WO2017183711A1 - Method for capturing lectin target molecule - Google Patents

Method for capturing lectin target molecule Download PDF

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Publication number
WO2017183711A1
WO2017183711A1 PCT/JP2017/015991 JP2017015991W WO2017183711A1 WO 2017183711 A1 WO2017183711 A1 WO 2017183711A1 JP 2017015991 W JP2017015991 W JP 2017015991W WO 2017183711 A1 WO2017183711 A1 WO 2017183711A1
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Prior art keywords
lectin
target molecule
sugar chain
afp
reactive
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PCT/JP2017/015991
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French (fr)
Japanese (ja)
Inventor
郁恵 内藤
将也 豊永
佐藤　英雄
伸之 伊勢
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富士レビオ株式会社
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Priority to JP2018513225A priority Critical patent/JP6910699B2/en
Publication of WO2017183711A1 publication Critical patent/WO2017183711A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin

Definitions

  • the present invention relates to a method for capturing a lectin target molecule, a complex containing the lectin target molecule, a reagent for capturing the lectin target molecule, a kit, and the like.
  • ⁇ -fetoprotein is divided into three fractions, a non-binding fraction (L1), a weak binding fraction (L2), and a binding fraction (L3), depending on the binding properties with lentil bean glutinin (Lens). Classified into minutes. Of these, AFP-L3 is used as a liver cancer marker. Development of an excellent measurement method for AFP-L3 is required for the diagnosis of liver cancer.
  • Prostate-specific antigen is also used as a prostate cancer marker.
  • PSA Prostate-specific antigen
  • the PSA measurement value rises even in prostatic hypertrophy and prostatitis, low specificity is a problem.
  • changes in the sugar chain modification of PSA have been reported in prostate cancer, and it is expected to improve the accuracy of cancer diagnosis by capturing this change in sugar chain modification.
  • Patent Document 1 discloses a method for measuring fucosylated AFP using a lectin.
  • Patent Document 2 discloses a method for measuring PSA having a sugar chain containing a ⁇ -N-acetylgalactosamine residue using a lectin.
  • Non-Patent Document 1 discloses a method for measuring AFP-L3 using a lectin.
  • Non-Patent Document 2 discloses that the malignancy of prostate cancer can be determined by measuring the amount of fucosylated PSA.
  • An object of the present invention is to improve the measurement of lectin target molecules (eg, AFP-L3 and PSA) using lectins.
  • lectin target molecules eg, AFP-L3 and PSA
  • the present invention is as follows.
  • [1] A method for capturing a lectin target molecule, comprising binding a lectin to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.
  • [2] The method according to [1], wherein the lectin is a legume lectin or a mushroom collectin.
  • [3] The method of [1] or [2], wherein the lectin is a fucose-specific lectin.
  • [4] The method according to any one of [1] to [3], wherein the lectin is lentil agglutinin (LCA), concanavalin A (ConA), or yellow chawantake lectin (AAL).
  • LCA lentil agglutinin
  • ConA concanavalin A
  • AAL yellow chawantake lectin
  • a complex comprising a lectin target molecule, a lectin, and a lectin-reactive sugar chain-containing entity.
  • the method of the present invention can enhance the binding of a lectin to a lectin target molecule by using the lectin in the presence of a lectin-reactive sugar chain-containing entity. Therefore, the method of the present invention is useful, for example, for measurement, extraction, concentration, purification, detection and quantification of lectin target molecules. More specifically, when the method of the present invention using a lectin in the presence of a lectin-reactive sugar chain-containing entity is used for measuring a lectin target molecule, it is more sensitive and stable than using a lectin alone. Lectin target molecules can be measured.
  • the complex of the present invention is useful, for example, for carrying out the capture method of the present invention.
  • the reagent and kit of the present invention are useful, for example, for simple implementation of the capture method of the present invention.
  • FIG. 1 shows the confirmation of molecular weight by SDS-PAGE and LCA reactivity by lectin blot for purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D.
  • FIG. 2 is a diagram showing confirmation of LCA reactivity by ELISA for purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D.
  • FIG. 3 shows specific detection of AFP-L3 by LCA in the presence of LCA-reactive F (ab ′) 2 fragment antibody A.
  • FIG. 4 shows specific detection of AFP-L3 by LCA in the presence of LCA-reactive F (ab ′) 2 fragment antibody B.
  • FIG. 1 shows the confirmation of molecular weight by SDS-PAGE and LCA reactivity by lectin blot for purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D.
  • FIG. 2 is a diagram showing confirmation of LCA reactivity by EL
  • FIG. 5 shows the results of detection of AFP-L3 and AFP-L1 by LCA in the presence of LCA non-reactive F (ab ′) 2 fragment antibody C.
  • FIG. 6 shows the results of detection of AFP-L3 and AFP-L1 by LCA in the presence of LCA non-reactive F (ab ′) 2 fragment antibody D.
  • FIG. 7 is a view showing the molecular weight by SDS-PAGE and confirmation of LCA reactivity by lectin blot for a sugar chain-depleted mouse IgG F (ab ′) 2 fragment.
  • Figure 8 is a sugar chain-containing mouse IgG F (ab ') 2 fragments and deglycosylation mouse IgG F (ab') for 2 fragments, a diagram illustrating a confirmation of LCA reactive by ELISA.
  • FIG. 10 is a diagram showing detection results of -L3 and AFP-L1.
  • Anti-TNF ⁇ antibody (P): anti-TNF ⁇ antibody having an N-type sugar chain binding consensus sequence in the variable region; anti-TNF ⁇ antibody (N): anti-TNF ⁇ antibody having no N-type sugar chain-binding consensus sequence in the variable region; anti-IL- 10 antibody (P ′): anti-IL-10 antibody having N-type sugar chain binding consensus sequence in variable region; anti-IL-10 antibody (N ′): anti-IL having no N-type sugar chain binding consensus sequence in variable region -10 antibody.
  • P anti-TNF ⁇ antibody having an N-type sugar chain binding consensus sequence in the variable region
  • anti-TNF ⁇ antibody anti-TNF ⁇ antibody having no N-type sugar chain-binding consensus sequence in the variable region
  • anti-IL- 10 antibody P ′
  • anti-IL-10 antibody anti-IL having no N-type sugar chain binding consensus sequence in variable region
  • FIG. 11 shows specific detection of AFP-L3 by LCA in the presence of a lectin-reactive antigen (HBs antigen).
  • FIG. 12 is a diagram showing the confirmation of the molecular weight by SDS-PAGE and LCA reactivity by lectin blot for the sugar chain-removed HBs antigen.
  • FIG. 13 is a diagram showing confirmation of LCA reactivity by ELISA for sugar chain-containing HBs antigen and sugar chain-removed HBs antigen.
  • FIG. 14 is a diagram showing the detection results of AFP-L3 and AFP-L1 by LCA in the presence of sugar chain-containing HBs antigen-HRP showing LCA reactivity or sugar chain-removed HBs antigen-HRP not showing LCA reactivity. It is.
  • FIG. 15 shows the detection of AFP-binding glycans by Aurolia aurantia lectin (AAL) in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type glycan-binding consensus sequence.
  • AAL Aurolia aurantia lectin
  • FIG. 16 is a diagram showing detection of an AFP-linked sugar chain by concanavalin A (ConA) in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain-binding consensus sequence.
  • FIG. 17 is a diagram showing detection of PSA-binding sugar chains by AAL in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain-binding consensus sequence.
  • FIG. 18 is a diagram showing specific detection of AFP-L3 by LCA in the presence of mouse IgG F (ab ′) 2 -HRP having an N-type sugar chain binding consensus sequence.
  • Co-addition conditions A solution containing LCA and mouse IgG F (ab ′) 2 antibody A-HRP was incubated in AFP capture wells; Wash conditions: Washed and unreacted LCA after incubating a solution containing LCA in AFP capture wells Was removed. Subsequently, mouse IgG F (ab ′) 2 antibody A-HRP was added to the AFP capture well from which unreacted LCA was removed, and then incubated.
  • FIG. 19 shows specific detection of AFP-L3 by LCA in the presence of HBs antigen-HRP.
  • Co-addition conditions Incubate solution containing LCA and HBs antigen-HRP in AFP capture wells; Wash conditions: Incubate solution containing LCA in AFP capture wells and wash to remove unreacted LCA from AFP capture wells . Then, in the AFP capture well from which unreacted LCA was removed, incubation was carried out after adding a solution containing HBs antigen-HRP.
  • FIG. 20 shows specific detection of AFP-L3 by AAL in the presence of HBs antigen-HRP.
  • FIG. 21 is a diagram showing specific detection of AFP-L3 by LCA under conditions using LCA-reactive antibody fragment A or B as a solid phase. (+): LCA reactivity; (-): LCA non-reactive FIG.
  • FIG. 22 shows the detection of hemopexin (HPX) -bound AAL-reactive fucosylated glycans in liver cancer patient serum by mouse IgG F (ab ′) 2 -HRP having an N-type glycan-binding consensus sequence in the presence of AAL.
  • FIG. 1% BSA-PBS control; HCC-1, HCC-2: liver cancer patient sera 2 cases; NHS-1, NHS-2: healthy subject sera 2 cases.
  • FIG. 23 shows detection of transferrin (TF) -bound AAL-reactive fucosylated sugar chain in liver cancer patient serum by mouse IgG F (ab ′) 2 -HRP having N-type sugar chain binding consensus sequence in the presence of AAL.
  • FIG. 1% BSA-PBS control; HCC-1, HCC-2: liver cancer patient sera 2 cases; NHS-1, NHS-2: healthy subject sera 2 cases.
  • the present invention provides a method for capturing a lectin target molecule, which comprises binding a lectin to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.
  • a lectin is a protein that recognizes a specific sugar chain structure and exhibits binding activity.
  • lectins for example, those derived from animals (eg, vertebrates, invertebrates), plants (eg, legumes, gramineae), fungi (eg, mushrooms, moths) are known.
  • legume lectins include pea lectin (Concanavalin A: ConA), lentil agglutinin (Lens culinaris agglutinin: LCA), pea lectin (Pisum sativum), and the like.
  • examples of the mushroom collectin include herochawantake lectin (Aurelia aurantia lectin: AAL).
  • the lectin used in the present invention can also be appropriately selected according to the sugar chain structure of the lectin target molecule.
  • the lectin used in the present invention include a fucose-specific lectin having an affinity for fucose, a galectin having an affinity for galactose, a sialic acid-reactive lectin, and the like, but a fucose-specific lectin is preferable.
  • a fucose-specific lectin for example, a lectin that recognizes a sugar chain structure (core fucose structure) in which fucose is ⁇ 1,6 added to GlcNAc that is the root of an N-type sugar chain (eg, LCA, pea sactivum (PSA)).
  • lectin that recognizes a sugar chain structure in which fucose is added at any of ⁇ 1, 2, ⁇ 1, 3, ⁇ 1, 4, ⁇ 1, 6 to GlcNAc of N-type sugar chain, O-type sugar chain and glycolipid sugar chain [Example, AAL, Aspergillus oryzae Lectin: AOL].
  • a lectin target molecule refers to a target molecule that has the ability to bind to a lectin and is to be captured in the present invention.
  • the target molecule is a molecule composed of or containing a specific sugar chain to which a lectin binds.
  • the lectin target molecule is a glycoprotein.
  • the glycoprotein as a lectin target molecule include glycoproteins derived from mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms, or viruses.
  • the glycoprotein as a lectin target molecule may be a glycoprotein for which measurement, extraction, concentration or purification is desired.
  • a glycoprotein is a protein having an N-type sugar chain binding consensus sequence.
  • the N-type sugar chain-binding consensus sequence is an amino acid sequence composed of amino acid residues of Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline).
  • N-type sugar chains are known to bind to asparagine (Asn) residues in N-type sugar chain binding consensus sequences. More specifically, examples of the lectin target molecule include, but are not limited to, AFP-L3, PSA, hemopexin (HPX), and transferrin (TF).
  • PSA is known as a modified protein of a sugar chain such as an N-type sugar chain.
  • the lectin-reactive sugar chain-containing entity means a molecule containing a lectin-reactive sugar chain or a solid phase to which a lectin-reactive sugar chain is added.
  • the lectin-reactive sugar chain-containing entity is different from the lectin target molecule to be captured by the method of the present invention.
  • the lectin-reactive sugar chain-containing entity is not an antibody whose glycin reacts with lectin but has lectin or a lectin target molecule as an antigen.
  • Examples of the molecule containing a lectin-reactive sugar chain include biomolecules and artificial synthetic products (eg, artificial synthetic molecules such as artificial synthetic polymers).
  • Examples of the solid phase include particles (eg, magnetic particles); membranes (eg, nitrocellulose membrane, filter paper), supports such as columns; and containers such as plates (eg, multiwell plates) and tubes.
  • Examples of the solid phase material include glass, plastic, polysaccharide matrix, and metal.
  • the lectin-reactive sugar chain-containing entity can be appropriately selected according to the type of lectin used. For example, such a selection can be performed by evaluating the binding ability of a sugar chain or a sugar chain-containing entity to a lectin (see Example 2, for example).
  • the lectin-reactive sugar chain-containing entity is a biomolecule containing a lectin-reactive sugar chain.
  • biomolecules include polypeptides and nucleic acids.
  • the lectin-reactive sugar chain-containing entity is a glycoprotein.
  • glycoproteins as lectin-reactive sugar chain-containing entities include glycoproteins derived from mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms, or viruses.
  • glycoproteins as lectin-reactive sugar chain-containing entities include, for example, immunoglobulins (eg, antibodies), secretory proteins, membrane-bound proteins, hormones, cytokines, chemokines, virus-derived antigens, enzymes, extracellular matrices, Examples include extracellular vesicle membrane proteins, tumor-specific antigens, and partial peptides thereof.
  • immunoglobulins eg, antibodies
  • secretory proteins eg, secretory proteins, membrane-bound proteins, hormones, cytokines, chemokines, virus-derived antigens, enzymes, extracellular matrices
  • extracellular matrices examples include extracellular vesicle membrane proteins, tumor-specific antigens, and partial peptides thereof.
  • the glycoprotein as the lectin-reactive sugar chain-containing entity is a protein having an N-type sugar chain binding consensus sequence.
  • the N-type sugar chain-binding consensus sequence is an amino acid sequence composed of amino acid residues of Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline).
  • N-type sugar chains are known to bind to asparagine (Asn) residues in N-type sugar chain binding consensus sequences.
  • examples of the protein having an N-type sugar chain binding consensus sequence include hepatitis B virus surface antigen, human chorionic gonadotropin ⁇ chain, luteinizing hormone, PSA antigen, HE4 antigen, MUC1 antigen, thyroglobulin, And antibodies having an N-type sugar chain-binding consensus sequence in the variable region.
  • the glycoprotein as the lectin-reactive sugar chain-containing entity is an antibody having an N-type sugar chain binding consensus sequence in the variable region.
  • antibodies having an N-type sugar chain binding consensus sequence in the variable region include natural full-length antibodies and modified antibodies.
  • the modified antibody include an antibody fragment having a variable region (eg, Fab, F (ab ′) 2 ), and a single chain antibody.
  • antibodies having an N-type sugar chain binding consensus sequence in the variable region include IgG, IgM, IgA, IgD, IgE, and IgY.
  • the antibody having an N-type sugar chain binding consensus sequence in the variable region may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.
  • a lectin is bound to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.
  • the method of the present invention is carried out in a system comprising at least three components of a lectin target molecule and a lectin and lectin-reactive sugar chain-containing entity, and contains a lectin and a lectin-reactive sugar chain as means for capturing the lectin target molecule.
  • the entity is used together.
  • the method of the present invention is performed in a system in which a lectin and a lectin-reactive sugar chain-containing entity are added to a liquid sample containing a lectin target molecule.
  • the origin of the liquid sample is not particularly limited, and may be a biological sample derived from a living organism or an environmental sample. Examples of organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens), insects, and the like.
  • a biological sample can also be a blood-related sample (eg, whole blood, serum, plasma), saliva, urine, milk, tissue or cell extract, or a mixture thereof, which is the blood itself or a sample derived from blood. Also good.
  • blood-related sample eg, whole blood, serum, plasma
  • saliva, urine, milk, tissue or cell extract, or a mixture thereof which is the blood itself or a sample derived from blood.
  • environmental samples include samples derived from soil, seawater, and fresh water.
  • a liquid sample subjected to pretreatment may be used. Examples of such pretreatment include centrifugation, fractionation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.
  • the amount of the lectin used in the method of the present invention is not particularly limited as long as it is an amount capable of capturing the target lectin in the presence of the lectin-reactive sugar chain-containing entity, and is, for example, 0.1 ⁇ g / mL to 100 mg / mL. .
  • the amount of lectin is preferably 1 ⁇ g / mL or more, more preferably 5 ⁇ g / mL or more, and even more preferably 10 ⁇ g / mL or more.
  • the amount of lectin is also preferably 20 mg / mL or less, more preferably 10 mg / mL or less, and even more preferably 2 mg / mL or less. Such an amount of lectin may be added to the liquid sample.
  • the amount of the lectin-reactive sugar chain-containing entity used in the method of the present invention is not particularly limited as long as it is an amount capable of supporting capture of the lectin target molecule by the lectin, and is, for example, 0.001 ⁇ g / mL to 100 mg / mL. .
  • the amount of the lectin-reactive sugar chain-containing entity is preferably 0.005 ⁇ g / mL or more, more preferably 0.02 ⁇ g / mL or more, and even more preferably 0.1 ⁇ g / mL or more.
  • the amount of the lectin-reactive sugar chain-containing entity is also preferably 20 mg / mL or less, more preferably 5 mg / mL or less, and even more preferably 1 mg / mL or less. Such an amount of lectin-reactive sugar chain-containing entity may be added to the liquid sample.
  • the method of the present invention is a method for measuring a lectin target molecule.
  • the lectin target molecule can be measured after binding of the lectin to the lectin target molecule.
  • the measurement method of the present invention can be carried out according to an immunological technique, and can be carried out by a technique similar to the immunological technique by using a lectin-reactive sugar chain-containing entity and a lectin instead of an antibody. it can.
  • Examples of the immunological technique to which the measurement method of the present invention is applied include enzyme immunoassay (EIA) (eg, chemiluminescence EIA (CLEIA), enzyme-adsorbed EIA (ELISA)), fluorescence immunoassay, chemiluminescence immunoassay Method, electrochemiluminescence immunoassay, agglutination, immunostaining, flowmetry, biolayer interferometry, In Situ PLA method, chemically amplified luminescence proximity homogenous assay, line blotting, Western blotting It is done.
  • EIA enzyme immunoassay
  • CLIA chemiluminescence EIA
  • ELISA enzyme-adsorbed EIA
  • fluorescence immunoassay eg, chemiluminescence immunoassay Method
  • electrochemiluminescence immunoassay eg.g, electrochemiluminescence immunoassay, agglutination, immunostaining, flowmetry,
  • an affinity substance specific for the lectin target molecule may be further used.
  • the affinity substance specific for the lectin target molecule used in the method of the present invention binds to the lectin target molecule at a site (eg, peptide region) different from the lectin binding site (sugar chain) in the lectin target molecule.
  • Examples of the affinity substance specific for the lectin target molecule include antibodies and aptamers, and antibodies are preferred.
  • the amount of the affinity substance specific for the lectin target molecule used in the method of the present invention is not particularly limited as long as it is a sufficient amount to bind the lectin target molecule, and is, for example, 0.0001 ⁇ g / mL to 10 mg / mL. is there.
  • the amount of the affinity substance is preferably 0.001 ⁇ g / mL or more, more preferably 0.005 ⁇ g / mL or more, and even more preferably 0.01 ⁇ g / mL or more.
  • the amount of affinity substance is also preferably 2 mg / mL or less, more preferably 0.5 mg / mL or less, and even more preferably 0.1 mg / mL or less. Such an amount of affinity substance may be added to the liquid sample.
  • the measurement method of the present invention may be performed in a sandwich manner.
  • an affinity substance specific for the lectin target molecule may be used as the solid phase immobilization means, and the lectin and the lectin reactive sugar chain-containing entity may be used in combination as the detection means.
  • a lectin and a lectin-reactive sugar chain-containing entity may be used together as a solid phase immobilization means, and an affinity substance specific for the lectin target molecule may be used as a detection means.
  • a lectin-reactive sugar chain-containing entity or lectin, or an affinity substance specific for a lectin target molecule may be labeled with a labeling substance.
  • labeling substances include enzymes (eg, peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase) and affinity substances (eg, streptavidin and biotin, which are complementary to each other in the sense strand and the antisense strand nucleic acid.
  • fluorescent substance or protein eg, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein
  • luminescent substance eg, luciferin, aequorin, acridinium ester, tris (2,2'- Bipyridyl) ruthenium, luminol
  • radioactive materials eg, 3 H, 14 C, 32 P, 35 S, 125 I.
  • the lectin-reactive sugar chain-containing entity or an affinity substance specific to the lectin-reactive sugar chain-containing entity is labeled.
  • the measurement method of the present invention when performed in a sandwich manner, can be performed by the following steps: (1) binding a lectin target molecule to an affinity substance specific for the lectin target molecule to obtain a first complex including the lectin target molecule and an affinity substance specific for the lectin target molecule; (2) A lectin is bound to the first complex in the presence of a lectin-reactive sugar chain-containing entity, and a lectin target molecule, an affinity substance specific for the lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity Obtaining a second complex comprising; and (3) measuring the amount of lectin target molecules contained in the second complex.
  • the lectin target molecules can be those contained in the liquid sample as described above. Therefore, as a specific embodiment of the step (1), the step (1) may be performed by adding an affinity substance specific for the lectin target molecule to the liquid sample containing the lectin target molecule.
  • an affinity substance specific for the lectin target molecule in an amount as described above can be used, but preferably specific for an excess equivalent of the lectin target molecule relative to the expected lectin target molecule.
  • a typical affinity substance may be used.
  • an affinity substance specific for the lectin target molecule it is preferable to use a substance immobilized on a solid phase as described above.
  • Binding of a lectin target molecule and an affinity substance specific for the lectin target molecule can be achieved by incubating both in solution (eg, buffer), thereby allowing the lectin target molecule and lectin target molecule to bind to each other. A first complex containing a specific affinity substance is obtained.
  • step (2) a second complex is obtained.
  • the step (1) by adding the lectin and the lectin-reactive sugar chain-containing entity to the solution containing the first complex obtained in the step (1), the step (2) May be performed.
  • the amount of lectin as described above and the amount of lectin-reactive sugar chain-containing entity as described above can be used, but preferably in excess equivalent to the expected lectin target molecule. Lectins and excess equivalents of lectin-reactive sugar chain-containing entities may be used.
  • Either the lectin or the lectin-reactive sugar chain-containing entity may be labeled with a labeling substance, but preferably the lectin-reactive sugar chain-containing entity may be labeled with a labeling substance.
  • Binding of the lectin to the first complex in the presence of the lectin-reactive sugar chain-containing entity can be achieved by incubating the three in solution (eg, buffer), whereby the lectin target molecule, A second complex comprising an affinity substance specific for the lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity is obtained.
  • the substance specific to the lectin target molecule that is immobilized on the solid phase is used, the second complex is immobilized on the solid phase.
  • a washing step including removal and addition of the liquid phase may be provided.
  • step (3) the amount of the lectin target molecule can be measured using the signal from the labeling substance in the second complex as an index.
  • the present invention also provides a complex comprising a lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity.
  • the complex of the present invention may further contain an affinity substance specific for the lectin target molecule.
  • the complex of the present invention can be prepared by incubating these components in a solution and binding them.
  • the definition, examples and preferred examples of the lectin target molecule, the lectin, the lectin-reactive sugar chain-containing entity, and the affinity substance specific for the lectin target molecule are as described above.
  • the complex of the present invention is useful for the implementation of the capture method of the present invention, for example.
  • the present invention further provides a reagent for capturing a lectin target molecule, which contains a lectin and a lectin-reactive sugar chain-containing entity in a mixed form.
  • the capture reagent of the present invention includes a composition of a lectin and a lectin-reactive sugar chain-containing entity. Since the lectin and the lectin-reactive sugar chain-containing entity contained in the reagent of the present invention are in a mixed form, in the capture reagent of the present invention, a complex is formed between the lectin and the lectin-reactive sugar chain-containing entity. May be.
  • either the lectin or the lectin-reactive sugar chain-containing entity may be labeled with a labeling substance, but preferably the lectin-reactive sugar chain-containing entity is labeled. It may be labeled with a substance.
  • the reagent of the present invention may contain the labeling substance in an unmixed form with the lectin and the lectin-reactive sugar chain-containing entity. Good.
  • the reagent of the present invention may further contain an affinity substance specific for the lectin target molecule.
  • the affinity substance specific for the lectin target molecule may be labeled with a labeling substance.
  • the affinity substance specific for the lectin target molecule may be immobilized on the solid phase as described above.
  • the reagent of the present invention can be suitably used for measuring lectin target molecules in a sandwich manner.
  • the reagent of the present invention contains an affinity substance specific for a lectin target molecule in either a mixed form or a non-mixed form with a lectin and a lectin-reactive sugar chain-containing entity, but preferably in a non-mixed form.
  • affinity substances specific for lectin target molecules, lectins, lectin-reactive sugar chain-containing entities, and lectin target molecules are as described above.
  • the reagent of the present invention is useful for, for example, simple implementation of the capture method of the present invention, and can be suitably used for the measurement method of the present invention, for example.
  • the present invention also provides a kit for capturing a lectin target molecule comprising a lectin and a lectin-reactive sugar chain-containing entity.
  • the lectin and lectin-reactive sugar chain-containing entity included in the kit of the present invention is in a mixed form (ie, in the form of a composition) or in an unmixed form (ie, a form isolated from each other). Therefore, the component contained in the kit of this invention is provided with the form accommodated in the different container (for example, tube, plate), respectively, or the form accommodated in the different divisions of the same container.
  • kits of the present invention When the kit of the present invention is used for measurement of a lectin target molecule, either a lectin or a lectin-reactive sugar chain-containing entity may be labeled with a labeling substance, but preferably a lectin-reactive sugar chain-containing entity is labeled It may be labeled with a substance. Alternatively, when neither the lectin nor the lectin-reactive sugar chain-containing entity is labeled with a labeling substance, the kit of the present invention can be used as an affinity substance (labeled with a labeling substance and / or a lectin target molecule). May be included).
  • the kit of the present invention may further contain an affinity substance specific for the lectin target molecule.
  • the kit of the present invention can be suitably used for measuring a lectin target molecule in a sandwich manner.
  • the affinity substance specific for the lectin target molecule may be immobilized on the solid phase as described above.
  • the definition, examples and preferred examples of lectin target molecules, lectins, lectin-reactive sugar chain-containing entities, and affinity substances specific for the lectin target molecules are as described above.
  • the kit of the present invention is useful for, for example, simple implementation of the capture method of the present invention, and can be suitably used for, for example, the measurement method of the present invention.
  • Example 1 Preparation of Antibody Fragment and Preparation of Antibody Fragment-HRP Mouse IgG1, which is not an anti-LCA antibody, was digested with pepsin and LCA-reactive mouse IgG F (ab ′) 2 fragment and non-LCA-reactive mouse IgG F ( ab ') 2 fragments were prepared.
  • preparation was performed as follows.
  • Mouse IgG1 antibodies A, B, C and D that are not anti-LCA antibodies were digested with pepsin. Digestion was performed by adding pepsin (SIGMA) to mouse IgG1 antibodies A, B, C and D buffer-exchanged to citrate buffer, reacting at 37 ° C. for 1 hour, and stopping the reaction by adding Tris buffer. .
  • SIGMA pepsin
  • Gel filtration was performed with Superdex 200 increase 10 / 300GL equilibrated with PBS to obtain four types of purified mouse IgG F (ab ′) 2 fragments.
  • Purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D were labeled with horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • HRP was labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached protocol to obtain 4 types of purified antibody fragments—HRP (mouse IgG F (ab ′) 2 -HRP).
  • Example 2 Confirmation of purity, molecular weight, and lectin reactivity of antibody fragments The purity, molecular weight, and lectin reactivity of the four antibody fragments prepared in Example 1 were confirmed.
  • confirmation was performed as follows. (1) The molecular weights of purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D were confirmed by SDS-PAGE (FIG. 1 left). (2) LCA reactivity of purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D was confirmed by lectin blot (right side of FIG. 1). (3) purified mouse IgG F (ab ') 2 fragment antibodies A, B, and LCA reactive C and D were confirmed by ELISA. Purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D were diluted with PBS so as to be 5 ⁇ g / mL each.
  • Antibody A had an N-type sugar chain binding consensus sequence in the heavy chain of the antibody variable region. This indicates that the F (ab ′) 2 fragment is modified with an N-type sugar chain. Therefore, F (ab ′) 2 fragment antibody A is considered to exhibit lectin reactivity via an N-type sugar chain.
  • Antibody B is the same as antibody A.
  • Antibody C did not have an N-type sugar chain binding consensus sequence in the heavy or light chain of the antibody variable region. This indicates that the F (ab ′) 2 fragment is not modified with an N-type sugar chain. Therefore, F (ab ′) 2 fragment antibody C is considered not to show lectin reactivity.
  • Antibody D is the same as antibody C.
  • Example 3 Construction of AFP-L3 measurement system using lectin in the presence of lectin-reactive antibody fragments Using mouse IgG F (ab ') 2 -HRP prepared in Example 1, immunological measurement of AFP-L3 A system was constructed.
  • the construction was performed as follows.
  • Anti-AFP F (ab ′) 2 was diluted with PBS so as to be 10 ⁇ g / mL.
  • 50 ⁇ L of the diluted solution was incubated at 4 ° C. for 24 hours to immobilize the antibody.
  • the antibody-immobilized well was washed with 250 ⁇ L of PBST three times, and then blocked with 150 ⁇ L of 10% blocking reagent at 37 ° C. for 1 hour.
  • the antibody-immobilized well was washed 3 times with 250 ⁇ L of PBST.
  • mouse IgG F (ab ′) 2 -HRP prepared in Example 1 was diluted with 10% blocking reagent to 5 ⁇ g / mL, and LCA (Vector) was further added to 100 ⁇ g / mL.
  • a mixed solution was prepared, and the mixed solution was added to an antibody-immobilized well and incubated at 37 ° C. for 1 hour.
  • the antibody-immobilized well was washed 3 times with 250 ⁇ L of PBST.
  • 50 ⁇ L of TMB substrate solution (Dako) was added and reacted at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
  • LCA was able to specifically detect AFP-L3 in the presence of LCA-reactive antibody fragment A or B (Table 2, FIGS. 3 to 6).
  • lectin can specifically detect AFP-L3 in the presence of a labeled lectin-reactive antibody fragment.
  • Example 4 Confirmation of Involvement of Lectin-Reactive Sugar Chain in Lectin-Reactive Antibody Fragment in AFP-L3 Detection
  • the sugar chain of mouse IgG F (ab ′) 2 antibody B prepared in Example 1 is N-glycosidase F It was confirmed whether or not the sugar chain-removed mouse IgG F (ab ′) 2 antibody B maintained AFP-L3 reactivity in the presence of LCA.
  • N-glycosidase F is an enzyme that specifically cleaves a GlcNAc-Asn bond between an N-linked sugar chain and a protein.
  • Example 5 Detection of AFP-L3 by a lectin in the presence of mouse IgG having an N-type sugar chain binding consensus sequence in the variable region The presence or absence of an N-type sugar chain binding consensus sequence in the variable region of the antibody is useful for AFP-L3 detection It was examined whether or not it affected.
  • Example 1 An anti-TNF ⁇ antibody (P) with and without an N-type sugar chain binding consensus sequence (Asn-Ser-Ser) in the variable region (N), and an N-type sugar chain binding consensus sequence (N) in the variable region
  • the anti-IL-10 antibody (P ′) having Asn-Asp-Thr) and the anti-IL-10 antibody not having N (N ′) were labeled with HRP in the same manner as in Example 1.
  • Example 3 As in Example 3, the AFP-L3 reactivity in the presence of LCA was confirmed. The AFP concentration was examined at 5 ⁇ g / mL.
  • LCA was able to specifically detect AFP-L3 in the presence of an antibody having an N-type sugar chain binding consensus sequence (Table 4, FIG. 10).
  • the lectin can specifically detect AFP-L3 in the presence of an antibody having an N-type sugar chain binding consensus sequence in the variable region (regardless of the type of antigen to which the present antibody binds).
  • Example 6 Construction of AFP-L3 measurement system with lectin in the presence of lectin-reactive antigen Using HBs antigen (hepatitis B virus surface antigen), a glycoprotein having an N-type sugar chain-binding consensus sequence other than mouse IgG Thus, an immunological measurement system for AFP-L3 was constructed.
  • HBs antigen hepatitis B virus surface antigen
  • Example 3 the construction was performed as follows. (1) HRP labeling was performed on HBs antigens having an N-type sugar chain binding consensus sequence in the same manner as in Example 1. (2) As in Example 3, the AFP-L3 reactivity in the presence of LCA was confirmed. The AFP concentration was examined by diluting from 1000 ng / mL to 2 n. HBs antigen-HRP was reacted at 2 ⁇ g / mL.
  • Example 7 Confirmation of involvement of lectin-reactive sugar chain in antigen in detection of AFP-L3 By removing the sugar chain of HBs antigen with N-glycosidase F, AFP in the presence of LCA in the presence of sugar chain-removed HBs antigen -Confirmed to maintain L3 reactivity.
  • N-glycosidase F treatment of HBs antigen was performed.
  • the N-glycosidase F treatment was performed using a reaction solution attached to N-glycosidase F and PNGase F (New England Biolabs) according to the protocol attached to PNGase F to remove sugar chains.
  • the sugar chain-removed HBs antigen was labeled with HRP in the same manner as in Example 1.
  • HRP As in Example 2, the degree of purification, molecular weight, and LCA reactivity were confirmed.
  • AFP-L3 reactivity in the presence of LCA was confirmed. The AFP concentration was examined at 1 ⁇ g / mL.
  • HBs antigen-HRP was added at 1 ⁇ g / mL.
  • Example 8 Detection of AFP-L3 by lectin in the presence of lectin-reactive glycoprotein LCA was able to detect AFP-L3 not only in the presence of antibodies but also in the presence of antigen (Example 7). Therefore, another glycoprotein having an N-type sugar chain binding consensus sequence in its amino acid sequence was labeled with HRP, and it was confirmed whether LCA could detect AFP-L3 in the presence of the other glycoprotein.
  • Example 3 confirmation was performed as follows. (1) HCG labeling was performed on hCG ⁇ (human chorionic gonadotropin ⁇ chain) and LH (luteinizing hormone) each having an N-type sugar chain binding consensus sequence in the amino acid sequence in the same manner as in Example 1. (2) As in Example 3, the AFP-L3 reactivity of LCA was confirmed. The AFP concentration was examined at 1 ⁇ g / mL.
  • a numerical value shows the measured value of OD450.
  • Example 9 Construction of detection system for AFP-binding sugar chain by various lectins in the presence of lectin-reactive labeled antibody or lectin-reactive labeled antigen Mouse IgG F (ab ') 2 -HRP having N-type sugar chain-binding consensus sequence
  • a detection system for an AFP-binding sugar chain having the following structural formula was constructed by using Aleuria aurantia lectin (AAL) and concanavalin A (ConA) as lectins.
  • AAL Aleuria aurantia lectin
  • ConA concanavalin A
  • AFP-L3 and AFP-L1 were each diluted with a 10% blocking reagent to 1 ⁇ g / mL, and the reaction was performed using the diluted solution.
  • the antibody-immobilized well was washed 3 times with 250 ⁇ L of PBST.
  • the 1 [mu] g / mL respectively mouse IgG F (ab ') 2 -HRP and 10% blocking reagent the HBs antigen -HRP prepared in Example 6 with an N-type sugar chain binding consensus sequence prepared in Example 1 Then, dilute the blocking reagent in the same way, and then prepare a mixed solution to which either AAL (J-Oil Mills) is added at 10 ⁇ g / mL or ConA (Vector) is added at 10 ⁇ g / mL. Incubated at 37 ° C for 1 hour in addition to phased wells. (3) The antibody-immobilized well was washed 3 times with 250 ⁇ L of PBST. 50 ⁇ L of TMB substrate solution was added to the well after washing, and the reaction was performed at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
  • AAL J-Oil Mills
  • ConA Vector
  • AAL and ConA can detect AFP-linked glycans in the same manner as LCA in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type glycan-binding consensus sequence. (Table 9, FIGS. 15 and 16).
  • AFP-linked sugar chains can be detected not only for LCA but also for other lectins in the presence of glycoprotein.
  • Example 10 Construction of a detection system for PSA-binding sugar chains by lectins in the presence of lectin-reactive labeled antibodies or lectin-reactive labeled antigens AAL-reactive sugar chains are bound to PSA (prostate specific antigen) It has been reported.
  • a PSA-binding AAL-reactive fucosylated glycan having the following structural formula using AAL as a lectin in the presence of mouse IgG F (ab ′) 2 -HRP and HBs antigen-HRP having an N-type glycan-binding consensus sequence
  • a detection system was constructed.
  • the construction was performed as follows. (1) Anti-PSA F (ab ′) 2 was diluted with PBS so as to be 10 ⁇ g / mL. In the U96 MaxiSorp Nunc-Immuno Plate, 50 ⁇ L of the diluted solution was incubated at 4 ° C. for 24 hours to immobilize the antibody. (2) The antibody-immobilized well was washed with 250 ⁇ L of PBST three times, and then blocked with 150 ⁇ L of 10% blocking reagent at 37 ° C. for 1 hour.
  • the antibody-immobilized well was washed 3 times with PBST 250 ⁇ L of PBST, and Lumipulse PSA-N standard PSA solution (Fujirebio) 100 ng / mL, 40 ng / mL, 5 ng / mL, 0.5 ng / mL, 0 ng / ML was added to antibody-immobilized wells and incubated at 37 ° C for 1 hour.
  • the antibody-immobilized well was washed 3 times with 250 ⁇ L of PBST.
  • Mouse IgG F (ab ′) 2 -HRP having the N-type sugar chain binding consensus sequence prepared in Example 1 and HBs antigen-HRP prepared in Example 6 were each 1 ⁇ g / mL with 10% blocking reagent. AAL was further added to a concentration of 10 ⁇ g / mL. The solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour. (5) The antibody-immobilized well was washed 3 times with 250 ⁇ L of PBST. 50 ⁇ L of TMB substrate solution was added to the well after washing, and the reaction was performed at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
  • AAL can detect PSA-bound AAL-reactive sugar chains in a concentration-dependent manner in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain-binding consensus sequence. (Table 10, FIG. 17).
  • lectins can detect not only AFP-L3 but also other lectin reactive glycoproteins such as PSA in the presence of lectin reactive labeled antibodies or lectin reactive labeled antigens.
  • Example 11 Enhancement of binding force between lectin and target molecule by glycoprotein (1) This example was performed to prove the enhancement of the binding force between the lectin and the target molecule by the glycoprotein.
  • Anti-AFP F (ab ′) 2 was diluted with PBS so as to be 10 ⁇ g / mL.
  • F16 MaxiSorp Nunc-Immuno Plate Thermo
  • 100 ⁇ L of the diluted solution was incubated at 4 ° C. for 24 hours to immobilize the antibody.
  • the antibody-immobilized well was washed with 300 ⁇ L of PBST three times, and then blocked with 200 ⁇ L of 10% blocking reagent at 37 ° C. for 1 hour.
  • the antibody-immobilized well was washed 3 times with 300 ⁇ L of PBST.
  • the AFP-L3 solution and the AFP-L1 solution were each diluted with a 10% blocking reagent to 500 ng / mL.
  • 100 ⁇ L of the diluted solution was added and incubated at 37 ° C. for 1 hour.
  • the antibody-immobilized well was washed 3 times with 300 ⁇ L of PBST. Under washing conditions, LCA was diluted with a 10% blocking reagent so as to be 100 ⁇ g / mL, and 100 ⁇ L of the diluted solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
  • mouse IgG F (ab ′) 2 antibody A-HRP prepared in Example 1 was diluted with 10% blocking reagent to 5 ⁇ g / mL or HBs antigen-HRP at 2 ⁇ g / mL, and LCA was further diluted. Each was added so as to be 100 ⁇ g / mL. 100 ⁇ L of the diluted solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour. (6) The antibody-immobilized well was washed 3 times with 300 ⁇ L of PBST. To the well after washing, 100 ⁇ L of TMB substrate solution was added and reacted at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
  • AFP-L3 was significantly attenuated by adding a washing step after the reaction between AFP-L3 and lectin (LCA) (FIGS. 18 and 19).
  • AFP-L3 was reacted with lectin (LCA) in the presence of labeled glycoprotein (mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain binding consensus sequence) in the presence of simultaneous addition conditions. Only AFP-L3 could be detected specifically (FIGS. 18 and 19).
  • Example 12 Enhancement of binding force between lectin and target molecule by glycoprotein (2) This example was performed to prove the enhancement of the binding force between the lectin and the target molecule by the glycoprotein. Washing after the reaction of the target molecule (AFP-linked fucosylated sugar chain) and lectin (AAL), and the conditions (washing conditions) detected by glycoprotein (HBs antigen-HRP) and the target molecule (AFP-linked fucosylated sugar chain) It was examined whether there was a difference in target molecule detection ability under the conditions (simultaneous addition conditions) in which lectin (AAL) was reacted in the presence of glycoprotein (HBs antigen-HRP). The number of washings was adjusted between conditions.
  • the examination was conducted as follows. (1) In the same manner as in Example 11, 100 ⁇ L of AFP-L3 and AFP-L1 diluted to 500 ng / mL were added to wells on which anti-AFP F (ab ′) 2 had been immobilized, respectively. Incubated for hours. (2) The antibody-immobilized well was washed 3 times with 300 ⁇ L of PBST. Under washing conditions, AAL was diluted with a 10% blocking reagent so as to be 10 ⁇ g / mL, 100 ⁇ L of the diluted AAL solution was added to the antibody-immobilized well, and the resulting solution was incubated at 37 ° C. for 1 hour.
  • HBs antigen-HRP prepared in Example 6 was diluted with 10% blocking reagent so as to be 2 ⁇ g / mL, and 100 ⁇ L of the diluted solution was incubated at 37 ° C. for 1 hour.
  • the HBs antigen-HRP prepared in Example 6 was diluted with a 10% blocking reagent to 2 ⁇ g / mL, and AAL was further added to 10 ⁇ g / mL in the diluted HBs antigen-HRP solution. 100 ⁇ L of the diluted solution was added to each antibody-immobilized well and incubated at 37 ° C. for 1 hour. (4) The solution obtained in (3) was washed 3 times with 300 ⁇ L of PBST. 100 ⁇ L of TMB substrate solution was added to the wells after washing and allowed to react at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
  • Example 13 Construction of detection system for AFP-L3 using mouse IgG F (ab ′) 2 having an N-type sugar chain binding consensus sequence as a solid phase This experiment was conducted using mouse IgG F ( This was done to prove whether ab ′) 2 can capture AFP-L3 on the solid phase.
  • Mouse IgG F (ab ′) 2 fragment antibodies A and B having an N-type sugar chain binding consensus sequence and mouse IgG F (ab ′) 2 fragment antibodies C and D having no N-type sugar chain binding consensus sequence Then, AFP-L3 and AFP-L1 were reacted with these immobilized antibodies in the presence of LCA to examine whether mouse IgG F (ab ′) 2 can specifically capture AFP-L3.
  • the examination was conducted as follows.
  • (1) The purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D prepared in Example 1 were diluted with PBS so as to be 10 ⁇ g / mL.
  • 50 ⁇ L of the diluted solution was incubated at 37 ° C. for 1 hour to be immobilized.
  • (2) The antibody-immobilized well was washed with 250 ⁇ L of PBST three times, and blocked with 150 ⁇ L of 10% blocking reagent at 37 ° C. for 24 hours.
  • the antibody-immobilized well was washed with 250 ⁇ L of PBST three times, and the AFP-L3 solution and the AFP-L1 solution, whose concentrations were converted from the measured values of Mutus Wako AFP-L3, were each 5 ⁇ g / mL with a 10% blocking reagent.
  • the solution was further diluted by adding LCA to 100 ⁇ g / mL and anti-AFP F (ab ′) 2 -HRP to 1 ⁇ g / mL.
  • 50 ⁇ L of the prepared solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
  • the antibody-immobilized well was washed 3 times with 250 ⁇ L of PBST. Next, 50 ⁇ L of TMB substrate solution was added and reacted at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
  • LCA was able to specifically detect AFP-L3 under conditions in which LCA-reactive antibody fragment A or B was used as a solid phase (FIG. 21).
  • a lectin target molecule can be captured by using a solid-phased lectin-reactive sugar chain-containing entity together with a lectin.
  • Example 14 Construction of hemopexin (HPX) -linked sugar chain and transferrin (TF) -linked sugar chain detection system using mouse IgG-HRP having an N-type sugar chain-binding consensus sequence in the variable region. Whether mouse IgG having a chain-binding consensus sequence can be reacted with AAL in the presence of AAL to detect HPX-bound AAL-reactive fucosylated glycans and TF-bound AAL-reactive fucosylated glycans in liver cancer patient serum investigated.
  • HPX hemopexin
  • TF transferrin
  • Example 2 Mouse IgG having an N-type sugar chain binding consensus sequence in the amino acid sequence of the variable region was labeled with HRP in the same manner as in Example 1.
  • N-glycosidase F (ROCHE) was added to the anti-HPX antibody whose buffer was exchanged with a tris acid buffer, and reacted at 37 ° C. for 24 hours. Thereafter, ultrafiltration at 100 KDa was performed to remove N glycosidase.
  • a lectin target molecule can be captured by using a solid-phased lectin-reactive sugar chain-containing entity together with a lectin.

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Abstract

The present invention provides an improved method for measuring lectin target molecules (such as AFP-L3, PSA, hemopexin and transferrin) using lectin. More specifically, the present invention provides: a method for capturing a lectin target molecule, including causing lectin to bind to the lectin target molecule in the presence of a lectin-reactive sugar-chain containing entity; a complex including a lectin target molecule, lectin and a lectin-reactive sugar-chain containing entity; a reagent for capturing a lectin target molecule, including lectin and a lectin-reactive sugar-chain containing entity in a mixed form; and a kit for capturing a lectin target molecule, including lectin and a lectin-reactive sugar-chain containing entity.

Description

レクチン標的分子の捕捉方法Method for capturing lectin target molecules
　本発明は、レクチン標的分子の捕捉方法、レクチン標的分子を含む複合体、レクチン標的分子の捕捉用試薬およびキットなどに関する。 The present invention relates to a method for capturing a lectin target molecule, a complex containing the lectin target molecule, a reagent for capturing the lectin target molecule, a kit, and the like.
　α－フェトプロテイン（ＡＦＰ）は、レンズマメアグルチニン（Ｌｅｎｓ　ｃｕｌｉｎａｒｉｓ　ａｇｇｌｕｔｉｎｉｎ：ＬＣＡ）との結合性によって非結合画分（Ｌ１）、弱結合性画分（Ｌ２）、結合性画分（Ｌ３）の３画分に分類される。このうちＡＦＰ－Ｌ３は、肝癌マーカーとして利用されている。肝癌の診断のためＡＦＰ－Ｌ３の優れた測定法の開発が求められている。 α-fetoprotein (AFP) is divided into three fractions, a non-binding fraction (L1), a weak binding fraction (L2), and a binding fraction (L3), depending on the binding properties with lentil bean glutinin (Lens). Classified into minutes. Of these, AFP-L3 is used as a liver cancer marker. Development of an excellent measurement method for AFP-L3 is required for the diagnosis of liver cancer.
　また、前立腺特異抗原（Ｐｒｏｓｔａｔｅ－ｓｐｅｃｉｆｉｃ　ａｎｔｉｇｅｎ：ＰＳＡ）は、前立腺癌マーカーとして利用されている。しかし、前立腺肥大や前立腺炎においてもＰＳＡ測定値が上昇するため、特異性が低いことが課題となっている。近年、前立腺癌においてＰＳＡの糖鎖修飾変化が報告されており、この糖鎖修飾の変化を捉えることで癌診断の精度を向上させることが期待されている。 Prostate-specific antigen (PSA) is also used as a prostate cancer marker. However, since the PSA measurement value rises even in prostatic hypertrophy and prostatitis, low specificity is a problem. In recent years, changes in the sugar chain modification of PSA have been reported in prostate cancer, and it is expected to improve the accuracy of cancer diagnosis by capturing this change in sugar chain modification.
　特許文献１は、レクチンを用いたフコシル化ＡＦＰの測定法を開示している。
　特許文献２は、レクチンを用いたβ－Ｎ－アセチルガラクトサミン残基を含有する糖鎖をもつＰＳＡの測定方法を開示している。
　非特許文献１は、レクチンを用いたＡＦＰ－Ｌ３の測定法を開示している。
　非特許文献２は、フコシル化ＰＳＡ量を測定することで前立腺癌の悪性度を判定できることを開示している。 Patent Document 1 discloses a method for measuring fucosylated AFP using a lectin.
Patent Document 2 discloses a method for measuring PSA having a sugar chain containing a β-N-acetylgalactosamine residue using a lectin.
Non-Patent Document 1 discloses a method for measuring AFP-L3 using a lectin.
Non-Patent Document 2 discloses that the malignancy of prostate cancer can be determined by measuring the amount of fucosylated PSA.
国際公開第９９／３９２０９号International Publication No. 99/39209 国際公開第２０１０／９０２６４号International Publication No. 2010/90264
　本発明の目的は、レクチンを用いたレクチン標的分子（例、ＡＦＰ－Ｌ３およびＰＳＡ）の測定を改善することである。 An object of the present invention is to improve the measurement of lectin target molecules (eg, AFP-L3 and PSA) using lectins.
　本発明者らは、鋭意検討した結果、レクチン反応性糖鎖含有実体の存在下においてレクチンを用いると、レクチンのレクチン標的分子への結合が増強することなどを見出した。本知見は、レクチンによるレクチン標的分子の測定をはじめとする、レクチン標的分子の捕捉方法全般に応用することができる。本発明者らは、かかる知見に基づき、本発明を完成するに至った。上記特許文献および非特許文献は、レクチンとレクチン標的分子（ＡＦＰ－Ｌ３およびＰＳＡ）との結合を増強する方法を記載も示唆もしていない。 As a result of intensive studies, the present inventors have found that, when a lectin is used in the presence of a lectin-reactive sugar chain-containing entity, the binding of the lectin to the lectin target molecule is enhanced. This knowledge can be applied to all methods of capturing lectin target molecules, including measurement of lectin target molecules using lectins. Based on this finding, the present inventors have completed the present invention. The above-mentioned patent documents and non-patent documents do not describe or suggest a method for enhancing the binding between a lectin and a lectin target molecule (AFP-L3 and PSA).
　すなわち、本発明は以下のとおりである。
〔１〕レクチン反応性糖鎖含有実体の存在下において、レクチンをレクチン標的分子に結合させることを含む、レクチン標的分子の捕捉方法。
〔２〕レクチンがマメ科レクチンまたはキノコレクチンである、〔１〕の方法。
〔３〕レクチンがフコース特異的レクチンである、〔１〕又は〔２〕の方法。
〔４〕レクチンが、レンズマメアグルチニン（ＬＣＡ）、コンカナバリンＡ（ＣｏｎＡ）またはヒイロチャワンタケレクチン（ＡＡＬ）である、〔１〕～〔３〕のいずれかの方法。
〔５〕レクチン標的分子が糖タンパク質である、〔１〕～〔４〕のいずれかの方法。
〔６〕糖タンパク質がＡＦＰ－Ｌ３またはＰＳＡである、〔５〕の方法。
〔７〕糖タンパク質がヘモペキシン（ＨＰＸ）またはトランスフェリン（ＴＦ）である、〔５〕の方法。
〔８〕前記実体がＮ型糖鎖結合コンセンサス配列を有する糖タンパク質である、〔１〕～〔７〕のいずれかの方法。
〔９〕Ｎ型糖鎖結合コンセンサス配列を有する糖タンパク質がＮ型糖鎖結合コンセンサス配列を可変領域に有する免疫グロブリンである、〔８〕の方法。
〔１０〕前記実体が、Ｂ型肝炎ウイルス表面抗原（ＨＢｓ抗原）、ヒト絨毛性ゴナドトロピンα鎖（ｈＣＧα）または黄体形成ホルモン（ＬＨ）である、〔１〕～〔８〕のいずれかの方法。
〔１１〕レクチンのレクチン標的分子への結合後にレクチン標的分子を測定することを含むレクチン標的分子の測定方法である、〔１〕～〔１０〕のいずれかの方法。
〔１２〕レクチン標的分子に特異的な親和性物質をさらに用いる、〔１１〕の方法。
〔１３〕以下を含む方法である、〔１〕～〔１２〕のいずれかの方法：
（１）レクチン標的分子を、レクチン標的分子に特異的な親和性物質に結合させて、レクチン標的分子およびレクチン標的分子に特異的な親和性物質を含む第１複合体を得ること；
（２）レクチン反応性糖鎖含有実体の存在下においてレクチンを第１複合体と結合させて、レクチン標的分子、レクチン標的分子に特異的な親和性物質、レクチンおよびレクチン反応性糖鎖含有実体を含む第２複合体を得ること；ならびに
（３）第２複合体に含まれるレクチン標的分子の量を測定すること。
〔１４〕レクチン標的分子、レクチンおよびレクチン反応性糖鎖含有実体を含む複合体。
〔１５〕レクチンおよびレクチン反応性糖鎖含有実体を混合形態で含む、レクチン標的分子の捕捉用試薬。
〔１６〕レクチンおよびレクチン反応性糖鎖含有実体を含む、レクチン標的分子の捕捉用キット。 That is, the present invention is as follows.
[1] A method for capturing a lectin target molecule, comprising binding a lectin to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.
[2] The method according to [1], wherein the lectin is a legume lectin or a mushroom collectin.
[3] The method of [1] or [2], wherein the lectin is a fucose-specific lectin.
[4] The method according to any one of [1] to [3], wherein the lectin is lentil agglutinin (LCA), concanavalin A (ConA), or yellow chawantake lectin (AAL).
[5] The method according to any one of [1] to [4], wherein the lectin target molecule is a glycoprotein.
[6] The method of [5], wherein the glycoprotein is AFP-L3 or PSA.
[7] The method of [5], wherein the glycoprotein is hemopexin (HPX) or transferrin (TF).
[8] The method according to any one of [1] to [7], wherein the entity is a glycoprotein having an N-type sugar chain binding consensus sequence.
[9] The method according to [8], wherein the glycoprotein having an N-type sugar chain binding consensus sequence is an immunoglobulin having an N-type sugar chain binding consensus sequence in a variable region.
[10] The method according to any one of [1] to [8], wherein the entity is hepatitis B virus surface antigen (HBs antigen), human chorionic gonadotropin α chain (hCGα) or luteinizing hormone (LH).
[11] The method according to any one of [1] to [10], which is a method for measuring a lectin target molecule, comprising measuring the lectin target molecule after binding of the lectin to the lectin target molecule.
[12] The method of [11], further using an affinity substance specific for the lectin target molecule.
[13] The method according to any one of [1] to [12], which is a method comprising:
(1) binding a lectin target molecule to an affinity substance specific for the lectin target molecule to obtain a first complex including the lectin target molecule and an affinity substance specific for the lectin target molecule;
(2) A lectin is bound to the first complex in the presence of a lectin-reactive sugar chain-containing entity, and a lectin target molecule, an affinity substance specific for the lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity Obtaining a second complex comprising; and (3) measuring the amount of lectin target molecules contained in the second complex.
[14] A complex comprising a lectin target molecule, a lectin, and a lectin-reactive sugar chain-containing entity.
[15] A reagent for capturing a lectin target molecule, comprising a lectin and a lectin-reactive sugar chain-containing entity in a mixed form.
[16] A kit for capturing a lectin target molecule, comprising a lectin and a lectin-reactive sugar chain-containing entity.
　本発明の方法は、レクチン反応性糖鎖含有実体の存在下においてレクチンを使用することにより、レクチンのレクチン標的分子への結合を増強できる。したがって、本発明の方法は、例えば、レクチン標的分子の測定、抽出、濃縮、精製、検出、定量に有用である。より具体的には、レクチン反応性糖鎖含有実体の存在下でレクチンを使用する本発明の方法をレクチン標的分子の測定に利用した場合、レクチン単独の使用に比し、高感度かつ安定的にレクチン標的分子を測定することができる。
　本発明の複合体は、例えば、本発明の捕捉方法の実施に有用である。
　本発明の試薬およびキットは、例えば、本発明の捕捉方法の簡便な実施に有用である。 The method of the present invention can enhance the binding of a lectin to a lectin target molecule by using the lectin in the presence of a lectin-reactive sugar chain-containing entity. Therefore, the method of the present invention is useful, for example, for measurement, extraction, concentration, purification, detection and quantification of lectin target molecules. More specifically, when the method of the present invention using a lectin in the presence of a lectin-reactive sugar chain-containing entity is used for measuring a lectin target molecule, it is more sensitive and stable than using a lectin alone. Lectin target molecules can be measured.
The complex of the present invention is useful, for example, for carrying out the capture method of the present invention.
The reagent and kit of the present invention are useful, for example, for simple implementation of the capture method of the present invention.
図１は、精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤについて、ＳＤＳ－ＰＡＧＥによる分子量、およびレクチンブロットによるＬＣＡ反応性の確認を示す図である。FIG. 1 shows the confirmation of molecular weight by SDS-PAGE and LCA reactivity by lectin blot for purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D. 図２は、精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤについて、ＥＬＩＳＡによるＬＣＡ反応性の確認を示す図である。FIG. 2 is a diagram showing confirmation of LCA reactivity by ELISA for purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D. 図３は、ＬＣＡ反応性Ｆ（ａｂ’）２フラグメント抗体Ａの存在下におけるＬＣＡによるＡＦＰ－Ｌ３の特異的な検出を示す図である。FIG. 3 shows specific detection of AFP-L3 by LCA in the presence of LCA-reactive F (ab ′) 2 fragment antibody A. 図４は、ＬＣＡ反応性Ｆ（ａｂ’）２フラグメント抗体Ｂの存在下におけるＬＣＡによるＡＦＰ－Ｌ３の特異的な検出を示す図である。FIG. 4 shows specific detection of AFP-L3 by LCA in the presence of LCA-reactive F (ab ′) 2 fragment antibody B. 図５は、ＬＣＡ非反応性Ｆ（ａｂ’）２フラグメント抗体Ｃの存在下におけるＬＣＡによるＡＦＰ－Ｌ３およびＡＦＰ－Ｌ１の検出の結果を示す図である。FIG. 5 shows the results of detection of AFP-L3 and AFP-L1 by LCA in the presence of LCA non-reactive F (ab ′) 2 fragment antibody C. 図６は、ＬＣＡ非反応性Ｆ（ａｂ’）２フラグメント抗体Ｄの存在下におけるＬＣＡによるＡＦＰ－Ｌ３およびＡＦＰ－Ｌ１の検出の結果を示す図である。FIG. 6 shows the results of detection of AFP-L3 and AFP-L1 by LCA in the presence of LCA non-reactive F (ab ′) 2 fragment antibody D. 図７は、糖鎖除去マウスＩｇＧ　Ｆ（ａｂ’）２フラグメントについて、ＳＤＳ－ＰＡＧＥによる分子量、およびレクチンブロットによるＬＣＡ反応性の確認を示す図である。FIG. 7 is a view showing the molecular weight by SDS-PAGE and confirmation of LCA reactivity by lectin blot for a sugar chain-depleted mouse IgG F (ab ′) 2 fragment. 図８は、糖鎖含有マウスＩｇＧ　Ｆ（ａｂ’）２フラグメントおよび糖鎖除去マウスＩｇＧ　Ｆ（ａｂ’）２フラグメントについて、ＥＬＩＳＡによるＬＣＡ反応性の確認を示す図である。Figure 8 is a sugar chain-containing mouse IgG F (ab ') 2 fragments and deglycosylation mouse IgG F (ab') for 2 fragments, a diagram illustrating a confirmation of LCA reactive by ELISA. 図９は、ＬＣＡ反応性を示す糖鎖含有マウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰ、またはＬＣＡ反応性を示さない糖鎖除去マウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰの存在下におけるＬＣＡによるＡＦＰ－Ｌ３およびＡＦＰ－Ｌ１の検出結果を示す図である。9, AFP by LCA in the presence of a sugar chain-containing mouse IgG F (ab ') 2 -HRP or deglycosylation mouse IgG F showing no LCA reactivity (ab,') 2 -HRP indicating the LCA reactivity FIG. 10 is a diagram showing detection results of -L3 and AFP-L1. 図１０は、Ｎ型糖鎖結合コンセンサス配列を有する抗体の存在下におけるＬＣＡによるＡＦＰ－Ｌ３およびＡＦＰ－Ｌ１の検出結果を示す図である。抗ＴＮＦα抗体（Ｐ）：Ｎ型糖鎖結合コンセンサス配列を可変領域に有する抗ＴＮＦα抗体；抗ＴＮＦα抗体（Ｎ）：Ｎ型糖鎖結合コンセンサス配列を可変領域に有しない抗ＴＮＦα抗体；抗ＩＬ－１０抗体（Ｐ’）：Ｎ型糖鎖結合コンセンサス配列を可変領域に有する抗ＩＬ－１０抗体；抗ＩＬ－１０抗体（Ｎ’）：Ｎ型糖鎖結合コンセンサス配列を可変領域に有しない抗ＩＬ－１０抗体。FIG. 10 is a view showing the detection results of AFP-L3 and AFP-L1 by LCA in the presence of an antibody having an N-type sugar chain binding consensus sequence. Anti-TNFα antibody (P): anti-TNFα antibody having an N-type sugar chain binding consensus sequence in the variable region; anti-TNFα antibody (N): anti-TNFα antibody having no N-type sugar chain-binding consensus sequence in the variable region; anti-IL- 10 antibody (P ′): anti-IL-10 antibody having N-type sugar chain binding consensus sequence in variable region; anti-IL-10 antibody (N ′): anti-IL having no N-type sugar chain binding consensus sequence in variable region -10 antibody. 図１１は、レクチン反応性抗原（ＨＢｓ抗原）の存在下におけるＬＣＡによるＡＦＰ－Ｌ３の特異的な検出を示す図である。FIG. 11 shows specific detection of AFP-L3 by LCA in the presence of a lectin-reactive antigen (HBs antigen). 図１２は、糖鎖除去ＨＢｓ抗原について、ＳＤＳ－ＰＡＧＥによる分子量、およびレクチンブロットによるＬＣＡ反応性の確認を示す図である。FIG. 12 is a diagram showing the confirmation of the molecular weight by SDS-PAGE and LCA reactivity by lectin blot for the sugar chain-removed HBs antigen. 図１３は、糖鎖含有ＨＢｓ抗原および糖鎖除去ＨＢｓ抗原について、ＥＬＩＳＡによるＬＣＡ反応性の確認を示す図である。FIG. 13 is a diagram showing confirmation of LCA reactivity by ELISA for sugar chain-containing HBs antigen and sugar chain-removed HBs antigen. 図１４は、ＬＣＡ反応性を示す糖鎖含有ＨＢｓ抗原－ＨＲＰ、またはＬＣＡ反応性を示さない糖鎖除去ＨＢｓ抗原－ＨＲＰの存在下におけるＬＣＡによるＡＦＰ－Ｌ３およびＡＦＰ－Ｌ１の検出結果を示す図である。FIG. 14 is a diagram showing the detection results of AFP-L3 and AFP-L1 by LCA in the presence of sugar chain-containing HBs antigen-HRP showing LCA reactivity or sugar chain-removed HBs antigen-HRP not showing LCA reactivity. It is. 図１５は、Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰの存在下におけるヒイロチャワンタケレクチン（Ａｌｅｕｒｉａ　ａｕｒａｎｔｉａ　Ｌｅｃｔｉｎ：ＡＡＬ）によるＡＦＰ結合糖鎖の検出を示す図である。FIG. 15 shows the detection of AFP-binding glycans by Aurolia aurantia lectin (AAL) in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type glycan-binding consensus sequence. FIG. 図１６は、Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰの存在下におけるコンカナバリンＡ（ＣｏｎＡ）によるＡＦＰ結合糖鎖の検出を示す図である。FIG. 16 is a diagram showing detection of an AFP-linked sugar chain by concanavalin A (ConA) in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain-binding consensus sequence. 図１７は、Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰの存在下におけるＡＡＬによるＰＳＡ結合糖鎖の検出を示す図である。FIG. 17 is a diagram showing detection of PSA-binding sugar chains by AAL in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain-binding consensus sequence. 図１８は、Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰの存在下におけるＬＣＡによるＡＦＰ－Ｌ３の特異的な検出を示す図である。同時添加条件：ＡＦＰ捕捉ウェルにおいてＬＣＡおよびマウスＩｇＧ　Ｆ（ａｂ’）２抗体Ａ－ＨＲＰを含む溶液をインキュベートした；洗浄条件：ＡＦＰ捕捉ウェルにおいてＬＣＡを含む溶液をインキュベートした後に洗浄して未反応ＬＣＡを除去した。次いで、未反応ＬＣＡが除去されたＡＦＰ捕捉ウェルに、マウスＩｇＧ　Ｆ（ａｂ’）２抗体Ａ－ＨＲＰを添加した後にインキュベートした。FIG. 18 is a diagram showing specific detection of AFP-L3 by LCA in the presence of mouse IgG F (ab ′) 2 -HRP having an N-type sugar chain binding consensus sequence. Co-addition conditions: A solution containing LCA and mouse IgG F (ab ′) 2 antibody A-HRP was incubated in AFP capture wells; Wash conditions: Washed and unreacted LCA after incubating a solution containing LCA in AFP capture wells Was removed. Subsequently, mouse IgG F (ab ′) 2 antibody A-HRP was added to the AFP capture well from which unreacted LCA was removed, and then incubated. 図１９は、ＨＢｓ抗原－ＨＲＰの存在下におけるＬＣＡによるＡＦＰ－Ｌ３の特異的な検出を示す図である。同時添加条件：ＡＦＰ捕捉ウェルにおいてＬＣＡおよびＨＢｓ抗原－ＨＲＰを含む溶液をインキュベートした；洗浄条件：ＡＦＰ捕捉ウェルにおいてＬＣＡを含む溶液をインキュベートした後に洗浄して、ＡＦＰ捕捉ウェルから未反応ＬＣＡを除去した。次いで、未反応ＬＣＡが除去されたＡＦＰ捕捉ウェルにおいて、ＨＢｓ抗原－ＨＲＰを含む溶液を添加した後にインキュベートした。FIG. 19 shows specific detection of AFP-L3 by LCA in the presence of HBs antigen-HRP. Co-addition conditions: Incubate solution containing LCA and HBs antigen-HRP in AFP capture wells; Wash conditions: Incubate solution containing LCA in AFP capture wells and wash to remove unreacted LCA from AFP capture wells . Then, in the AFP capture well from which unreacted LCA was removed, incubation was carried out after adding a solution containing HBs antigen-HRP. 図２０は、ＨＢｓ抗原－ＨＲＰの存在下におけるＡＡＬによるＡＦＰ－Ｌ３の特異的な検出を示す図である。同時添加条件：ＡＦＰ捕捉ウェルにおいてＡＡＬおよびＨＢｓ抗原－ＨＲＰを含む溶液をインキュベートした；洗浄条件：ＡＦＰ捕捉ウェルにおいてＡＡＬを含む溶液をインキュベートした後に洗浄して、ＡＦＰ捕捉ウェルから未反応ＡＡＬを除去した。次いで、未反応ＡＡＬが除去されたＡＦＰ捕捉ウェルに、ＨＢｓ抗原－ＨＲＰを添加した後にインキュベートした。FIG. 20 shows specific detection of AFP-L3 by AAL in the presence of HBs antigen-HRP. Co-addition conditions: AAL and HBs antigen-HRP containing solutions were incubated in AFP capture wells; Wash conditions: AAL containing solutions were incubated in AFP capture wells and then washed to remove unreacted AAL from AFP capture wells . The AFP capture well from which unreacted AAL was removed was then incubated after adding HBs antigen-HRP. 図２１は、ＬＣＡ反応性抗体フラグメントＡまたはＢを固相とした条件下におけるＬＣＡによるＡＦＰ－Ｌ３の特異的な検出を示す図である。（＋）：ＬＣＡ反応性；（－）：ＬＣＡ非反応性FIG. 21 is a diagram showing specific detection of AFP-L3 by LCA under conditions using LCA-reactive antibody fragment A or B as a solid phase. (+): LCA reactivity; (-): LCA non-reactive 図２２は、ＡＡＬの存在下におけるＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰによる肝臓癌患者血清中のヘモペキシン（ＨＰＸ）結合ＡＡＬ反応性フコシル化糖鎖の検出を示す図である。１％ＢＳＡ－ＰＢＳ：対照；ＨＣＣ－１、ＨＣＣ－２：肝臓癌患者血清２例；ＮＨＳ－１、ＮＨＳ－２：健常人血清２例。FIG. 22 shows the detection of hemopexin (HPX) -bound AAL-reactive fucosylated glycans in liver cancer patient serum by mouse IgG F (ab ′) 2 -HRP having an N-type glycan-binding consensus sequence in the presence of AAL. FIG. 1% BSA-PBS: control; HCC-1, HCC-2: liver cancer patient sera 2 cases; NHS-1, NHS-2: healthy subject sera 2 cases. 図２３は、ＡＡＬの存在下におけるＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰによる肝臓癌患者血清中のトランスフェリン（ＴＦ）結合ＡＡＬ反応性フコシル化糖鎖の検出を示す図である。１％ＢＳＡ－ＰＢＳ：対照；ＨＣＣ－１、ＨＣＣ－２：肝臓癌患者血清２例；ＮＨＳ－１、ＮＨＳ－２：健常人血清２例。FIG. 23 shows detection of transferrin (TF) -bound AAL-reactive fucosylated sugar chain in liver cancer patient serum by mouse IgG F (ab ′) 2 -HRP having N-type sugar chain binding consensus sequence in the presence of AAL. FIG. 1% BSA-PBS: control; HCC-1, HCC-2: liver cancer patient sera 2 cases; NHS-1, NHS-2: healthy subject sera 2 cases.
　本発明は、レクチン反応性糖鎖含有実体の存在下において、レクチンをレクチン標的分子に結合させることを含む、レクチン標的分子の捕捉方法を提供する。 The present invention provides a method for capturing a lectin target molecule, which comprises binding a lectin to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.
　レクチンは、特定の糖鎖構造を認識して結合活性を示すタンパク質である。レクチンとしては、例えば、動物（例、脊椎動物、無脊椎動物）、植物（例、マメ科、イネ科）、菌類（例、キノコ、麹）由来のものが知られている。マメ科レクチンとしては、例えば、ナタマメレクチン（Ｃｏｎｃａｎａｖａｌｉｎ　Ａ：ＣｏｎＡ）、レンズマメアグルチニン（Ｌｅｎｓ　ｃｕｌｉｎａｒｉｓ　ａｇｇｌｕｔｉｎｉｎ：ＬＣＡ）、エンドウマメレクチン（Ｐｉｓｕｍ　ｓａｔｉｖｕｍ）などが挙げられる。キノコレクチンとしては、例えば、ヒイロチャワンタケレクチン（Ａｌｅｕｒｉａ　ａｕｒａｎｔｉａ　Ｌｅｃｔｉｎ：ＡＡＬ）などが挙げられる。 A lectin is a protein that recognizes a specific sugar chain structure and exhibits binding activity. As lectins, for example, those derived from animals (eg, vertebrates, invertebrates), plants (eg, legumes, gramineae), fungi (eg, mushrooms, moths) are known. Examples of legume lectins include pea lectin (Concanavalin A: ConA), lentil agglutinin (Lens culinaris agglutinin: LCA), pea lectin (Pisum sativum), and the like. Examples of the mushroom collectin include herochawantake lectin (Aurelia aurantia lectin: AAL).
　本発明で用いられるレクチンはまた、レクチン標的分子の糖鎖構造に応じて適宜選択することができる。本発明で用いられるレクチンとしては、例えば、フコースに対する親和性を有するフコース特異的レクチン、ガラクトースに対する親和性を有するガレクチン、シアル酸反応性レクチンなどが挙げられるが、フコース特異的レクチンが好ましい。フコース特異的レクチンとしては、例えば、Ｎ型糖鎖の根本のＧｌｃＮＡｃにフコースがα１，６付加した糖鎖構造（コアフコース構造）を認識するレクチン〔例、ＬＣＡ、エンドウマメレクチン（Ｐｉｓｕｍ　ｓａｔｉｖｕｍ：ＰＳＡ）〕、並びにＮ型糖鎖、Ｏ型糖鎖及び糖脂質糖鎖のＧｌｃＮＡｃにフコースをα１，２、α１，３、α１，４、α１，６のいずれかで付加した糖鎖構造を認識するレクチン〔例、ＡＡＬ、麹菌レクチン（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ　Ｌｅｃｔｉｎ：ＡＯＬ）〕が挙げられる。 The lectin used in the present invention can also be appropriately selected according to the sugar chain structure of the lectin target molecule. Examples of the lectin used in the present invention include a fucose-specific lectin having an affinity for fucose, a galectin having an affinity for galactose, a sialic acid-reactive lectin, and the like, but a fucose-specific lectin is preferable. As the fucose-specific lectin, for example, a lectin that recognizes a sugar chain structure (core fucose structure) in which fucose is α1,6 added to GlcNAc that is the root of an N-type sugar chain (eg, LCA, pea sactivum (PSA)). And a lectin that recognizes a sugar chain structure in which fucose is added at any of α1, 2, α1, 3, α1, 4, α1, 6 to GlcNAc of N-type sugar chain, O-type sugar chain and glycolipid sugar chain [Example, AAL, Aspergillus oryzae Lectin: AOL].
　レクチン標的分子とは、レクチンと結合する能力を有する、本発明において捕捉されるべき標的分子をいう。標的分子は、レクチンが結合する特定の糖鎖からなる、又は当該特定の糖鎖を含有する分子である。 A lectin target molecule refers to a target molecule that has the ability to bind to a lectin and is to be captured in the present invention. The target molecule is a molecule composed of or containing a specific sugar chain to which a lectin binds.
　好ましくは、レクチン標的分子は、糖タンパク質である。レクチン標的分子としての糖タンパク質としては、例えば、哺乳動物、鳥類、爬虫類、両生類、魚類、植物、昆虫、微生物、またはウイルス由来の糖タンパク質が挙げられる。レクチン標的分子としての糖タンパク質は、測定、抽出、濃縮または精製が所望される糖タンパク質であってもよい。具体的には、糖タンパク質は、Ｎ型糖鎖結合コンセンサス配列を有するタンパク質である。Ｎ型糖鎖結合コンセンサス配列は、Ａｓｎ－Ｘ－ＳｅｒまたはＡｓｎ－Ｘ－Ｔｈｒのアミノ酸残基（Ｘはプロリン以外のアミノ酸残基）から構成されるアミノ酸配列である。Ｎ型糖鎖は、Ｎ型糖鎖結合コンセンサス配列中のアスパラギン（Ａｓｎ）残基に結合することが知られている。より具体的には、レクチン標的分子としては、例えば、ＡＦＰ－Ｌ３、ＰＳＡ、ヘモペキシン（ＨＰＸ）、およびトランスフェリン（ＴＦ）が挙げられるが、これらに限定されるものではない。ＰＳＡは、Ｎ型糖鎖等の糖鎖の修飾タンパク質として知られている。 Preferably, the lectin target molecule is a glycoprotein. Examples of the glycoprotein as a lectin target molecule include glycoproteins derived from mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms, or viruses. The glycoprotein as a lectin target molecule may be a glycoprotein for which measurement, extraction, concentration or purification is desired. Specifically, a glycoprotein is a protein having an N-type sugar chain binding consensus sequence. The N-type sugar chain-binding consensus sequence is an amino acid sequence composed of amino acid residues of Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline). N-type sugar chains are known to bind to asparagine (Asn) residues in N-type sugar chain binding consensus sequences. More specifically, examples of the lectin target molecule include, but are not limited to, AFP-L3, PSA, hemopexin (HPX), and transferrin (TF). PSA is known as a modified protein of a sugar chain such as an N-type sugar chain.
　レクチン反応性糖鎖含有実体とは、レクチン反応性糖鎖を含有する分子、またはレクチン反応性糖鎖が付加されている固相をいう。レクチン反応性糖鎖含有実体は、本発明の方法で捕捉されるべきレクチン標的分子とは異なる。レクチン反応性糖鎖含有実体は、その糖鎖がレクチンと反応するのであって、レクチンまたはレクチン標的分子を抗原とする抗体ではない。レクチン反応性糖鎖を含有する分子としては、例えば、生体分子、および人工合成物（例、人工合成高分子等の人工合成分子）が挙げられる。固相としては、例えば、粒子（例、磁性粒子）；メンブレン（例、ニトロセルロース膜、ろ紙）、カラム等の支持体；並びにプレート（例、マルチウェルプレート）、チューブ等の容器が挙げられる。固相の材料としては、例えば、ガラス、プラスチック、多糖体のマトリックス、および金属が挙げられる。レクチン反応性糖鎖含有実体は、用いられるレクチンの種類に応じて適宜選択することができる。例えば、このような選択は、レクチンとの糖鎖または糖鎖含有実体の結合能力を評価することにより行うことができる（例、実施例２を参照）。 The lectin-reactive sugar chain-containing entity means a molecule containing a lectin-reactive sugar chain or a solid phase to which a lectin-reactive sugar chain is added. The lectin-reactive sugar chain-containing entity is different from the lectin target molecule to be captured by the method of the present invention. The lectin-reactive sugar chain-containing entity is not an antibody whose glycin reacts with lectin but has lectin or a lectin target molecule as an antigen. Examples of the molecule containing a lectin-reactive sugar chain include biomolecules and artificial synthetic products (eg, artificial synthetic molecules such as artificial synthetic polymers). Examples of the solid phase include particles (eg, magnetic particles); membranes (eg, nitrocellulose membrane, filter paper), supports such as columns; and containers such as plates (eg, multiwell plates) and tubes. Examples of the solid phase material include glass, plastic, polysaccharide matrix, and metal. The lectin-reactive sugar chain-containing entity can be appropriately selected according to the type of lectin used. For example, such a selection can be performed by evaluating the binding ability of a sugar chain or a sugar chain-containing entity to a lectin (see Example 2, for example).
　好ましくは、レクチン反応性糖鎖含有実体は、レクチン反応性糖鎖を含有する生体分子である。生体分子としては、例えば、ポリペプチドおよび核酸が挙げられる。より好ましくは、レクチン反応性糖鎖含有実体は、糖タンパク質である。レクチン反応性糖鎖含有実体としての糖タンパク質としては、例えば、哺乳動物、鳥類、爬虫類、両生類、魚類、植物、昆虫、微生物、またはウイルス由来の糖タンパク質が挙げられる。レクチン反応性糖鎖含有実体としての糖タンパク質としてはまた、例えば、免疫グロブリン（例、抗体）、分泌型タンパク質、膜結合型タンパク質、ホルモン、サイトカイン、ケモカイン、ウイルス由来抗原、酵素、細胞外マトリックス、細胞外小胞膜タンパク質、腫瘍特異抗原、及びそれらの部分ペプチドが挙げられる。 Preferably, the lectin-reactive sugar chain-containing entity is a biomolecule containing a lectin-reactive sugar chain. Examples of biomolecules include polypeptides and nucleic acids. More preferably, the lectin-reactive sugar chain-containing entity is a glycoprotein. Examples of glycoproteins as lectin-reactive sugar chain-containing entities include glycoproteins derived from mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms, or viruses. Examples of glycoproteins as lectin-reactive sugar chain-containing entities include, for example, immunoglobulins (eg, antibodies), secretory proteins, membrane-bound proteins, hormones, cytokines, chemokines, virus-derived antigens, enzymes, extracellular matrices, Examples include extracellular vesicle membrane proteins, tumor-specific antigens, and partial peptides thereof.
　好ましくは、レクチン反応性糖鎖含有実体としての糖タンパク質は、Ｎ型糖鎖結合コンセンサス配列を有するタンパク質である。Ｎ型糖鎖結合コンセンサス配列は、Ａｓｎ－Ｘ－ＳｅｒまたはＡｓｎ－Ｘ－Ｔｈｒのアミノ酸残基（Ｘはプロリン以外のアミノ酸残基）から構成されるアミノ酸配列である。Ｎ型糖鎖は、Ｎ型糖鎖結合コンセンサス配列中のアスパラギン（Ａｓｎ）残基に結合することが知られている。より具体的には、Ｎ型糖鎖結合コンセンサス配列を有するタンパク質としては、例えば、Ｂ型肝炎ウイルス表面抗原、ヒト絨毛性ゴナドトロピンα鎖、黄体形成ホルモン、ＰＳＡ抗原、ＨＥ４抗原、ＭＵＣ１抗原、サイログロブリン、ならびにＮ型糖鎖結合コンセンサス配列を可変領域に有する抗体が挙げられる。 Preferably, the glycoprotein as the lectin-reactive sugar chain-containing entity is a protein having an N-type sugar chain binding consensus sequence. The N-type sugar chain-binding consensus sequence is an amino acid sequence composed of amino acid residues of Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline). N-type sugar chains are known to bind to asparagine (Asn) residues in N-type sugar chain binding consensus sequences. More specifically, examples of the protein having an N-type sugar chain binding consensus sequence include hepatitis B virus surface antigen, human chorionic gonadotropin α chain, luteinizing hormone, PSA antigen, HE4 antigen, MUC1 antigen, thyroglobulin, And antibodies having an N-type sugar chain-binding consensus sequence in the variable region.
　より好ましくは、レクチン反応性糖鎖含有実体としての糖タンパク質は、Ｎ型糖鎖結合コンセンサス配列を可変領域に有する抗体である。Ｎ型糖鎖結合コンセンサス配列を可変領域に有する抗体としては、例えば、天然の全長抗体および改変抗体が挙げられる。改変抗体としては、例えば、可変領域を有する抗体フラグメント（例、Ｆａｂ、Ｆ（ａｂ’）２）、および単鎖抗体が挙げられる。また、Ｎ型糖鎖結合コンセンサス配列を可変領域に有する抗体としては、例えば、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、およびＩｇＹが挙げられる。Ｎ型糖鎖結合コンセンサス配列を可変領域に有する抗体は、ポリクローナル抗体またはモノクローナル抗体であってもよいが、好ましくはモノクローナル抗体である。 More preferably, the glycoprotein as the lectin-reactive sugar chain-containing entity is an antibody having an N-type sugar chain binding consensus sequence in the variable region. Examples of antibodies having an N-type sugar chain binding consensus sequence in the variable region include natural full-length antibodies and modified antibodies. Examples of the modified antibody include an antibody fragment having a variable region (eg, Fab, F (ab ′) 2 ), and a single chain antibody. Examples of antibodies having an N-type sugar chain binding consensus sequence in the variable region include IgG, IgM, IgA, IgD, IgE, and IgY. The antibody having an N-type sugar chain binding consensus sequence in the variable region may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.
　本発明の方法は、レクチン反応性糖鎖含有実体の存在下において、レクチンがレクチン標的分子に結合される。換言すれば、本発明の方法は、レクチン標的分子、ならびにレクチンおよびレクチン反応性糖鎖含有実体という少なくとも三成分を含む系において行われ、レクチン標的分子の捕捉手段としてレクチンおよびレクチン反応性糖鎖含有実体が併用される。レクチン反応性糖鎖含有実体の存在下においてレクチンを使用することにより、レクチンのレクチン標的分子への結合を増強できる。したがって、本発明の方法は、例えば、レクチン標的分子の測定、抽出、濃縮、精製、定量、検出に有用である。 In the method of the present invention, a lectin is bound to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity. In other words, the method of the present invention is carried out in a system comprising at least three components of a lectin target molecule and a lectin and lectin-reactive sugar chain-containing entity, and contains a lectin and a lectin-reactive sugar chain as means for capturing the lectin target molecule. The entity is used together. By using a lectin in the presence of a lectin-reactive sugar chain-containing entity, the binding of the lectin to the lectin target molecule can be enhanced. Therefore, the method of the present invention is useful, for example, for measurement, extraction, concentration, purification, quantification, and detection of lectin target molecules.
　レクチン標的分子は、液体サンプルに含まれるものが使用される。換言すれば、本発明の方法は、レクチン標的分子を含む液体サンプルに、レクチンおよびレクチン反応性糖鎖含有実体を加えた系において行われる。液体サンプルの由来は特に限定されず、生物由来の生物学的サンプルであってもよく、または環境サンプルなどであってもよい。生物学的サンプルが由来する生物としては、例えば、哺乳動物（例、ヒト、サル、マウス、ラット、ウサギ、ウシ、ブタ、ウマ、ヤギ、ヒツジ）、鳥類（例、ニワトリ）等の動物、昆虫、微生物、植物、菌類、魚類が挙げられるが、好ましくは哺乳動物、菌類、魚類であり、より好ましくは哺乳動物であり、さらにより好ましくはヒトである。生物学的サンプルはまた、血液自体または血液に由来するサンプルである血液関連サンプル（例、全血、血清、血漿）、唾液、尿、乳汁、組織または細胞抽出液、あるいはこれらの混合物であってもよい。環境サンプルとしては、土壌、海水、淡水由来のサンプルが挙げられる。液体サンプルは、予備処理に付されたものを用いてもよい。このような予備処理としては、例えば、遠心分離、分画、抽出、ろ過、沈殿、加熱、凍結、冷蔵、及び攪拌が挙げられる。 As the lectin target molecule, one contained in a liquid sample is used. In other words, the method of the present invention is performed in a system in which a lectin and a lectin-reactive sugar chain-containing entity are added to a liquid sample containing a lectin target molecule. The origin of the liquid sample is not particularly limited, and may be a biological sample derived from a living organism or an environmental sample. Examples of organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens), insects, and the like. , Microorganisms, plants, fungi, and fish, preferably mammals, fungi, and fish, more preferably mammals, and even more preferably humans. A biological sample can also be a blood-related sample (eg, whole blood, serum, plasma), saliva, urine, milk, tissue or cell extract, or a mixture thereof, which is the blood itself or a sample derived from blood. Also good. Examples of environmental samples include samples derived from soil, seawater, and fresh water. A liquid sample subjected to pretreatment may be used. Examples of such pretreatment include centrifugation, fractionation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.
　本発明の方法において用いられるレクチンの量は、レクチン反応性糖鎖含有実体の存在下で標的レクチンを捕捉できる量である限り特に限定されないが、例えば、０．１μｇ／ｍＬ～１００ｍｇ／ｍＬである。レクチンの量は、好ましくは１μｇ／ｍＬ以上、より好ましくは５μｇ／ｍＬ以上、さらにより好ましくは１０μｇ／ｍＬ以上である。レクチンの量はまた、好ましくは２０ｍｇ／ｍＬ以下、より好ましくは１０ｍｇ／ｍＬ以下、さらにより好ましくは２ｍｇ／ｍＬ以下である。このような量のレクチンが、液体サンプルに添加されてもよい。 The amount of the lectin used in the method of the present invention is not particularly limited as long as it is an amount capable of capturing the target lectin in the presence of the lectin-reactive sugar chain-containing entity, and is, for example, 0.1 μg / mL to 100 mg / mL. . The amount of lectin is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 10 μg / mL or more. The amount of lectin is also preferably 20 mg / mL or less, more preferably 10 mg / mL or less, and even more preferably 2 mg / mL or less. Such an amount of lectin may be added to the liquid sample.
　本発明の方法において用いられるレクチン反応性糖鎖含有実体の量は、レクチンによるレクチン標的分子の捕捉を支援できる量である限り特に限定されないが、例えば、０．００１μｇ／ｍＬ～１００ｍｇ／ｍＬである。レクチン反応性糖鎖含有実体の量は、好ましくは０．００５μｇ／ｍＬ以上、より好ましくは０．０２μｇ／ｍＬ以上、さらにより好ましくは０．１μｇ／ｍＬ以上である。レクチン反応性糖鎖含有実体の量はまた、好ましくは２０ｍｇ／ｍＬ以下、より好ましくは５ｍｇ／ｍＬ以下、さらにより好ましくは１ｍｇ／ｍＬ以下である。このような量のレクチン反応性糖鎖含有実体が、液体サンプルに添加されてもよい。 The amount of the lectin-reactive sugar chain-containing entity used in the method of the present invention is not particularly limited as long as it is an amount capable of supporting capture of the lectin target molecule by the lectin, and is, for example, 0.001 μg / mL to 100 mg / mL. . The amount of the lectin-reactive sugar chain-containing entity is preferably 0.005 μg / mL or more, more preferably 0.02 μg / mL or more, and even more preferably 0.1 μg / mL or more. The amount of the lectin-reactive sugar chain-containing entity is also preferably 20 mg / mL or less, more preferably 5 mg / mL or less, and even more preferably 1 mg / mL or less. Such an amount of lectin-reactive sugar chain-containing entity may be added to the liquid sample.
　好ましい実施形態では、本発明の方法は、レクチン標的分子の測定方法である。本発明の測定方法では、レクチンのレクチン標的分子への結合後にレクチン標的分子を測定することができる。本発明の測定方法は、免疫学的手法に準じて行うことができ、抗体の代わりにレクチン反応性糖鎖含有実体およびレクチンを併用することにより、免疫学的手法と同様の手法により行うことができる。本発明の測定方法が準じる免疫学的手法としては、例えば、酵素免疫測定法（ＥＩＡ）（例、化学発光ＥＩＡ（ＣＬＥＩＡ）、酵素吸着ＥＩＡ（ＥＬＩＳＡ））、蛍光免疫測定法、化学発光免疫測定法、電気化学発光免疫測定法、凝集法、免疫染色、フローメトリー法、バイオレイヤー干渉法、Ｉｎ　Ｓｉｔｕ　ＰＬＡ法、化学増幅型ルミネッセンス・プロキシミティ・ホモジニアス・アッセイ、ラインブロット法、ウエスタンブロット法が挙げられる。本発明の測定方法は、定性的または定量的に行われ、レクチン標的分子の有無または量を評価することができる。 In a preferred embodiment, the method of the present invention is a method for measuring a lectin target molecule. In the measurement method of the present invention, the lectin target molecule can be measured after binding of the lectin to the lectin target molecule. The measurement method of the present invention can be carried out according to an immunological technique, and can be carried out by a technique similar to the immunological technique by using a lectin-reactive sugar chain-containing entity and a lectin instead of an antibody. it can. Examples of the immunological technique to which the measurement method of the present invention is applied include enzyme immunoassay (EIA) (eg, chemiluminescence EIA (CLEIA), enzyme-adsorbed EIA (ELISA)), fluorescence immunoassay, chemiluminescence immunoassay Method, electrochemiluminescence immunoassay, agglutination, immunostaining, flowmetry, biolayer interferometry, In Situ PLA method, chemically amplified luminescence proximity homogenous assay, line blotting, Western blotting It is done. The measurement method of the present invention is performed qualitatively or quantitatively, and the presence or amount of a lectin target molecule can be evaluated.
　本発明の方法では、レクチン標的分子に特異的な親和性物質がさらに用いられてもよい。本発明の方法で用いられるレクチン標的分子に特異的な親和性物質は、レクチン標的分子中のレクチン結合部位（糖鎖）とは異なる部位（例、ペプチド領域）でレクチン標的分子に結合する。レクチン標的分子に特異的な親和性物質としては、例えば、抗体およびアプタマーが挙げられるが、抗体が好ましい。 In the method of the present invention, an affinity substance specific for the lectin target molecule may be further used. The affinity substance specific for the lectin target molecule used in the method of the present invention binds to the lectin target molecule at a site (eg, peptide region) different from the lectin binding site (sugar chain) in the lectin target molecule. Examples of the affinity substance specific for the lectin target molecule include antibodies and aptamers, and antibodies are preferred.
　本発明の方法において用いられるレクチン標的分子に特異的な親和性物質の量は、レクチン標的分子を十分に結合できる量である限り特に限定されないが、例えば、０．０００１μｇ／ｍＬ～１０ｍｇ／ｍＬである。親和性物質の量は、好ましくは０．００１μｇ／ｍＬ以上、より好ましくは０．００５μｇ／ｍＬ以上、さらにより好ましくは０．０１μｇ／ｍＬ以上である。親和性物質の量はまた、好ましくは２ｍｇ／ｍＬ以下、より好ましくは０．５ｍｇ／ｍＬ以下、さらにより好ましくは０．１ｍｇ／ｍＬ以下である。このような量の親和性物質が、液体サンプルに添加されてもよい。 The amount of the affinity substance specific for the lectin target molecule used in the method of the present invention is not particularly limited as long as it is a sufficient amount to bind the lectin target molecule, and is, for example, 0.0001 μg / mL to 10 mg / mL. is there. The amount of the affinity substance is preferably 0.001 μg / mL or more, more preferably 0.005 μg / mL or more, and even more preferably 0.01 μg / mL or more. The amount of affinity substance is also preferably 2 mg / mL or less, more preferably 0.5 mg / mL or less, and even more preferably 0.1 mg / mL or less. Such an amount of affinity substance may be added to the liquid sample.
　レクチン標的分子に特異的な親和性物質がさらに用いられる場合、本発明の測定方法は、サンドイッチ様式で行われてもよい。例えば、固相化手段として、レクチン標的分子に特異的な親和性物質が用いられ、検出手段として、レクチンおよびレクチン反応性糖鎖含有実体が併用されてもよい。あるいは、固相化手段として、レクチンおよびレクチン反応性糖鎖含有実体が併用され、検出手段として、レクチン標的分子に特異的な親和性物質が用いられてもよい。 When an affinity substance specific for the lectin target molecule is further used, the measurement method of the present invention may be performed in a sandwich manner. For example, an affinity substance specific for the lectin target molecule may be used as the solid phase immobilization means, and the lectin and the lectin reactive sugar chain-containing entity may be used in combination as the detection means. Alternatively, a lectin and a lectin-reactive sugar chain-containing entity may be used together as a solid phase immobilization means, and an affinity substance specific for the lectin target molecule may be used as a detection means.
　本発明の測定方法では、レクチン反応性糖鎖含有実体もしくはレクチン、またはレクチン標的分子に特異的な親和性物質が標識物質で標識されていてもよい。標識物質としては、例えば、酵素（例、ペルオキシダーゼ、アルカリホスファターゼ、ルシフェラーゼ、βガラクトシダーゼ）、親和性物質（例、ストレプトアビジンおよびビオチンのうちの一方、互いに相補的なセンス鎖およびアンチセンス鎖の核酸のうちの一方）、蛍光物質またはタンパク質（例、フルオレセイン、フルオレセインイソチオシアネート、ローダミン、緑色蛍光タンパク質、赤色蛍光タンパク質）、発光物質（例、ルシフェリン、エクオリン、アクリジニウムエステル、トリス（２，２’－ビピリジル）ルテニウム、ルミノール）、放射性物質（例、３Ｈ、１４Ｃ、３２Ｐ、３５Ｓ、１２５Ｉ）が挙げられる。好ましくは、レクチン反応性糖鎖含有実体、またはレクチン反応性糖鎖含有実体に特異的な親和性物質が標識される。 In the measurement method of the present invention, a lectin-reactive sugar chain-containing entity or lectin, or an affinity substance specific for a lectin target molecule may be labeled with a labeling substance. Examples of labeling substances include enzymes (eg, peroxidase, alkaline phosphatase, luciferase, β-galactosidase) and affinity substances (eg, streptavidin and biotin, which are complementary to each other in the sense strand and the antisense strand nucleic acid. One of them), fluorescent substance or protein (eg, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substance (eg, luciferin, aequorin, acridinium ester, tris (2,2'- Bipyridyl) ruthenium, luminol), radioactive materials (eg, 3 H, 14 C, 32 P, 35 S, 125 I). Preferably, the lectin-reactive sugar chain-containing entity or an affinity substance specific to the lectin-reactive sugar chain-containing entity is labeled.
　より具体的には、本発明の測定方法は、サンドイッチ様式で行われる場合、以下の工程により行うことができる：
（１）レクチン標的分子を、レクチン標的分子に特異的な親和性物質に結合させて、レクチン標的分子およびレクチン標的分子に特異的な親和性物質を含む第１複合体を得ること；
（２）レクチン反応性糖鎖含有実体の存在下においてレクチンを第１複合体と結合させて、レクチン標的分子、レクチン標的分子に特異的な親和性物質、レクチンおよびレクチン反応性糖鎖含有実体を含む第２複合体を得ること；ならびに
（３）第２複合体に含まれるレクチン標的分子の量を測定すること。 More specifically, the measurement method of the present invention, when performed in a sandwich manner, can be performed by the following steps:
(1) binding a lectin target molecule to an affinity substance specific for the lectin target molecule to obtain a first complex including the lectin target molecule and an affinity substance specific for the lectin target molecule;
(2) A lectin is bound to the first complex in the presence of a lectin-reactive sugar chain-containing entity, and a lectin target molecule, an affinity substance specific for the lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity Obtaining a second complex comprising; and (3) measuring the amount of lectin target molecules contained in the second complex.
　工程（１）では、レクチン標的分子は、上述したような液体サンプルに含まれるものを使用することができる。したがって、工程（１）の具体的な実施形態として、レクチン標的分子を含む液体サンプルに、レクチン標的分子に特異的な親和性物質を添加することにより、工程（１）が行われてもよい。工程（１）では、上述したような量のレクチン標的分子に特異的な親和性物質を用いることができるが、好ましくは、予想されるレクチン標的分子に対して、過剰当量のレクチン標的分子に特異的な親和性物質を用いてもよい。レクチン標的分子に特異的な親和性物質としては、上述したような固相に固定されたものを用いるのが好ましい。レクチン標的分子とレクチン標的分子に特異的な親和性物質との結合は、両者を溶液（例、緩衝液）中でインキュベートすることにより達成することができ、これによりレクチン標的分子およびレクチン標的分子に特異的な親和性物質を含む第１複合体が得られる。 In step (1), the lectin target molecules can be those contained in the liquid sample as described above. Therefore, as a specific embodiment of the step (1), the step (1) may be performed by adding an affinity substance specific for the lectin target molecule to the liquid sample containing the lectin target molecule. In the step (1), an affinity substance specific for the lectin target molecule in an amount as described above can be used, but preferably specific for an excess equivalent of the lectin target molecule relative to the expected lectin target molecule. A typical affinity substance may be used. As an affinity substance specific for the lectin target molecule, it is preferable to use a substance immobilized on a solid phase as described above. Binding of a lectin target molecule and an affinity substance specific for the lectin target molecule can be achieved by incubating both in solution (eg, buffer), thereby allowing the lectin target molecule and lectin target molecule to bind to each other. A first complex containing a specific affinity substance is obtained.
　工程（２）では、第２複合体が得られる。工程（１）の具体的な実施形態として、工程（１）で得られた上記第１複合体を含む溶液に、レクチン、およびレクチン反応性糖鎖含有実体を添加することにより、工程（２）が行われてもよい。工程（２）では、上述したような量のレクチン、および上述したような量のレクチン反応性糖鎖含有実体を用いることができるが、好ましくは、予想されるレクチン標的分子に対して、過剰当量のレクチンおよび過剰当量のレクチン反応性糖鎖含有実体を用いてもよい。レクチンまたはレクチン反応性糖鎖含有実体のいずれかが標識物質で標識されていてもよいが、好ましくはレクチン反応性糖鎖含有実体が標識物質で標識されていてもよい。レクチン反応性糖鎖含有実体の存在下におけるレクチンの第１複合体への結合は、三者を溶液（例、緩衝液）中でインキュベートすることにより達成することができ、これによりレクチン標的分子、レクチン標的分子に特異的な親和性物質、レクチンおよびレクチン反応性糖鎖含有実体を含む第２複合体が得られる。レクチン標的分子に特異的な親和性物質として固相に固定されたものを用いた場合、第２複合体は固相に固定されている。この場合、工程（２）の後に、液相の除去および添加から構成される洗浄工程が設けられてもよい。 In step (2), a second complex is obtained. As a specific embodiment of the step (1), by adding the lectin and the lectin-reactive sugar chain-containing entity to the solution containing the first complex obtained in the step (1), the step (2) May be performed. In step (2), the amount of lectin as described above and the amount of lectin-reactive sugar chain-containing entity as described above can be used, but preferably in excess equivalent to the expected lectin target molecule. Lectins and excess equivalents of lectin-reactive sugar chain-containing entities may be used. Either the lectin or the lectin-reactive sugar chain-containing entity may be labeled with a labeling substance, but preferably the lectin-reactive sugar chain-containing entity may be labeled with a labeling substance. Binding of the lectin to the first complex in the presence of the lectin-reactive sugar chain-containing entity can be achieved by incubating the three in solution (eg, buffer), whereby the lectin target molecule, A second complex comprising an affinity substance specific for the lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity is obtained. When the substance specific to the lectin target molecule that is immobilized on the solid phase is used, the second complex is immobilized on the solid phase. In this case, after the step (2), a washing step including removal and addition of the liquid phase may be provided.
　工程（３）では、第２複合体における標識物質からのシグナルを指標に、レクチン標的分子の量を測定することができる。 In step (3), the amount of the lectin target molecule can be measured using the signal from the labeling substance in the second complex as an index.
　本発明はまた、レクチン標的分子、レクチンおよびレクチン反応性糖鎖含有実体を含む複合体を提供する。本発明の複合体は、レクチン標的分子に特異的な親和性物質をさらに含んでいてもよい。本発明の複合体は、これらの構成因子を溶液中でインキュベートして結合させることにより、作製することができる。 The present invention also provides a complex comprising a lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity. The complex of the present invention may further contain an affinity substance specific for the lectin target molecule. The complex of the present invention can be prepared by incubating these components in a solution and binding them.
　本発明の複合体において、レクチン標的分子、レクチン、レクチン反応性糖鎖含有実体、およびレクチン標的分子に特異的な親和性物質の定義、例および好ましい例は、上述したとおりである。 In the complex of the present invention, the definition, examples and preferred examples of the lectin target molecule, the lectin, the lectin-reactive sugar chain-containing entity, and the affinity substance specific for the lectin target molecule are as described above.
　本発明の複合体は、例えば、本発明の捕捉方法の実施に有用である。 The complex of the present invention is useful for the implementation of the capture method of the present invention, for example.
　本発明はさらに、レクチンおよびレクチン反応性糖鎖含有実体を混合形態で含む、レクチン標的分子の捕捉用試薬を提供する。換言すれば、本発明の捕捉用試薬は、レクチンおよびレクチン反応性糖鎖含有実体の組成物を含む。本発明の試薬に含まれるレクチンおよびレクチン反応性糖鎖含有実体は混合形態にあることから、本発明の捕捉用試薬ではレクチンとレクチン反応性糖鎖含有実体との間で複合体が形成されていてもよい。 The present invention further provides a reagent for capturing a lectin target molecule, which contains a lectin and a lectin-reactive sugar chain-containing entity in a mixed form. In other words, the capture reagent of the present invention includes a composition of a lectin and a lectin-reactive sugar chain-containing entity. Since the lectin and the lectin-reactive sugar chain-containing entity contained in the reagent of the present invention are in a mixed form, in the capture reagent of the present invention, a complex is formed between the lectin and the lectin-reactive sugar chain-containing entity. May be.
　本発明の試薬がレクチン標的分子の測定に用いられる場合、レクチンまたはレクチン反応性糖鎖含有実体のいずれかが標識物質で標識されていてもよいが、好ましくはレクチン反応性糖鎖含有実体が標識物質で標識されていてもよい。あるいは、レクチンまたはレクチン反応性糖鎖含有実体のいずれも標識物質で標識されていない場合、本発明の試薬は、レクチンおよびレクチン反応性糖鎖含有実体と非混合形態で標識物質を含んでいてもよい。 When the reagent of the present invention is used for measurement of a lectin target molecule, either the lectin or the lectin-reactive sugar chain-containing entity may be labeled with a labeling substance, but preferably the lectin-reactive sugar chain-containing entity is labeled. It may be labeled with a substance. Alternatively, when neither the lectin nor the lectin-reactive sugar chain-containing entity is labeled with a labeling substance, the reagent of the present invention may contain the labeling substance in an unmixed form with the lectin and the lectin-reactive sugar chain-containing entity. Good.
　本発明の試薬は、レクチン標的分子に特異的な親和性物質をさらに含んでいてもよい。レクチン標的分子に特異的な親和性物質は、標識物質で標識されていてもよい。また、レクチン標的分子に特異的な親和性物質は、上述したような固相に固定されていてもよい。この場合、本発明の試薬は、サンドイッチ様式においてレクチン標的分子を測定するために好適に用いることができる。本発明の試薬は、レクチン標的分子に特異的な親和性物質を、レクチンおよびレクチン反応性糖鎖含有実体と混合形態または非混合形態のいずれかで含むが、非混合形態で含むことが好ましい。 The reagent of the present invention may further contain an affinity substance specific for the lectin target molecule. The affinity substance specific for the lectin target molecule may be labeled with a labeling substance. Moreover, the affinity substance specific for the lectin target molecule may be immobilized on the solid phase as described above. In this case, the reagent of the present invention can be suitably used for measuring lectin target molecules in a sandwich manner. The reagent of the present invention contains an affinity substance specific for a lectin target molecule in either a mixed form or a non-mixed form with a lectin and a lectin-reactive sugar chain-containing entity, but preferably in a non-mixed form.
　本発明の試薬において、レクチン標的分子、レクチン、レクチン反応性糖鎖含有実体、およびレクチン標的分子に特異的な親和性物質の定義、例および好ましい例は、上述したとおりである。 In the reagent of the present invention, definitions, examples and preferred examples of affinity substances specific for lectin target molecules, lectins, lectin-reactive sugar chain-containing entities, and lectin target molecules are as described above.
　本発明の試薬は、例えば、本発明の捕捉方法の簡便な実施に有用であり、例えば、本発明の測定方法に好適に用いることができる。 The reagent of the present invention is useful for, for example, simple implementation of the capture method of the present invention, and can be suitably used for the measurement method of the present invention, for example.
　本発明はまた、レクチンおよびレクチン反応性糖鎖含有実体を含む、レクチン標的分子の捕捉用キットを提供する。本発明のキットに含まれるレクチンおよびレクチン反応性糖鎖含有実体は、混合形態（すなわち、組成物の形態）にあるか、または非混合形態（すなわち、互いに隔離された形態）にある。よって、本発明のキットに含まれる構成成分は、それぞれ異なる容器（例、チューブ、プレート）に収容された形態、または同一容器の異なる区画に収容された形態で提供される。 The present invention also provides a kit for capturing a lectin target molecule comprising a lectin and a lectin-reactive sugar chain-containing entity. The lectin and lectin-reactive sugar chain-containing entity included in the kit of the present invention is in a mixed form (ie, in the form of a composition) or in an unmixed form (ie, a form isolated from each other). Therefore, the component contained in the kit of this invention is provided with the form accommodated in the different container (for example, tube, plate), respectively, or the form accommodated in the different divisions of the same container.
　本発明のキットがレクチン標的分子の測定に用いられる場合、レクチンまたはレクチン反応性糖鎖含有実体のいずれかが標識物質で標識されていてもよいが、好ましくはレクチン反応性糖鎖含有実体が標識物質で標識されていてもよい。あるいは、レクチンまたはレクチン反応性糖鎖含有実体のいずれも標識物質で標識されていない場合、本発明のキットは、標識物質および／またはレクチン標的分子に特異的な親和性物質（標識物質で標識されていてもよい）を含んでいてもよい。 When the kit of the present invention is used for measurement of a lectin target molecule, either a lectin or a lectin-reactive sugar chain-containing entity may be labeled with a labeling substance, but preferably a lectin-reactive sugar chain-containing entity is labeled It may be labeled with a substance. Alternatively, when neither the lectin nor the lectin-reactive sugar chain-containing entity is labeled with a labeling substance, the kit of the present invention can be used as an affinity substance (labeled with a labeling substance and / or a lectin target molecule). May be included).
　本発明のキットは、レクチン標的分子に特異的な親和性物質をさらに含んでいてもよい。この場合、本発明のキットは、サンドイッチ様式においてレクチン標的分子を測定するために好適に用いることができる。レクチン標的分子に特異的な親和性物質は、上述したような固相に固定されていてもよい。 The kit of the present invention may further contain an affinity substance specific for the lectin target molecule. In this case, the kit of the present invention can be suitably used for measuring a lectin target molecule in a sandwich manner. The affinity substance specific for the lectin target molecule may be immobilized on the solid phase as described above.
　本発明のキットにおいて、レクチン標的分子、レクチン、レクチン反応性糖鎖含有実体、およびレクチン標的分子に特異的な親和性物質の定義、例および好ましい例は、上述したとおりである。 In the kit of the present invention, the definition, examples and preferred examples of lectin target molecules, lectins, lectin-reactive sugar chain-containing entities, and affinity substances specific for the lectin target molecules are as described above.
　本発明のキットは、例えば、本発明の捕捉方法の簡便な実施に有用であり、例えば、本発明の測定方法に好適に用いることができる。 The kit of the present invention is useful for, for example, simple implementation of the capture method of the present invention, and can be suitably used for, for example, the measurement method of the present invention.
　以下、本発明を実施例により詳細に説明するが、本発明は、これらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these examples.
実施例１：抗体フラグメントの調製、及び抗体フラグメント－ＨＲＰの調製
　抗ＬＣＡ抗体ではないマウスＩｇＧ１をペプシンで消化し、ＬＣＡ反応性マウスＩｇＧ　Ｆ（ａｂ’）２フラグメントとＬＣＡ非反応性マウスＩｇＧ　Ｆ（ａｂ’）２フラグメントを調製した。 Example 1: Preparation of Antibody Fragment and Preparation of Antibody Fragment-HRP Mouse IgG1, which is not an anti-LCA antibody, was digested with pepsin and LCA-reactive mouse IgG F (ab ′) 2 fragment and non-LCA-reactive mouse IgG F ( ab ') 2 fragments were prepared.
　具体的には、調製は、以下のとおり行った。
（１）抗ＬＣＡ抗体ではないマウスＩｇＧ１抗体Ａ、Ｂ、ＣおよびＤをペプシンで消化した。消化は、クエン酸緩衝液にバッファー交換したマウスＩｇＧ１抗体Ａ、Ｂ、ＣおよびＤにペプシン（ＳＩＧＭＡ）を添加し、３７℃１時間反応させ、トリス緩衝液の添加により反応停止させることにより行った。
（２）ＰＢＳで平衡化したＳｕｐｅｒｄｅｘ２００　ｉｎｃｒｅａｓｅ　１０／３００ＧＬでゲルろ過を行い、４種の精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメントを得た。
（３）精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤを、西洋わさびペルオキシダーゼ（ＨＲＰ）で標識した。ＨＲＰの標識は、Ｐｅｒｏｘｉｄａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２（同仁化学研究所）を用いて、添付のプロトコールに従って行い、４種の精製抗体フラグメント－ＨＲＰ（マウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰ）を得た。 Specifically, preparation was performed as follows.
(1) Mouse IgG1 antibodies A, B, C and D that are not anti-LCA antibodies were digested with pepsin. Digestion was performed by adding pepsin (SIGMA) to mouse IgG1 antibodies A, B, C and D buffer-exchanged to citrate buffer, reacting at 37 ° C. for 1 hour, and stopping the reaction by adding Tris buffer. .
(2) Gel filtration was performed with Superdex 200 increase 10 / 300GL equilibrated with PBS to obtain four types of purified mouse IgG F (ab ′) 2 fragments.
(3) Purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D were labeled with horseradish peroxidase (HRP). HRP was labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached protocol to obtain 4 types of purified antibody fragments—HRP (mouse IgG F (ab ′) 2 -HRP).
実施例２：抗体フラグメントの精製度、分子量およびレクチン反応性の確認
　実施例１で調製した４種の抗体フラグメントについて、精製度、分子量およびレクチン反応性を確認した。 Example 2: Confirmation of purity, molecular weight, and lectin reactivity of antibody fragments The purity, molecular weight, and lectin reactivity of the four antibody fragments prepared in Example 1 were confirmed.
　具体的には、確認は、以下のとおり行った。
（１）精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤの分子量をＳＤＳ－ＰＡＧＥにて確認した（図１左）。
（２）精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤのＬＣＡ反応性をレクチンブロットにて確認した（図１右）。
（３）精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤのＬＣＡ反応性をＥＬＩＳＡにて確認した。精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤを各５μｇ／ｍＬとなるようにＰＢＳで希釈した。Ｕ９６　ＭａｘｉＳｏｒｐ　Ｎｕｎｃ－Ｉｍｍｕｎｏ　Ｐｌａｔｅ（Ｔｈｅｒｍｏ）において、希釈された溶液５０μＬを４℃２４時間インキュベートして、上記抗体を固相化した。抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した後、１０％ブロッキング試薬（Ｃａｒｂｏｆｒｅｅ　ｂｌｏｃｋｉｎｇ　ｓｏｌｕｔｉｏｎ；Ｖｅｃｔｏｒ）１５０μＬで３７℃１時間ブロッキングした。抗体固相化ウェルをＰＢＳＴ　２５０μＬで３回洗浄した。次いで、１０％ブロッキング試薬でＬＣＡ－ＨＲＰ（Ｊ－オイルミルズ）を５μｇ／ｍＬとなるように希釈した溶液　５０μＬを加え、抗体固相化ウェルにおいて溶液を３７℃１時間インキュベートした。ＰＢＳＴ　２５０μＬでウェルを３回洗浄した。洗浄後のウェルに５０μＬのＴＭＢ基質溶液（Ｄａｋｏ）を加え室温で５分反応を行った。１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した（図２）。 Specifically, confirmation was performed as follows.
(1) The molecular weights of purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D were confirmed by SDS-PAGE (FIG. 1 left).
(2) LCA reactivity of purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D was confirmed by lectin blot (right side of FIG. 1).
(3) purified mouse IgG F (ab ') 2 fragment antibodies A, B, and LCA reactive C and D were confirmed by ELISA. Purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D were diluted with PBS so as to be 5 μg / mL each. In U96 MaxiSorp Nunc-Immuno Plate (Thermo), 50 μL of the diluted solution was incubated at 4 ° C. for 24 hours to immobilize the antibody. The antibody-immobilized well was washed with 250 μL of PBST three times, and then blocked with 150 μL of 10% blocking reagent (Carboxer) at 37 ° C. for 1 hour. The antibody-immobilized well was washed 3 times with 250 μL of PBST. Subsequently, 50 μL of a solution obtained by diluting LCA-HRP (J-Oil Mills) to 5 μg / mL with 10% blocking reagent was added, and the solution was incubated at 37 ° C. for 1 hour in the antibody-immobilized well. The wells were washed 3 times with 250 μL PBST. To the well after washing, 50 μL of TMB substrate solution (Dako) was added and reacted at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm (FIG. 2).
　その結果、抗体フラグメントＡおよびＢはＬＣＡ反応性であるが、抗体フラグメントＣおよびＤはＬＣＡ非反応性であることが確認された（表１、図１、２）。 As a result, it was confirmed that antibody fragments A and B were LCA-reactive, but antibody fragments C and D were non-LCA-reactive (Table 1, FIGS. 1 and 2).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　用いた抗体を解析した。
　抗体Ａは、Ｎ型糖鎖結合コンセンサス配列を抗体可変領域の重鎖に有していた。このことは、Ｆ（ａｂ’）２フラグメントがＮ型糖鎖で修飾されていることを示す。よって、Ｆ（ａｂ’）２フラグメント抗体Ａは、Ｎ型糖鎖を介してレクチン反応性を示すと考えられる。
　抗体Ｂは、抗体Ａと同様である。
　抗体Ｃは、Ｎ型糖鎖結合コンセンサス配列を抗体可変領域の重鎖にも軽鎖にも有していなかった。このことは、Ｆ（ａｂ’）２フラグメントはＮ型糖鎖で修飾されていないことを示す。よって、Ｆ（ａｂ’）２フラグメント抗体Ｃは、レクチン反応性を示さないと考えられる。
　抗体Ｄは、抗体Ｃと同様である。 The antibody used was analyzed.
Antibody A had an N-type sugar chain binding consensus sequence in the heavy chain of the antibody variable region. This indicates that the F (ab ′) 2 fragment is modified with an N-type sugar chain. Therefore, F (ab ′) 2 fragment antibody A is considered to exhibit lectin reactivity via an N-type sugar chain.
Antibody B is the same as antibody A.
Antibody C did not have an N-type sugar chain binding consensus sequence in the heavy or light chain of the antibody variable region. This indicates that the F (ab ′) 2 fragment is not modified with an N-type sugar chain. Therefore, F (ab ′) 2 fragment antibody C is considered not to show lectin reactivity.
Antibody D is the same as antibody C.
実施例３：レクチン反応性抗体フラグメントの存在下におけるレクチンによるＡＦＰ－Ｌ３測定系の構築
　実施例１で調製したマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰを用いて、ＡＦＰ－Ｌ３の免疫学的測定系を構築した。 Example 3: Construction of AFP-L3 measurement system using lectin in the presence of lectin-reactive antibody fragments Using mouse IgG F (ab ') 2 -HRP prepared in Example 1, immunological measurement of AFP-L3 A system was constructed.
　具体的には、構築は、以下のとおり行った。
（１）ａｎｔｉ－ＡＦＰ　Ｆ（ａｂ’）２を１０μｇ／ｍＬとなるようにＰＢＳで希釈した。Ｕ９６　ＭａｘｉＳｏｒｐ　Ｎｕｎｃ－Ｉｍｍｕｎｏ　Ｐｌａｔｅにおいて、希釈された溶液　５０μＬを４℃２４時間インキュベートして、上記抗体を固相化した。
（２）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した後、１０％ブロッキング試薬　１５０μＬで３７℃１時間ブロッキングした。
（３）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した。次いで、ミュータスワコーＡＦＰ－Ｌ３（和光純薬工業）の測定値から濃度を換算したＡＦＰ－Ｌ３溶液とＡＦＰ－Ｌ１溶液を、それぞれ１０％ブロッキング試薬で、１０００ｎｇ／ｍＬから２ｎ希釈した溶液　５０μＬを抗体固相化ウェルに加え３７℃１時間インキュベートした。
（４）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した。次いで、実施例１で調製したマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰをそれぞれ１０％ブロッキング試薬で５μｇ／ｍＬとなるように希釈し、さらにＬＣＡ（Ｖｅｃｔｏｒ）を１００μｇ／ｍＬとなるように加えた混合液を調製し、その混合液を抗体固相化ウェルに加え３７℃１時間インキュベートした。
（５）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した。次いで、５０μＬのＴＭＢ基質溶液（Ｄａｋｏ）を加え室温で５分反応させた。１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した。 Specifically, the construction was performed as follows.
(1) Anti-AFP F (ab ′) 2 was diluted with PBS so as to be 10 μg / mL. In the U96 MaxiSorp Nunc-Immuno Plate, 50 μL of the diluted solution was incubated at 4 ° C. for 24 hours to immobilize the antibody.
(2) The antibody-immobilized well was washed with 250 μL of PBST three times, and then blocked with 150 μL of 10% blocking reagent at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Next, 50 μL of a solution obtained by diluting the AFP-L3 solution and the AFP-L1 solution, whose concentrations were converted from the measured values of Mutus Wako AFP-L3 (Wako Pure Chemical Industries, Ltd.) with a 10% blocking reagent from 1000 ng / mL to 2 n, respectively. It was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Next, mouse IgG F (ab ′) 2 -HRP prepared in Example 1 was diluted with 10% blocking reagent to 5 μg / mL, and LCA (Vector) was further added to 100 μg / mL. A mixed solution was prepared, and the mixed solution was added to an antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(5) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Next, 50 μL of TMB substrate solution (Dako) was added and reacted at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
　その結果、ＬＣＡは、ＬＣＡ反応性抗体フラグメントＡまたはＢの存在下でＡＦＰ－Ｌ３を特異的に検出することができた（表２、図３～６）。 As a result, LCA was able to specifically detect AFP-L3 in the presence of LCA-reactive antibody fragment A or B (Table 2, FIGS. 3 to 6).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　以上より、レクチンは、標識されたレクチン反応性抗体フラグメントの存在下でＡＦＰ－Ｌ３を特異的に検出できることが示された。 From the above, it was shown that lectin can specifically detect AFP-L3 in the presence of a labeled lectin-reactive antibody fragment.
実施例４：ＡＦＰ－Ｌ３検出における、レクチン反応性抗体フラグメント中のレクチン反応性糖鎖の関与の確認
　実施例１で調製したマウスＩｇＧ　Ｆ（ａｂ’）２抗体Ｂの糖鎖をＮ－グリコシダーゼＦで除去することにより、糖鎖除去マウスＩｇＧ　Ｆ（ａｂ’）２抗体ＢがＬＣＡ共存下でのＡＦＰ－Ｌ３反応性を維持しているか否かを確認した。 Example 4 Confirmation of Involvement of Lectin-Reactive Sugar Chain in Lectin-Reactive Antibody Fragment in AFP-L3 Detection The sugar chain of mouse IgG F (ab ′) 2 antibody B prepared in Example 1 is N-glycosidase F It was confirmed whether or not the sugar chain-removed mouse IgG F (ab ′) 2 antibody B maintained AFP-L3 reactivity in the presence of LCA.
　具体的には、確認は、以下のとおり行った。
（１）実施例１で調製したマウスＩｇＧ　Ｆ（ａｂ’）２抗体ＢのＮ－グリコシダーゼＦ処理を実施した。Ｎ－グリコシダーゼＦ処理は、トリス塩酸緩衝液にバッファー交換したマウスＩｇＧ　Ｆ（ａｂ’）２抗体ＢにＮ－グリコシダーゼＦ（ＲＯＣＨＥ）を添加し、３７℃２４時間反応させた。Ｎ－グリコシダーゼＦは、Ｎ結合型糖鎖とタンパク質とのＧｌｃＮＡｃ－Ａｓｎ結合を特異的に切断する酵素である。
（２）ＰＢＳで平衡化したＳｕｐｅｒｄｅｘ２００　ｉｎｃｒｅａｓｅ　１０／３００ＧＬでゲルろ過を行い、糖鎖除去マウスＩｇＧ　Ｆ（ａｂ’）２フラグメントを得た。
（３）糖鎖除去マウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰを、実施例１と同様に調製した。
（４）実施例２と同様に、精製度、分子量およびＬＣＡ反応性を確認した（図７、８）。
（５）実施例３と同様に、標識抗体の存在下におけるＬＣＡのＡＦＰ－Ｌ３反応性を確認した（図９、表３）。ＡＦＰ濃度は１μｇ／ｍＬで検討した。 Specifically, confirmation was performed as follows.
(1) The mouse IgG F (ab ′) 2 antibody B prepared in Example 1 was treated with N-glycosidase F. In N-glycosidase F treatment, N-glycosidase F (ROCHE) was added to mouse IgG F (ab ′) 2 antibody B buffer-exchanged with Tris-HCl buffer, and reacted at 37 ° C. for 24 hours. N-glycosidase F is an enzyme that specifically cleaves a GlcNAc-Asn bond between an N-linked sugar chain and a protein.
(2) Gel filtration was performed with Superdex 200 increase 10 / 300GL equilibrated with PBS to obtain a sugar chain-removed mouse IgG F (ab ′) 2 fragment.
(3) Sugar chain-removed mouse IgG F (ab ′) 2 -HRP was prepared in the same manner as in Example 1.
(4) As in Example 2, the degree of purification, molecular weight, and LCA reactivity were confirmed (FIGS. 7 and 8).
(5) As in Example 3, the AFP-L3 reactivity of LCA in the presence of labeled antibody was confirmed (FIG. 9, Table 3). The AFP concentration was examined at 1 μg / mL.
　その結果、ＬＣＡ反応性を消失した糖鎖除去標識抗体フラグメント（図７、８）の存在下では、ＬＣＡはＡＦＰ－Ｌ３を検出できなかった（表３、図９）。 As a result, LCA could not detect AFP-L3 in the presence of the sugar chain-removed labeled antibody fragment (FIGS. 7 and 8) that lost LCA reactivity (Table 3 and FIG. 9).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　以上より、抗体の可変領域中の糖鎖が、レクチンによるＡＦＰ－Ｌ３の検出に重要であることが示された。 From the above, it was shown that the sugar chain in the variable region of the antibody is important for detection of AFP-L3 by lectin.
実施例５：可変領域中にＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧの存在下におけるレクチンによるＡＦＰ－Ｌ３検出
　抗体の可変領域中のＮ型糖鎖結合コンセンサス配列の有無がＡＦＰ－Ｌ３検出に影響するか否かを検討した。 Example 5: Detection of AFP-L3 by a lectin in the presence of mouse IgG having an N-type sugar chain binding consensus sequence in the variable region The presence or absence of an N-type sugar chain binding consensus sequence in the variable region of the antibody is useful for AFP-L3 detection It was examined whether or not it affected.
　具体的には、検討は、以下のとおり行った。
（１）可変領域にＮ型糖鎖結合コンセンサス配列（Ａｓｎ－Ｓｅｒ－Ｓｅｒ）を有する抗ＴＮＦα抗体（Ｐ）と有さない抗ＴＮＦα抗体（Ｎ）、可変領域にＮ型糖鎖結合コンセンサス配列（Ａｓｎ－Ａｓｐ－Ｔｈｒ）を有する抗ＩＬ－１０抗体（Ｐ’）と有さない抗ＩＬ－１０抗体（Ｎ’）を、それぞれ実施例１と同様にＨＲＰ標識を行った。
（２）実施例３と同様に、ＬＣＡ共存下でのＡＦＰ－Ｌ３反応性を確認した。ＡＦＰ濃度は５μｇ／ｍＬで検討した。 Specifically, the examination was conducted as follows.
(1) An anti-TNFα antibody (P) with and without an N-type sugar chain binding consensus sequence (Asn-Ser-Ser) in the variable region (N), and an N-type sugar chain binding consensus sequence (N) in the variable region The anti-IL-10 antibody (P ′) having Asn-Asp-Thr) and the anti-IL-10 antibody not having N (N ′) were labeled with HRP in the same manner as in Example 1.
(2) As in Example 3, the AFP-L3 reactivity in the presence of LCA was confirmed. The AFP concentration was examined at 5 μg / mL.
　その結果、ＬＣＡは、Ｎ型糖鎖結合コンセンサス配列を有する抗体の存在下で、ＡＦＰ－Ｌ３を特異的に検出することができた（表４、図１０）。 As a result, LCA was able to specifically detect AFP-L3 in the presence of an antibody having an N-type sugar chain binding consensus sequence (Table 4, FIG. 10).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　以上より、レクチンは、Ｎ型糖鎖結合コンセンサス配列を可変領域に有する抗体（本抗体が結合する抗原の種類を問わない）の存在下でＡＦＰ－Ｌ３を特異的に検出できることが示された。 From the above, it was shown that the lectin can specifically detect AFP-L3 in the presence of an antibody having an N-type sugar chain binding consensus sequence in the variable region (regardless of the type of antigen to which the present antibody binds).
実施例６：レクチン反応性抗原の存在下におけるレクチンによるＡＦＰ－Ｌ３測定系の構築
　マウスＩｇＧ以外のＮ型糖鎖結合コンセンサス配列を有する糖タンパク質であるＨＢｓ抗原（Ｂ型肝炎ウイルス表面抗原）を用いて、ＡＦＰ－Ｌ３の免疫学的測定系を構築した。 Example 6: Construction of AFP-L3 measurement system with lectin in the presence of lectin-reactive antigen Using HBs antigen (hepatitis B virus surface antigen), a glycoprotein having an N-type sugar chain-binding consensus sequence other than mouse IgG Thus, an immunological measurement system for AFP-L3 was constructed.
　具体的には、構築は、以下のとおり行った。
（１）Ｎ型糖鎖結合コンセンサス配列を有するＨＢｓ抗原を、それぞれ実施例１と同様にＨＲＰ標識を行った。
（２）実施例３と同様に、ＬＣＡ共存下でのＡＦＰ－Ｌ３反応性を確認した。ＡＦＰ濃度は１０００ｎｇ／ｍＬから２ｎ希釈して検討した。ＨＢｓ抗原－ＨＲＰは２μｇ／ｍＬで反応させた。 Specifically, the construction was performed as follows.
(1) HRP labeling was performed on HBs antigens having an N-type sugar chain binding consensus sequence in the same manner as in Example 1.
(2) As in Example 3, the AFP-L3 reactivity in the presence of LCA was confirmed. The AFP concentration was examined by diluting from 1000 ng / mL to 2 n. HBs antigen-HRP was reacted at 2 μg / mL.
　その結果、ＬＣＡは、Ｎ型糖鎖結合コンセンサス配列を有するＨＢｓ抗原の共存下で、ＡＦＰ－Ｌ３を特異的に検出することができた（表５、図１１）。 As a result, LCA was able to specifically detect AFP-L3 in the presence of HBs antigen having an N-type sugar chain binding consensus sequence (Table 5, FIG. 11).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　以上より、レクチンは、抗体のみならず、レクチン反応性抗原の存在下でも、ＡＦＰ－Ｌ３を特異的に検出できることが示された。 From the above, it was shown that lectin can specifically detect AFP-L3 not only in the antibody but also in the presence of lectin-reactive antigen.
実施例７：ＡＦＰ－Ｌ３検出における、抗原中のレクチン反応性糖鎖の関与の確認
　ＨＢｓ抗原の糖鎖をＮ－グリコシダーゼＦで除去することにより、糖鎖除去ＨＢｓ抗原がＬＣＡ共存下でのＡＦＰ－Ｌ３反応性を維持するか確認した。 Example 7: Confirmation of involvement of lectin-reactive sugar chain in antigen in detection of AFP-L3 By removing the sugar chain of HBs antigen with N-glycosidase F, AFP in the presence of LCA in the presence of sugar chain-removed HBs antigen -Confirmed to maintain L3 reactivity.
　具体的には、確認は、以下のとおり行った。
（１）ＨＢｓ抗原のＮ－グリコシダーゼＦ処理を実施した。Ｎ－グリコシダーゼＦ処理は、Ｎ－グリコシダーゼＦとＰＮＧａｓｅＦ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ）付属の反応溶液を用いて、ＰＮＧａｓｅＦ添付のプロトコールに従って行い、糖鎖除去を行った。
（２）糖鎖除去ＨＢｓ抗原を、実施例１と同様にＨＲＰで標識した。
（３）実施例２と同様に、精製度、分子量およびＬＣＡ反応性を確認した。
（４）実施例６と同様に、ＬＣＡ共存下でのＡＦＰ－Ｌ３反応性を確認した。ＡＦＰ濃度は１μｇ／ｍＬで検討した。ＨＢｓ抗原－ＨＲＰは、１μｇ／ｍＬで添加した。 Specifically, confirmation was performed as follows.
(1) N-glycosidase F treatment of HBs antigen was performed. The N-glycosidase F treatment was performed using a reaction solution attached to N-glycosidase F and PNGase F (New England Biolabs) according to the protocol attached to PNGase F to remove sugar chains.
(2) The sugar chain-removed HBs antigen was labeled with HRP in the same manner as in Example 1.
(3) As in Example 2, the degree of purification, molecular weight, and LCA reactivity were confirmed.
(4) As in Example 6, AFP-L3 reactivity in the presence of LCA was confirmed. The AFP concentration was examined at 1 μg / mL. HBs antigen-HRP was added at 1 μg / mL.
　その結果、ＬＣＡは、ＬＣＡ反応性を消失した糖鎖除去標識抗原（図１２、１３）の存在下では、ＡＦＰ－Ｌ３を検出できなかった（表６、図１４）。 As a result, LCA could not detect AFP-L3 in the presence of the glycan-removed labeled antigen (FIGS. 12, 13) that lost LCA reactivity (Table 6, FIG. 14).
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　以上より、糖タンパク質抗原中の糖鎖が、レクチンによるＡＦＰ－Ｌ３の検出に重要であることが示された。 From the above, it was shown that the sugar chain in the glycoprotein antigen is important for detection of AFP-L3 by lectin.
実施例８：レクチン反応性糖タンパク質の存在下におけるレクチンによるＡＦＰ－Ｌ３検出
　抗体のみならず抗原の存在下においても、ＬＣＡはＡＦＰ－Ｌ３を検出することが可能であった（実施例７）。そこで、アミノ酸配列にＮ型糖鎖結合コンセンサス配列を有する他の糖タンパク質をＨＲＰ標識し、他の糖タンパク質の存在下においてＬＣＡがＡＦＰ－Ｌ３を検出できるか確認した。 Example 8: Detection of AFP-L3 by lectin in the presence of lectin-reactive glycoprotein LCA was able to detect AFP-L3 not only in the presence of antibodies but also in the presence of antigen (Example 7). Therefore, another glycoprotein having an N-type sugar chain binding consensus sequence in its amino acid sequence was labeled with HRP, and it was confirmed whether LCA could detect AFP-L3 in the presence of the other glycoprotein.
　具体的には、確認は、以下のとおり行った。
（１）アミノ酸配列中にＮ型糖鎖結合コンセンサス配列を有するｈＣＧα（ヒト絨毛性ゴナドトロピンα鎖）とＬＨ（黄体形成ホルモン）を、それぞれ実施例１と同様にＨＲＰ標識を行った。
（２）実施例３と同様に、ＬＣＡのＡＦＰ－Ｌ３反応性を確認した。ＡＦＰ濃度は１μｇ／ｍＬで検討した。 Specifically, confirmation was performed as follows.
(1) HCG labeling was performed on hCGα (human chorionic gonadotropin α chain) and LH (luteinizing hormone) each having an N-type sugar chain binding consensus sequence in the amino acid sequence in the same manner as in Example 1.
(2) As in Example 3, the AFP-L3 reactivity of LCA was confirmed. The AFP concentration was examined at 1 μg / mL.
　その結果、ＬＣＡは、ｈＣＧαまたはＬＨの存在下で、ＡＦＰ－Ｌ３を特異的に検出することができた（表７、８）。 As a result, LCA was able to specifically detect AFP-L3 in the presence of hCGα or LH (Tables 7 and 8).
Figure JPOXMLDOC01-appb-T000007
 数値は、ＯＤ４５０の測定値を示す。
Figure JPOXMLDOC01-appb-T000007
 A numerical value shows the measured value of OD450.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　以上より、レクチンは、レクチン反応性糖タンパク質の存在下で、ＡＦＰ－Ｌ３を特異的に検出できることが示された。 From the above, it was shown that lectin can specifically detect AFP-L3 in the presence of lectin-reactive glycoprotein.
実施例９：レクチン反応性標識抗体またはレクチン反応性標識抗原の存在下における各種レクチンによるＡＦＰ結合糖鎖の検出系の構築
　Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰの存在下で、ヒイロチャワンタケレクチン（Ａｌｅｕｒｉａ　ａｕｒａｎｔｉａ　Ｌｅｃｔｉｎ：ＡＡＬ）とコンカナバリンＡ（ＣｏｎＡ）をレクチンとして用いることにより、下記構造式を有するＡＦＰ結合糖鎖の検出系を構築した。 Example 9: Construction of detection system for AFP-binding sugar chain by various lectins in the presence of lectin-reactive labeled antibody or lectin-reactive labeled antigen Mouse IgG F (ab ') 2 -HRP having N-type sugar chain-binding consensus sequence Alternatively, in the presence of HBs antigen-HRP, a detection system for an AFP-binding sugar chain having the following structural formula was constructed by using Aleuria aurantia lectin (AAL) and concanavalin A (ConA) as lectins.
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
　上記構造式については、既報（Ｎａｋａｇａｗａ　ｅｔ　ａｌ．，Ｊ　Ｐｒｏｔｅｏｍｅ　Ｒｅｓ．　２００８　Ｊｕｎ；７（６）：２２２２－３３）により、培養細胞（Ｈｕｈ７）と肝細胞癌検体中に６０％以上含まれると報告されている構造式を代表例として示した。 The above structural formula is reported to be contained in cultured cells (Huh7) and hepatocellular carcinoma by more than 60% according to a previous report (Nakagawa et al., J Proteome Res. 2008 Jun; 7 (6): 2222-33). The structural formula is shown as a representative example.
　具体的には、構築は、以下のとおり行った。
（１）実施例３と同様に、ＡＦＰ－Ｌ３とＡＦＰ－Ｌ１を１０％ブロッキング試薬でそれぞれ１μｇ／ｍＬとなるように希釈し、その希釈溶液を用いて反応を行った。
（２）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した。次いで、実施例１で調製したＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰと実施例６で調製したＨＢｓ抗原－ＨＲＰを１０％ブロッキング試薬でそれぞれ１μｇ／ｍＬとなるようにブロッキング試薬希釈し、さらにＡＡＬ（Ｊ－オイルミルズ）を１０μｇ／ｍＬ、またはＣｏｎＡ（Ｖｅｃｔｏｒ）を１０μｇ／ｍＬとなるようにいずれかを加えた混合液を調製し、その混合液を抗体固相化ウェルに加え３７℃１時間インキュベートした。
（３）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した。洗浄後のウェルに５０μＬのＴＭＢ基質溶液を加え室温で５分反応を行った。１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した。 Specifically, the construction was performed as follows.
(1) In the same manner as in Example 3, AFP-L3 and AFP-L1 were each diluted with a 10% blocking reagent to 1 μg / mL, and the reaction was performed using the diluted solution.
(2) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Then, the 1 [mu] g / mL respectively mouse IgG F (ab ') 2 -HRP and 10% blocking reagent the HBs antigen -HRP prepared in Example 6 with an N-type sugar chain binding consensus sequence prepared in Example 1 Then, dilute the blocking reagent in the same way, and then prepare a mixed solution to which either AAL (J-Oil Mills) is added at 10 μg / mL or ConA (Vector) is added at 10 μg / mL. Incubated at 37 ° C for 1 hour in addition to phased wells.
(3) The antibody-immobilized well was washed 3 times with 250 μL of PBST. 50 μL of TMB substrate solution was added to the well after washing, and the reaction was performed at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
　その結果、ＡＡＬおよびＣｏｎＡは、Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰの存在下で、ＬＣＡと同様にＡＦＰ結合糖鎖を検出することができた（表９、図１５、１６）。 As a result, AAL and ConA can detect AFP-linked glycans in the same manner as LCA in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type glycan-binding consensus sequence. (Table 9, FIGS. 15 and 16).
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　以上より、ＬＣＡに限らず他のレクチンについても、糖タンパク質の存在下で、ＡＦＰ結合糖鎖を検出できることが示された。 From the above, it was shown that AFP-linked sugar chains can be detected not only for LCA but also for other lectins in the presence of glycoprotein.
実施例１０：レクチン反応性標識抗体またはレクチン反応性標識抗原の存在下におけるレクチンによるＰＳＡ結合糖鎖の検出系の構築
　ＡＡＬ反応性糖鎖がＰＳＡ（前立腺特異的抗原）に結合していることが報告されている。Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰとＨＢｓ抗原－ＨＲＰの存在下で、ＡＡＬをレクチンとして用いた、下記構造式を有するＰＳＡ結合ＡＡＬ反応性フコシル化糖鎖の検出系を構築した。 Example 10: Construction of a detection system for PSA-binding sugar chains by lectins in the presence of lectin-reactive labeled antibodies or lectin-reactive labeled antigens AAL-reactive sugar chains are bound to PSA (prostate specific antigen) It has been reported. A PSA-binding AAL-reactive fucosylated glycan having the following structural formula using AAL as a lectin in the presence of mouse IgG F (ab ′) 2 -HRP and HBs antigen-HRP having an N-type glycan-binding consensus sequence A detection system was constructed.
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
　上記構造式については、既報（Ｔａｊｉｒｉ　ｅｔ　ａｌ．，Ｊ　Ｇｌｙｃｏｂｉｏｌｏｇｙ．　２００８　Ｊａｎ；１８（１）：２－８）により、前立腺癌検３例を解析し、Ｒｅｌａｔｉｖｅ　ａｂｕｎｄａｎｃｅが共通して最も高いと報告されている構造式を代表例として示した。 As for the above structural formula, three cases of prostate cancer were analyzed and reported as having the highest relative abundance according to the previous report (Tajiri et al., J Glycobiology. 2008 Jan; 18 (1): 2-8). The structural formula is shown as a representative example.
　具体的には、構築は、以下のとおり行った。
（１）ａｎｔｉ－ＰＳＡ　Ｆ（ａｂ’）２を１０μｇ／ｍＬとなるようにＰＢＳで希釈した。Ｕ９６　ＭａｘｉＳｏｒｐ　Ｎｕｎｃ－Ｉｍｍｕｎｏ　Ｐｌａｔｅにおいて、希釈された溶液　５０μＬを４℃２４時間インキュベートして、上記抗体を固相化した。
（２）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した後、１０％ブロッキング試薬　１５０μＬで３７℃１時間ブロッキングした。
（３）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬのＰＢＳＴで３回洗浄し、ルミパルスＰＳＡ－Ｎ標準ＰＳＡ溶液（富士レビオ）１００ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、５ｎｇ／ｍＬ、０．５ｎｇ／ｍＬ、０ｎｇ／ｍＬを抗体固相化ウェルに５０μＬ加え、３７℃１時間インキュベートした。
（４）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した。実施例１で調製したＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰと実施例６で調製したＨＢｓ抗原－ＨＲＰを、１０％ブロッキング試薬でそれぞれ１μｇ／ｍＬとなるように希釈し、さらにＡＡＬを１０μｇ／ｍＬとなるようにそれぞれ加えた。その溶液を抗体固相化ウェルに加え３７℃１時間インキュベートした。
（５）抗体固相化ウェルを、ＰＢＳＴ　２５０μＬで３回洗浄した。洗浄後のウェルに５０μＬのＴＭＢ基質溶液を加え室温で５分反応を行った。１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した。 Specifically, the construction was performed as follows.
(1) Anti-PSA F (ab ′) 2 was diluted with PBS so as to be 10 μg / mL. In the U96 MaxiSorp Nunc-Immuno Plate, 50 μL of the diluted solution was incubated at 4 ° C. for 24 hours to immobilize the antibody.
(2) The antibody-immobilized well was washed with 250 μL of PBST three times, and then blocked with 150 μL of 10% blocking reagent at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with PBST 250 μL of PBST, and Lumipulse PSA-N standard PSA solution (Fujirebio) 100 ng / mL, 40 ng / mL, 5 ng / mL, 0.5 ng / mL, 0 ng / ML was added to antibody-immobilized wells and incubated at 37 ° C for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Mouse IgG F (ab ′) 2 -HRP having the N-type sugar chain binding consensus sequence prepared in Example 1 and HBs antigen-HRP prepared in Example 6 were each 1 μg / mL with 10% blocking reagent. AAL was further added to a concentration of 10 μg / mL. The solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(5) The antibody-immobilized well was washed 3 times with 250 μL of PBST. 50 μL of TMB substrate solution was added to the well after washing, and the reaction was performed at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
　その結果、ＡＡＬは、Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰの存在下で、ＰＳＡ結合ＡＡＬ反応性糖鎖を濃度依存的に検出することができた（表１０、図１７）。 As a result, AAL can detect PSA-bound AAL-reactive sugar chains in a concentration-dependent manner in the presence of mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain-binding consensus sequence. (Table 10, FIG. 17).
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　以上より、レクチンは、レクチン反応性標識抗体またはレクチン反応性標識抗原の存在下で、ＡＦＰ－Ｌ３のみならず、ＰＳＡ等の他のレクチン反応性糖タンパク質を検出できることが示された。 From the above, it was shown that lectins can detect not only AFP-L3 but also other lectin reactive glycoproteins such as PSA in the presence of lectin reactive labeled antibodies or lectin reactive labeled antigens.
実施例１１：糖タンパク質によるレクチンと標的分子との結合力の増強（１）
　本実施例は、糖タンパク質によるレクチンと標的分子との結合力の増強について証明するため行った。標的分子（ＡＦＰ－Ｌ３）とレクチン（ＬＣＡ）の反応後に洗浄し、糖タンパク質（Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰ）で検出した条件（洗浄条件）と、標的分子（ＡＦＰ－Ｌ３）を糖タンパク質（Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰ）の存在下でレクチン（ＬＣＡ）と反応させた条件（同時添加条件）で、標的分子の検出能に差があるか検討した。洗浄回数は、条件間でそろえた。 Example 11: Enhancement of binding force between lectin and target molecule by glycoprotein (1)
This example was performed to prove the enhancement of the binding force between the lectin and the target molecule by the glycoprotein. Conditions after washing with the target molecule (AFP-L3) and lectin (LCA) and detecting with a glycoprotein (mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain binding consensus sequence) (Washing conditions) and target molecule (AFP-L3) in the presence of glycoprotein (mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having N-type sugar chain binding consensus sequence) lectin (LCA) It was examined whether there was a difference in the ability to detect the target molecule under the reaction conditions (simultaneous addition conditions). The number of washings was adjusted between conditions.
　具体的には、検討は、以下のとおり行った。
（１）ａｎｔｉ－ＡＦＰ　Ｆ（ａｂ’）２を１０μｇ／ｍＬとなるようにＰＢＳで希釈した。Ｆ１６　ＭａｘｉＳｏｒｐ　Ｎｕｎｃ－Ｉｍｍｕｎｏ　Ｐｌａｔｅ（Ｔｈｅｒｍｏ）において、希釈された溶液　１００μＬを４℃２４時間インキュベートして、上記抗体を固相化した。
（２）抗体固相化ウェルを、ＰＢＳＴ　３００μＬで３回洗浄した後、１０％ブロッキング試薬　２００μＬで３７℃１時間ブロッキングした。
（３）抗体固相化ウェルを、ＰＢＳＴ　３００μＬで３回洗浄した。その後、ミュータスワコーＡＦＰ－Ｌ３の測定値から濃度を換算したＡＦＰ－Ｌ３溶液とＡＦＰ－Ｌ１溶液をそれぞれ１０％ブロッキング試薬で５００ｎｇ／ｍＬとなるように希釈した。その希釈された溶液　１００μＬを加え３７℃１時間インキュベートした。
（４）抗体固相化ウェルを、ＰＢＳＴ　３００μＬで３回洗浄した。洗浄条件では、ＬＣＡを１００μｇ／ｍＬとなるように１０％ブロッキング試薬で希釈し、その希釈溶液　１００μＬを抗体固相化ウェルに加え３７℃１時間インキュベートした。同時添加条件では、１０％ブロッキング試薬のみを抗体固相化ウェルに１００μＬ加え、３７℃１時間インキュベートした。
（５）抗体固相化ウェルを、ＰＢＳＴ　３００μＬで３回洗浄した。洗浄条件では、実施例１で調製したマウスＩｇＧ　Ｆ（ａｂ’）２抗体Ａ－ＨＲＰ　５μｇ／ｍＬまたは実施例６で調製したＨＢｓ抗原－ＨＲＰを２μｇ／ｍＬとなるように１０％ブロッキング試薬で希釈し、その希釈溶液　１００μＬを抗体固相化ウェルに加え３７℃１時間インキュベートした。同時添加条件では、実施例１で調製したマウスＩｇＧ　Ｆ（ａｂ’）２抗体Ａ－ＨＲＰ　５μｇ／ｍＬまたはＨＢｓ抗原－ＨＲＰを２μｇ／ｍＬとなるように１０％ブロッキング試薬で希釈し、さらにＬＣＡを１００μｇ／ｍＬとなるようにそれぞれ添加した。その希釈された溶液　１００μＬを抗体固相化ウェルに加え、３７℃１時間インキュベートした。
（６）抗体固相化ウェルを、ＰＢＳＴ　３００μＬで３回洗浄した。洗浄後のウェルにＴＭＢ基質溶液　１００μＬを加え室温で５分反応を行った。１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した。 Specifically, the examination was conducted as follows.
(1) Anti-AFP F (ab ′) 2 was diluted with PBS so as to be 10 μg / mL. In F16 MaxiSorp Nunc-Immuno Plate (Thermo), 100 μL of the diluted solution was incubated at 4 ° C. for 24 hours to immobilize the antibody.
(2) The antibody-immobilized well was washed with 300 μL of PBST three times, and then blocked with 200 μL of 10% blocking reagent at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Thereafter, the AFP-L3 solution and the AFP-L1 solution, the concentrations of which were converted from the measured values of Mutus Wako AFP-L3, were each diluted with a 10% blocking reagent to 500 ng / mL. 100 μL of the diluted solution was added and incubated at 37 ° C. for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Under washing conditions, LCA was diluted with a 10% blocking reagent so as to be 100 μg / mL, and 100 μL of the diluted solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour. Under the simultaneous addition conditions, 100 μL of 10% blocking reagent alone was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(5) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Under washing conditions, mouse IgG F (ab ′) 2 antibody A-HRP prepared in Example 1 5 μg / mL or HBs antigen-HRP prepared in Example 6 was diluted with 10% blocking reagent so as to be 2 μg / mL. Then, 100 μL of the diluted solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour. Under the simultaneous addition conditions, mouse IgG F (ab ′) 2 antibody A-HRP prepared in Example 1 was diluted with 10% blocking reagent to 5 μg / mL or HBs antigen-HRP at 2 μg / mL, and LCA was further diluted. Each was added so as to be 100 μg / mL. 100 μL of the diluted solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(6) The antibody-immobilized well was washed 3 times with 300 μL of PBST. To the well after washing, 100 μL of TMB substrate solution was added and reacted at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
　その結果、ＡＦＰ－Ｌ３とレクチン（ＬＣＡ）との反応後に洗浄ステップを入れることで、ＡＦＰ－Ｌ３に対する反応性は著しく減弱した（図１８、１９）。ＡＦＰ－Ｌ３を、標識糖タンパク質（Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２－ＨＲＰまたはＨＢｓ抗原－ＨＲＰ）の存在下でレクチン（ＬＣＡ）と反応させた同時添加条件でのみ、ＡＦＰ－Ｌ３を特異的に検出することができた（図１８、１９）。 As a result, the reactivity to AFP-L3 was significantly attenuated by adding a washing step after the reaction between AFP-L3 and lectin (LCA) (FIGS. 18 and 19). AFP-L3 was reacted with lectin (LCA) in the presence of labeled glycoprotein (mouse IgG F (ab ′) 2 -HRP or HBs antigen-HRP having an N-type sugar chain binding consensus sequence) in the presence of simultaneous addition conditions. Only AFP-L3 could be detected specifically (FIGS. 18 and 19).
　以上より、レクチン（ＬＣＡ）を糖タンパク質の存在下で標的分子（ＡＦＰ－Ｌ３）と反応させることにより、レクチンと標的分子との結合力が増強することが示された。 From the above, it was shown that the binding force between the lectin and the target molecule is enhanced by reacting the lectin (LCA) with the target molecule (AFP-L3) in the presence of glycoprotein.
実施例１２：糖タンパク質によるレクチンと標的分子との結合力の増強（２）
　本実施例は、糖タンパク質によるレクチンと標的分子との結合力の増強について証明するため行った。標的分子（ＡＦＰ結合フコシル化糖鎖）とレクチン（ＡＡＬ）の反応後に洗浄し、糖タンパク質（ＨＢｓ抗原－ＨＲＰ）で検出した条件（洗浄条件）と、標的分子（ＡＦＰ結合フコシル化糖鎖）を糖タンパク質（ＨＢｓ抗原－ＨＲＰ）の存在下でレクチン（ＡＡＬ）と反応させた条件（同時添加条件）で、標的分子の検出能に差があるか検討した。洗浄回数は、条件間でそろえた。 Example 12: Enhancement of binding force between lectin and target molecule by glycoprotein (2)
This example was performed to prove the enhancement of the binding force between the lectin and the target molecule by the glycoprotein. Washing after the reaction of the target molecule (AFP-linked fucosylated sugar chain) and lectin (AAL), and the conditions (washing conditions) detected by glycoprotein (HBs antigen-HRP) and the target molecule (AFP-linked fucosylated sugar chain) It was examined whether there was a difference in target molecule detection ability under the conditions (simultaneous addition conditions) in which lectin (AAL) was reacted in the presence of glycoprotein (HBs antigen-HRP). The number of washings was adjusted between conditions.
　具体的には、検討は、以下のとおり行った。
（１）実施例１１と同様に、ａｎｔｉ－ＡＦＰ　Ｆ（ａｂ’）２を固相化したウェルにそれぞれ５００ｎｇ／ｍＬとなるように希釈したＡＦＰ－Ｌ３とＡＦＰ－Ｌ１を１００μＬ加え、３７℃１時間インキュベートした。
（２）抗体固相化ウェルを３００μＬのＰＢＳＴで３回洗浄した。洗浄条件では、ＡＡＬを１０μｇ／ｍＬとなるように１０％ブロッキング試薬で希釈し、希釈されたＡＡＬ溶液　１００μＬを抗体固相化ウェルに加え、得られた溶液を３７℃１時間インキュベートした。同時添加条件では、抗体固相化ウェルに１０％ブロッキング試薬のみを１００μＬ加え、得られた溶液を３７℃１時間インキュベートした。
（３）抗体固相化ウェルをＰＢＳＴ　３００μＬで３回洗浄した。洗浄条件では、実施例６で調製したＨＢｓ抗原－ＨＲＰを２μｇ／ｍＬとなるように１０％ブロッキング試薬で希釈し、希釈された溶液　１００μＬを３７℃１時間インキュベートした。同時添加条件では、実施例６で調製したＨＢｓ抗原－ＨＲＰを２μｇ／ｍＬとなるように１０％ブロッキング試薬で希釈し、希釈されたＨＢｓ抗原－ＨＲＰ溶液に、さらにＡＡＬを１０μｇ／ｍＬとなるようにそれぞれ添加し、抗体固相化ウェルに希釈された溶液　１００μＬを加え３７℃１時間インキュベートした。
（４）（３）で得られた溶液を３００μＬのＰＢＳＴで３回洗浄した。洗浄後のウェルに１００μＬのＴＭＢ基質溶液を加え室温で５分反応させた。１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した。 Specifically, the examination was conducted as follows.
(1) In the same manner as in Example 11, 100 μL of AFP-L3 and AFP-L1 diluted to 500 ng / mL were added to wells on which anti-AFP F (ab ′) 2 had been immobilized, respectively. Incubated for hours.
(2) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Under washing conditions, AAL was diluted with a 10% blocking reagent so as to be 10 μg / mL, 100 μL of the diluted AAL solution was added to the antibody-immobilized well, and the resulting solution was incubated at 37 ° C. for 1 hour. Under the simultaneous addition conditions, 100 μL of 10% blocking reagent alone was added to the antibody-immobilized well, and the resulting solution was incubated at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Under washing conditions, HBs antigen-HRP prepared in Example 6 was diluted with 10% blocking reagent so as to be 2 μg / mL, and 100 μL of the diluted solution was incubated at 37 ° C. for 1 hour. Under the simultaneous addition conditions, the HBs antigen-HRP prepared in Example 6 was diluted with a 10% blocking reagent to 2 μg / mL, and AAL was further added to 10 μg / mL in the diluted HBs antigen-HRP solution. 100 μL of the diluted solution was added to each antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(4) The solution obtained in (3) was washed 3 times with 300 μL of PBST. 100 μL of TMB substrate solution was added to the wells after washing and allowed to react at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
　その結果、ＡＦＰ結合フコシル化糖鎖とレクチン（ＡＡＬ）との反応後に洗浄ステップを入れることで、ＡＦＰ結合フコシル化糖鎖に対する反応性は著しく減弱した（図２０）。ＡＦＰ結合フコシル化糖鎖を、標識糖タンパク質（ＨＢｓ抗原－ＨＲＰ）の存在下でレクチン（ＡＡＬ）と反応させた同時添加条件でのみ、ＡＦＰ結合フコシル化糖鎖を検出することができた（図２０）。 As a result, by adding a washing step after the reaction between the AFP-bound fucosylated sugar chain and lectin (AAL), the reactivity with respect to the AFP-bound fucosylated sugar chain was remarkably attenuated (FIG. 20). The AFP-bound fucosylated sugar chain could be detected only under the simultaneous addition conditions in which the AFP-bound fucosylated sugar chain was reacted with lectin (AAL) in the presence of the labeled glycoprotein (HBs antigen-HRP) (FIG. 20).
　以上より、レクチン（ＡＡＬ）を糖タンパク質の存在下で標的分子（ＡＦＰ結合フコシル化糖鎖）と反応させることにより、レクチンとその標的分子との結合力が増強することが示された。 From the above, it was shown that the binding force between a lectin and its target molecule is enhanced by reacting the lectin (AAL) with a target molecule (AFP-linked fucosylated sugar chain) in the presence of glycoprotein.
実施例１３：Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２を固相に用いた、ＡＦＰ－Ｌ３の検出系の構築
　本実験は、実施例１で調製したマウスＩｇＧ　Ｆ（ａｂ’）２が固相上でＡＦＰ－Ｌ３を捕捉できるかどうかを証明するため行った。Ｎ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体ＡおよびＢ、ならびにＮ型糖鎖結合コンセンサス配列を有しないマウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体ＣおよびＤを固相化し、次いでＡＦＰ－Ｌ３およびＡＦＰ－Ｌ１をＬＣＡ共存下でこれらの固相化抗体と反応させることにより、マウスＩｇＧ　Ｆ（ａｂ’）２がＡＦＰ－Ｌ３を特異的に捕捉できるかどうか検討した。 Example 13: Construction of detection system for AFP-L3 using mouse IgG F (ab ′) 2 having an N-type sugar chain binding consensus sequence as a solid phase This experiment was conducted using mouse IgG F ( This was done to prove whether ab ′) 2 can capture AFP-L3 on the solid phase. Mouse IgG F (ab ′) 2 fragment antibodies A and B having an N-type sugar chain binding consensus sequence and mouse IgG F (ab ′) 2 fragment antibodies C and D having no N-type sugar chain binding consensus sequence Then, AFP-L3 and AFP-L1 were reacted with these immobilized antibodies in the presence of LCA to examine whether mouse IgG F (ab ′) 2 can specifically capture AFP-L3.
　具体的には、検討は以下のとおり行った。
（１）実施例１で調製した精製マウスＩｇＧ　Ｆ（ａｂ’）２フラグメント抗体Ａ、Ｂ、ＣおよびＤを１０μｇ／ｍＬとなるようにＰＢＳで希釈した。Ｕ９６　ＭａｘｉＳｏｒｐ　Ｎｕｎｃ－Ｉｍｍｕｎｏ　Ｐｌａｔｅにおいて、希釈された溶液　５０μＬを３７℃１時間インキュベートして固相化した。
（２）抗体固相化ウェルをＰＢＳＴ　２５０μＬで３回洗浄し、１０％ブロッキング試薬　１５０μＬで３７℃２４時間ブロッキングした。
（３）抗体固相化ウェルをＰＢＳＴ　２５０μＬで３回洗浄し、ミュータスワコーＡＦＰ－Ｌ３の測定値から濃度を換算したＡＦＰ－Ｌ３溶液とＡＦＰ－Ｌ１溶液をそれぞれ１０％ブロッキング試薬で５μｇ／ｍＬとなるように希釈し、さらにＬＣＡを１００μｇ／ｍＬとａｎｔｉ－ＡＦＰ　Ｆ（ａｂ’）２－ＨＲＰを１μｇ／ｍＬとなるように添加して溶液を調製した。調製された溶液　５０μＬを抗体固相化ウェルに加え３７℃１時間インキュベートした。
（４）抗体固相化ウェルをＰＢＳＴ　２５０μＬで３回洗浄した。次いで、ＴＭＢ基質溶液　５０μＬを加え室温で５分反応させた。１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した。 Specifically, the examination was conducted as follows.
(1) The purified mouse IgG F (ab ′) 2 fragment antibodies A, B, C and D prepared in Example 1 were diluted with PBS so as to be 10 μg / mL. In a U96 MaxiSorp Nunc-Immuno Plate, 50 μL of the diluted solution was incubated at 37 ° C. for 1 hour to be immobilized.
(2) The antibody-immobilized well was washed with 250 μL of PBST three times, and blocked with 150 μL of 10% blocking reagent at 37 ° C. for 24 hours.
(3) The antibody-immobilized well was washed with 250 μL of PBST three times, and the AFP-L3 solution and the AFP-L1 solution, whose concentrations were converted from the measured values of Mutus Wako AFP-L3, were each 5 μg / mL with a 10% blocking reagent. The solution was further diluted by adding LCA to 100 μg / mL and anti-AFP F (ab ′) 2 -HRP to 1 μg / mL. 50 μL of the prepared solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Next, 50 μL of TMB substrate solution was added and reacted at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
　その結果、ＬＣＡは、ＬＣＡ反応性抗体フラグメントＡまたはＢを固相とした条件下でＡＦＰ－Ｌ３を特異的に検出することができた（図２１）。 As a result, LCA was able to specifically detect AFP-L3 under conditions in which LCA-reactive antibody fragment A or B was used as a solid phase (FIG. 21).
　以上より、固相化されたレクチン反応性糖鎖含有実体をレクチンと併用することによりレクチン標的分子を捕捉できることが示された。 From the above, it was shown that a lectin target molecule can be captured by using a solid-phased lectin-reactive sugar chain-containing entity together with a lectin.
実施例１４：可変領域にＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ－ＨＲＰを用いた、ヘモペキシン（ＨＰＸ）結合糖鎖とトランスフェリン（ＴＦ）結合糖鎖の検出系の構築
　可変領域にＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧを用いて、ＡＡＬと共存下で反応させ、肝臓癌患者血清中のＨＰＸ結合ＡＡＬ反応性フコシル化糖鎖とＴＦ結合ＡＡＬ反応性フコシル化糖鎖を検出できるかどうか検討した。 Example 14: Construction of hemopexin (HPX) -linked sugar chain and transferrin (TF) -linked sugar chain detection system using mouse IgG-HRP having an N-type sugar chain-binding consensus sequence in the variable region. Whether mouse IgG having a chain-binding consensus sequence can be reacted with AAL in the presence of AAL to detect HPX-bound AAL-reactive fucosylated glycans and TF-bound AAL-reactive fucosylated glycans in liver cancer patient serum investigated.
　具体的には、検討は以下のとおり行った。
（１）可変領域のアミノ酸配列中にＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧを、実施例１と同様にＨＲＰ標識を行った。
（２）抗ＨＰＸ抗体（Ｒ＆Ｄ　ＳＹＳＴＥＭＳ　Ｃｌｏｎｅ＃６９８８１３）と抗ＴＦ抗体（ａｂｃａｍ　Ｃｌｏｎｅ＃２Ａ２）のＮグリコシダーゼ処理を実施した。Ｎグリコシダーゼ処理は、トリス酸緩衝液にバッファー交換した抗ＨＰＸ抗体に、ＮグリコシダーゼＦ（ＲＯＣＨＥ社）を添加し、３７℃２４時間反応させた。その後、１００ＫＤａの限界ろ過を実施し、Ｎグリコシダーゼを除去した。
（３）Ｎグリコシダーゼ処理を実施した抗ＨＰＸ抗体と抗ＴＦ抗体を２μｇ／ｍＬとなるようにＰＢＳで希釈し、Ｃ９６　ＭａｘｉＳｏｕｐ　Ｎｕｎｃ－Ｉｍｍｕｎｏ　Ｐｌａｔｅ（Ｔｈｅｒｍｏ社）に５０μＬ／ｗｅｌｌで分注し、４℃２４時間固相化した。
（４）２５０μＬの１％ＢＳＡ－ＰＢＳを２００μＬ添加し、４℃２４時間ブロッキングした。
（５）３００μＬのＰＢＳＴで３回洗浄し、１％ＢＳＡ－ＰＢＳで５００倍希釈した肝臓癌患者血清２例（ＨＣＣ－１，ＨＣＣ－２）と健常人血清２例（ＮＨＳ－１，ＮＨＳ－２）を、５０μＬ加え３７℃１時間インキュベートした。
（６）３００μＬのＰＢＳＴで３回洗浄し、可変領域にＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧ－ＨＲＰを２μｇ／ｍＬとなるように、希釈液（自社製）で希釈し、さらにＡＡＬ（Ｖｅｃｔｏｒ社）を２０μｇ／ｍＬとなるようにそれぞれ加え、３７℃１時間インキュベートした。
（７）３００μＬのＰＢＳＴで３回洗浄し、５０μＬのＴＭＢ基質溶液（Ｔｈｅｒｍｏ社）を加え室温で２分反応させ、１Ｎ硫酸で反応停止後、４５０ｎｍで吸光度を測定した。 Specifically, the examination was conducted as follows.
(1) Mouse IgG having an N-type sugar chain binding consensus sequence in the amino acid sequence of the variable region was labeled with HRP in the same manner as in Example 1.
(2) N-glycosidase treatment of anti-HPX antibody (R & D SYSTEMS Clone # 6988813) and anti-TF antibody (abcam Clone # 2A2) was performed. In the N-glycosidase treatment, N-glycosidase F (ROCHE) was added to the anti-HPX antibody whose buffer was exchanged with a tris acid buffer, and reacted at 37 ° C. for 24 hours. Thereafter, ultrafiltration at 100 KDa was performed to remove N glycosidase.
(3) The N-glycosidase-treated anti-HPX antibody and anti-TF antibody were diluted with PBS to 2 μg / mL, and dispensed at 50 μL / well into C96 MaxiSoup Nunc-Immuno Plate (Thermo) at 4 ° C. Solidified for 24 hours.
(4) 200 μL of 250 μL of 1% BSA-PBS was added and blocked at 4 ° C. for 24 hours.
(5) Two liver cancer patient sera (HCC-1, HCC-2) and two healthy human sera (NHS-1, NHS-) washed three times with 300 μL PBST and diluted 500-fold with 1% BSA-PBS 2) was added and incubated at 37 ° C. for 1 hour.
(6) Wash 3 times with 300 μL of PBST, dilute mouse IgG-HRP having an N-type sugar chain binding consensus sequence in the variable region to 2 μg / mL with a diluent (manufactured in-house), and further add AAL ( Vector) was added to 20 μg / mL and incubated at 37 ° C. for 1 hour.
(7) Washed 3 times with 300 μL of PBST, added 50 μL of TMB substrate solution (Thermo), reacted at room temperature for 2 minutes, stopped with 1N sulfuric acid, and measured absorbance at 450 nm.
　その結果、可変領域にＮ型糖鎖結合コンセンサス配列を有するマウスＩｇＧを用いて、ＡＡＬと共存下、肝臓癌患者血清中のＨＰＸ結合ＡＡＬ反応性フコシル化糖鎖（図２２）とＴＦ結合ＡＡＬ反応性フコシル化糖鎖（図２３）を検出できた。 As a result, using mouse IgG having an N-type sugar chain binding consensus sequence in the variable region, HPX-bound AAL-reactive fucosylated sugar chain (FIG. 22) and TF-bound AAL reaction in liver cancer patient serum in the presence of AAL. Sex fucosylated sugar chain (FIG. 23) could be detected.
　以上より、固相化されたレクチン反応性糖鎖含有実体をレクチンと併用することによりレクチン標的分子を捕捉できることが示された。 From the above, it was shown that a lectin target molecule can be captured by using a solid-phased lectin-reactive sugar chain-containing entity together with a lectin.

Claims (16)

  1. 　レクチン反応性糖鎖含有実体の存在下において、レクチンをレクチン標的分子に結合させることを含む、レクチン標的分子の捕捉方法。 A method for capturing a lectin target molecule, comprising binding a lectin to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.
  2. 　レクチンがマメ科レクチンまたはキノコレクチンである、請求項１記載の方法。 The method according to claim 1, wherein the lectin is a legume lectin or a mushroom lectin.
  3. 　レクチンがフコース特異的レクチンである、請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the lectin is a fucose-specific lectin.
  4. 　レクチンが、レンズマメアグルチニン（ＬＣＡ）、コンカナバリンＡ（ＣｏｎＡ）またはヒイロチャワンタケレクチン（ＡＡＬ）である、請求項１～３のいずれか一項記載の方法。 4. The method according to any one of claims 1 to 3, wherein the lectin is lentil agglutinin (LCA), concanavalin A (ConA), or yellow chawantake lectin (AAL).
  5. 　レクチン標的分子が糖タンパク質である、請求項１～４のいずれか一項記載の方法。 The method according to any one of claims 1 to 4, wherein the lectin target molecule is a glycoprotein.
  6. 　糖タンパク質がＡＦＰ－Ｌ３またはＰＳＡである、請求項５記載の方法。 The method according to claim 5, wherein the glycoprotein is AFP-L3 or PSA.
  7. 　糖タンパク質がヘモペキシン（ＨＰＸ）またはトランスフェリン（ＴＦ）である、請求項５記載の方法。 The method according to claim 5, wherein the glycoprotein is hemopexin (HPX) or transferrin (TF).
  8. 　前記実体がＮ型糖鎖結合コンセンサス配列を有する糖タンパク質である、請求項１～７のいずれか一項記載の方法。 The method according to any one of claims 1 to 7, wherein the entity is a glycoprotein having an N-type sugar chain binding consensus sequence.
  9. 　Ｎ型糖鎖結合コンセンサス配列を有する糖タンパク質がＮ型糖鎖結合コンセンサス配列を可変領域に有する免疫グロブリンである、請求項８記載の方法。 The method according to claim 8, wherein the glycoprotein having an N-type sugar chain binding consensus sequence is an immunoglobulin having an N-type sugar chain binding consensus sequence in a variable region.
  10. 　前記実体が、Ｂ型肝炎ウイルス表面抗原（ＨＢｓ抗原）、ヒト絨毛性ゴナドトロピンα鎖（ｈＣＧα）または黄体形成ホルモン（ＬＨ）である、請求項１～８のいずれか一項記載の方法。 The method according to any one of claims 1 to 8, wherein the entity is hepatitis B virus surface antigen (HBs antigen), human chorionic gonadotropin α chain (hCGα) or luteinizing hormone (LH).
  11. 　レクチンのレクチン標的分子への結合後にレクチン標的分子を測定することを含むレクチン標的分子の測定方法である、請求項１～１０のいずれか一項記載の方法。 The method according to any one of claims 1 to 10, which is a method for measuring a lectin target molecule, comprising measuring the lectin target molecule after binding of the lectin to the lectin target molecule.
  12. 　レクチン標的分子に特異的な親和性物質をさらに用いる、請求項１１記載の方法。 The method according to claim 11, wherein an affinity substance specific for the lectin target molecule is further used.
  13. 　以下を含む方法である、請求項１～１２のいずれか一項記載の方法：
    （１）レクチン標的分子を、レクチン標的分子に特異的な親和性物質に結合させて、レクチン標的分子およびレクチン標的分子に特異的な親和性物質を含む第１複合体を得ること；
    （２）レクチン反応性糖鎖含有実体の存在下においてレクチンを第１複合体と結合させて、レクチン標的分子、レクチン標的分子に特異的な親和性物質、レクチンおよびレクチン反応性糖鎖含有実体を含む第２複合体を得ること；ならびに
    （３）第２複合体に含まれるレクチン標的分子の量を測定すること。     The method according to any one of claims 1 to 12, which is a method comprising:
    (1) binding a lectin target molecule to an affinity substance specific for the lectin target molecule to obtain a first complex including the lectin target molecule and an affinity substance specific for the lectin target molecule;
    (2) A lectin is bound to the first complex in the presence of a lectin-reactive sugar chain-containing entity, and a lectin target molecule, an affinity substance specific for the lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity Obtaining a second complex comprising; and (3) measuring the amount of lectin target molecules contained in the second complex.
  14. 　レクチン標的分子、レクチンおよびレクチン反応性糖鎖含有実体を含む複合体。 A complex comprising a lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity.
  15. 　レクチンおよびレクチン反応性糖鎖含有実体を混合形態で含む、レクチン標的分子の捕捉用試薬。 A reagent for capturing a lectin target molecule, which contains a lectin and a lectin-reactive sugar chain-containing entity in a mixed form.
  16. 　レクチンおよびレクチン反応性糖鎖含有実体を含む、レクチン標的分子の捕捉用キット。 A kit for capturing a lectin target molecule, comprising a lectin and a lectin-reactive sugar chain-containing entity.
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