Method for immunologically detecting detection target substance, method for suppressing non-specific reaction, fucose-containing composition, and reagent kit

Abstract

The present invention addresses the problem of providing: a method for immunologically detecting a detection target substance having a sugar chain in a biological sample, in which the occurrence of a non-specific reaction is suppressed; a method for suppressing a non-specific reaction in a method for immunologically detecting a detection target substance having a sugar chain in a biological sample; a fucose-containing composition; and a reagent kit. According to the present invention, there is provided a method for immunologically detecting a detection target substance having a sugar chain in a biological sample, the method including reacting lectin with the detection target substance in the presence of fucose.

Description

Method for immunologically detecting a target substance, method for suppressing non-specific reactions, fucose-containing composition, and reagent kit

[0001] The present invention relates to a method for immunologically detecting a target substance having a sugar chain in a biological sample. The present invention further relates to a method for suppressing non-specific reactions in a method for immunologically detecting a target substance having a sugar chain in a biological sample. The present invention further relates to a fucose-containing composition and a reagent kit for use in the above method.

[0002] Lectins have specific binding activity to sugar chains and are used to detect glycoproteins. BC2LC is a lectin derived from the gram-negative bacterium Burkholderia cenocepacia. In particular, rBC2LCN, a recombinant protein in which the N-terminal domain is expressed in Escherichia coli, has high affinity for the O-glycan H type 3 (Fucα1-2Galβ1-3GalNAc). BC2LC is also a lectin used to detect undifferentiated cells and is known to be useful for detecting cancer cells.

[0003] On the other hand, Patent Document 1 describes a method for reducing the influence of contaminants in lectin-based immunoassays. In Patent Document 1, the influence of contaminants is reduced by adding a sugar chain compound that competes with the contaminants in a biological sample in binding between a labeled lectin and an analyte. All of the lectins used in the examples of Patent Document 1 are N-type sugar chain-recognizing lectins, and the sugar chain compound is a galactose derivative or a fucose derivative.

[0004] Patent Document 2 describes a method for preventing non-specific reactions in immunological tests, in which cells are immobilized on a support using lectins, and non-specific reactions are prevented by using two molecules of N-acetylglucosamine.

[0005] Patent Document 3 describes a method for separating cells with different degrees of undifferentiation using lectins, and describes that adding fucose during cell separation enables efficient separation of target cells.

[0006] Patent No. 6260541 JP 8-240592 JP 2022-122595

[0007] BC2LCN-reactive α1-antichymotrypsin (BC2LCN-reactive AACT) is known as a marker that shows significantly higher values ​​in pancreatic cancer patients than in healthy individuals and patients with benign diseases. The BC2LCN-reactive AACT measurement system sometimes shows nonspecific reactions in samples from non-pancreatic cancer patients, which poses a challenge.

[0008] An object of the present invention is to provide a method for immunologically detecting a target substance having a sugar chain in a biological sample, wherein non-specific reactions are suppressed.A further object of the present invention is to provide a method for suppressing non-specific reactions in a method for immunologically detecting a target substance having a sugar chain in a biological sample.A further object of the present invention is to provide a fucose-containing composition and a reagent kit for use in the above method.

[0009] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found that, in the step of reacting a target substance in a biological sample with a lectin, non-specific reactions can be suppressed by reacting the target substance with the lectin in the presence of fucose. In particular, the addition of fucose showed a greater inhibitory effect on non-specific reactions than the addition of galactose, N-acetylgalactosamine, or a fucose derivative contained in the lectin's recognition site, demonstrating that the addition of fucose is effective in suppressing non-specific reactions. The present invention was completed based on the above findings.

[0010] That is, the present invention provides the following: <1> A method for immunologically detecting a target substance having a sugar chain in a biological sample, the method comprising reacting the target substance with a lectin in the presence of fucose. <2> The method according to <1>, wherein the lectin is a lectin that recognizes O-type sugar chains. <3> The method according to <1> or <2>, wherein the lectin is a lectin that recognizes Fucα1-2Galβ1-3GlcNAc, which is an H type 1 structure, or Fucα1-2Galβ1-3GalNAc, which is an H type 3 structure. <4> The method according to any one of <1> to <3>, wherein the lectin is BC2LCN, wherein BC2LCN is a protein that contains the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained by deleting, substituting, inserting, and / or adding 1 to 10 amino acids from the amino acid sequence shown in SEQ ID NO: 1, and that specifically recognizes the sugar chain structure of Fucα1-2Galβ1-3GlcNAc or Fucα1-2Galβ1-3GalNAc. <5> The method according to any one of <1> to <4>, wherein the detection target substance is a glycoprotein, a glycopeptide, or a degradation product thereof. <6> The method according to any one of <1> to <5>, wherein the biological sample is a blood-derived sample. <7> The method according to any one of <1> to <6>, wherein the lectin is a solid-phase lectin, and the method comprises, after a reaction between the lectin and the target substance, reacting the target substance bound to the solid-phase lectin with a binding substance that specifically binds to the target substance, and detecting the binding substance bound to the target substance by detecting the target substance. <8> A method for suppressing a non-specific reaction in a method for immunologically detecting a target substance having a sugar chain in a biological sample, the method comprising reacting a lectin with the target substance in the presence of fucose. <9> A fucose-containing composition for use in a method for detecting a target substance having a sugar chain in a biological sample using a lectin. <10> A reagent kit for detecting a target substance having a sugar chain in a biological sample, the reagent kit comprising the fucose-containing composition according to <9> and a lectin. <11> The reagent kit according to <10>, wherein the lectin is a solid-phase lectin.<12> The reagent kit according to <10> or <11>, wherein the lectin is a non-immobilized lectin, and the reagent kit further comprises a solid-phase carrier. <13> The reagent kit according to any one of <10> to <12>, further comprising a binding substance that specifically binds to a detection target substance, the binding substance being optionally labeled.

[0011] According to the present invention, in a method for immunologically detecting a detection target substance having a sugar chain in a biological sample, non-specific reactions can be suppressed.

[0012] The present invention will be described in detail below. In this specification, the word "to" is used to mean that the numerical values ​​before and after it are included as the lower limit and upper limit.

[0013] <Method for immunologically detecting a target substance> The present invention relates to a method for immunologically detecting a target substance having a sugar chain in a biological sample, the method comprising reacting a lectin with the target substance in the presence of fucose.

[0014] When reacting a detection target substance having a sugar chain with a lectin, it is expected that proteins having sugar chains other than sugar chains (Fucα-1,2Galβ1-3GalNAc) that have high affinity for lectins such as BC2LCN may also weakly bind to the lectin, potentially causing a nonspecific reaction. In the present invention, it is expected that by reacting the lectin with the detection target substance in the presence of fucose, fucose competes with nonspecific reactants, thereby suppressing the nonspecific reaction.

[0015] A sugar chain refers to a group of compounds having a structure in which monosaccharides are linked by glycosidic bonds in a chain form (a linear chain or a branched chain having dendritic branches). Examples of monosaccharides that constitute sugar chains include hexoses such as glucose, galactose, and mannose; deoxyhexoses such as L-fucose; hexosamines such as N-acetylglucosamine and N-acetylgalactosamine; sialic acids such as N-acetylneuraminic acid and N-glycolylneuraminic acid; and pentoses such as xylose and L-arabinose. The number of monosaccharides that constitute a sugar chain is not particularly limited and is generally about 20 to tens of thousands, for example, 2 to 20,000.

[0016] Lectin refers to a protein that recognizes and binds to the partial structure or the entire structure of a sugar chain bound to complex carbohydrates such as glycoproteins, glycolipids, proteoglycans, glycopeptides, lipopolysaccharides, peptidoglycans, and glycosides such as steroid compounds.

[0017] The lectins used in the present invention recognize and bind to the partial or entire structure of a sugar chain possessed by a substance to be detected. In some embodiments of the present invention, the detection specificity of the substance to be detected can be further increased by using two molecules of one type of lectin for detection. In some embodiments of the present invention, two types of lectins (hereinafter sometimes referred to as lectin 1 and lectin 2) are used, and in such cases, the sugar chain structure recognized by lectin 1 and the sugar chain structure recognized by lectin 2 may be the same or different. By using lectin 1 and lectin 2 that bind to different sugar chain structures, the detection specificity of the substance to be detected can be further increased.

[0018] The lectin may be a single lectin or a mixture of multiple types of lectins. When a mixture of multiple types of lectins is used as the lectin, the sugar chain structures recognized by the lectins contained in the mixture may be the same or different.

[0019] Although lectins inherently have sugar chains, in the present invention, it may be preferable to use a lectin that does not have a sugar chain as the lectin, as long as the binding ability to the detection target substance is not reduced. In the present invention, when two types of lectins (lectin 1 and lectin 2) are used, at least one of lectin 1 and lectin 2 may be a lectin that does not have a sugar chain, or both lectin 1 and lectin 2 may be lectins that do not have a sugar chain.

[0020] By using lectins without sugar chains as lectin 1 and lectin 2, it is possible to prevent the formation of complexes between lectins 1 and 1, complexes between lectins 2 and 1, and to detect only the complex of "lectin 1-substance to be detected-lectin 2" with high sensitivity. By using a lectin without sugar chains as one of lectins 1 and 2, it is also possible to suppress the formation of complexes between lectins 1 and 1, complexes between lectins 2 and 1, and to detect the complex of "lectin 1-substance to be detected-lectin 2" with high sensitivity.

[0021] The term "glycosylated lectin" encompasses not only lectins without any sugar chain modification, but also lectins without sugar chains that can cause lectin-lectin binding via sugar chains. More specifically, any lectin that is not modified with a sugar chain having a sugar chain structure recognized by lectin 1 and / or a sugar chain having a sugar chain structure recognized by lectin 2 can be used as the lectin 1 without sugar chains. This is because even if lectin 1 has a sugar chain, if the sugar chain does not contain a sugar chain structure recognized by lectin 1 and / or lectin 2, a complex between lectins 1 and / or a complex between lectin 1 and lectin 2 will not be formed. Similarly, any lectin that is not modified with a sugar chain having a sugar chain structure recognized by lectin 1 and / or a sugar chain having a sugar chain structure recognized by lectin 2 can be used as the lectin 2 without sugar chains.

[0022] Specific examples of lectins without sugar chains include recombinant lectins expressed in prokaryotic cells and modified lectins obtained by modifying the sugar chain structure of natural proteins.

[0023] (Recombinant lectins) Because prokaryotic cells do not have a membrane structure that can modify sugar chains on proteins synthesized within the cells, recombinant lectins expressed in prokaryotic cells as host cells do not have sugar chains. As explained below, recombinant lectins can be mass-produced easily and at low cost using common genetic engineering techniques.

[0024] An example of a method for producing a recombinant lectin is described below. First, a base sequence containing a base sequence encoding the amino acid sequence of a lectin capable of binding to a target sugar chain structure is inserted into an appropriate expression vector according to a standard method to obtain a recombinant expression vector. The expression vector may also contain a base sequence containing only the base sequence encoding the amino acid sequence of the sugar chain structure-binding portion of the amino acid sequence of a lectin capable of binding to a target sugar chain structure.

[0025] The expression vector is not particularly limited as long as it has the function of expressing and producing a recombinant lectin in various host cells, and examples of the expression vector include a plasmid vector, a phage vector, and a virus vector. Specific examples include plasmid vectors such as pTrcHis2 vector, pcDNA3.1 / myc-His vector (Invitrogen), pUC119 (Takara Bio), pBR322 (Takara Bio), pBluescript II KS+ (Stratagene), Pqe-tri (Qiagen), pET, pGEM-3Z, pGEX, and pMAL; bacteriophage vectors such as λENBL3 (Stratagene) and λDASHII (Funakoshi); and cosmid vectors such as Charomid DNA (Fujifilm Wako Pure Chemical Industries, Ltd.) and Lorist6 (Fujifilm Wako Pure Chemical Industries, Ltd.). Other examples include plasmids derived from Escherichia coli (for example, pTrc99A, pKK223, pET3a), bacteriophages such as λ phage, as well as pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNA I / Neo, p3xFLAG-CMV-14, pCAT3, pcDNA3.1, pCMV, and the like.

[0026] To facilitate detection and purification of recombinant lectins, the lectins may be expressed as fusion proteins with tag peptides or other proteins, such as FLAG tag, 3xFLAG tag, and His tag (His tag, e.g., 6xHis tag).

[0027] The resulting recombinant expression vector is used to transform (transduce) suitable host cells to prepare transformants. Host cells that express and produce recombinant proteins without glycosylation are used. Examples of host cells include prokaryotes, specifically Escherichia coli and Bacillus species (e.g., B. subtilis, B. brevis, B. borstellens, etc.). Examples of E. coli that can be used include BL21, BL21(DE3), K-12, DH1, DH5, DH5α, M15, HB101, C600, XL-1 Blue, JM109, JM105, JM127, XL1-Blue, VCS257, and TOP10. Alternatively, competent cells, which have a higher efficiency of introducing plasmid or phage DNA, may be used. Examples of competent cells include E. coli DH5α competent cells and E. coli JM109 competent cells (manufactured by Takara Bio Inc.).

[0028] Transformation of host cells with a recombinant expression vector can be carried out using conventionally known methods. For example, when the host cell is Escherichia coli, transformation can be carried out by the heat shock method (chemical transformation method), electroporation, etc. When using commercially available competent cells, transformation can be carried out according to the product protocol.

[0029] To confirm that a transformant (transducant) expresses and produces a recombinant lectin, a conventional hybridization method such as Southern hybridization or colony hybridization using a probe can be applied. The following method can also be used.

[0030] When the recombinant lectin is not secreted into the culture medium of the transformant, for example, when it is expressed as a transmembrane protein, the resulting transformant is treated by a standard method for disrupting or lysing cells (e.g., ultrasonication, treatment with a homogenizer, treatment with a membrane-dissolving agent such as a surfactant, etc.) to obtain a lysate. The lysate can then be further purified, if necessary, and then subjected to a standard immunoassay (dot Western blotting, Western blotting, etc.) using an antibody against the tag peptide to confirm that the tag peptide is expressed in the lysate.

[0031] Furthermore, when the recombinant lectin is secreted into the culture medium of the transformant, the culture medium (culture supernatant) is subjected to the same confirmation procedure as that performed for the lysate.

[0032] The transformant is cultured in a nutrient medium to produce the recombinant lectin. The culture is carried out by a conventionally known method, and the temperature, pH of the medium, and culture time can be appropriately set. When the host cell is Escherichia coli, the transformant can be cultured in a commonly used liquid medium under standard conditions for culturing Escherichia coli.

[0033] Recombinant lectin can be obtained from the culture obtained by culturing as follows. Specifically, when the recombinant lectin is present in the periplasm or cytoplasm of the transformant, the bacterial cells or cells are recovered from the culture by methods such as filtration or centrifugation and resuspended in an appropriate buffer. The cell walls and / or cell membranes of the recovered cells are then disrupted by, for example, surfactant treatment, ultrasonic treatment, lysozyme treatment, or freeze-thawing, and a crude extract containing the recombinant lectin is obtained by methods such as centrifugation or filtration. When the recombinant lectin is secreted into the culture medium of the transformant, a culture medium (culture supernatant) is obtained. The recombinant lectin is then isolated and purified from the crude extract or culture medium (culture supernatant) using commonly used methods to avoid contamination with sugars (glycans).

[0034] Methods for isolating and purifying recombinant lectins include, for example, methods that utilize solubility such as salting out and solvent precipitation, methods that utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, methods that utilize charge such as ion exchange chromatography, methods that utilize specific affinity such as affinity chromatography, methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography, and methods that utilize differences in isoelectric point such as isoelectric focusing. The purified recombinant lectin can be confirmed, for example, by ELISA using an anti-His antibody.

[0035] (Modified Lectin) A modified lectin can be obtained by treating a natural lectin with an acid or a glycolytic enzyme.

[0036] By treating native lectins with acids such as periodic acid or its salts (sodium salt, potassium salt, etc.), trifluoromethanesulfonic acid, etc., the hydroxyl groups of the sugar chains can be oxidized, thereby changing the overall structure of the sugar chains or partial structures within the sugar chains. Furthermore, sugar chain structures can also be removed by beta-decomposition using alkali treatment. This alters the sugar chain structures present in the sugar chains of native lectins that are recognized by the native lectins or sugar chain structures recognized by other lectins, thereby converting the native lectins into modified lectins that do not induce lectin-to-lectin binding via sugar chains. The acid treatment can be performed by known techniques.

[0037] Furthermore, sugar chains can be removed from native lectins by treating them with glycolytic enzymes such as glycanases (N-glycanase, O-glycanase, etc.), mannosidase, galactosidase, keratanase, chondroitinase, sialidase, fucosidase, N-acetylglucosaminidase, and N-acetylhexosaminidase. Alternatively, the sugar chains of native lectins can be cleaved by glycolytic enzyme treatment to change the overall structure of the sugar chain or a partial structure within the sugar chain. By doing so, the sugar chain structure recognized by the native lectin or the sugar chain structure recognized by other lectins, present in the sugar chains of the native lectin, can be modified, thereby converting the native lectin into a modified lectin that does not induce lectin-lectin binding via the sugar chain. The enzyme treatment can be performed by a conventionally known method.

[0038] However, modified lectins obtained by acid treatment, alkali treatment, or glycolytic enzyme treatment may lose or weaken their binding ability to sugar chains due to protein denaturation, so it is more preferable to use the above-mentioned recombinant lectins as lectins.

[0039] The lectin of the present invention is preferably a lectin that recognizes O-type sugar chains. Examples of lectins that recognize O-type sugar chains include BC2LCN and dog jelly lectin (MALII). The lectin of the present invention is more preferably a lectin that recognizes Fucα1-2Galβ1-3GlcNAc, which is an H type 1 structure, or Fucα1-2Galβ1-3GalNAc, which is an H type 3 structure.

[0040] In the sugar chain structure of Fucα1-2Galβ1-3GlcNAc, the hydroxyl group at position 4 of GlcNAc may be substituted with a monosaccharide (preferably fucose) or a branched or unbranched oligosaccharide chain (preferably a sugar chain consisting of 2 to 5 sugars). Furthermore, on the surface of undifferentiated stem cells, the sugar chain structure is a sugar chain bound to the non-reducing end of a glycoprotein, glycolipid, sugar, or the like at position 1 of GlcNAc as a membrane component. Therefore, the sugar chain structure secreted into the culture supernatant of undifferentiated stem cells also has an OH group, or the non-reducing end of another sugar, protein, lipid, or other molecule bound at position 1 of GlcNAc. That is, the sugar chain structure can be represented by the following formula (1):

[0041]

[0042] (In the formula, R1 represents an OH group or any sugar chain, for example, a 4αFuc group. R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)

[0043] In the sugar chain structure of Fucα1-2Galβ1-3GalNAc, the hydroxyl group at position 1 of GalNAc may be substituted with a branched or unbranched oligosaccharide chain (preferably a sugar chain consisting of 2 to 5 sugars). Furthermore, on the surface of undifferentiated stem cells, this sugar chain is a sugar chain that is bound to the non-reducing end of a glycoprotein, glycolipid, sugar, or the like at position 1 of GalNAc as a membrane component. Therefore, the sugar chain structure secreted into the culture supernatant of undifferentiated stem cells also has an OH group, or the non-reducing end of another sugar, protein, lipid, or other molecule bound at position 1 of GalNAc. That is, the sugar chain structure can be represented by the following formula (2):

[0044]

[0045] (In the formula, R1 represents an OH group or any sugar chain, for example, a Galβ1-4Glc group. R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)

[0046] In the present invention, the lectin is particularly preferably BC2LCN, which is a protein that contains the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained by deleting, substituting, inserting, and / or adding 1 to 10 amino acids from the amino acid sequence shown in SEQ ID NO: 1, and specifically recognizes the sugar chain structure Fucα1-2Galβ1-3GlcNAc or Fucα1-2Galβ1-3GalNAc.

[0047] rBC2LCN refers to a recombinant lectin found in the gram-negative bacterium Burkholderia cenocepacia, expressed in Escherichia coli. This lectin corresponds to the N-terminal domain of a protein called BC2L-C (GenBank / NCBI-GI accession number: YP_002232818). BC2LCN (rBC2LCN) shows structural similarity to TNF-like proteins and is known to form trimers. Analysis using glycan arrays has revealed that this lectin exhibits binding specificity not only for "Fucα1-2Galβ1-3GlcNAc (H type 1 glycan)" and "Fucα1-2Galβ1-3GalNAc (H type 3 glycan)," but also for "Lewis b glycan (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc)" and "Globo H glycan (Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc)," which are glycan structures containing H type 1 or H type 3 glycans.

[0048] Because BC2LCN does not contain a sugar chain, it can be mass-produced using transformed bacteria. Specifically, the BC2LCN gene encoding the amino acid sequence (SEQ ID NO: 1) of GenBank / NCBI-GI registration number: YP_002232818 (Genome ID: 206562055) is used, and after appropriate optimization for the host, it can be expressed in transformed Escherichia coli and purified by conventional protein purification methods.

[0049] SEQ ID NO: 1: Met Pro Leu Leu Ser Ala Ser Ile Val Ser Ala Pro Val Val Thr Ser Glu Thr Tyr Val Asp Ile Pro Gly Leu Tyr Leu Asp Val Ala Lys Ala Gly Ile Arg Asp Gly Lys Leu Gln Val Ile Leu Asn Val Pro Thr Pro Tyr Ala Thr Gly Asn Asn Phe Pro Gly Ile Tyr Phe Ala Ile Ala Thr Asn Gln Gly Val Val Ala Asp Gly Cys Phe Thr Tyr Ser Ser Lys Val Pro Glu Ser Thr Gly Arg Met Pro Phe Thr Leu Val Ala Thr Ile Asp Val Gly Ser Gly Val Thr Phe Val Lys Gly Gln Trp Lys Ser Val Arg Gly Ser Ala Met His Ile Asp Ser Tyr Ala Ser Leu Ser Ala Ile Trp Gly Thr Ala Ala Pro Ser Ser Gln Gly Ser Gly Asn Gln Gly Ala Glu Thr Gly Gly Thr Gly Ala Gly Asn Ile Gly Gly Gly

[0050] BC2LCN does not need to be the full length corresponding to SEQ ID NO: 1, and even if some amino acids in SEQ ID NO: 1 are partially deleted, substituted, inserted and / or added, it is sufficient that the BC2LCN maintains the property of specifically recognizing "Fucα1-2Galβ1-3GlcNAc / GalNAc", i.e., the sugar chain structure represented by (Formula 1) or (Formula 2).

[0051] Specifically, BC2LCN or a variant thereof can be expressed, for example, as follows: "A protein that comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained by deleting, substituting, inserting and / or adding 1 to 10 amino acids from the amino acid sequence shown in SEQ ID NO: 1, and that specifically recognizes the sugar chain structure of Fucα1-2Galβ1-3GlcNAc or Fucα1-2Galβ1-3GalNAc."

[0052] Furthermore, if the sugar chain structure is represented using the above (Formula 1) and (Formula 2), it can be expressed as "a protein that contains the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which 1 to 10 amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, inserted and / or added, and that specifically recognizes the sugar chain structure shown in (Formula 1) or (Formula 2)." Here, 1 to 10 preferably means 1 to 7, more preferably 1 to 5, and even more preferably 1 to 3.

[0053] In the present invention, a detection target substance having a sugar chain in a biological sample is immunologically detected. The biological sample is preferably a sample derived from a human subject. Examples of biological samples include, but are not limited to, blood-derived samples (blood, plasma, serum, etc.), urine, saliva, lymph, cerebrospinal fluid, pleural effusion, ascites, tears, semen, bladder washings, tissue extracts, tissue sections, tissue biopsy samples, and samples prepared from these. The biological sample is preferably a blood-derived sample, more preferably serum or plasma, and particularly preferably serum.

[0054] Examples of the substance to be detected include glycoconjugates selected from the group consisting of glycoproteins, glycopeptides, glycolipids, proteoglycans, lipopolysaccharides, peptidoglycans, and glycosides in which a sugar chain is bound to a steroid compound, or degradation products thereof. The substance to be detected is preferably a glycoprotein, a glycopeptide, or degradation products thereof.

[0055] In the present invention, a lectin is reacted with a substance to be detected in the presence of fucose. Fucose is 6-deoxy-galactose. The concentration of fucose in a reaction solution containing the substance to be detected is preferably 0.01 to 1 mmol / L, more preferably 0.02 to 0.5 mmol / L.

[0056] The present invention can be implemented in the following manner: (1) A biological sample is mixed with fucose. The resulting mixture is brought into contact with an immobilized lectin. As a result, the target substance in the biological sample binds to the immobilized lectin, forming a complex 1 between the lectin and the target substance. Next, after removing any substances that are not bound to the immobilized lectin, a labeled antibody that binds to the target substance is brought into contact with the complex 1 between the target substance and the lectin, forming a complex 2 between the lectin, the target substance, and the labeled antibody. After removing the unbound labeled antibody, the target substance in the biological sample can be detected by detecting the immobilized label.

[0057] (2) A biological sample and fucose are brought into contact with the immobilized lectin. As a result, the target substance in the biological sample binds to the immobilized lectin, forming a complex 1 between the lectin and the target substance. Next, after removing any substances that are not bound to the immobilized lectin, a labeled antibody that binds to the target substance is brought into contact with the complex of the target substance and the lectin, forming a complex 2 between the lectin, the target substance, and the labeled antibody. After removing the unbound labeled antibody, the target substance in the biological sample can be detected by detecting the immobilized label.

[0058] (3) A biological sample is contacted with an immobilized antibody (this antibody is an antibody that binds to the target substance). As a result, the target substance in the biological sample binds to the immobilized antibody, forming a complex 1 between the antibody and the target substance. Next, after removing any substances that do not bind to the immobilized antibody, a labeled lectin is contacted with the complex of the antibody and the target substance in the presence of fucose, forming a complex 2 between the antibody, the target substance, and the labeled lectin. After removing the unbound labeled lectin, the target substance in the biological sample can be detected by detecting the immobilized label.

[0059] (4) A biological sample, an immobilized lectin, and a labeled antibody that binds to a target substance are contacted in the presence of fucose, thereby forming a complex 2 of the lectin, the target substance, and the labeled antibody. After removing unbound labeled antibody, the target substance in the biological sample can be detected by detecting the immobilized label.

[0060] (5) A biological sample is mixed with fucose. The resulting mixture is brought into contact with an immobilized lectin. As a result, the target substance in the biological sample binds to the immobilized lectin, forming a complex 1 between the lectin and the target substance. Next, after removing substances that are not bound to the immobilized lectin, a labeled lectin that binds to the target substance is brought into contact with complex 1 between the target substance and the lectin, forming complex 2 between the lectin, the target substance, and the labeled lectin. After removing the unbound labeled lectin, the target substance in the biological sample can be detected by detecting the immobilized label.

[0061] (6) A biological sample and fucose are brought into contact with the immobilized lectin. As a result, the substance to be detected in the biological sample binds to the immobilized lectin, forming a complex 1 between the lectin and the substance to be detected. Next, after removing substances that are not bound to the immobilized lectin, a labeled lectin that binds to the substance to be detected is brought into contact with complex 1 between the substance to be detected and the lectin, forming complex 2 between the lectin, the substance to be detected, and the labeled lectin. After removing the unbound labeled lectin, the substance to be detected in the biological sample can be detected by detecting the immobilized label.

[0062] (7) A biological sample, an immobilized lectin, and a labeled lectin that binds to a target substance are contacted in the presence of fucose, thereby forming a complex 2 of the lectin, the target substance, and the labeled lectin. After removing the unbound labeled lectin, the target substance in the biological sample can be detected by detecting the immobilized label.

[0063] (8) In the above (5) to (7), an unlabeled lectin can be used instead of the labeled lectin.

[0064] (9) In the above (1) to (8), a non-immobilized lectin or antibody can be used instead of the immobilized lectin or antibody.

[0065] In one example of the present invention, the lectin is a solid-phase lectin, and after the reaction between the lectin and the substance to be detected, the substance to be detected bound to the solid-phase lectin is reacted with a binding substance (antibody, lectin, etc.) that specifically binds to the substance to be detected, and the binding substance bound to the substance to be detected is detected, thereby making it possible to detect the substance to be detected.

[0066] (Sandwich method) As described above, in the present invention, preferably, a sandwich method using a "lectin," a "substance to be detected," and a "binding substance (antibody, lectin, etc.) that specifically binds to the substance to be detected" can be used.

[0067] The sandwich method includes a complex formation step and a detection step. The complex formation step is a step in which a target substance to be detected, a lectin, and a binding substance (antibody, lectin, etc.) that specifically binds to the target substance to be detected are simultaneously or sequentially contacted to form a complex between the target substance to be detected, the lectin, and the binding substance (antibody, lectin, etc.) that specifically binds to the target substance to be detected. The detection step is a step in which the target substance to be detected is detected by detecting the "complex between the lectin and the binding substance (antibody, lectin, etc.) that specifically binds to the target substance to be detected" formed in the complex formation step.

[0068] In the complex formation procedure, the target substance, the lectin, and the binding substance (antibody, lectin, etc.) that specifically binds to the target substance are contacted, and the reactions may be simultaneous or sequential. The complex formation procedure may be performed by a method that performs B / F separation using a solid phase carrier, or by a method that performs B / F separation without using a solid phase carrier.

[0069] The amount (concentration) of the binding substance that specifically binds to the detection target substance and is reacted with the biological sample is appropriately set depending on the type of detection target substance, the required measurement sensitivity, the measurement method, the measurement device, etc.

[0070] An example of a method for performing B / F separation using a solid phase carrier can include a first step of contacting a lectin 1 bound to a solid phase carrier with a substance to be detected to obtain a complex 1 composed of the lectin 1 and the substance to be detected, and a second step of contacting the complex 1 with a labeled antibody to obtain a complex 2 composed of the lectin 1, the substance to be detected, and the labeled antibody.

[0071] An example of a method for performing B / F separation using a solid phase carrier can include a first step of contacting a lectin 1 bound to a solid phase carrier with a substance to be detected to obtain a complex 1 composed of the lectin 1 and the substance to be detected, and a second step of contacting the complex 1 with a labeled lectin 2 to obtain a complex 2 composed of the lectin 1, the substance to be detected, and the labeled lectin 2. In this case, a lectin having no sugar chain may be used as the lectin 1 bound to the solid phase carrier, or both the lectin 1 and the lectin 2 may have no sugar chain.

[0072] Examples of solid-phase supports for immobilizing binding substances such as lectins or antibodies include insoluble solid-phase supports used in conventional immunoassays. Specifically, substrates used in conventional protein immobilization methods, such as glass slides, ELISA plates, microtiter plates, beads (e.g., magnetic beads), microparticles (e.g., latex particles), filters, tubes, films, and membranes, can be used. Examples of substrate materials include synthetic polymers such as polycarbonate, polystyrene, polyurethane, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber, as well as inorganic materials such as glass (e.g., porous glass), ground glass, silicon, ceramics, alumina, silica gel, activated carbon, and metal oxides. These supports with immobilized binding substances such as lectins or antibodies can be packed into columns and used, for example, in lectin affinity column methods.

[0073] The method for immobilizing a binding substance such as a lectin or an antibody on a solid support is not particularly limited, and known methods such as chemical binding (immobilization by covalent bonding) and physical adsorption can be applied. It is also possible to immobilize a lectin on a solid support using a very strong binding reaction such as the avidin-biotin reaction. In this case, a biotinylated lectin, in which biotin is bound to a lectin, can be immobilized on a streptavidin-coated streptavidin plate. Alternatively, a lectin can be immobilized on a solid support via various linkers commonly used in this field.

[0074] A method for separating complex 2 between a lectin, a target substance to be detected, and a binding substance may, for example, be performed using a solid-phase carrier by B / F separation. After a first step in which a sample is reacted with lectin 1 immobilized on a solid-phase carrier, a washing step for removing unnecessary substances from the solid-phase surface may be performed before a second step in which complex 1 (immobilized lectin 1-target substance to be detected) is reacted with the binding substance. A washing step may also be performed after the second step and before the detection step. The washing step removes contaminants in the sample and unreacted binding substances from the solid-phase surface, allowing only complex 2 (immobilized lectin 1-target substance to be detected-binding substance) to be separated on the solid-phase surface.

[0075] Methods for separating the complex 2 of the lectin, the target substance, and the binding substance include, for example, electrophoretic methods such as chromatography, high-performance liquid chromatography, capillary electrophoresis, capillary tip electrophoresis, LBA (Liquid-phase Binding Assay), and LBA-EATA (Liquid-phase Binding Assay and Electrokinetic Analyte Transport Assay). These methods also allow B / F separation to be performed without using a solid phase carrier. Specific conditions are appropriately set depending on the type and properties of the sample, target substance, lectin, and binding substance. For example, when separation is performed using HPLC, the separation may be performed according to the method described in Anal. Chem. 65, 5, 613-616 (1993), etc. When capillary electrophoresis is used, the separation may be performed according to the method described in J. Chromatogr. 593 253-258 (1992), Anal. Chem. 64 1926-1932 (1992) or the like.

[0076] (Lectin Electrophoresis) In the present invention, lectin electrophoresis can be used, which utilizes the interaction between a "lectin" and a "substance to be detected." In the present invention, a gel containing fucose can be used to separate the substance to be detected, or a complex of the substance to be detected and a binding substance that specifically binds to the substance to be detected.

[0077] Specifically, lectin electrophoresis includes a complex formation step, a separation step, and a detection step. The complex formation step is a step in which a target substance is contacted with a binding substance (e.g., antibody or lectin) that specifically binds to the target substance to form a complex between the target substance and the binding substance (e.g., antibody or lectin). The separation step is a step in which the "complex of the target substance and the binding substance (e.g., antibody or lectin) that specifically binds to the target substance" formed in the complex formation step is separated by electrophoresis using the interaction between the "lectin" and the target substance. The detection step is a step in which the target substance is detected by detecting the "complex of the target substance and the binding substance (e.g., antibody or lectin) that specifically binds to the target substance" separated in the separation step. The binding substance that specifically binds to the target substance is preferably labeled with the labeling substance described above.

[0078] The amount (concentration) of the binding substance that specifically binds to the detection target substance and is reacted with the biological sample is appropriately set depending on the type of detection target substance, the required measurement sensitivity, the measurement method, the measurement device, etc.

[0079] In the present invention, the sandwich method is preferred.

[0080] A method using an automated immunoassay analyzer may be applied to the present invention, such as the Mu-TAS Wako i30 (Fujifilm Wako Pure Chemical Industries, Ltd.) or the Mu-TAS Wako i50 (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0081] The Mu-TAS Wako i30 (FUJIFILM Wako Pure Chemical Industries, Ltd.) or Mu-TAS Wako i50 is an immunoassay method based on the LBA-EATA method, and details thereof are described in Japanese Patent No. 4862093. The contents of Japanese Patent No. 4862093 are incorporated herein by reference. The immunoassay method includes the steps of: (i) contacting a sample containing an analyte with a conjugate of an affinity molecule (such as an antibody) having affinity for the analyte and a charged carrier molecule (such as DNA) in the presence of a polyanionic polymer to form a complex of the analyte and the conjugate; (ii) concentrating the complex in the presence of the polyanionic polymer by using a concentration channel of a microfluidic device comprising a concentration channel with microscale dimensions; (iii) separating the complex from unbound conjugate in the presence of the polyanionic polymer by using a separation channel of a microfluidic device comprising a separation channel with microscale dimensions; and (iv) detecting the complex to identify the presence of the analyte or measure the amount of the analyte in the sample.

[0082] A specific example of automated immunoassay using the Mu-TAS Wako i30 (FUJIFILM Wako Pure Chemical Corporation) or the Mu-TAS Wako i50 will be described below. The Mu-TAS Wako i30 (FUJIFILM Wako Pure Chemical Corporation) or the Mu-TAS Wako i50 is provided with an isotachophoresis zone and a zone electrophoresis zone.

[0083] In a first example, a target substance can be separated and detected by isotachophoresis followed by lectin electrophoresis. In isotachophoresis, electrophoresis is performed while forming a complex between a "first antibody against the target substance labeled with DNA (charged carrier molecule)," the "target substance," and a "second antibody against the target substance labeled with fluorescence (labeling substance)." In the subsequent lectin electrophoresis, the complex containing the target substance interacts with lectin. In the present invention, fucose can be contained in the gel for lectin electrophoresis. The mobility of the complex is slowed by the interaction with lectin, thereby allowing the target substance to be separated.

[0084] As a second example, the sandwich method may be performed using a Mu-TAS Wako i30 (FUJIFILM Wako Pure Chemical Industries, Ltd.) or a Mu-TAS Wako i50. Specific examples of the sandwich method include the following (1) to (3).

[0085] (1) A complex is formed between a DNA (charged carrier molecule)-labeled lectin, a substance to be detected, and an antibody against the substance to be detected that is labeled with a fluorescent label (labeling substance), and this complex is detected. In the present invention, the reaction between the DNA (charged carrier molecule)-labeled lectin and the substance to be detected is carried out in the presence of fucose.

[0086] (2) A complex is formed between a DNA (charged carrier molecule)-labeled antibody, a substance to be detected, and a lectin labeled with a fluorescent substance (labeling substance), and this complex is detected. In the present invention, the reaction between the substance to be detected and the lectin labeled with a fluorescent substance (labeling substance) is carried out in the presence of fucose.

[0087] (3) A complex between the target substance and the lectin is formed by reacting the target substance with the lectin in the presence of fucose. Then, the complex is contacted with a DNA (charged carrier molecule)-labeled antibody and an antibody against the target substance labeled with a fluorescent substance (labeling substance) to form a complex between the target substance, the lectin, the DNA (charged carrier molecule)-labeled antibody, and the antibody against the target substance labeled with a fluorescent substance (labeling substance), which is then detected.

[0088] (Antibodies and Labeled Antibodies) In one example of the present invention, an antibody can be used as a binding substance that specifically binds to a substance to be detected. The antibody may be a commercially available product or one appropriately prepared by a conventional method, and may be a monoclonal antibody or a polyclonal antibody. Furthermore, these may be used alone or in appropriate combination.

[0089] When the antibody is a monoclonal antibody, its origin is not particularly limited, and it may be a commercially available product or one produced by a method known per se using cell fusion technology, genetic recombination technology, or the like [Eur. J Immunol, 6, 511 (1976)].

[0090] When the antibody is a polyclonal antibody, its origin is not particularly limited, and examples thereof include those having the above-mentioned properties derived from rabbit, rat, mouse, sheep, goat, horse, etc. Commercially available products or polyclonal antibodies obtained by the method described in, for example, "Introduction to Immunology Experiments, 2nd Edition, Nao Matsuhashi et al., Academic Press Center, 1981" may be used.

[0091] The antibody may be Fab, Fab', or F(ab') of the antibody. ２ , Fv, Fd, single-chain Fv (scFv), disulfide-linked Fv (sdFv), V Ｌ , V Ｈ , diabody ((V Ｌ -V Ｈ ) ２ Or (V Ｈ -V Ｌ ) ２ ), triabody (trivalent antibody), tetrabody (tetravalent antibody), minibody ((scF Ｖ -C Ｈ 3) ２ ), IgG-delta-CH2, scFv-Fc, (scFv) ２ It may be an Fc fragment or the like.

[0092] The antibody may be labeled with a detectable labeling substance. The labeling substance used to label the antibody may be, for example, the following substances, but is not particularly limited, and any labeling substance commonly used in this field may be used.

[0093] Enzymes such as peroxidase, alkaline phosphatase, β-galactosidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase; ９９ｍ Tc, １３１ I, １２５ I, １４ C. ３ H. ３２ P, ３５ Radioactive isotopes such as S;

[0094] Fluorescent substances such as HiLyte 647 (manufactured by AhaSpec), fluorescein, dansyl, fluorescamine, coumarin, naphthylamine, fluorescein isothiocyanate (FITC), rhodamine, rhodamine X isothiocyanate, sulforhodamine 101, Lucifer Yellow, acridine, acridine isothiocyanate, riboflavin, or derivatives thereof; luminescent substances such as luciferin, isoluminol, luminol, bis(2,4,6-trifluorophenyl)oxalate;

[0095] Substances that absorb in the ultraviolet region, such as phenol, naphthol, anthracene, or derivatives thereof; substances that have properties as spin labeling agents, such as compounds having an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxyl;

[0096] HiLyte dyes such as HiLyte Fluor 647, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 680, and HiLyte Fluor 750 (all trade names of HiLyte Bioscience, Inc.); Alexa Fluor Dye 350, Alexa Fluor Dye 430, Alexa Fluor Dye 488, Alexa Fluor Dye 532, Alexa Fluor Dye 546, Alexa Fluor Dye 555, and Alexa Fluor Dye Alexa dyes such as Alexa Fluor Dye 568, Alexa Fluor Dye 594, Alexa Fluor Dye 633, Alexa Fluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye 680, Alexa Fluor Dye 700, and Alexa Fluor Dye 750 (all trade names of Molecular Probes);

[0097] CyDye dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 (all trade names of Amersham Biosciences); dyes such as Coomassie Brilliant Blue R250 and Methyl Orange

[0098] The method for binding a labeling substance to an antibody can be carried out by appropriately utilizing a labeling method used in conventional immunoassays and the like, and examples thereof include those described in "Medical Chemistry Experiment Lectures," Vol. 8, edited by Yamamura Yuichi, 1st ed., Nakayama Shoten, 1971; "Illustrated Fluorescent Antibodies," by Kawao Akira, 1st ed., Soft Science Co., Ltd., 1983; and "Enzyme Immunoassay," edited by Ishikawa Eiji, Kawai Tadashi, and Muroi Kiyoshi, 2nd ed., Igaku Shoin, 1982. The labeling substance may be directly bound to the antibody, or may be indirectly bound to the antibody via a suitable spacer (e.g., via one or several amino acids, or via a combination of one or several amino acids and a linker, or a substance having affinity, such as an avidin (e.g., streptavidin, tamavidin) and a biotin).

[0099] (Labeled Lectin) In another example of the present invention, a lectin can be used as a binding substance that specifically binds to a detection target substance. When a lectin is used as a binding substance that specifically binds to a detection target substance, the lectin may be a labeled lectin or an unlabeled lectin.

[0100] Labeling substances used to label lectins include the same labeling substances as those used to label the antibodies. Lectins can be labeled using any suitable labeling method employed in conventional immunoassays and the like. The labeling substance may be directly bound to the lectin, or may be indirectly bound to the lectin via an appropriate spacer (e.g., via one or several amino acids, or via a combination of one or several amino acids and a linker, or avidins (e.g., streptavidin, tamavidin) and a substance having affinity for the lectin, such as biotin).

[0101] (Detection Procedure) The "complex 2 of lectin, detection target substance, and binding substance" formed in the complex formation procedure can be detected by detecting a labeling substance. The "complex 2 of lectin, detection target substance, and binding substance" can also be detected without using a labeling substance, for example, by a measurement method that utilizes a property inherent in the complex, specifically, a method such as a homogeneous immunoassay system such as surface plasmon resonance.

[0102] The method for measuring a signal derived from a labeling substance varies depending on the type of labeling substance, but may be carried out according to a predetermined method depending on the properties of the labeling substance that can be detected by some method. For example, when the labeling substance is an enzyme, measurement may be carried out according to a conventional immunoassay method, such as the method described in "Enzyme Immunoassay Method" (Protein, Nucleic Acid, Enzyme, Special Issue No. 31, edited by Kitagawa Tsunehiro, Minamihara Toshio, Tsuji Akio, and Ishikawa Eiji, pp. 51-63, Kyoritsu Shuppan Co., Ltd., 1987). When the labeling substance is a radioactive substance, measurement may be carried out according to a conventional method used in RIA, using an appropriate measuring instrument such as an immersion GM counter, a liquid scintillation counter, a well-type scintillation counter, or an HPLC counter, depending on the type and intensity of radiation emitted by the radioactive substance (see, for example, Medical Chemistry Experiment Course, Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971). Furthermore, when the labeling substance is a fluorescent substance, the measurement may be carried out in accordance with a standard method used in FIA using a measuring device such as a fluorometer, for example, the method described in "Illustrated Fluorescent Antibodies, by Akira Kawao, 1st Edition, Soft Science Co., Ltd., 1983," and when the labeling substance is a luminescent substance, the measurement may be carried out in accordance with a standard method using a measuring device such as a photocounter, for example, the method described in "Enzyme Immunoassay" (Protein, Nucleic Acid, Enzyme, Special Issue No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, and Eiji Ishikawa, pp. 252-263, Kyoritsu Shuppan Co., Ltd., 1987). Furthermore, when the labeling substance is a substance that absorbs in the ultraviolet region, measurement may be performed by a conventional method using a measuring instrument such as a spectrophotometer, and when the labeling substance has spin properties, measurement may be performed by a conventional method using an electron spin resonance apparatus, for example, in accordance with the method described in "Enzyme Immunoassay" (Protein, Nucleic Acid, Enzyme, Special Issue No. 31, edited by Kitagawa Tsunehiro, Minamihara Toshio, Tsuji Akio, and Ishikawa Eiji, pp. 264-271, Kyoritsu Shuppan Co., Ltd., 1987).

[0103] In the present invention, all separation / measurement devices, various reagents, etc. commonly used in the field of immunological assay methods known per se can be used. The concentrations of the antibodies and reagents used in the measurement may be appropriately selected from the concentration ranges commonly used in this field. The measurement conditions (reaction temperature, reaction time, pH during the reaction, measurement wavelength, measurement device, etc.) may be appropriately selected according to methods known per se.

[0104] Research to date has revealed the diverse functions of glycans. Glycans are known to play important roles, particularly in cancer (metastasis, tumor markers, etc.), immunity (immunoreceptor regulation, immune cell differentiation, antibody drugs, etc.), fertilization, development and differentiation (regenerative medicine, etc.), infectious diseases (influenza, Helicobacter pylori, cholera toxin, etc.), biomedicine, the brain, blood typing, etc. Therefore, the method of the present invention, which can detect specific glycan structures, will be particularly useful in the fields of research (discovery, development, etc.) of disease-related biomarkers such as cancer markers (SLX antigen, CA19-9 antigen, etc.), diagnosis and determination of cancer and other diseases by detecting biomarkers (e.g., determination of cancer malignancy, evaluation of cancer metastasis potential, etc.), elucidation of the mechanisms of disease onset and development of treatments for each disease, research on mesenchymal stem cell markers, differentiation markers, etc., quality control and development of biopharmaceuticals, and cell quality control.

[0105] The method of the present invention is not limited to manual methods, but can also be applied to measurement systems using automated analyzers to allow for easy and rapid measurements. There are no particular restrictions on the combination of reagents, etc., when performing measurements manually or using an automated analyzer; the best combination of reagents, etc., may be selected and used according to the environment and model of the automated analyzer to be used, or taking other factors into consideration. Furthermore, the method of the present invention can also be applied to Micro-TAS (Micro-Total Analysis Systems: μ-TAS, μ Total Analysis System).

[0106] <Method for suppressing non-specific reactions> In the method for immunologically detecting a target substance of the present invention, non-specific reactions can be suppressed by reacting a lectin with the target substance in the presence of fucose. That is, according to the present invention, there is provided a method for suppressing non-specific reactions in a method for immunologically detecting a target substance having a sugar chain in a biological sample, the method comprising reacting a lectin with the target substance in the presence of fucose. Specific and preferred aspects of the method for suppressing non-specific reactions are the same as those described above in <Method for immunologically detecting a target substance of the present invention>.

[0107] <Fucose-Containing Composition> The present invention is characterized by reacting a lectin with a target substance to be detected in the presence of fucose. In the present invention, fucose can be used as a reagent for use in a method for detecting a target substance having a sugar chain in a biological sample using a lectin. That is, the present invention provides a fucose-containing composition for use in a method for detecting a target substance having a sugar chain in a biological sample using a lectin. Use of the fucose-containing composition of the present invention can suppress nonspecific reactions in methods for immunologically detecting a target substance to be detected. In one example, the fucose-containing composition is, for example, a sample dilution solution, a pretreatment solution, or an electrophoresis buffer solution. In another example, the fucose-containing composition contains a binding substance (such as a lectin or an antibody) that specifically binds to the target substance to be detected.

[0108] The fucose-containing composition of the present invention may contain reagents commonly used in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., which do not inhibit the stability of coexisting reagents or the reaction between the target substance and lectin. The concentration of these reagents may also be appropriately selected from the range of concentrations commonly used in this field.

[0109] <Reagent Kit> According to the present invention, there is provided a reagent kit for detecting a target substance having a sugar chain in a biological sample, which comprises the fucose-containing composition of the present invention described above and a lectin. Note that fucose and the lectin may be contained in the same reagent. That is, the lectin may be contained in the fucose-containing composition. Furthermore, the lectin may be labeled with a labeling substance. In one example, the lectin is a solid-phase lectin. In another example, the lectin is a non-solid-phase lectin, and the reagent kit further comprises a solid-phase carrier. Details and preferred examples of the lectin and the solid-phase carrier are as described above in this specification.

[0110] The reagent kit may further include a binding substance that specifically binds to the target substance, and that may be labeled. Details and preferred examples of the binding substance that may be labeled are as described above in this specification.

[0111] The preferred concentrations of these reagents may be appropriately selected from the ranges of concentrations usually used in this field.

[0112] The reagent kit of the present invention may contain a color-developing reagent for detecting a labeled substance, a reaction stop solution for the color-developing reaction, and a washing solution. The reagent kit of the present invention may further contain reagents commonly used in this field, such as buffers, reaction accelerators, stabilizers such as sugars, proteins, salts, and surfactants, preservatives, etc., which do not inhibit the stability of coexisting reagents or the reaction between the target substance and the lectin. The concentrations of these reagents may also be selected appropriately from the concentration range commonly used in this field.

[0113] The reagent kit of the present invention may further include a standard used to prepare a calibration curve for the substance to be detected. The standard may be a commercially available substance or a substance produced according to a known method.

[0114] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0115] Comparative Example 1 Measurement of BC2LCN-reactive AACT in Various Specimens (1) Preparation of Measurement Samples A 50 mmol / L MOPS (Dojindo Laboratories, Inc.) buffer solution containing 2% BSA (Merck) was prepared and used as specimen diluent 1. Serum specimens from pancreatic cancer patients (n=8) and non-pancreatic cancer patients (n=16) were diluted 100-fold with specimen diluent 1 to prepare measurement samples.

[0116] (2) Preparation of Standard Curve Samples BC2LCN-reactive AACT purified from pancreatic cancer cell culture supernatant was diluted with sample diluent 1 to concentrations of 0, 2, 10, 70, 120, and 200 μg / mL to prepare standard curve samples.

[0117] (3) Preparation of BC2LCN-immobilized plate BC2LCN (AiLecS1) lectin, recombinant, solution (rBC2LCN, Fujifilm Wako Pure Chemical Industries, Ltd.) 5 μg / mL (50 mmol / L sodium carbonate buffer, pH 9.7) was immobilized on a NUNC-IMMUNO MODULE plate (Thermo Fisher Scientific) by a standard method, and then blocked with phosphate buffer containing 1% Block Ace (KAC Corporation) to obtain a plate on which rBC2LCN was immobilized.

[0118] (4) Preparation of peroxidase (POD)-labeled anti-AACT antibody solution. Mice were immunized with alpha-1 antichymotrypsin (AACT) and human plasma (Athers Research & Technology) to produce anti-AACT antibodies (monoclonal antibodies). The anti-AACT antibodies were converted to Fab' fragments by standard methods and then labeled with peroxidase (POD) by standard methods (see Ishikawa Eiji, Enzyme Labeling Methods, Academic Press Center, 1991, p. 62). The POD-labeled anti-AACT antibody was prepared at 5 nmol / L in 50 mmol / L MES (Dojindo Laboratories) buffer containing 2% BSA.

[0119] (5) Coloring solution and washing solution, etc. The following reagent solutions were prepared: Coloring solution: 3,3',5,5'-tetramethylbenzidine (TMB) solution (Fujifilm Wako Pure Chemical Industries, Ltd.), Reaction stop solution: 1 mol / L HCl, Washing solution: Phosphate buffer containing 0.1% polyoxyethylene (20) sorbitan monolaurate (Tween 20).

[0120] (6) Measurement Method: 50 μL of the measurement sample and calibration curve sample prepared in (1) were added to an rBC2LCN-coated plate and allowed to react at room temperature for 1 hour. The wells were then washed three times with 300 μL of washing solution. Next, 100 μL of POD-labeled anti-AACT antibody solution was added and allowed to react at room temperature for 1 hour, after which the wells were washed five times with 300 μL of washing solution. 100 μL of color-developing solution was added and allowed to react at room temperature for 30 minutes. The reaction was then stopped by adding 100 μL of reaction stop solution. Measurements were performed at a dominant wavelength of 450 nm and a secondary wavelength of 620 nm. A calibration curve was created using the calibration curve sample, and the BC2LCN-reactive AACT concentration (measured value) in the measurement sample was calculated.

[0121] (7) Results The average BC2LCN-reactive AACT concentration in the pancreatic cancer patient samples was 9.0 μg / mL. Of the 16 non-pancreatic cancer patient samples, 8 samples exhibited a non-specific reaction, resulting in a high BC2LCN-reactive AACT concentration (higher than 9.0). Hereinafter, the 8 samples that exceeded the average value of the pancreatic cancer patient samples were referred to as "false-positive non-pancreatic cancer patient samples," and the 8 samples that did not exceed the average value of the pancreatic cancer patient samples (less than 9) were referred to as "normal non-pancreatic cancer patient samples."

[0122] Example 1: Verification of the Inhibitory Effect of Fucose (Monosaccharide) on Nonspecific Reactions The inhibitory effect of fucose, a monosaccharide, on the nonspecific reactions confirmed in Comparative Example 1 was verified. Measurements were performed using the same serum samples (serum samples from pancreatic cancer patients (n=8), normal non-pancreatic cancer patients (n=8), and false-positive non-pancreatic cancer patients (n=8)) and calibration curve samples as in Comparative Example 1 in the same manner as in Comparative Example 1, except that specimen dilution 2 was prepared by adding L(-)-fucose (Fujifilm Wako Pure Chemical Industries, Ltd.) to specimen dilution 1 at a concentration of 0.1 mmol / L (fucose concentration in the specimen dilution) instead of the specimen dilution 1 described above. The BC2LCN-reactive AACT concentrations were calculated, and the average values ​​were calculated for each specimen type: the specimens from pancreatic cancer patients, the normal non-pancreatic cancer patients, and the false-positive non-pancreatic cancer patients. According to the following formula, the average BC2LCN-reactive AACT concentrations of serum samples prepared with specimen diluent 1, which are the results of Comparative Example 1, were compared with the average BC2LCN-reactive AACT concentrations of serum samples prepared with specimen diluent 2, which are the results of Example 1, to calculate the relative value to Comparative Example 1. The results are shown in Table 1.

[0123] Relative value to Comparative Example 1 = [mean value of BC2LCN-reactive AACT concentrations (n=8) of each serum specimen using specimen diluent 2 (containing fucose) / mean value of BC2LCN-reactive AACT concentrations (n=8) of each serum specimen using specimen diluent 1 (not containing fucose)] x 100 (%)

[0124] As shown in Table 1, the addition of fucose decreased the measured values, and the measured values ​​of non-pancreatic cancer patient samples tended to be lower than those of pancreatic cancer patient samples. These results demonstrate that the coexistence of the monosaccharide fucose suppresses non-specific reactions in the detection of lectins and glycoproteins. That is, the relative value in false-positive samples decreased to 72%, a greater decrease than the relative value of 87% in pancreatic cancer patient samples, indicating that non-specific reactions were suppressed. Furthermore, the relative value in normal samples decreased significantly to 57%, making it easier to distinguish between pancreatic cancer and non-pancreatic cancer.

[0125]

[0126] Synthesis Example 1 Preparation of Fucose Derivatives With reference to Hagiwara Metal, J. Org. Chem. 2011, 76, 13, 5229-5239 and Unverzagt C, et al., Chem. Eur. J. 2009, 15, 12292-12302, a fucose derivative, 4AcGlu-2AcGlcNAc-AcGlcNAcO(NH)CCl, was synthesized from L(-)-fucose (Fujifilm Wako Pure Chemical Industries, Ltd.). ３ -3AcFuc (structure shown below) was synthesized.

[0127]

[0128] The structure of the obtained compound was analyzed by NMR, and it was confirmed that the target compound was obtained. The molecular weight was 1,297.

[0129] Comparative Examples 2 to 5: Verification of the Inhibitory Effect of Various Sugars on Nonspecific Reactions The inhibitory effect of various sugars on the nonspecific reactions confirmed in Comparative Example 1 was verified. Specimen dilution 3 was prepared by adding 0.1 mmol / L D(+)-galactose (Gal) (Fujifilm Wako Pure Chemical Corporation) to the above specimen dilution 1; specimen dilution 4 was prepared by adding 0.1 mmol / L N-acetyl-D-galactosamine (GalNAc) (Fujifilm Wako Pure Chemical Corporation) to specimen dilution 1; specimen dilution 5 was prepared by adding 0.1 mmol / L ethyl 2,3,4-tri-O-benzyl-β-L-thiofcopyranoside (also referred to as "Fuc derivative 1") (Fujifilm Wako Pure Chemical Corporation); and specimen dilution 6 was prepared by adding 0.1 mmol / L of the fucose derivative prepared in Synthesis Example 1 (also referred to as "Fuc derivative 2") to specimen dilution 1.

[0130] The same serum samples and calibration curve samples from the same false-positive specimens (n=8) from non-pancreatic cancer patients as in Comparative Example 1 were measured in the same manner as in Comparative Example 1, except that specimen dilutions 3, 4, 5, and 6 were used instead of specimen dilution 1, and the BC2LCN-reactive AACT concentrations were calculated. The average BC2LCN-reactive AACT concentrations of the serum samples prepared with specimen dilution 1, which is the result of Comparative Example 1, were compared with the average BC2LCN-reactive AACT concentrations of the serum samples prepared with specimen dilutions 3 to 6, which are the results of Comparative Examples 2 to 5. The results are shown in Table 2.

[0131] As can be seen from Table 2, D(+)-galactose (Gal), ethyl 2,3,4-tri-O-benzyl-β-L-thiofucopyranoside (Fuc derivative 1), and the fucose derivative (Fuc derivative 2) prepared in Synthesis Example 1 increased the measured values ​​of false-positive samples from non-pancreatic cancer patients. Furthermore, N-acetyl-D-galactosamine (GalNAc) only slightly reduced the measured values ​​of false-positive samples from non-pancreatic cancer patients compared to L(-)-fucose tested in Example 1. These results demonstrate that the suppression of non-specific reactions in the detection of lectins and glycoproteins is insufficient except for the monosaccharide fucose.

[0132]

Claims

1. A method for immunologically detecting a target substance having a sugar chain in a biological sample, the method comprising reacting a lectin with the target substance in the presence of fucose.

2. The method according to claim 1, wherein the lectin is a lectin that recognizes O-type glycans.

3. The method according to claim 1, wherein the lectin recognizes Fucα1-2Galβ1-3GlcNAc, which is an H type 1 structure, or Fucα1-2Galβ1-3GalNAc, which is an H type 3 structure.

4. The method of claim 1, wherein the lectin is BC2LCN. Here, BC2LCN is a protein that contains the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which 1 to 10 amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, inserted and / or added, and that specifically recognizes the sugar chain structure of Fucα1-2Galβ1-3GlcNAc or Fucα1-2Galβ1-3GalNAc.

5. The method according to claim 1, wherein the substance to be detected is a glycoprotein, a glycopeptide, or a degradation product thereof.

6. The method of claim 1, wherein the biological sample is a blood-derived sample.

7. The method according to claim 1, wherein the lectin is a solid-phase lectin, and after the reaction between the lectin and the target substance, the target substance bound to the solid-phase lectin is reacted with a binding substance that specifically binds to the target substance, and the binding substance bound to the target substance is detected, thereby detecting the target substance.

8. A method for suppressing non-specific reactions in a method for immunologically detecting a target substance having a sugar chain in a biological sample, the method comprising reacting a lectin with the target substance in the presence of fucose.

9. A fucose-containing composition for use in a method for detecting a target substance having a sugar chain in a biological sample using a lectin.

10. A reagent kit for detecting a target substance having a sugar chain in a biological sample, comprising the fucose-containing composition according to claim 9 and a lectin.

11. The reagent kit according to claim 10, wherein the lectin is a solid-phase lectin.

12. The reagent kit according to claim 10, wherein the lectin is a non-immobilized lectin, and the reagent kit further comprises a solid phase carrier.

13. A reagent kit according to any one of claims 10 to 12, further comprising a binding substance that specifically binds to the substance to be detected, the binding substance optionally being labeled.