antibody of the present invention is a polyclonal antibody that does not bind to cellulose and therefore does not recognize the glucan structure of β1,4 glucan of cellulose.
　本発明の抗ＰＳＫポリクローナル抗体は、ラミナリンのβ１，６グルカンを認識しない。β１，６グルカンは、ラミナリン又はデキストランに存在する。デキストランは、グルコースのみからなる多糖類の一種である。本発明の抗ＰＳＫポリクローナル抗体は、ラミナリンに結合せず、従ってラミナリンのβ１，６グルカンを認識しないポリクローナル抗体である。 The anti-PSK polyclonal antibody of the present invention does not recognize β1,6 glucan of laminarin. β1,6 glucan is present in laminarin or dextran. Dextran is a kind of polysaccharide consisting only of glucose. The anti-PSK polyclonal antibody of the present invention is a polyclonal antibody that does not bind to laminarin and therefore does not recognize β1,6 glucan of laminarin.
　本明細書において、「ＰＳＫに特異的に結合する」とは、ＰＳＫに結合し、少なくともラミナリン及びセルロースのグルカンに実質的に結合しないことを意味する。また、ラミナリン及びセルロースのグルカンに実質的に結合しないとは、ＰＳＫの分析方法に本発明の抗体を用いた場合、実用的なレベルで非特異的な反応が生じないことを意味する。具体的には、例えば、Ｂｉａｃｏｒｅを用い、固相化した抗ＰＳＫポリクローナル抗体と、ＰＳＫ又はラミナリン若しくはセルロースとの結合を調べることによって、判定することができる。より具体的には、抗ＰＳＫポリクローナル抗体のＰＳＫに対する結合力を１００とした場合の、セルロースのβ１，４グルカンのグルカン構造、又はラミナリンのβ１，３グルカン、及びβ１，６グルカンのグルカン構造に対する結合力が３０％以下であることが好ましく、２０％以下であることが好ましく、１０％以下であることが最も好ましい。 In the present specification, “specifically binds to PSK” means that it binds to PSK and does not substantially bind to at least laminarin and cellulose glucan. The fact that it does not substantially bind to laminarin and cellulose glucan means that nonspecific reaction does not occur at a practical level when the antibody of the present invention is used in the PSK analysis method. Specifically, for example, the determination can be made by examining the binding of the immobilized anti-PSK polyclonal antibody to PSK, laminarin or cellulose using Biacore. More specifically, binding to the β1,4 glucan glucan structure of cellulose or the β1,3 glucan of laminarin and the glucan structure of β1,6 glucan when the binding strength of the anti-PSK polyclonal antibody to PSK is 100 The force is preferably 30% or less, more preferably 20% or less, and most preferably 10% or less.
（ＰＳＫのタンパク部分）
　本発明の抗ポリクローナル抗体が認識してもよいＰＳＫのタンパク部分は、ＰＳＫの約１８～３８％のタンパク質を占め、タンパク質の構成アミノ酸は、アスパラギン酸やグルタミン酸等の酸性アミノ酸と、バリンやロイシン等の中性アミノ酸が多く、リジンやアルギニン等の塩基性アミノ酸は少ない。 (Protein part of PSK)
The protein portion of PSK that may be recognized by the anti-polyclonal antibody of the present invention occupies about 18 to 38% of the protein of PSK, and the amino acids constituting the protein include acidic amino acids such as aspartic acid and glutamic acid, valine, leucine and the like. There are many neutral amino acids and few basic amino acids such as lysine and arginine.
（ＰＳＫに特異的なグルカン構造）
　本発明抗ポリクローナル抗体が認識してもよいＰＳＫに特異的なグルカン構造は、β１→３、β１→４及びβ１→６結合を含む分枝構造であり、主な構成単糖はグルコースやマンノースである。前記ＰＳＫに特異的なグルカン構造は、セルロースのβ１，４グルカンのグルカン構造、又はラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造とは異なるものである。 (Glucan structure specific to PSK)
The glucan structure specific to PSK that may be recognized by the anti-polyclonal antibody of the present invention is a branched structure containing β1 → 3, β1 → 4 and β1 → 6 bonds, and the main constituent monosaccharides are glucose and mannose. is there. The glucan structure specific to PSK is different from the glucan structure of β1,4 glucan of cellulose or the glucan structure of β1,3 glucan and β1,6 glucan of laminarin.
（カワラタケの熱水・アルカリ抽出物）
　従来の、ＰＳＫを免疫して得られる従来のポリクローナル抗体は、ラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造、並びにセルロースのβ１，４グルカンのグルカン構造に結合する抗体を含んでいた。この理由は、従来免疫に用いられていたＰＳＫが、ラミナリンのβ１，３グルカン及びβ１，６グルカン、並びにセルロースのβ１，４グルカンと同一又は構造の類似したグルカンを含んでいたためと考えられる。このようなグルカン構造を含むことは、特異性の高い抗体を取得するための免疫原としては、好ましくない。 (Hot water / alkaline extract of Kawaratake)
Conventional polyclonal antibodies obtained by immunization with PSK include antibodies that bind to the laminarin β1,3 glucan and β1,6 glucan glucan structures and the cellulose β1,4 glucan glucan structure. This is probably because PSK, which has been used for immunization, contained glucan having the same or similar structure to β1,3 glucan and β1,6 glucan of laminarin and β1,4 glucan of cellulose. Containing such a glucan structure is not preferable as an immunogen for obtaining a highly specific antibody.
　一方、本発明の抗ＰＳＫポリクローナル抗体は、ラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造、並びにセルロースのβ１，４グルカンのグルカン構造に結合する抗体を実質的に含まない。この理由は、免疫に用いたカワラタケの熱水・アルカリ抽出物が、ラミナリンのβ１，３グルカン及びβ１，６グルカン、並びにセルロースのβ１，４グルカンと同一又は構造の類似したグルカンを実質的に含まないためであると考えられる。 On the other hand, the anti-PSK polyclonal antibody of the present invention does not substantially contain an antibody that binds to the laminarin β1,3 glucan and β1,6 glucan glucan structures and the cellulose β1,4 glucan glucan structure. The reason for this is that the hot water / alkaline extract of Kawaratake used for immunization substantially contains laminarin β1,3 glucan and β1,6 glucan, and cellulose β1,4 glucan identical or similar in structure to glucan. This is thought to be because there is not.
　本発明の抗ＰＳＫポリクローナル抗体を取得するために用いられるカワラタケの熱水・アルカリ抽出物は、例えば、以下のように製造することができる。 The hot water / alkaline extract of Kawaratake used to obtain the anti-PSK polyclonal antibody of the present invention can be produced, for example, as follows.
　カワラタケ菌を常法に従って、培地に接種し、培養する。得られた菌苔をホモジェナイズし、種菌を調整する。前記種菌を常法に従って培養し、培養後に乾燥させ、乾燥菌糸体を得る。この乾燥菌糸体から、熱水及びアルカリ溶液を用いて抽出を行う。得られた熱水抽出液とアルカリ抽出液とを混合し、希塩酸で中和し、更に濃縮する。塩析を行い、沈殿を回収する。得られた沈殿から、凍結乾燥体として、熱水・アルカリ抽出物を得ることができる。 ¡Inoculate and cultivate Kawaratake fungus according to a conventional method. The obtained fungus moss is homogenized to prepare the inoculum. The inoculum is cultured according to a conventional method and dried after the culture to obtain a dried mycelium. Extraction is performed from the dried mycelium using hot water and an alkaline solution. The obtained hot water extract and alkali extract are mixed, neutralized with dilute hydrochloric acid, and further concentrated. Salt out and collect the precipitate. From the obtained precipitate, a hot water / alkali extract can be obtained as a lyophilized product.
　熱水及びアルカリ溶液による抽出は、まず熱水による抽出を行い、遠心して得られた残渣からアルカリ水溶液による抽出を行う。得られた熱水抽出液とアルカリ水溶液抽出液とを混合し、塩析及び沈殿等の工程を経て熱水・アルカリ抽出物とする。 In the extraction with hot water and an alkaline solution, extraction with hot water is first performed, and the aqueous solution is extracted from the residue obtained by centrifugation. The obtained hot water extract and the aqueous alkaline solution are mixed and subjected to processes such as salting out and precipitation to obtain a hot water / alkaline extract.
　熱水の温度は、５０℃以上であれば、特に限定されないが、７０℃以上が好ましく、８０℃以上がより好ましく、９５～９９℃が最も好ましい。また、熱水による抽出時間も、特に限定されるものではないが、１０分～６時間が好ましく、１時間～３時間がより好ましく、３０分～９０分が最も好ましい。 The temperature of hot water is not particularly limited as long as it is 50 ° C or higher, but is preferably 70 ° C or higher, more preferably 80 ° C or higher, and most preferably 95 to 99 ° C. The extraction time with hot water is not particularly limited, but is preferably 10 minutes to 6 hours, more preferably 1 hour to 3 hours, and most preferably 30 minutes to 90 minutes.
　アルカリ溶液は、特に限定されるものではないが、例えばアルカリ金属の水酸化物を用いることが可能であり、水酸化ナトリウム水溶液が好ましい。例えば水酸化ナトリウム水溶液を用いる場合、その濃度は、０．２Ｍ以上であれば特に限定されるものではないが、０．３Ｍ～３Ｍが好ましく、０．５Ｍ～１．５Ｍがより好ましく、０．７Ｍ～１．３Ｍが最も好ましい。前記アルカリ溶液の温度は、４℃以上であれば、特に限定されないが、２５℃以上が好ましく、５０℃以上がより好ましく、９５～９９℃が最も好ましい。また、アルカリ溶液による抽出時間も、特に限定されるものではないが、１０分～６時間が好ましく、１時間～３時間がより好ましく、３０分～９０分が最も好ましい。 The alkali solution is not particularly limited. For example, an alkali metal hydroxide can be used, and a sodium hydroxide aqueous solution is preferable. For example, when an aqueous sodium hydroxide solution is used, the concentration is not particularly limited as long as it is 0.2 M or higher, but is preferably 0.3 M to 3 M, more preferably 0.5 M to 1.5 M, and Most preferred is 7M to 1.3M. The temperature of the alkaline solution is not particularly limited as long as it is 4 ° C. or higher, but is preferably 25 ° C. or higher, more preferably 50 ° C. or higher, and most preferably 95 to 99 ° C. The extraction time with an alkaline solution is not particularly limited, but is preferably 10 minutes to 6 hours, more preferably 1 hour to 3 hours, and most preferably 30 minutes to 90 minutes.
　従来のＰＳＫの抽出方法は、例えば特許文献２の実施例に記載のように、０．１Ｍ水酸化ナトリウム水溶液のみで抽出するものである。このような抽出法において得られたＰＳＫは、ラミナリンのβ１，３グルカン及びβ１，６グルカン、並びにセルロースのβ１，４グルカンと同一又は構造の類似したグルカンを含むものと考えられる。一方、本発明の抗ポリクローナル抗体を取得するために用いる熱水・アルカリ抽出物のように、水抽出及びアルカリ抽出を組み合わせることにより、ラミナリンのβ１，３グルカン及びβ１，６グルカン、並びにセルロースのβ１，４グルカンと同一又は構造の類似したグルカンを含まないものを得ることができると考えられる。 A conventional method for extracting PSK is, for example, extraction with a 0.1 M aqueous sodium hydroxide solution as described in the Examples of Patent Document 2. The PSK obtained by such an extraction method is considered to contain glucan having the same or similar structure to β1,3 glucan and β1,6 glucan of laminarin and β1,4 glucan of cellulose. On the other hand, by combining water extraction and alkali extraction like the hot water / alkaline extract used to obtain the anti-polyclonal antibody of the present invention, β1,3 glucan and β1,6 glucan of laminarin, and β1 of cellulose , 4 glucan is considered to be obtained that does not contain the same or similar structure glucan.
（抗原結合性断片）
　本発明の抗原結合性断片は、本発明の抗ＰＳＫポリクローナル抗体の抗原結合部位を含み、ＰＳＫへの結合性を有する限り、特に限定されるものではないが、例えばＦａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ断片、ディアボディー、単一鎖抗体分子、及びマルチ特異性抗体を挙げることができる。これらの抗原結合性断片は、例えば、抗体を常法によりタンパク質分解酵素（例えば、ペプシン又はパパイン等）によって消化し、続いて、常法のタンパク質の分離精製の方法により精製することにより、得ることができる。また、ディアボディー、単一鎖抗体分子、及び抗体断片から形成されたマルチ特異性抗体は、遺伝子組換えにより調製することができる。 (Antigen-binding fragment)
The antigen-binding fragment of the present invention is not particularly limited as long as it contains the antigen-binding site of the anti-PSK polyclonal antibody of the present invention and has binding ability to PSK. For example, Fab, Fab ′, F (ab ') 2 , Fv fragments, diabodies, single chain antibody molecules, and multispecific antibodies. These antigen-binding fragments can be obtained, for example, by digesting an antibody with a proteolytic enzyme (for example, pepsin or papain) by a conventional method, and subsequently purifying the protein by a conventional method for separating and purifying proteins. Can do. In addition, multispecific antibodies formed from diabodies, single chain antibody molecules, and antibody fragments can be prepared by genetic recombination.
［２］ＰＳＫの免疫学的分析方法
　本発明のＰＳＫの免疫学的分析方法は、ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片を用いることを特徴とするものである。ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片としては、前記の「［１］抗ＰＳＫポリクローナル抗体」の項目に記載の抗体を用いることができる。例えば、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体を用いてもよく、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体を用いてもよい。ＰＳＫの免疫学的分析方法を用いることにより、ＰＳＫを正確に分析することができる。 [2] Immunological analysis method of PSK The immunological analysis method of PSK of the present invention is characterized by using an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof. As the anti-PSK polyclonal antibody or antigen-binding fragment thereof that specifically binds to PSK, the antibody described in the item “[1] Anti-PSK polyclonal antibody” can be used. For example, an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific for PSK may be used, reacts with PSK, and the glucan structure of β1,4 glucan of cellulose, and of laminarin. An anti-PSK polyclonal antibody that does not react with the glucan structure of β1,3 glucan and β1,6 glucan may be used. By using the immunological analysis method for PSK, PSK can be accurately analyzed.
　ＰＳＫを免疫学的に分析する方法としては、本発明の抗ＰＳＫポリクローナル抗体又はその抗原結合性断片を用いて、ＰＳＫを定量的又は半定量的に決定することができるか、あるいは、ＰＳＫの存在の有無を判定することができる限り、特に限定されるものではない。例えば、酵素免疫測定法、免疫組織染色法、表面プラズモン共鳴法（ＳＰＲ法：Ｂｉａｃｏｒｅ法）、ラテックス凝集免疫測定法、化学発光免疫測定法、蛍光抗体法、放射免疫測定法、免疫沈降法、ウエスタンブロット法、イムノクロマトグラフ法、磁気ビーズ凝集法、又は磁気ビーズ酵素免疫法を挙げることができる。 As a method for immunologically analyzing PSK, PSK can be quantitatively or semi-quantitatively determined using the anti-PSK polyclonal antibody of the present invention or an antigen-binding fragment thereof, or the presence of PSK. There is no particular limitation as long as the presence or absence can be determined. For example, enzyme immunoassay, immunohistochemical staining, surface plasmon resonance (SPR method: Biacore method), latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation, western A blotting method, an immunochromatography method, a magnetic bead aggregation method, or a magnetic bead enzyme immunization method can be mentioned.
　なお、本明細書における「分析」には、分析対象物質の量を定量的又は半定量的に決定する「測定」と、分析対象物質の存在の有無を判定する「検出」との両方の意味が含まれる。 In this specification, “analysis” means both “measurement” that quantitatively or semi-quantitatively determines the amount of an analyte, and “detection” that determines the presence or absence of the analyte. Is included.
（被検試料）
　ＰＳＫの免疫学的分析方法に用いることのできる被検試料としては、ＰＳＫを含有する可能性のある試料であれば特に限定されるものではないが、特には生理活性を有するＰＳＫを含有する可能性のある医薬品若しくは飲食物、又はＰＳＫが投与された患者の生体試料又は生体由来試料を挙げることができる。医薬品又は飲食物としては、具体的には、医薬組成物、若しくは医薬製剤、又はそれらの原料となる菌類由来の熱水・アルカリ抽出物、あるいは健康食品、若しくは機能性食品、又はそれらの原料となる菌類由来の熱水・アルカリ抽出物を挙げることができる。また、生体試料又は生体由来試料としては、例えば、尿、血液、血清、血漿、便、髄液、唾液、細胞、組織、若しくは器官、又はそれらの調整物（例えば、生検標本）等を挙げることができる。 (Test sample)
The test sample that can be used in the immunological analysis method of PSK is not particularly limited as long as it is a sample that may contain PSK, but in particular, it may contain PSK having physiological activity. Examples include biological samples or biological samples of patients who have sex medicine or food or drink, or PSK. Specifically, as pharmaceuticals or foods and drinks, pharmaceutical compositions, pharmaceutical preparations, hot water / alkaline extracts derived from fungi used as raw materials thereof, health foods, functional foods, or raw materials thereof And hot water / alkaline extract derived from fungi. Examples of biological samples or biological samples include urine, blood, serum, plasma, feces, spinal fluid, saliva, cells, tissues, organs, or preparations thereof (for example, biopsy specimens). be able to.
　ＰＳＫの免疫学的分析方法を用いることにより、医薬品や飲食物並びにＰＳＫを服用後の血液や組織などから、特定の熱水・アルカリ抽出物を定性的・定量的に検出できる。従って、生理活性を有するＰＳＫの摂取量（投与量）を把握できるために大変有用である。例えば、ＰＳＫ投与後の血中濃度や腫瘍へのＰＳＫの到達度合を簡易かつ高精度に判別できるため、体内動態や薬効を簡易かつ高精度に評価できる。 By using the immunological analysis method of PSK, a specific hot water / alkaline extract can be detected qualitatively and quantitatively from medicines, foods and drinks, blood and tissues after taking PSK. Therefore, it is very useful because the intake (dose) of physiologically active PSK can be grasped. For example, since the blood concentration after PSK administration and the degree of PSK reaching the tumor can be determined easily and with high accuracy, pharmacokinetics and drug efficacy can be evaluated easily and with high accuracy.
　更に、本発明のＰＳＫの免疫学的分析方法により、血中濃度の測定を行うことにより、ＰＳＫの薬効の予測およびＰＳＫの投与至適濃度を把握するためのデータ測定にも利用することができる。例えば、ＰＳＫの薬効の予測を行う場合、ＰＳＫ投与の一定時間後に、ＰＳＫが投与された患者のＰＳＫの血中濃度を測定する。得られた血中濃度から、ある一定以上の血中濃度であれば、ＰＳＫの薬効が高いことを予測可能であり、一定以下の血中濃度であれば、ＰＳＫの薬効が低いことを予測可能である。また、患者によって、ＰＳＫの投与至適濃度が異なることがある。 Furthermore, by measuring the blood concentration by the immunological analysis method of PSK of the present invention, it can be used for the measurement of data for predicting the drug effect of PSK and grasping the optimum concentration of PSK. . For example, when predicting the efficacy of PSK, the blood concentration of PSK in a patient to whom PSK is administered is measured after a certain time after PSK administration. From the obtained blood concentration, if the blood concentration is above a certain level, it can be predicted that the PSK will have high efficacy, and if it is below a certain level, it can be predicted that the PSK will have low efficacy. It is. In addition, the optimal concentration of PSK may vary depending on the patient.
　従って、ＰＳＫの投与至適濃度を把握することは、患者の治療において重要である。ＰＳＫの投与至適濃度を把握するためには、ＰＳＫの投与の一定時間後に、ＰＳＫが投与された患者のＰＳＫの血中濃度を測定する。得られた血中濃度が低く薬効が期待できない場合は、ＰＳＫの投与量を増加させ、逆に血中濃度が高い場合は、ＰＳＫの投与量を減少させることができる。 Therefore, grasping the optimal concentration of PSK is important in the treatment of patients. In order to grasp the optimal concentration of PSK, the blood concentration of PSK in a patient to whom PSK is administered is measured after a certain time after the administration of PSK. When the obtained blood concentration is low and a drug effect cannot be expected, the dose of PSK can be increased. Conversely, when the blood concentration is high, the dose of PSK can be decreased.
　本発明のＰＳＫの免疫学的分析方法の好ましい１つの実施態様においては、ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片の１種類を用いて、ＰＳＫを分析することができる。例えばこのような実施態様としては、免疫組織染色法、表面プラズモン共鳴法（ＳＰＲ法：Ｂｉａｃｏｒｅ法）、ラテックス凝集免疫測定法、蛍光抗体法、放射免疫測定法、免疫沈降法、又はウエスタンブロット法を挙げることができる。 In one preferred embodiment of the immunological analysis method for PSK of the present invention, PSK can be analyzed using an anti-PSK polyclonal antibody that specifically binds to PSK or one of its antigen-binding fragments. . For example, as such an embodiment, immunohistological staining method, surface plasmon resonance method (SPR method: Biacore method), latex agglutination immunoassay method, fluorescent antibody method, radioimmunoassay method, immunoprecipitation method, or Western blot method may be used. Can be mentioned.
　本発明のＰＳＫの免疫学的分析方法の別の好ましい１つの実施態様においては、ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片と、ＰＳＫと反応する第２の抗体又はその抗原結合性断片とを使用して、ＰＳＫを分析することができる。本実施態様で用いる抗ＰＳＫポリクローナル抗体は、本発明の抗ＰＳＫポリクローナル抗体である。本実施態様としては、具体的には、例えば抗ＰＳＫポリクローナル抗体及び第２の抗体を用いたサンドイッチ法を挙げることができる。更に、抗ＰＳＫポリクローナル抗体及び第２の抗体を混合して用いる免疫組織染色法、表面プラズモン共鳴法（ＳＰＲ法：Ｂｉａｃｏｒｅ法）、ラテックス凝集免疫測定法、蛍光抗体法、放射免疫測定法、免疫沈降法、又はウエスタンブロット法を挙げることができる。また、本実施態様においては、２種類以上の第２の抗体を用いることもできる。 In another preferred embodiment of the method for immunological analysis of PSK of the present invention, an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof, and a second antibody that reacts with PSK or its PSK can be analyzed using antigen-binding fragments. The anti-PSK polyclonal antibody used in this embodiment is the anti-PSK polyclonal antibody of the present invention. Specific examples of this embodiment include a sandwich method using an anti-PSK polyclonal antibody and a second antibody. Furthermore, immunohistological staining method using a mixture of anti-PSK polyclonal antibody and second antibody, surface plasmon resonance method (SPR method: Biacore method), latex agglutination immunoassay method, fluorescent antibody method, radioimmunoassay method, immunoprecipitation method Or Western blotting. In this embodiment, two or more types of second antibodies can also be used.
　第２の抗体は、ＰＳＫに結合することのできるものであれば、特に限定されないが、例えば、ＰＳＫを認識し、且つ他の多糖類のβ１，３グルカン、β１，４グルカン、及びβ１，６グルカンを認識する非特許文献１に記載のポリクローナル抗体、又はＰＳＫに特異的に結合する抗ＰＳＫ抗体を挙げることができるが、ＰＳＫ特異的抗体が好ましい。 The second antibody is not particularly limited as long as it can bind to PSK. For example, it recognizes PSK and other polysaccharides β1,3 glucan, β1,4 glucan, and β1,6 The polyclonal antibody described in Non-Patent Document 1 that recognizes glucan or the anti-PSK antibody that specifically binds to PSK can be mentioned, and a PSK-specific antibody is preferred.
　ＰＳＫ特異的な抗体としては、例えば、本発明のＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体（例えば、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体、又はＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体）；ＰＳＫを認識し、且つデキストラン、グリコーゲン、ラミナリン、及びセルロースのβ１，３グルカン、β１，４グルカン、及びβ１，６グルカンを認識しないモノクローナル抗体；又はＰＳＫの生理活性部位を認識するモノクローナル抗体を挙げることができるが、最も好ましくは、ＰＳＫの生理活性部位を認識するモノクローナル抗体である。活性を有するＰＳＫを正確に測定することができるからである。 Examples of the PSK-specific antibody include an anti-PSK polyclonal antibody that specifically binds to the PSK of the present invention (for example, an anti-PSK polyclonal antibody that recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK). Or an anti-PSK polyclonal antibody that reacts with PSK and does not react with the β1,4 glucan glucan structure of cellulose and the β1,3 glucan and β1,6 glucan glucan structures of laminarin); and recognizes PSK and dextran, Examples include monoclonal antibodies that do not recognize β1,3 glucan, β1,4 glucan, and β1,6 glucan of glycogen, laminarin, and cellulose; or monoclonal antibodies that recognize a physiologically active site of PSK, most preferably Physiological activity of PSK Place is a monoclonal antibody that recognizes. This is because PSK having activity can be accurately measured.
　第２の抗体は、本発明の抗ＰＳＫポリクローナル抗体を除いては、常法に従って調製することができる。例えば、モノクローナル抗体は、ＫｏｅｈｌｅｒとＭｉｌｓｔｅｉｎの方法（Ｎａｔｕｒｅ　２５６：４９５－４９７，１９７５）に従って、作製することができる。抗ＰＳＫ抗体を取得するための免疫抗原は、カワラタケ菌ＣＭ１０１株〔ＦＥＲＭ－Ｐ２４１２（ＡＴＣＣ２０５４７）〕の菌糸体を水系溶媒、例えば、熱水又はアルカリ溶液（例えば、アルカリ金属の水酸化物、特には水酸化ナトリウムの水溶液）で抽出し、精製した後に乾燥して得たものであり、抗腫瘍活性を有するものであれば、特に限定せずに用いることができる。すなわち、免疫原として、ＰＳＫを用いること以外は、通常のモノクローナル抗体調製法に従って、調製することができる。しかしながら、第２の抗体を得る場合にも、免疫原として、前記のカワラタケの熱水・アルカリ抽出物を用いることが好ましい。 The second antibody can be prepared according to a conventional method except for the anti-PSK polyclonal antibody of the present invention. For example, monoclonal antibodies can be prepared according to the method of Koehler and Milstein (Nature 256: 495-497, 1975). The immunizing antigen for obtaining the anti-PSK antibody is obtained by using a mycelium of Kawaratake fungus CM101 strain [FERM-P2412 (ATCC20547)] in an aqueous solvent such as hot water or an alkaline solution (for example, alkali metal hydroxide, particularly It can be used without particular limitation as long as it is obtained by extraction with an aqueous solution of sodium hydroxide), purification and drying, and it has antitumor activity. That is, it can be prepared according to a conventional monoclonal antibody preparation method except that PSK is used as an immunogen. However, when obtaining the second antibody, it is preferable to use the above-mentioned hot water / alkaline extract of Kawaratake as an immunogen.
　ＰＳＫの生理活性部位を認識するモノクローナル抗体としては、例えば以下の抗体を挙げることができる。
　（モノクローナル抗体（Ａ））
　モノクローナル抗体（Ａ）は、後述の参考例に記載の２Ｇ９抗体に代表されるものである。その重鎖可変領域ドメインは、好ましくは配列番号６で表されるアミノ酸配列（ＳＹＧＭＳ）からなるＨ－ＣＤＲ１のポリペプチド、配列番号１０で表されるアミノ酸配列（ＴＩＳＳＧＧＳＹＴＹＹＰＤＳＶＫＧ）からなるＨ－ＣＤＲ２のポリペプチド、配列番号１４で表されるアミノ酸配列（ＲＩＴＴＶＶＡＲＳＦＹＦＤＹ）からなるＨ－ＣＤＲ３のポリペプチドを含む。また、この抗体の軽鎖可変領域ドメインは、好ましくは配列番号２２で表されるアミノ酸配列（ＲＡＳＫＳＶＳＴＳＧＹＳＹＭＨ）からなるＬ－ＣＤＲ１のポリペプチド、配列番号２６で表されるアミノ酸配列（ＬＶＳＮＬＥＳ）からなるＬ－ＣＤＲ２のポリペプチド、配列番号３０で表されるアミノ酸配列（ＱＨＩＲＥＬＴＲＳ）からなるＬ－ＣＤＲ３のポリペプチドを含む。 Examples of monoclonal antibodies that recognize PSK bioactive sites include the following antibodies.
(Monoclonal antibody (A))
The monoclonal antibody (A) is typified by the 2G9 antibody described in Reference Examples described later. The heavy chain variable region domain is preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 (SYGMS), and an H-CDR2 polymorphism consisting of the amino acid sequence represented by SEQ ID NO: 10 (TISSGGSYTYYPDSVKG). The peptide includes an H-CDR3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 (RITTVVARSFYFDY). The light chain variable region domain of this antibody is preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22 (RASKSVSSTSGYSYMH), and an L-sequence consisting of the amino acid sequence represented by SEQ ID NO: 26 (LVSNLES). -A polypeptide of CDR2, an L-CDR3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 30 (QHIRELTRS).
　更に、モノクローナル抗体（Ａ）の重鎖可変領域ドメインは、より好ましくは、配列番号６で表されるアミノ酸配列からなるＨ－ＣＤＲ１のポリペプチド、配列番号１０で表されるアミノ酸配列からなるＨ－ＣＤＲ２のポリペプチド、配列番号１４で表されるアミノ酸配列からなるＨ－ＣＤＲ３のポリペプチド、並びに重鎖可変領域フレームワークのポリペプチドを含み、最も好ましくは、配列番号２で表されるアミノ酸配列からなる重鎖可変領域ドメインのポリペプチドである。また、この抗体の軽鎖可変領域ドメインは、より好ましくは配列番号２２で表されるアミノ酸配列からなるＬ－ＣＤＲ１のポリペプチド、配列番号２６で表されるアミノ酸配列からなるＬ－ＣＤＲ２のポリペプチド、配列番号３０で表されるアミノ酸配列からなるＬ－ＣＤＲ３のポリペプチド、並びに軽鎖可変領域フレームワークのポリペプチドを含み、最も好ましくは、配列番号１８で表されるアミノ酸配列からなる軽鎖可変領域ドメインのポリペプチドである。 Further, the heavy chain variable region domain of the monoclonal antibody (A) is more preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and an H-- consisting of the amino acid sequence represented by SEQ ID NO: 10. A polypeptide of CDR2, an H-CDR3 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 14, and a heavy chain variable region framework polypeptide, most preferably from the amino acid sequence represented by SEQ ID NO: 2. A heavy chain variable region domain polypeptide. The light chain variable region domain of this antibody is more preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22, and an L-CDR2 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 26 An L-CDR3 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 30 and a light chain variable region framework polypeptide, most preferably a light chain variable comprising the amino acid sequence represented by SEQ ID NO: 18. A domain domain polypeptide.
　モノクローナル抗体（Ａ）は、例えば２Ｇ９抗体とエピトープへの結合が競合する抗体を含み、特には、２Ｇ９抗体が結合するＰＳＫのエピトープと同一のエピトープに結合する抗体を挙げることができる。 The monoclonal antibody (A) includes, for example, an antibody that competes with the 2G9 antibody for binding to the epitope, and particularly includes an antibody that binds to the same epitope as the PSK epitope to which the 2G9 antibody binds.
　２Ｇ９抗体が結合するＰＳＫのエピトープは、ＰＳＫの細胞障害活性を示す生理活性部位に存在するエピトープであるか、あるいはその近傍のエピトープである可能性が高く、２Ｇ９抗体がそのエピトープに結合することにより、ＰＳＫの細胞障害活性を示す生理活性部位の活性を抑制させることができるエピトープである。また、２Ｇ９抗体が結合するＰＳＫのエピトープは、ＰＳＫのＴＧＦ－β１阻害活性を示す生理活性部位に存在するエピトープであるか、あるいはその近傍のエピトープである可能性が高く、２Ｇ９抗体がそのエピトープに結合することにより、ＰＳＫのＴＧＦ－β１阻害活性を示す生理活性部位の活性を抑制させることができるエピトープである。 The PSK epitope to which the 2G9 antibody binds is likely to be an epitope present in the physiologically active site exhibiting the cytotoxic activity of PSK or an epitope in the vicinity thereof, and the 2G9 antibody binds to the epitope. , An epitope capable of suppressing the activity of a physiologically active site exhibiting cytotoxic activity of PSK. In addition, the PSK epitope to which the 2G9 antibody binds is likely to be an epitope present in the physiologically active site exhibiting TGF-β1 inhibitory activity of PSK, or an epitope in the vicinity thereof, and the 2G9 antibody is considered to be the epitope. By binding, it is an epitope that can suppress the activity of a physiologically active site showing TGF-β1 inhibitory activity of PSK.
　本明細書において、「エピトープへの結合が競合する抗体」とは、２つの抗体を用いたエピトープの競合試験において、競合作用を示したすべての抗体を含む。２つの抗体を用いたエピトープの競合試験において、競合率を計算することができ、「エピトープへの結合が競合する抗体」は、１％～１００％の競合率を示すことがあり、具体的には１０％以上、２０％以上、３０％以上、４０％以上、５０％以上、６０％以上、７０％以上、８０％以上又は９０％以上の競合率を示す抗体を含む。 In this specification, “an antibody that competes for binding to an epitope” includes all antibodies that have shown a competitive action in an epitope competition test using two antibodies. In an epitope competition test using two antibodies, the competition rate can be calculated. “Antibodies that compete for binding to the epitope” may exhibit a competition rate of 1% to 100%, specifically Includes antibodies exhibiting a competitive rate of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
　また、「同一のエピトープに結合する」とは、抗体の抗原結合部位が結合するエピトープが同一であることを意味し、この抗体は２つの抗体を用いたエピトープの競合試験において、競合作用を示す。「同一のエピトープに結合する」抗体のエピトープの競合試験における競合率は特に限定されるものではない。これは、エピトープの競合試験における競合率は、２つの抗体の力価、結合定数、解離定数、及び親和定数等により決定されるためである。従って、「同一のエピトープに結合する」抗体は、１％～１００％の競合率を示すことがあり、具体的には１０％以上、２０％以上、３０％以上、４０％以上、５０％以上、６０％以上、７０％以上、８０％以上又は９０％以上の競合率を示す抗体を含むことができる。 In addition, “binding to the same epitope” means that the epitope to which the antigen-binding site of the antibody binds is the same, and this antibody exhibits a competitive action in an epitope competition test using two antibodies. . The competition rate in the epitope competition test of an antibody “binding to the same epitope” is not particularly limited. This is because the competition rate in the epitope competition test is determined by the titer, binding constant, dissociation constant, affinity constant, and the like of the two antibodies. Therefore, an antibody that “binds to the same epitope” may exhibit a competition rate of 1% to 100%, specifically 10% or more, 20% or more, 30% or more, 40% or more, 50% or more. , 60% or higher, 70% or higher, 80% or higher, or 90% or higher.
　エピトープの競合試験は、以下の方法により行うことができる。ＰＳＫを１μｇ／ｗｅｌｌの濃度で、９６ウェルプレートに４℃で一晩コート後、１％ＢＳＡでブロッキングしてＰＳＫを固相化したプレートを作製する。例えば、０．１μｇ／ｍＬ、０．５μｇ／ｍＬ、１μｇ／ｍＬ、又は５μｇ／ｍＬの第一の抗体を添加して、２５℃で３時間インキュベートする。ＴＢＳＴで各ウェルを３回洗浄した後、０．５μｇ／ｍＬ濃度に調製したＨＲＰ標識した第二の抗体溶液を添加して、２５℃で１時間インキュベートする。ＴＢＳＴで各ウェルを３回洗浄した後、基質であるＡＢＳＴを加え、１５分間程度発色させた。Ｐｅｒｏｘｉｄａｓｅ　Ｓｔｏｐ　Ｓｏｌｕｔｉｏｎで発色反応を停止させた後、プレートリーダーを用いて、４０５ｎｍの吸光度を測定し、競合率を計算する。 The epitope competition test can be performed by the following method. PSK is coated at a concentration of 1 μg / well on a 96-well plate overnight at 4 ° C., and then blocked with 1% BSA to prepare a plate on which PSK is immobilized. For example, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, or 5 μg / mL first antibody is added and incubated at 25 ° C. for 3 hours. After washing each well 3 times with TBST, a second HRP-labeled antibody solution prepared to a concentration of 0.5 μg / mL is added and incubated at 25 ° C. for 1 hour. Each well was washed three times with TBST, then ABST as a substrate was added, and color was developed for about 15 minutes. After stopping the color reaction with Peroxidase Stop Solution, the absorbance at 405 nm is measured using a plate reader, and the competition rate is calculated.
　（モノクローナル抗体（Ｂ））
　モノクローナル抗体（Ｂ）は、後述の参考例に記載の５Ｇ５抗体に代表されるものである。その重鎖可変領域ドメインは、好ましくは配列番号３８で表されるアミノ酸配列（ＧＹＴＭＮ）からなるＨ－ＣＤＲ１のポリペプチド、配列番号４２で表されるアミノ酸配列（ＬＩＮＰＹＮＧＧＴＳＹＮＱＫＦＫＧ）からなるＨ－ＣＤＲ２のポリペプチド、配列番号４６で表されるアミノ酸配列（ＧＧＫＦＡＴＧＴＳＹ）からなるＨ－ＣＤＲ３のポリペプチドを含む。また、この抗体の軽鎖可変領域ドメインは、好ましくは配列番号５４で表されるアミノ酸配列（ＲＳＳＴＧＡＶＴＴＳＮＹＡＮ）からなるＬ－ＣＤＲ１のポリペプチド、配列番号５８で表されるアミノ酸配列（ＧＴＮＮＲＡＰ）からなるＬ－ＣＤＲ２のポリペプチド、配列番号６２で表されるアミノ酸配列（ＡＬＷＹＳＮＨＷＶ）からなるＬ－ＣＤＲ３のポリペプチドを含む。 (Monoclonal antibody (B))
The monoclonal antibody (B) is typified by the 5G5 antibody described in Reference Examples described later. The heavy chain variable region domain is preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 38 (GYTMN), and an H-CDR2 polymorphism consisting of the amino acid sequence represented by SEQ ID NO: 42 (LINPYNGGTSYNQKFKG). The peptide includes an H-CDR3 polypeptide consisting of the amino acid sequence (GGKFAGTTSY) represented by SEQ ID NO: 46. The light chain variable region domain of this antibody is preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 54 (RSSTGAVTTSNYAN) and an L-sequence consisting of the amino acid sequence represented by SEQ ID NO: 58 (GTNNRAP). -A polypeptide of CDR2, an L-CDR3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 62 (ALWYSNHWV).
　更に、モノクローナル抗体（Ｂ）の重鎖可変領域ドメインは、より好ましくは、配列番号３８で表されるアミノ酸配列からなるＨ－ＣＤＲ１のポリペプチド、配列番号４２で表されるアミノ酸配列からなるＨ－ＣＤＲ２のポリペプチド、配列番号４６で表されるアミノ酸配列からなるＨ－ＣＤＲ３のポリペプチド、並びに重鎖可変領域フレームワークのポリペプチドを含み、最も好ましくは、配列番号３４で表されるアミノ酸配列からなる重鎖可変領域ドメインのポリペプチドである。また、この抗体の軽鎖可変領域ドメインは、より好ましくは配列番号５４で表されるアミノ酸配列からなるＬ－ＣＤＲ１のポリペプチド、配列番号５８で表されるアミノ酸配列からなるＬ－ＣＤＲ２のポリペプチド、配列番号６２で表されるアミノ酸配列からなるＬ－ＣＤＲ３のポリペプチド、並びに軽鎖可変領域フレームワークのポリペプチドを含み、最も好ましくは、配列番号５０で表されるアミノ酸配列からなる軽鎖可変領域ドメインのポリペプチドである。 Furthermore, the heavy chain variable region domain of the monoclonal antibody (B) is more preferably an H-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 38, and an H-- consisting of the amino acid sequence represented by SEQ ID NO: 42. A CDR2 polypeptide, an H-CDR3 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 46, and a heavy chain variable region framework polypeptide, most preferably from the amino acid sequence represented by SEQ ID NO: 34 A heavy chain variable region domain polypeptide. The light chain variable region domain of this antibody is more preferably an L-CDR1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 54 and an L-CDR2 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 58 An L-CDR3 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 62, and a light chain variable region framework polypeptide, most preferably a light chain variable comprising the amino acid sequence represented by SEQ ID NO: 50 A domain domain polypeptide.
　モノクローナル抗体（Ｂ）は、例えば５Ｇ５抗体とエピトープへの結合が競合する抗体を含み、特には、５Ｇ５抗体が、結合するＰＳＫのエピトープと同一のエピトープに結合する抗体を挙げることができる。 Monoclonal antibody (B) includes, for example, an antibody that competes with the 5G5 antibody for binding to the epitope, and in particular, an antibody that binds to the same epitope as the epitope of PSK to which the 5G5 antibody binds.
　５Ｇ５抗体が結合するＰＳＫのエピトープは、ＰＳＫの細胞障害活性を示す生理活性部位に存在するエピトープであるか、あるいはその近傍のエピトープである可能性が高く、５Ｇ５抗体がそのエピトープに結合することにより、ＰＳＫの細胞障害活性を示す生理活性部位の活性を抑制させることができるエピトープである。また、５Ｇ５抗体が結合するＰＳＫのエピトープは、ＰＳＫのＴＧＦ－β１阻害活性を示す生理活性部位に存在するエピトープであるか、あるいはその近傍のエピトープである可能性が高く、５Ｇ５抗体がそのエピトープに結合することにより、ＰＳＫのＴＧＦ－β１阻害活性を示す生理活性部位の活性を抑制させることができるエピトープである。 The PSK epitope to which the 5G5 antibody binds is likely to be an epitope present in the physiologically active site exhibiting the cytotoxic activity of PSK or an epitope in the vicinity thereof, and the 5G5 antibody binds to the epitope. , An epitope capable of suppressing the activity of a physiologically active site exhibiting cytotoxic activity of PSK. In addition, the PSK epitope to which the 5G5 antibody binds is likely to be an epitope present in the physiologically active site exhibiting TGF-β1 inhibitory activity of PSK, or an epitope in the vicinity thereof, and the 5G5 antibody is considered to be the epitope. By binding, it is an epitope that can suppress the activity of a physiologically active site showing TGF-β1 inhibitory activity of PSK.
　モノクローナル抗体（Ａ）及びモノクローナル抗体（Ｂ）の、Ｈ－ＣＤＲ１のポリペプチド、Ｈ－ＣＤＲ２のポリペプチド、Ｈ－ＣＤＲ３のポリペプチド、Ｌ－ＣＤＲ１のポリペプチド、Ｌ－ＣＤＲ２のポリペプチド、及びＬ－ＣＤＲ３のポリペプチドは、それぞれ１又は数個のアミノ酸が欠失、置換、挿入又は付加されてもよい。それらの欠失、置換、挿入又は付加されたポリペプチドを含む重鎖可変領域ドメイン及び軽鎖可変領域ドメインから形成される抗原結合部位が結合するエピトープは、２Ｇ９抗体又は５Ｇ５抗体が結合するエピトープと同一であり、その結合によりＰＳＫの細胞障害活性を抑制することができる。 Monoclonal antibody (A) and monoclonal antibody (B), H-CDR1 polypeptide, H-CDR2 polypeptide, H-CDR3 polypeptide, L-CDR1 polypeptide, L-CDR2 polypeptide, and L -CDR3 polypeptides may each have one or several amino acids deleted, substituted, inserted or added. The epitope to which the antigen-binding site formed from the heavy chain variable region domain and the light chain variable region domain containing the polypeptide having the deletion, substitution, insertion or addition thereof binds to the epitope to which the 2G9 antibody or 5G5 antibody binds It is the same, and its binding can suppress the cytotoxic activity of PSK.
　また、モノクローナル抗体（Ａ）及びモノクローナル抗体（Ｂ）の、重鎖可変領域ドメイン又は軽鎖可変領域ドメインのポリペプチドも、それぞれ１又は数個のアミノ酸が欠失、置換、挿入又は付加されてもよい。それらの欠失、置換、挿入又は付加されたポリペプチドを含む重鎖可変領域ドメイン及び軽鎖可変領域ドメインから形成される抗原結合部位が結合するエピトープは、２Ｇ９抗体又は５Ｇ５抗体が結合するエピトープと同一であり、その結合によりＰＳＫの細胞障害活性を抑制することができる。 In addition, the heavy chain variable region domain or light chain variable region domain polypeptide of the monoclonal antibody (A) and the monoclonal antibody (B) may be deleted, substituted, inserted, or added, respectively. Good. The epitope to which the antigen-binding site formed from the heavy chain variable region domain and the light chain variable region domain containing the polypeptide having the deletion, substitution, insertion or addition thereof binds to the epitope to which the 2G9 antibody or 5G5 antibody binds It is the same, and its binding can suppress the cytotoxic activity of PSK.
　より具体的には、アミノ酸の欠失、置換、挿入、又は付加は、それぞれのポリペプチドにおいて３個以下が好ましく、２個以下がより好ましく、１個が最も好ましい。またアミノ酸の置換の場合、限定されるものではないが、親水性のアミノ酸は親水性のアミノ酸と、疎水性のアミン酸は親水性のアミノ酸と、塩基性のアミノ酸は塩基性のアミノ酸と、酸性アミノ酸は酸性のアミノ酸と、置換されることが好ましい。このような性質の似たアミノ酸の置換の場合、タンパク質の立体構造が維持されることが多く、従って抗ＰＳＫ抗体の抗原結合部位の立体構造も維持され、抗ＰＳＫ抗体は、ＰＳＫと結合することができる。 More specifically, amino acid deletion, substitution, insertion, or addition is preferably 3 or less, more preferably 2 or less, and most preferably 1 in each polypeptide. In the case of amino acid substitution, the hydrophilic amino acid is a hydrophilic amino acid, the hydrophobic amino acid is a hydrophilic amino acid, the basic amino acid is a basic amino acid, and an acidic amino acid is not limited. The amino acid is preferably substituted with an acidic amino acid. In the case of substitution of amino acids having similar properties, the three-dimensional structure of the protein is often maintained, and thus the three-dimensional structure of the antigen-binding site of the anti-PSK antibody is also maintained, and the anti-PSK antibody binds to PSK. Can do.
　本発明のＰＳＫの免疫学的分析方法の好ましい実施態様として、サンドイッチ法を利用する場合、例えば、サンドイッチ法は、抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片又は第２の抗体若しくはその抗原結合性断片のいずれか一方の抗体若しくはその抗原結合性断片を固定化した不溶性担体、ＰＳＫを含む可能性のある被検試料、残る一方の抗体若しくはその抗原結合性断片に標識を付した標識抗体を接触させ、不溶性担体上に固定化抗体とＰＳＫと標識抗体との複合体を形成する工程（以下、複合体形成工程と称する）と、複合体における標識からの信号を分析する工程と、を含む。複合体形成工程において、抗体等が固相化された不溶性担体、ＰＳＫを含む可能性のある被検試料、及び標識抗体の接触の順番は、特に限定されるものではなく、不溶性担体及び被検試料を先に接触させ、次に標識抗体を接触させてもよく、標識抗体及び被検試料を接触させ、次に不溶性担体を接触させてもよく、不溶性担体、被検試料、及び標識抗体を一緒に接触させてもよい。 As a preferred embodiment of the immunological analysis method for PSK of the present invention, when a sandwich method is used, for example, the sandwich method is performed by using an anti-PSK polyclonal antibody or an antigen-binding fragment thereof or a second antibody or an antigen-binding fragment thereof. An insoluble carrier on which any one of the antibodies or antigen-binding fragments thereof is immobilized, a test sample that may contain PSK, and the remaining one of the antibodies or antigen-binding fragments thereof are contacted with a labeled antibody. A step of forming a complex of an immobilized antibody, PSK and a labeled antibody on an insoluble carrier (hereinafter referred to as a complex formation step), and a step of analyzing a signal from the label in the complex. In the complex formation step, the order of contact of the insoluble carrier on which the antibody or the like is immobilized, the test sample that may contain PSK, and the labeled antibody is not particularly limited, and the insoluble carrier and the test are not limited. The sample may be contacted first, and then the labeled antibody may be contacted, the labeled antibody and the test sample may be contacted, and then the insoluble carrier may be contacted, and the insoluble carrier, test sample, and labeled antibody may be contacted. They may be contacted together.
　本明細書等における「第２の抗体」とは、ＰＳＫに結合することのできるものであれば、特に限定されないが、例えば、ＰＳＫを認識し、且つ他の多糖類のβ１，３グルカン、β１，４グルカン、及びβ１，６グルカンを認識する非特許文献１に記載のポリクローナル抗体、又はＰＳＫに特異的に結合する抗ＰＳＫ抗体を挙げることができるが、ＰＳＫに特異的に結合する抗ＰＳＫ抗体が好ましい。 The “second antibody” in the present specification and the like is not particularly limited as long as it can bind to PSK. For example, it recognizes PSK and other polysaccharides β1,3 glucan, β1 , 4 glucan, and polyclonal antibody described in Non-Patent Document 1 that recognizes β1,6 glucan, or anti-PSK antibody that specifically binds to PSK, but anti-PSK antibody that specifically binds to PSK Is preferred.
　より具体的には、本発明のＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体（例えば、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体、又はＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体）；ＰＳＫを認識し、且つデキストラン、グリコーゲン、ラミナリン、及びセルロースのβ１，３グルカン、β１，４グルカン、及びβ１，６グルカンを認識しないモノクローナル抗体；又はＰＳＫの生理活性部位を認識するモノクローナル抗体を挙げることができるが、最も好ましくは、ＰＳＫの生理活性部位を認識するモノクローナル抗体である。 More specifically, an anti-PSK polyclonal antibody that specifically binds to the PSK of the present invention (for example, an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK, or PSK An anti-PSK polyclonal antibody that reacts and does not react with the β1,4 glucan glucan structure of cellulose and the β1,3 glucan and β1,6 glucan glucan structures of laminarin; and recognizes PSK and dextran, glycogen, laminarin, And a monoclonal antibody that does not recognize β1,3 glucan, β1,4 glucan, and β1,6 glucan of cellulose; or a monoclonal antibody that recognizes a physiologically active site of PSK, most preferably, a physiological activity of PSK Monok that recognizes parts It is Naru antibody.
　サンドイッチ法を利用する本発明の免疫学的分析方法は、第２の抗体又はその抗原結合性断片を不溶性担体に固定化した後、ＰＳＫを含む可能性のある被検試料と接触させ、続いて、抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片に標識を付した標識抗体と接触させ、不溶性担体上に、固定化抗体とＰＳＫと標識抗体とからなるサンドイッチ状複合体を形成させ、抗ＰＳＫポリクローナル抗体の標識からの信号を検出することができる。 In the immunological analysis method of the present invention using the sandwich method, a second antibody or an antigen-binding fragment thereof is immobilized on an insoluble carrier, and then contacted with a test sample that may contain PSK. The anti-PSK polyclonal antibody or the antigen-binding fragment thereof is contacted with a labeled antibody to form a sandwich complex composed of an immobilized antibody, PSK and the labeled antibody on an insoluble carrier, and the anti-PSK polyclonal antibody The signal from the label can be detected.
　なお、抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片を不溶性担体に固定化し、この抗体固定化不溶性担体とＰＳＫを含む可能性のある被検試料とを接触させ、続いて、第２の抗体又はその抗原結合性断片に標識を付した標識抗体と接触させ、不溶性担体上に、固定化抗体とＰＳＫと標識抗体とからなるサンドイッチ状複合体を形成させ、第２の抗体の標識の信号を検出してもよい。 The anti-PSK polyclonal antibody or antigen-binding fragment thereof is immobilized on an insoluble carrier, the antibody-immobilized insoluble carrier is brought into contact with a test sample that may contain PSK, and then the second antibody or its The antigen-binding fragment is contacted with a labeled antibody, and a sandwich-like complex composed of an immobilized antibody, PSK, and a labeled antibody is formed on an insoluble carrier, and a signal for labeling the second antibody is detected. May be.
　複合体形成工程は、抗ＰＳＫモノクローナル抗体若しくはその抗原結合性断片を固定化した不溶性担体と、ＰＳＫを含む可能性のある被検試料とを接触させ、そして抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片に標識を付した標識抗体を接触させる工程であることが好ましい。このとき、第２の抗体は抗ＰＳＫモノクローナル抗体である。 The complex formation step comprises contacting an insoluble carrier on which an anti-PSK monoclonal antibody or antigen-binding fragment thereof is immobilized with a test sample that may contain PSK, and then anti-PSK polyclonal antibody or antigen-binding fragment thereof. The step is preferably a step of contacting a labeled antibody with a label. At this time, the second antibody is an anti-PSK monoclonal antibody.
　すなわち、抗ＰＳＫモノクローナル抗体とＰＳＫと標識抗体（抗ＰＳＫポリクローナル抗体）とからなるサンドイッチ状複合体を形成させ、抗ＰＳＫポリクローナル抗体の標識の信号を検出することが最も好ましい。 That is, it is most preferable to form a sandwich complex composed of an anti-PSK monoclonal antibody, PSK and a labeled antibody (anti-PSK polyclonal antibody), and detect the signal of the anti-PSK polyclonal antibody label.
　なお、標識した第２の抗体又は抗ＰＳＫポリクローナル抗体に代えて、未標識の第２の抗体又は抗ＰＳＫポリクローナル抗体を用いてもよい。この場合、未標識の第２の抗体又は抗ＰＳＫポリクローナル抗体に結合する標識抗体を用いて信号を検出する。また、標識した第２の抗体又は抗ＰＳＫポリクローナル抗体に代えて、ビオチン標識した第２の抗体又は抗ＰＳＫポリクローナル抗体を用い、標識アビジンを用いて信号を検出することも可能である。 Note that an unlabeled second antibody or anti-PSK polyclonal antibody may be used instead of the labeled second antibody or anti-PSK polyclonal antibody. In this case, the signal is detected using a labeled antibody that binds to the unlabeled second antibody or anti-PSK polyclonal antibody. Further, instead of the labeled second antibody or anti-PSK polyclonal antibody, a biotin-labeled second antibody or anti-PSK polyclonal antibody can be used, and a signal can be detected using labeled avidin.
　本発明のサンドイッチ法による免疫学的分析方法に使用することのできる不溶性担体は特に限定されるものでなく、例えば、ポリエチレン、ポリスチレン、ポリプロピレン、ポリ塩化ビニル、ポリエステル、ポリアクリロニトリル、フッ素樹脂、架橋デキストラン、ポリサッカライド等の高分子、その他ニトロセルロース、紙、アガロース及びこれらの組み合わせ等を例示することができる。 The insoluble carrier that can be used in the immunological analysis method according to the sandwich method of the present invention is not particularly limited, and examples thereof include polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, and crosslinked dextran. And polymers such as polysaccharides, other nitrocellulose, paper, agarose, and combinations thereof.
　標識物質としては、酵素、蛍光物質、又は発光物質を使用するのが有利である。酵素としては、例えば、アルカリホスファターゼ、ペルオキシダーゼ、β－Ｄ－ガラクトシダーゼ等、また、蛍光物質としては、例えば、フルオレセインイソチオシアネート等、また、発光物質としては、例えば、アクリジニウムエステル、ルシフェリン等を使用することができる。 It is advantageous to use an enzyme, a fluorescent substance, or a luminescent substance as the labeling substance. Examples of enzymes include alkaline phosphatase, peroxidase, β-D-galactosidase, etc., examples of fluorescent materials include fluorescein isothiocyanate, and examples of luminescent materials include acridinium esters, luciferin, etc. can do.
［３］ＰＳＫの免疫学的分析用キット
　本発明のＰＳＫの免疫学的分析用キットは、ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片を含む。ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体は、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体であってもよく、また、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体であってもよい。 [3] Kit for immunological analysis of PSK The kit for immunological analysis of PSK of the present invention comprises an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof. The anti-PSK polyclonal antibody that specifically binds to PSK may be an anti-PSK polyclonal antibody that recognizes at least one of the protein portion of PSK and a glucan structure specific to PSK, and reacts with PSK, and It may be an anti-PSK polyclonal antibody that does not react with the glucan structure of β1,4 glucan of cellulose and the glucan structure of β1,3 glucan and β1,6 glucan of laminarin.
　本発明のＰＳＫの免疫学的分析用キットは、更に、ＰＳＫと反応する第２の抗体又はその抗原結合性断片を含んでもよい。 The PSK immunological analysis kit of the present invention may further contain a second antibody that reacts with PSK or an antigen-binding fragment thereof.
　本実施形態のＰＳＫの分析キットは、本発明の抗ＰＳＫポリクローナル抗体又はその抗体の抗原結合性断片を含むＰＳＫの免疫学的分析用キットである。ＰＳＫの免疫学的分析用キットは、本発明のＰＳＫの免疫学的分析方法に用いることができる。従って、本発明のＰＳＫの免疫学的分析用キットは、酵素免疫測定法、免疫組織染色法、表面プラズモン共鳴法（ＳＰＲ法：Ｂｉａｃｏｒｅ法）、ラテックス凝集免疫測定法、化学発光免疫測定法、蛍光抗体法、放射免疫測定法、免疫沈降法、ウエスタンブロット法、イムノクロマトグラフ法、磁気ビーズ凝集法、又は磁気ビーズ酵素免疫法等に用いるキットを含む。 The PSK analysis kit of this embodiment is a kit for immunological analysis of PSK containing the anti-PSK polyclonal antibody of the present invention or an antigen-binding fragment of the antibody. The kit for immunological analysis of PSK can be used for the immunological analysis method of PSK of the present invention. Therefore, the immunological analysis kit for PSK of the present invention comprises an enzyme immunoassay method, an immunohistochemical staining method, a surface plasmon resonance method (SPR method: Biacore method), a latex agglutination immunoassay method, a chemiluminescence immunoassay method, a fluorescence It includes kits used for antibody methods, radioimmunoassay methods, immunoprecipitation methods, Western blotting methods, immunochromatographic methods, magnetic bead agglutination methods, magnetic bead enzyme immunization methods, and the like.
　ＰＳＫの免疫学的分析用キットが、酵素免疫測定法、例えばサンドイッチ法のキットの場合、抗ＰＳＫポリクローナル抗体若しくは第２の抗体、又はそれらの抗原結合性断片が表面に固相化された担体（ポリエチレン、ポリスチレン、ポリプロピレン、ポリ塩化ビニル、ポリエステル、ポリアクリロニトリル、フッ素樹脂、架橋デキストラン、ポリサッカライド等の高分子、その他ニトロセルロース、紙、アガロース及びこれらの組み合わせ等）、標識された第１抗ＰＳＫポリクローナル抗体若しくは第２の抗体、又はそれらの抗原結合性断片、酵素、その発色基質、及びその他のＥＬＩＳＡ試薬（例えば、洗浄液）などを適宜組み合わせた構成にすることができる。 In the case where the kit for immunological analysis of PSK is an enzyme immunoassay, for example, a sandwich kit, a carrier having an anti-PSK polyclonal antibody or a second antibody, or an antigen-binding fragment thereof immobilized on its surface ( Polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharides and other polymers, nitrocellulose, paper, agarose and combinations thereof), labeled first anti-PSK polyclonal An antibody or a second antibody, or an antigen-binding fragment thereof, an enzyme, a chromogenic substrate thereof, and other ELISA reagents (for example, a washing solution) can be appropriately combined.
　ＰＳＫの分析キットが、免疫組織染色法のキットの場合、例えばビオチン化標識した本発明の抗ＰＳＫポリクローナル抗体、（ＨＲＰ）標識したストレプトアビジン、ＤＡＢ基質、あるいは、未標識の抗ＰＳＫポリクローナル抗体、ＨＲＰ標識した抗マウスＩｇＧ抗体、基質などを含むことができる。 When the PSK analysis kit is an immunohistochemical staining kit, for example, biotinylated anti-PSK polyclonal antibody of the present invention, (HRP) labeled streptavidin, DAB substrate, or unlabeled anti-PSK polyclonal antibody, HRP Labeled anti-mouse IgG antibodies, substrates, etc. can be included.
　ＰＳＫの分析キットが、ＳＰＲ分析法のキットの場合、本発明の抗ＰＳＫポリクローナル抗体が固定化されたセンサーチップなどを含む。 When the PSK analysis kit is an SPR analysis kit, it includes a sensor chip on which the anti-PSK polyclonal antibody of the present invention is immobilized.
　従って、ＰＳＫの分析キットは、用いる免疫学的手法に応じて、所望の形態で抗ＰＳＫポリクローナル抗体、あるいはその抗原結合性断片を含む。例えば、標識物質の具体例としては、酵素としてペルオキシダーゼ、アルカリホスファターゼ、β－Ｄ－ガラクトシダーゼ、又はグルコースオキシダーゼ等を、蛍光物質としてフルオレセインイソチアネート又は希土類金属キレート等を、放射性同位体として３Ｈ、１４Ｃ、又は１２５Ｉ等を、その他、ビオチン、アビジン、又は化学発光物質等を挙げることができる。酵素又は化学発光物質等の場合には、それ自体単独では測定可能なシグナルをもたらすことはできないことから、それぞれ対応する適当な基質等を選択して含むことが好ましい。 Therefore, the PSK analysis kit contains an anti-PSK polyclonal antibody or an antigen-binding fragment thereof in a desired form depending on the immunological technique used. For example, specific examples of the labeling substance include peroxidase, alkaline phosphatase, β-D-galactosidase or glucose oxidase as an enzyme, fluorescein isothiocyanate or rare earth metal chelate as a fluorescent substance, 3 H as a radioisotope, In addition to 14 C, 125 I and the like, biotin, avidin, a chemiluminescent substance, and the like can be given. In the case of an enzyme, a chemiluminescent substance, or the like, it is not possible to provide a measurable signal by itself, and therefore it is preferable to select and include a corresponding appropriate substrate.
　ＰＳＫの免疫学的分析用キットは、ＰＳＫを特異的に分析することができるものであり、そのことが記載された使用説明書などを含む。また、キットの包装などにＰＳＫを特異的に分析することができることが記載されていてもよい。 The kit for immunological analysis of PSK is capable of specifically analyzing PSK, and includes an instruction manual describing the fact. Moreover, it may be described that PSK can be specifically analyzed on the packaging of a kit or the like.
　本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能である。すなわち、請求項に示した範囲で適宜変更した技術的手段を組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。 The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope indicated in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.
　以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
《製造例１：カワラタケの熱水・アルカリ抽出物の製造》
　カワラタケ菌ＣＭ１０１株〔ＦＥＲＭ－Ｐ２４１２（ＡＴＣＣ２０５４７）〕を、グルコース５％、ペプトン０．２％、酵母エキス０．３％、ＫＨ２ＰＯ４０．１％及びＭｇＳＯ４・７Ｈ２Ｏの０．１％（いずれも重量％）からなる培地に接種し、１０日間培養し、培地表面に発育した菌苔を生理食塩水とともにホモジェナイズしたものを種菌とした。この種菌を上記と同一組成の培地２００ｍＬの入った１リットル容の培養用フラスコ１００本にそれぞれ接種し、２５～２７℃で２５日間培養した後、乾燥器中で乾燥し、乾燥菌糸体を得た。乾燥菌糸体収量は培養器一本当たり２．０～４．５ｇであった。この菌糸体１００ｇに蒸留水１リットルを加え、９７℃で１時間、撹拌しながら抽出した。次に、遠心残渣に対して、１規定の水酸化ナトリウム水溶液１リットルを加え、９７℃で１時間、撹拌しながら抽出した。塩酸で中和後、両抽出液を混合した。混合液を室温にまで冷却し、濾紙濾過により、抽出液を回収した。次いで、希塩酸で中和し、エバポレーターで液を濃縮した。濃縮液１リットルに対し飽和溶液となるように硫酸アンモニウムを加え、塩析を行い、生じた沈殿を遠心分離により分取した。分取した沈殿は蒸留水に再溶解し、透析及び限外濾過処理し、分子量５０００以下の低分子物質を除去した。次いで、凍結乾燥して、カワラタケの熱水・アルカリ抽出物粉末１５ｇを得た。 << Production Example 1: Production of hot water / alkaline extract of Kawaratake >>
Kawaratake fungus CM101 strain [FERM-P2412 (ATCC20547)] was prepared by adding glucose 5%, peptone 0.2%, yeast extract 0.3%, KH 2 PO 4 0.1% and MgSO 4 · 7H 2 O 0.1%. % (Both weight%) was inoculated, cultured for 10 days, and homogenized with physiologic saline on the surface of the medium was used as an inoculum. Each of these inoculums was inoculated into 100 1-liter culture flasks containing 200 mL of medium having the same composition as described above, cultured at 25-27 ° C. for 25 days, and then dried in a drier to obtain a dried mycelium. It was. The dry mycelium yield was 2.0 to 4.5 g per incubator. 1 liter of distilled water was added to 100 g of this mycelium, and extracted with stirring at 97 ° C. for 1 hour. Next, 1 liter of 1N aqueous sodium hydroxide solution was added to the centrifugal residue, and the mixture was extracted with stirring at 97 ° C. for 1 hour. After neutralization with hydrochloric acid, both extracts were mixed. The mixture was cooled to room temperature, and the extract was recovered by filter paper filtration. Next, the solution was neutralized with dilute hydrochloric acid and concentrated with an evaporator. Ammonium sulfate was added to 1 liter of the concentrated solution to form a saturated solution, salted out, and the resulting precipitate was collected by centrifugation. The fractionated precipitate was redissolved in distilled water and subjected to dialysis and ultrafiltration to remove low molecular weight substances having a molecular weight of 5000 or less. Subsequently, it was freeze-dried to obtain 15 g of hot water / alkali extract powder of Kawaratake.
《実施例１：抗ＰＳＫウサギポリクローナル抗体の作製》
　製造例１で得られたカワラタケの熱水・アルカリ抽出物粉末のＰＢＳ溶解液とＦｒｅｕｎｄ’ｓ　Ｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ、Ｔｏｋｙｏ）とを等量混合し、超音波発生器を用いて高粘性のエマルジョン液を作成した。前記エマルジョン液を雌性ニュージーランドホワイト・ウサギの後背部にＰＳＫの量として、０．５ｍｇ／羽になるように皮下注射した。２週後に、ＰＳＫ溶液とＦｒｅｕｎｄ’ｓ　Ｉｎｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）とのエマルジョン液を、ＰＳＫの量として、０．５ｍｇ／羽になるように、ウサギ後背部に皮下注射した。この注射を２週間隔で８回繰り返した。最終免疫後１週間経過した時点で、ケタミン・キシラジン麻酔下、頚動脈より全採血を行った。採取した血液は室温にて１時間静置後、４℃で一昼夜保存し、３０００ｒｐｍ、１０分間遠心分離し血清を得た。 Example 1: Preparation of anti-PSK rabbit polyclonal antibody
Equivalent amounts of PBS solution of Kawaratake hot water / alkaline extract powder obtained in Production Example 1 and Freund's Complete Adjuvant (Sigma-Aldrich, Tokyo) were mixed, and high-viscosity was produced using an ultrasonic generator. An emulsion was prepared. The emulsion solution was subcutaneously injected into the back of female New Zealand white rabbits in a PSK amount of 0.5 mg / feather. Two weeks later, an emulsion of PSK solution and Freund's Incomplete Adjuvant (Sigma-Aldrich) was injected subcutaneously into the back of the rabbit so that the amount of PSK was 0.5 mg / feather. This injection was repeated 8 times at 2-week intervals. One week after the final immunization, whole blood was collected from the carotid artery under ketamine / xylazine anesthesia. The collected blood was allowed to stand at room temperature for 1 hour, stored at 4 ° C. overnight, and centrifuged at 3000 rpm for 10 minutes to obtain serum.
　免疫血清の抗体価測定はＥＬＩＳＡ法によって測定した。すなわち、ＰＳＫを５０ｎｇ／ウェルの濃度でコートさせた９６ウェルのプレートに、得られた血清の１０００～２１８７０００倍希釈液を１００μｇ／Ｌ分注し、室温で３時間反応させた後、０．０５％Ｔｗｅｅｎ　８０添加ＴＢＳ（以下、「ＴＢＳ－Ｔ」とする）で３回洗浄した。次に、プレートの各ウェルにＨＲＰ標識抗ウサギＩｇＧ抗体溶液を加え、室温で１時間反応させた後、ＴＢＳ－Ｔで３回洗浄した。ＡＢＴＳ基質溶液を加えて５－１５分間発色させた後、プレートリーダーを用いて４０５－６３０ｎｍの吸光度を測定した。抗体価測定結果を図１に示す。以降の実験には、３個体中で最も力価が高かった個体Ｎｏ．２の抗血清を用いた。 The antibody titer of the immune serum was measured by ELISA. Specifically, a 96-well plate coated with PSK at a concentration of 50 ng / well was dispensed with a 100 to 2187,000-fold dilution of the obtained serum at 100 μg / L, reacted at room temperature for 3 hours, It was washed 3 times with% Tween 80-added TBS (hereinafter referred to as “TBS-T”). Next, an HRP-labeled anti-rabbit IgG antibody solution was added to each well of the plate, reacted at room temperature for 1 hour, and then washed 3 times with TBS-T. After adding ABTS substrate solution to develop color for 5-15 minutes, absorbance at 405-630 nm was measured using a plate reader. The antibody titer measurement results are shown in FIG. In the subsequent experiments, the individual No. Two antisera were used.
　次に、ＰＳＫ固相化カラムを用いたアフィニティークロマトにより、抗ＰＳＫポリクローナル抗体を精製した。定法に従い、ペプシン処理および還元処理を行い、その後にホースラディッシュペルオキシダーゼ（ＨＲＰ）標識を行った。得られたＦａｂ’－ＨＲＰが抗原認識能を維持していることは、前記のＥＬＩＳＡ法で確認した（表１）。 Next, the anti-PSK polyclonal antibody was purified by affinity chromatography using a PSK solid phase column. According to a conventional method, pepsin treatment and reduction treatment were performed, followed by horseradish peroxidase (HRP) labeling. It was confirmed by the aforementioned ELISA method that the obtained Fab′-HRP maintained the antigen recognition ability (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
《比較製造例１：従来のＰＳＫの製造》
　従来のＰＳＫの製造は、特許文献２の実施例２の方法に準じて行った。
　カワラタケ菌ＣＭ１０１株〔ＦＥＲＭ－Ｐ２４１２（ＡＴＣＣ２０５４７）〕を、グルコース５％、ペプトン０．２％、酵母エキス０．３％、ＫＨ２ＰＯ４０．１％及びＭｇＳＯ４・７Ｈ２Ｏの０．１％（いずれも重量％）からなる培地に接種し、１０日間培養し、培地表面に発育した菌苔を生理食塩水とともにホモジェナイズしたものを種菌とした。この種菌を上記と同一組成の培地２００ｍＬの入った１リットル容の培養用フラスコ１００本にそれぞれ接種し、２５～２７℃で２５日間培養した後、乾燥器中で乾燥し、乾燥菌糸体を得た。乾燥菌糸体収量は培養器一本当たり２．０～４．５ｇであった。この菌糸体１００ｇに０．１規定の水酸化ナトリウム水溶液３リットルを加え、９７℃で１時間、撹拌しながら抽出した。抽出終了後、室温にまで冷却し、濾紙濾過により、抽出液を回収した。次いで、希塩酸で中和し、エバポレーターで液を濃縮した。濃縮液１リットルに対し飽和溶液となるように硫酸アンモニウムを加え、塩析を行い、生じた沈殿を遠心分離により分取した。分取した沈殿は蒸留水に再溶解し、透析及び限外濾過処理し、分子量５０００以下の低分子物質を除去した。次いで、凍結乾燥して、タンパク多糖体粉末１５ｇを得た。 << Comparative Production Example 1: Production of conventional PSK >>
Conventional PSK was produced according to the method of Example 2 of Patent Document 2.
Kawaratake fungus CM101 strain [FERM-P2412 (ATCC20547)] was prepared by adding glucose 5%, peptone 0.2%, yeast extract 0.3%, KH 2 PO 4 0.1% and MgSO 4 · 7H 2 O 0.1%. % (Both weight%) was inoculated, cultured for 10 days, and homogenized with physiologic saline on the surface of the medium was used as an inoculum. Each of these inoculums was inoculated into 100 1-liter culture flasks containing 200 mL of medium having the same composition as described above, cultured at 25-27 ° C. for 25 days, and then dried in a drier to obtain a dried mycelium. It was. The dry mycelium yield was 2.0 to 4.5 g per incubator. To 100 g of this mycelium was added 3 liters of a 0.1N aqueous sodium hydroxide solution, and the mixture was extracted with stirring at 97 ° C. for 1 hour. After completion of extraction, the mixture was cooled to room temperature, and the extract was collected by filtration with filter paper. Next, the solution was neutralized with dilute hydrochloric acid and concentrated with an evaporator. Ammonium sulfate was added to 1 liter of the concentrated solution to form a saturated solution, salted out, and the resulting precipitate was collected by centrifugation. The fractionated precipitate was redissolved in distilled water and subjected to dialysis and ultrafiltration to remove low molecular weight substances having a molecular weight of 5000 or less. Subsequently, it was freeze-dried to obtain 15 g of protein polysaccharide powder.
《比較例１：従来の抗ＰＳＫウサギポリクローナル抗体の作製》
　製造例１で得られたカワラタケの熱水・アルカリ抽出物粉末に代えて、比較製造例１で得られたタンパク多糖体を用いたことを除いては、実施例１の操作を繰り返し、従来の抗ＰＳＫウサギポリクローナル抗体を得た。 << Comparative Example 1: Production of conventional anti-PSK rabbit polyclonal antibody >>
The procedure of Example 1 was repeated except that the protein polysaccharide obtained in Comparative Production Example 1 was used instead of the hot water / alkaline extract powder obtained in Production Example 1. An anti-PSK rabbit polyclonal antibody was obtained.
《実施例２：抗ＰＳＫウサギポリクローナル抗体の特異性評価》
　抗ＰＳＫウサギポリクローナル抗体のＰＳＫに対する特異性評価は、Ｂｉａｃｏｒｅを用いて行った。まず、Ｓｅｎｓｏｒ　Ｃｈｉｐ　ＣＭ５センサー表面に抗ウサギＩｇＧ抗体をアミノカップリング法で固相化した。次に、センサーチップに対して、比較例１で作成した従来の抗ＰＳＫウサギポリクローナル抗体（ｏｌｄ　ｒａｂｂｉｔ　ａｎｔｉ－ＰＳＫ　ｐＡｂ）又は実施例１で作製した抗ＰＳＫウサギポリクローナル抗体（ｎｅｗ　ｒａｂｂｉｔ　ａｎｔｉ－ＰＳＫ　ｐＡｂ）を一定量補足させた。これらのセンサーチップに対して１μｇ／ｍＬに調整したＰＳＫ、β１－４ｇｌｕｃａｎ、又はβ１－３，１－６ｇｌｕｃａｎをアナライトに用いて結合解析を行った。なお、β１－４ｇｌｕｃａｎとしてはセルロースを用い、β１－３，１－６ｇｌｕｃａｎとしては、ラミナリンを用いた。 << Example 2: Specificity evaluation of anti-PSK rabbit polyclonal antibody >>
The specificity of the anti-PSK rabbit polyclonal antibody for PSK was evaluated using Biacore. First, an anti-rabbit IgG antibody was immobilized on the surface of the Sensor Chip CM5 sensor by an amino coupling method. Next, the conventional anti-PSK rabbit polyclonal antibody prepared in Comparative Example 1 (old rabbit anti-PSK pAb) or the anti-PSK rabbit polyclonal antibody prepared in Example 1 (new rabbit anti-PSK pAb) is used against the sensor chip. A certain amount of was supplemented. Binding analysis was performed on these sensor chips using PSK, β1-4 glucan, or β1-3, 1-6 glucan adjusted to 1 μg / mL as an analyte. Note that cellulose was used as β1-4glucan, and laminarin was used as β1-3,1-6glucan.
　その結果、図２に示すように、従来のｏｌｄ　ｒａｂｂｉｔ　ａｎｔｉ－ＰＳＫ　ｐＡｂは、ＰＳＫ以外のグルカン類にも結合するのに対して、本発明のｎｅｗ　ｒａｂｂｉｔ　ａｎｔｉ－ＰＳＫ　ｐＡｂは、ＰＳＫに含まれる以外のβ１，３グルカン、β１，４グルカン、及びβ１，６グルカン構造に結合せず、ＰＳＫに特異的に結合することが分かった。 As a result, as shown in FIG. 2, the conventional old rabbit anti-PSK pAb also binds to glucans other than PSK, whereas the new rabbit anti-PSK pAb of the present invention is not included in PSK. It was found to bind specifically to PSK without binding to the β1,3 glucan, β1,4 glucan, and β1,6 glucan structures.
《参考例１：抗ＰＳＫモノクローナル抗体の作製》
　抗体の作製は、（１）抗原の免疫、（２）抗血清の抗体価の測定、（３）抗ＰＳＫモノクローナル抗体の作製の順に行った。以下、順に手順の概要を説明する。
（１）抗原の免疫：第１回免疫として、ＰＳＫのリン酸緩衝化生理食塩水（ｐｈｏｓｐｈａｔｅ　ｂｕｆｆｅｒｅｄ　ｓａｌｉｎｅ；以下、「ＰＢＳ」とする。）溶液とＦｒｅｕｎｄ’ｓ　Ｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（シグマ－アルドリッチ社製）とを等量混合し、超音波発生器を用いて高粘性のエマルジョン液を調製した。６週齢の雌性Ｂａｌｂ／ｃマウス（オリエンタル酵母株式会社）に、このエマルジョン液をＰＳＫ量が０．１ｍｇ／匹となるように、皮下注射した。１週間後に第２回目の免疫を行った。ＰＳＫのＰＢＳ溶液とＦｒｅｕｎｄ’ｓ　Ｉｎｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（シグマ－アルドリッチ社製）とを混合してエマルジョン液を調製した。ＰＳＫ量が０．１ｍｇ／匹となるように腹腔内注射した。１週間ごとに同じ手順で免疫を行い、第８回目の免疫後、尾静脈より採血して力価の測定を行った。抗体価の上昇が認められた個体について、ＰＳＫを腹腔内注射することによりブーストを行った後に、ハイブリドーマ取得のために細胞融合を行った。 << Reference Example 1: Preparation of anti-PSK monoclonal antibody >>
The antibody was prepared in the order of (1) immunization with antigen, (2) measurement of antibody titer of antiserum, and (3) preparation of anti-PSK monoclonal antibody. Hereinafter, the outline of the procedure will be described in order.
(1) Immunization of antigen: As the first immunization, PSK phosphate buffered saline solution (hereinafter referred to as “PBS”) and Freund's Complete Adjuvant (manufactured by Sigma-Aldrich) Were mixed in an equal amount, and a highly viscous emulsion liquid was prepared using an ultrasonic generator. This emulsion solution was subcutaneously injected into 6-week-old female Balb / c mice (Oriental Yeast Co., Ltd.) so that the PSK amount was 0.1 mg / animal. A second immunization was performed one week later. PSK in PBS and Freund's Incomplete Adjuvant (Sigma-Aldrich) were mixed to prepare an emulsion. The intraperitoneal injection was performed so that the PSK amount was 0.1 mg / animal. Immunization was performed in the same procedure every week, and after the eighth immunization, blood was collected from the tail vein and the titer was measured. Individuals in which an increase in antibody titer was observed were boosted by intraperitoneal injection of PSK, and then cell fusion was performed to obtain a hybridoma.
（２）抗血清の抗体価の測定：前記８回目の免疫後に、Ｂａｌｂ／ｃマウスから得られたそれぞれの血清（抗血清）の抗体価測定を、ＥＬＩＳＡ法により行った。手順を以下に示す。９６ウェルプレートに、ＰＳＫ溶液を１μｇ／ウェルずつ分注し、４℃、一晩反応させてＰＳＫを固相化した。１％ＢＳＡでブロッキング後、得られた血清の１，０００倍希釈液を各ウェルに５０μＬずつ分注し、２５℃で３時間反応させた。次に、ＴＢＳ－Ｔで、各ウェルを３回洗浄した後、１μｇ／ｍＬ濃度に調製したホースラディッシュペルオキシダーゼ（ＨＲＰ）標識抗マウスＩｇＭ抗体溶液を５０μＬ各ウェルに分注し、２５℃で１時間反応させた。ＴＢＳ－Ｔで各ウェルを３回洗浄した後、基質であるＡＢＳＴ（ＫＰＬ社）を加え、１５分間発色させた。５０μＬのＰｅｒｏｘｉｄａｓｅ　Ｓｔｏｐ　Ｓｏｌｕｔｉｏｎ（ＫＰＬ社）で発色反応を停止させた後、プレートリーダーを用いて、４０５ｎｍの吸光度を測定した。図３に、ＥＬＩＳＡ法による抗体価測定の結果を示しており、グラフの横軸には血清の希釈倍率、縦軸には吸光度（力価）を表す。 (2) Measurement of antibody titer of antiserum: After the eighth immunization, the antibody titer of each serum (antiserum) obtained from Balb / c mice was measured by ELISA. The procedure is shown below. The PSK solution was dispensed at 1 μg / well into a 96-well plate and reacted overnight at 4 ° C. to immobilize PSK. After blocking with 1% BSA, a 1,000-fold diluted solution of the obtained serum was dispensed into each well in an amount of 50 μL and reacted at 25 ° C. for 3 hours. Next, each well was washed three times with TBS-T, and then a horseradish peroxidase (HRP) -labeled anti-mouse IgM antibody solution prepared to a concentration of 1 μg / mL was dispensed into each well at 50 ° C. for 1 hour at 25 ° C. Reacted. Each well was washed 3 times with TBS-T, and then the substrate ABST (KPL) was added to cause color development for 15 minutes. After stopping the color reaction with 50 μL of Peroxidase Stop Solution (KPL), absorbance at 405 nm was measured using a plate reader. FIG. 3 shows the results of antibody titer measurement by ELISA. The horizontal axis of the graph represents the serum dilution ratio, and the vertical axis represents the absorbance (titer).
（３）抗ＰＳＫモノクローナル抗体の作製：ＰＳＫの免疫により、抗体の力価の上昇が認められた個体については、定法にてモノクローナル抗体作製を進めた。すなわち、ブーストの７日後、マウスの脾臓を摘出し脾細胞をマウスミエローマ細胞株Ｐ３Ｕ１と細胞融合させた。ＨＡＴ選択培養液の中で２～３週間培養し、ハイブリドーマコロニーを得た。これらの培養上清を回収して、前記（２）に記載のＥＬＩＳＡ法を用いて、ハイブリドーマのスクリーニングを行った。得られたＰＳＫ抗体を産生している陽性ハイブリドーマについては、同様のスクリーニングを２回繰り返し、抗体産生能や増殖性に優れたハイブリドーマを選抜した。その結果、約１００個の陽性ハイブリドーマから、２Ｇ９抗体及び５Ｇ５抗体を産生する２つのハイブリドーマを選択した。なお、２Ｇ９抗体及び５Ｇ５抗体はＩｇＭ抗体であることを確認した。 (3) Production of anti-PSK monoclonal antibody: Individuals in which an increase in antibody titer was recognized by PSK immunization were produced by a conventional method. That is, 7 days after the boost, the spleen of the mouse was removed and the spleen cells were fused with the mouse myeloma cell line P3U1. The hybridoma colony was obtained by culturing in a HAT selective culture solution for 2 to 3 weeks. These culture supernatants were collected and screened for hybridomas using the ELISA method described in (2) above. About the positive hybridoma which is producing the obtained PSK antibody, the same screening was repeated twice and the hybridoma excellent in antibody-producing ability and proliferation property was selected. As a result, two hybridomas producing 2G9 antibody and 5G5 antibody were selected from about 100 positive hybridomas. The 2G9 antibody and the 5G5 antibody were confirmed to be IgM antibodies.
　抗体の大量調製はマウス腹水で行った。具体的には５００μＬのプリスタンを雌性Ｂａｌｂ／ｃマウスの腹腔内に投与して、７～１０日後、マウス一匹あたり約１０７個のハイブリドーマを移植した。１～２週間後、腹水が溜まってきたら随時回収し、精製まで－８０℃で保存した。腹水からの抗体精製は以下のように行った。回収した腹水に最終濃度が２５ｍＭになるようにリン酸バッファー（ｐＨ７．５）を加え、０．４５μｍのフィルターに通過させた。これをＰｒｏｔｅｉｎＧカラムにアプライしてフロースルーを回収した。その後、定法によりＨｉＴｒａｐ　ＩｇＭカラム（アマシャム）あるいはＳｅｐｈａｒｏｓｅ　ＨＰカラム（アマシャム）でＩｇＭ画分を回収した。更にそのＩｇＭ画分をＳｅｐｈａｒｏｓｅ　２００ｐｇカラムで分画し、５量体のＩｇＭを精製した。得られた２Ｇ９抗体及び５Ｇ５抗体の力価を図４に示す。 Large-scale antibody preparation was performed in mouse ascites. Specifically, 500 μL of pristane was administered intraperitoneally to female Balb / c mice, and 7 to 10 days later, about 10 7 hybridomas per mouse were transplanted. After 1 to 2 weeks, when ascites collected, it was collected at any time and stored at −80 ° C. until purification. Antibody purification from ascites was performed as follows. Phosphate buffer (pH 7.5) was added to the collected ascites to a final concentration of 25 mM, and passed through a 0.45 μm filter. This was applied to a Protein G column and the flow-through was collected. Thereafter, the IgM fraction was collected by a HiTrap IgM column (Amersham) or Sepharose HP column (Amersham) by a conventional method. Further, the IgM fraction was fractionated with a Sepharose 200 pg column to purify pentamer IgM. The titers of the obtained 2G9 antibody and 5G5 antibody are shown in FIG.
《参考例２：抗ＰＳＫマウスモノクローナル抗体の特異性評価》
　抗ＰＳＫマウスモノクローナル抗体（２Ｇ９ｍＡｂ、５Ｇ５ｍＡｂ）のＰＳＫに対する特異性評価は、以下の競合ＥＬＩＳＡ法で行った。炭酸緩衝液に溶解したＰＳＫを１μｇ／ｇ／ウェルで９６ウェルプレートに固相化した。１μｇ／ｍＬの抗ＰＳＫ抗体と１０μｇ／ｍＬの各種グルカン類（ＰＳＫ、ｃｅｌｌｕｌｏｓｅ、ｌａｍｉｎａｒｉｎ、ｇｌｙｃｏｇｅｎ、ｄｅｘｔｒａｎ）とを混和後、９６ウェルプレートに添加して室温で１時間インキュベートした。検出は、ＨＲＰ標識抗マウスＩｇＭで行った。
　なお、ＰＳＫはβ１，３グルカン、β１，４グルカン、及びβ１，６グルカンを有しており、ｃｅｌｌｕｌｏｓｅはβ１，４グルカンを有しており、ｌａｍｉｎａｒｉｎはβ１，３グルカン及びβ１，６グルカンを有しており、ｇｌｙｃｏｇｅｎはβ１，４グルカンを有しており、ｄｅｘｔｒａｎはβ１，６グルカンを有している。 << Reference Example 2: Specificity evaluation of anti-PSK mouse monoclonal antibody >>
The specificity of anti-PSK mouse monoclonal antibodies (2G9 mAb, 5G5 mAb) for PSK was evaluated by the following competitive ELISA method. PSK dissolved in carbonate buffer was immobilized on a 96-well plate at 1 μg / g / well. After mixing 1 μg / mL anti-PSK antibody and 10 μg / mL various glucans (PSK, cellulose, laminarin, glycogen, dextran), they were added to a 96-well plate and incubated at room temperature for 1 hour. Detection was performed with HRP-labeled anti-mouse IgM.
PSK has β1,3 glucan, β1,4 glucan, and β1,6 glucan, cellulose has β1,4 glucan, and laminarin has β1,3 glucan and β1,6 glucan. Glycogen has β1,4 glucan, and dextran has β1,6 glucan.
　その結果、図５に示すように、２Ｇ９ｍＡｂと５Ｇ５ｍＡｂはＰＳＫを特異的に認識し、他の類似構造を持つグルカン類には結合しないことが分かった。 As a result, as shown in FIG. 5, it was found that 2G9 mAb and 5G5 mAb specifically recognize PSK and do not bind to other glucans having a similar structure.
《参考例３：２Ｇ９抗体及び５Ｇ５抗体の可変領域の配列決定》
　２Ｇ９抗体又は５Ｇ５抗体を産生するハイブリドーマから、定法によりｔｏｔａｌ　ＲＮＡを抽出し、オリゴｄＴプライマーを用いて逆転写反を行い、ｃＤＮＡを作製した。得られたｃＤＮＡから、可変領域遺伝子を増幅するためにｍｏｕｓｅ　Ｉｇ　ｐｒｉｍｅｒ　ｓｅｔ（Ｎｏｖａｇｅｎ社）を用いて、添付のプロトコールに従いＰＣＲを行った。得られた抗体可変領域遺伝子はｐＣＲ２．１ベクターにＴＡクローニングしてシーケンスの決定を行った。２Ｇ９抗体の重鎖可変領域ドメイン、及び軽鎖可変領域ドメインのヌクレオチドの塩基配列、及び５Ｇ５抗体の重鎖可変領域ドメイン、及び軽鎖可変領域ドメインのヌクレオチドの塩基配列を、それぞれ図７～図１０に示す。また、それぞれの抗体のＨ－ＦＲ１、Ｈ－ＣＤＲ１、Ｈ－ＦＲ２、Ｈ－ＣＤＲ２、Ｈ－ＦＲ３、Ｈ－ＣＤＲ３、及びＨ－ＦＲ４、並びにＬ－ＦＲ１、Ｌ－ＣＤＲ１、Ｌ－ＦＲ２、Ｌ－ＣＤＲ２、Ｌ－ＦＲ３、Ｌ－ＣＤＲ３、及びＬ－ＦＲ４のアミノ酸配列を以下に示す。 << Reference Example 3: Sequencing of variable regions of 2G9 antibody and 5G5 antibody >>
Total RNA was extracted from the hybridoma producing 2G9 antibody or 5G5 antibody by a conventional method, and reverse transcription was performed using an oligo dT primer to prepare cDNA. From the obtained cDNA, PCR was performed according to the attached protocol using a mouse Ig primer set (Novagen) to amplify the variable region gene. The obtained antibody variable region gene was TA cloned into the pCR2.1 vector and the sequence was determined. The nucleotide sequences of the heavy chain variable region domain and the light chain variable region domain of the 2G9 antibody, and the nucleotide sequences of the heavy chain variable region domain and the light chain variable region domain of the 5G5 antibody are shown in FIGS. Shown in In addition, H-FR1, H-CDR1, H-FR2, H-CDR2, H-FR3, H-CDR3, and H-FR4, and L-FR1, L-CDR1, L-FR2, L- The amino acid sequences of CDR2, L-FR3, L-CDR3, and L-FR4 are shown below.
　２Ｇ９抗体の重鎖可変領域ドメインのアミノ酸配列
Ｈ－ＦＲ１　：GVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFS（配列番号４）
Ｈ－ＣＤＲ１：SYGMS（配列番号６）
Ｈ－ＦＲ２　：WVRQTPDKRLEWVA（配列番号８）
Ｈ－ＣＤＲ２：TISSGGSYTYYPDSVKG（配列番号１０）
Ｈ－ＦＲ３　：RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR（配列番号１２）
Ｈ－ＣＤＲ３：RITTVVARSFYFDY（配列番号１４）
Ｈ－ＦＲ４　：WGQG（配列番号１６） Amino acid sequence H-FR1 of heavy chain variable region domain of 2G9 antibody: GVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFS (SEQ ID NO: 4)
H-CDR1: SYGMS (SEQ ID NO: 6)
H-FR2: WVRQTPDKRLEWVA (SEQ ID NO: 8)
H-CDR2: TISSGGSYTYYPDSVKG (SEQ ID NO: 10)
H-FR3: RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR (SEQ ID NO: 12)
H-CDR3: RITTVVARSFYFDY (SEQ ID NO: 14)
H-FR4: WGQG (SEQ ID NO: 16)
　２Ｇ９抗体の軽鎖可変領域ドメインのアミノ酸配列
Ｌ－ＦＲ１　：GSTGDIVLTQSPASLAVSLGQRATISY（配列番号２０）
Ｌ－ＣＤＲ１：RASKSVSTSGYSYMH（配列番号２２）
Ｌ－ＦＲ２　：WNQQKPGQPPRLLIY（配列番号２４）
Ｌ－ＣＤＲ２：LVSNLES（配列番号２６）
Ｌ－ＦＲ３　：GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC（配列番号２８）
Ｌ－ＣＤＲ３：QHIRELTRS（配列番号３０）
Ｌ－ＦＲ４　：EGGP（配列番号３２） Amino acid sequence L-FR1 of the light chain variable region domain of 2G9 antibody: GSTGDIVLTQSPASLAVSLGQRATISY (SEQ ID NO: 20)
L-CDR1: RASKSVSTSGYSYMH (SEQ ID NO: 22)
L-FR2: WNQQKPGQPPRLLIY (SEQ ID NO: 24)
L-CDR2: LVSNLES (SEQ ID NO: 26)
L-FR3: GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 28)
L-CDR3: QHIRELTRS (SEQ ID NO: 30)
L-FR4: EGGP (SEQ ID NO: 32)
　５Ｇ５抗体の重鎖可変領域ドメインのアミノ酸配列
Ｈ－ＦＲ１　：GVHSEVQLQQSGPELVKPGASMKISCKASGYSFT（配列番号３６）
Ｈ－ＣＤＲ１：GYTMN（配列番号３８）
Ｈ－ＦＲ２　：WVKQSHGKNLEWIG（配列番号４０）
Ｈ－ＣＤＲ２：LINPYNGGTSYNQKFKG（配列番号４２）
Ｈ－ＦＲ３　：KATLTVDKSSSTAYMELLSLTSEDSAVYYCAR（配列番号４４）
Ｈ－ＣＤＲ３：GGKFATGTSY（配列番号４６）
Ｈ－ＦＲ４　：WGQG（配列番号４８） Amino acid sequence H-FR1 of heavy chain variable region domain of 5G5 antibody: GVHSEVQLQQSGPELVKPGASMKISCKASGYSFT (SEQ ID NO: 36)
H-CDR1: GYTMN (SEQ ID NO: 38)
H-FR2: WVKQSHGKNLEWIG (SEQ ID NO: 40)
H-CDR2: LINPYNGGTSYNQKFKG (SEQ ID NO: 42)
H-FR3: KATLTVDKSSSTAYMELLSLTSEDSAVYYCAR (SEQ ID NO: 44)
H-CDR3: GGKFATGTSY (SEQ ID NO: 46)
H-FR4: WGQG (SEQ ID NO: 48)
　５Ｇ５抗体の軽鎖可変領域ドメインのアミノ酸配列
Ｌ－ＦＲ１　：GAISQAVVTQESALTTSPGETVTLTC（配列番号５２）
Ｌ－ＣＤＲ１：RSSTGAVTTSNYAN（配列番号５４）
Ｌ－ＦＲ２　：WVQEKPDHLFTGLIG（配列番号５６）
Ｌ－ＣＤＲ２：GTNNRAP（配列番号５８）
Ｌ－ＦＲ３　：GVPARFSGSLIGDKAALTITGAQTEDEAIYFC（配列番号６０）
Ｌ－ＣＤＲ３：ALWYSNHWV（配列番号６２）
Ｌ－ＦＲ４　：FGGG（配列番号６４） Amino acid sequence L-FR1 of the light chain variable region domain of the 5G5 antibody: GAISQAVVTQESALTTSPGETVTLTC (SEQ ID NO: 52)
L-CDR1: RSSTGAVTTSNYAN (SEQ ID NO: 54)
L-FR2: WVQEKPDHLFTGLIG (SEQ ID NO: 56)
L-CDR2: GTNNRAP (SEQ ID NO: 58)
L-FR3: GVPARFSGSLIGDKAALTITGAQTEDEAIYFC (SEQ ID NO: 60)
L-CDR3: ALWYSNHWV (SEQ ID NO: 62)
L-FR4: FGGG (SEQ ID NO: 64)
《実施例３：サンドイッチＥＬＩＳＡ系の構築》
　ＰＳＫの検出系はサンドイッチＥＬＩＳＡ法で行った。炭酸緩衝液に溶解した抗ＰＳＫモノクローナル抗体（２Ｇ９抗体及び５Ｇ５抗体）を５００ｎｇ／ウェルで９６ウェルプレートにコートした。１％ＢＳＡ含有ＰＢＳでブロッキング後、６２．５－５００ｎｇ／ｍＬのＰＳＫ溶液を添加して、１時間、室温でインキュベートした。ＴＢＳ－Ｔでプレートを洗浄後、ＨＲＰ標識抗ＰＳＫ　Ｆａｂ’抗体を添加して、更に３０分間、室温でインキュベートした。ＴＢＳ－Ｔでプレートを洗浄後、ＡＢＴＳ基質溶液を加えて５－１５分間発色させた後、プレートリーダーを用いて４０５－６３０ｎｍの吸光度を測定した。その結果、図６に示すように構築したＥＬＩＳＡ系は、感度良く、更に高い特異性をもってＰＳＫを検出することができた。 Example 3: Construction of sandwich ELISA system
The detection system for PSK was performed by sandwich ELISA. Anti-PSK monoclonal antibodies (2G9 antibody and 5G5 antibody) dissolved in carbonate buffer were coated on a 96-well plate at 500 ng / well. After blocking with PBS containing 1% BSA, 62.5-500 ng / mL PSK solution was added and incubated at room temperature for 1 hour. After washing the plate with TBS-T, HRP-labeled anti-PSK Fab ′ antibody was added and incubated at room temperature for an additional 30 minutes. After the plate was washed with TBS-T, ABTS substrate solution was added to develop color for 5-15 minutes, and then the absorbance at 405-630 nm was measured using a plate reader. As a result, the ELISA system constructed as shown in FIG. 6 was able to detect PSK with high sensitivity and higher specificity.
《実施例４：サンドイッチＥＬＩＳＡによるＰＳＫのマウス血中濃度の測定》
　Ｂａｌｂ／ｃマウス（雌、６週齢）にＰＳＫ（５０ｍｇ／ｋｇ）を単回、腹腔内投与した後、８時間後に採血を行い、上記サンドイッチＥＬＩＳＡ法で血中濃度を測定した。その結果、血中に取り込まれたＰＳＫを検出することが可能であった（表２）。 Example 4: Measurement of blood concentration of PSK in mouse by sandwich ELISA
After a single intraperitoneal administration of PSK (50 mg / kg) to Balb / c mice (female, 6 weeks old), blood was collected 8 hours later, and the blood concentration was measured by the sandwich ELISA method. As a result, it was possible to detect PSK taken up in blood (Table 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本発明のＰＳＫの免疫学的分析方法は、医薬の品質管理に用いることができる。また、本発明のＰＳＫの免疫学的分析方法は、ＰＳＫの体内動態の検査に用いることができる。
　以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。 The immunological analysis method of PSK of the present invention can be used for pharmaceutical quality control. In addition, the immunological analysis method for PSK of the present invention can be used for examination of pharmacokinetics of PSK.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

Claims (18)

  1. 　ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片を用いることを特徴とするＰＳＫの免疫学的分析方法。 A method for immunological analysis of PSK, comprising using an anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof.
  2. 　前記抗ＰＳＫポリクローナル抗体が、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する抗ＰＳＫポリクローナル抗体である、請求項１に記載のＰＳＫの免疫学的分析方法。 The method for immunological analysis of PSK according to claim 1, wherein the anti-PSK polyclonal antibody is an anti-PSK polyclonal antibody that recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK.
  3. 　前記抗ＰＳＫポリクローナル抗体が、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン及びβ１，６グルカンのグルカン構造に反応しない抗ＰＳＫポリクローナル抗体である、請求項１又は２に記載のＰＳＫの免疫学的分析方法。 The anti-PSK polyclonal antibody is an anti-PSK polyclonal antibody that reacts with PSK and does not react with the glucan structure of cellulose β1,4 glucan and the glucan structure of laminarin β1,3 glucan and β1,6 glucan. 3. The immunological analysis method for PSK according to 1 or 2.
  4. 　前記抗ＰＳＫポリクローナル抗体のＰＳＫに対する結合力を１００とした場合の、セルロースのβ１，４グルカンのグルカン構造、又はラミナリンのβ１，３グルカン、及びβ１，６グルカンのグルカン構造に対する結合力が３０％以下である、請求項３に記載のＰＳＫの免疫学的分析方法。 When the binding force of the anti-PSK polyclonal antibody to PSK is 100, the binding strength of cellulose β1,4 glucan glucan structure or laminarin β1,3 glucan and β1,6 glucan glucan structure is 30% or less. The immunological analysis method for PSK according to claim 3, wherein
  5. 　酵素免疫測定法、免疫組織染色法、表面プラズモン共鳴法、ラテックス凝集免疫測定法、化学発光免疫測定法、蛍光抗体法、放射免疫測定法、免疫沈降法、ウエスタンブロット法、イムノクロマトグラフ法、磁気ビーズ凝集法、又は磁気ビーズ酵素免疫法を用いる、請求項１～４のいずれか一項に記載のＰＳＫの免疫学的分析方法。 Enzyme immunoassay, immunohistochemical staining, surface plasmon resonance, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation, western blotting, immunochromatography, magnetic beads The method for immunological analysis of PSK according to any one of claims 1 to 4, wherein an agglutination method or a magnetic bead enzyme immunization method is used.
  6. 　前記ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片と、ＰＳＫと反応する第２の抗体又はその抗原結合性断片と、を使用する、請求項１～５のいずれか一項に記載のＰＳＫの免疫学的分析方法。 The anti-PSK polyclonal antibody or antigen-binding fragment thereof that specifically binds to PSK and the second antibody or antigen-binding fragment thereof that reacts with PSK are used. An immunological analysis method for PSK as described in 1. above.
  7. 　前記抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片、又は前記第２の抗体若しくはその抗原結合性断片のいずれか一方の抗体若しくはその抗原結合性断片を固定化した固定化抗体を有する不溶性担体、ＰＳＫを含む可能性のある被検試料、残る一方の抗体若しくはその抗原結合性断片に標識を付した標識抗体を接触させ、前記不溶性担体上に固定化抗体とＰＳＫと標識抗体との複合体を形成する複合体形成工程と、前記複合体における標識からの信号を分析する分析工程と、
    を含む、請求項６に記載のＰＳＫの免疫学的分析方法。     An insoluble carrier having an immobilized antibody on which either one of the anti-PSK polyclonal antibody or antigen-binding fragment thereof, or the second antibody or antigen-binding fragment thereof is immobilized, or PSK, A test antibody that may be contained, the remaining antibody or antigen-binding fragment thereof is contacted with a labeled antibody, and a complex of immobilized antibody, PSK, and labeled antibody is formed on the insoluble carrier. A complex formation step, an analysis step for analyzing a signal from a label in the complex,
    The immunological analysis method of PSK of Claim 6 containing this.
  8. 　前記複合体形成工程が、第２の抗体として抗ＰＳＫモノクローナル抗体若しくはその抗原結合性断片を固定化した不溶性担体と、ＰＳＫを含む可能性のある被検試料とを接触させ、そして前記抗ＰＳＫポリクローナル抗体若しくはその抗原結合性断片に標識を付した標識抗体を接触させる工程である、請求項７に記載のＰＳＫの免疫学的分析方法。 In the complex formation step, an anti-PSK monoclonal antibody or an insoluble carrier on which an antigen-binding fragment thereof is immobilized as a second antibody is contacted with a test sample that may contain PSK, and the anti-PSK polyclonal The method for immunological analysis of PSK according to claim 7, which is a step of contacting a labeled antibody with a label attached to the antibody or an antigen-binding fragment thereof.
  9. 　前記第２の抗体が、抗ＰＳＫモノクローナル抗体である、請求項６～８のいずれか一項に記載のＰＳＫの免疫学的分析方法。 The immunological analysis method for PSK according to any one of claims 6 to 8, wherein the second antibody is an anti-PSK monoclonal antibody.
  10. 　血中濃度の測定を行う、請求項１～９のいずれか一項に記載のＰＳＫの免疫学的分析方法。 10. The immunological analysis method for PSK according to any one of claims 1 to 9, wherein the blood concentration is measured.
  11. 　ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片。 Anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof.
  12. 　前記抗ＰＳＫポリクローナル抗体が、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する請求項１１に記載の抗ＰＳＫポリクローナル抗体又はその抗原結合性断片。 The anti-PSK polyclonal antibody or antigen-binding fragment thereof according to claim 11, wherein the anti-PSK polyclonal antibody recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK.
  13. 　前記抗ＰＳＫポリクローナル抗体が、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン、及びβ１，６グルカンのグルカン構造に反応しない、請求項１１又は１２に記載の抗ＰＳＫポリクローナル抗体又はその抗原結合性断片。 13. The anti-PSK polyclonal antibody reacts with PSK and does not react with the β1,4 glucan glucan structure of cellulose and the β1,3 glucan of laminarin and the glucan structure of β1,6 glucan. Anti-PSK polyclonal antibody or antigen-binding fragment thereof.
  14. 　前記抗原結合性断片が、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ断片、ディアボディー、単一鎖抗体分子、及びマルチ特異性抗体からなる群から選択される、請求項１１～１３のいずれか一項に記載の抗原結合性断片。 The antigen-binding fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2 , Fv fragment, deabody, single chain antibody molecule, and multispecific antibody. The antigen-binding fragment according to any one of the above.
  15. 　ＰＳＫに特異的に結合する抗ＰＳＫポリクローナル抗体又はその抗原結合性断片、
    を含むことを特徴とする、ＰＳＫの免疫学的分析用キット。     An anti-PSK polyclonal antibody that specifically binds to PSK or an antigen-binding fragment thereof,
    A kit for immunological analysis of PSK, comprising:
  16. 　前記抗ＰＳＫポリクローナル抗体が、ＰＳＫのタンパク部分およびＰＳＫに特異的なグルカン構造の少なくともいずれかを認識する、請求項１５に記載のＰＳＫの免疫学的分析用キット。 The PSK immunological analysis kit according to claim 15, wherein the anti-PSK polyclonal antibody recognizes at least one of a protein portion of PSK and a glucan structure specific to PSK.
  17. 　前記抗ＰＳＫポリクローナル抗体が、ＰＳＫに反応し、そしてセルロースのβ１，４グルカンのグルカン構造、並びにラミナリンのβ１，３グルカン、及びβ１，６グルカンのグルカン構造に反応しない、請求項１５又は１６に記載のＰＳＫの免疫学的分析用キット。 17. The anti-PSK polyclonal antibody reacts with PSK and does not react with the glucan structure of β1,4 glucan of cellulose and the β1,3 glucan of laminarin and the glucan structure of β1,6 glucan. Kit for immunological analysis of PSK.
  18. 　抗ＰＳＫモノクローナル抗体又はその抗原結合性断片を更に含む、請求項１５～１７のいずれか一項に記載のＰＳＫの免疫学的分析用キット。 The kit for immunological analysis of PSK according to any one of claims 15 to 17, further comprising an anti-PSK monoclonal antibody or an antigen-binding fragment thereof.
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