WO2017183711A1 - Method for capturing lectin target molecule - Google Patents

Method for capturing lectin target molecule Download PDF

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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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lectin
target molecule
afp
method
sugar chain
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PCT/JP2017/015991
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French (fr)
Japanese (ja)
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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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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/16Extraction; Separation; Purification by chromatography
    • C07K1/22Affinity chromatography or related techniques based upon selective absorption processes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566Immunoassay; 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/42Lectins, e.g. concanavalin, phytohaemagglutinin

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

Acquisition method of lectin target molecule

The present invention, method for capturing a lectin target molecule, a complex comprising a lectin target molecule relates to such capture reagents and kits lectin target molecule.

α- fetoprotein (AFP) is Lens culinaris agglutinin: unbound fraction by binding with (Lens culinaris agglutinin LCA) (L1), 3 strokes of weakly binding fraction (L2), coupled fraction (L3) It is classified to the minute. Among AFP-L3 it is used as a liver cancer marker. Development of excellent measurement of AFP-L3 for the diagnosis of liver cancer has been demanded.

Further, the prostate specific antigen (Prostate-specific antigen: PSA) is used as a prostate cancer marker. However, since the PSA measurements also rises in prostate hyperplasia or prostatitis, there lies a challenge low specificity. Recently, glycosylation changes in PSA in prostate cancer have been reported, to improve the accuracy of cancer diagnosis by capturing changes in the glycosylation is expected.

Patent Document 1 discloses a measurement method fucosylated AFP using lectin.
Patent Document 2 discloses a method of measuring PSA having a sugar chain containing a beta-N-acetylgalactosamine residues using lectins.
Non-Patent Document 1 discloses a method for measuring AFP-L3 with lectin.
Non-Patent Document 2 discloses that you can determine the malignancy of prostate cancer by measuring the fucosylated PSA amount.

International Publication No. WO 99/39209 WO 2010/90264

Tamano et al. , Biosci Biotechnol Biochem. 2005 Aug; 69 (8): 1616-9 Li et al., Theranostics. 2015 Jan; 5 (3): 267-76

An object of the present invention is to improve the measurement of lectin target molecules using lectins (eg, AFP-L3 and PSA).

The present inventors have made intensive studies, as a result, the use of lectins in the presence of lectin-reactive carbohydrate-containing substance, was found and to enhance binding to lectin target molecule lectin. This finding, including measurement of lectin target molecule by lectin, can be applied to the acquisition method in general lectin target molecule. The present inventors, based on these findings, the present invention has been completed. The patent and non-patent literature, neither describes a method of enhancing the binding of lectin and lectin target molecule (AFP-L3 and PSA) are also suggested.

That is, the present invention is as follows.
[1] in the presence of lectin-reactive carbohydrate-containing substance comprises binding the lectin in the lectin target molecule, the capture method of lectin target molecule.
[2] lectins are legume lectins or mushroom lectin, method [1].
The method of [3] Lectins are fucose-specific lectins, [1] or [2].
[4] lectin is Lens culinaris agglutinin (LCA), concanavalin A (ConA) or Aleuria aurantia lectin (AAL), the method of any of [1] to [3].
[5] lectin target molecule is a glycoprotein, the method of any of [1] to [4].
[6] glycoprotein is AFP-L3 or PSA, the method of [5].
[7] glycoprotein is hemopexin (HPX) or transferrin (TF), the method of [5].
[8] the entity is a glycoprotein having an N-type sugar chain binding consensus sequence, The method of any of [1] to [7].
[9] glycoproteins with N-type sugar chain binding consensus sequence is an immunoglobulin having an N-type sugar chain binding consensus sequence to the variable region, the method of [8].
[10] The entity, B type hepatitis virus surface antigen (HBs antigen), human chorionic gonadotropin α chain (HCGarufa) or luteinizing hormone (LH), the method of any of [1] to [8].
[11] a method for measuring the lectin target molecule comprising measuring lectin target molecule after binding to the lectin target molecule lectins The method of any of [1] to [10].
[12] Furthermore using specific affinity substance lectin target molecule, the method of [11].
[13] a method comprising the following method of any of [1] to [12]:
(1) a lectin target molecule by binding to the specific affinity substance lectin target molecule to obtain a first complex comprising a specific affinity substance lectin target molecule and a lectin target molecule;
(2) the lectin in the presence of lectin-reactive carbohydrate-containing substance is bound to the first complex, lectin target molecule, a lectin target molecule specific affinity substance, lectins and lectin-reactive carbohydrate-containing substance obtaining a second complex comprising; and (3) measuring the amount of lectin target molecules contained in the second mixture.
[14] lectin target molecules, lectins and lectin-reactive carbohydrate complex containing containing entity.
[15] lectins and lectin-reactive carbohydrate-containing substance in a mixed form, capture reagent lectin target molecule.
[16] lectins and lectin-reactive carbohydrate-containing entities, capture kit lectin target molecule.

The method of the present invention, by the use of lectins in the presence of lectin-reactive carbohydrate-containing substance, capable of enhancing binding to lectin target molecule lectin. Thus, the method of the invention, for example, measurement of the lectin target molecule, extraction, concentration, purification, detection, useful for quantitation. More specifically, if the method of the present invention using lectin in the presence of lectin-reactive carbohydrate-containing substance was used to measure lectin target molecule, as compared to the use of the lectin alone, high sensitivity and stable it is possible to measure the lectin target molecule.
Complexes of the present invention, for example, useful in the practice of the capture method of the present invention.
Reagents and kits of the present invention is useful, for example, a simple implementation of the acquisition method of the present invention.

1, purified mouse IgG F (ab ') 2 fragment antibodies A, B, the C and D, molecular weight by SDS-PAGE, and is a diagram illustrating a confirmation of LCA reactivity by lectin blot. 2, purified mouse IgG F (ab ') 2 fragment antibodies A, B, the C and D, is a diagram illustrating a confirmation of LCA reactive by ELISA. Figure 3 is a diagram showing a specific detection of AFP-L3 by LCA in the presence of LCA reactive F (ab ') 2 fragment antibodies in A. Figure 4 is a diagram showing a specific detection of AFP-L3 by LCA in the presence of LCA reactive F (ab ') 2 fragment antibodies in B. Figure 5 is a graph showing the results of the LCA non-reactive F (ab ') 2 Detection of the fragment antibody C AFP-L3 and AFP-L1 by LCA in the presence of. Figure 6 is a graph showing the results of the LCA non-reactive F (ab ') 2 fragment antibodies in D AFP-L3 and AFP-L1 by LCA in the presence of detection. 7, deglycosylation-mouse IgG F (ab ') for 2 fragments, molecular weight by SDS-PAGE, and is a diagram illustrating a confirmation of LCA reactivity by lectin blot. 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. 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 -L3 and is a diagram showing the detection results of the AFP-L1. Figure 10 is a diagram showing the detection results of the AFP-L3 and AFP-L1 by LCA in the presence of an antibody having N-type sugar chain binding consensus sequence. Anti TNFα antibodies (P): anti-TNFα antibody with an N-type sugar chain binding consensus sequence the variable region; anti-TNFα antibody (N): anti-TNFα antibody without the N-type sugar chain binding consensus sequence the variable region; anti-IL- 10 antibody (P '): anti-IL-10 antibody with an N-type sugar chain binding consensus sequence the variable region; anti-IL-10 antibody (N'): no N-type sugar chain binding consensus sequence the variable region of anti-IL -10 antibody. Figure 11 is a diagram showing a specific detection of AFP-L3 by LCA in the presence of a lectin-reactive antigen (HBs antigen). Figure 12, for deglycosylation HBs antigen, molecular weight by SDS-PAGE, and is a diagram illustrating a confirmation of LCA reactivity by lectin blot. 13, the sugar chain-containing HBs antigen and deglycosylation HBs antigen is a diagram illustrating a confirmation of LCA reactive by ELISA. Figure 14 is a diagram showing the detection results of the AFP-L3 and AFP-L1 by LCA in the presence of a sugar chain-containing HBs antigen -HRP or LCA not reactive deglycosylation HBs antigen -HRP, indicating the LCA reactivity it is. 15, N-type sugar-mouse IgG F (ab ') having a chain binding consensus sequence 2-HRP or Aleuria aurantia lectin in the presence of HBs antigen-HRP: Detection of AFP-linked sugar chains by (Aleuria aurantia Lectin AAL) It illustrates. Figure 16 is a diagram showing an AFP-linked sugar chains detection by mouse IgG F having N-type sugar chain binding consensus sequence (ab ') Concanavalin A (ConA) in the presence of 2-HRP or HBs antigen-HRP. Figure 17 is a diagram showing the detection of PSA-linked sugar chains by AAL in the presence of mouse IgG F (ab ') 2 -HRP or HBs antigen -HRP having N-type sugar chain binding consensus sequence. Figure 18 is a diagram showing a mouse IgG F (ab ') of AFP-L3 by LCA in the presence of 2-HRP specific detection with N-type sugar chain binding consensus sequence. Simultaneous adding conditions: In AFP capture wells were incubated with a solution containing the LCA and mouse IgG F (ab ') 2 antibody A-HRP; washing conditions: washing unreacted LCA after incubating the solution containing LCA in AFP capture well It was removed. Then, the AFP capture wells unreacted LCA was removed and incubated after adding mouse IgG F (ab ') 2 antibody A-HRP. Figure 19 is a diagram showing a specific detection of AFP-L3 by LCA in the presence of HBs antigen-HRP. Simultaneous adding conditions: In AFP capture wells were incubated with a solution containing the LCA and HBs antigen-HRP; washing conditions: washing after incubation a solution containing LCA in AFP capture wells to remove unreacted LCA from AFP capture wells . Then, the AFP capture wells unreacted LCA was removed and incubated after adding a solution containing an HBs antigen-HRP. Figure 20 is a diagram showing a specific detection of AFP-L3 by AAL in the presence of HBs antigen-HRP. Simultaneous adding conditions: In AFP capture wells were incubated with a solution containing the AAL and HBs antigen-HRP; washing conditions: washing after incubation a solution containing AAL in AFP capture wells to remove unreacted AAL from AFP capture wells . Then, the AFP capture wells unreacted AAL was removed and incubated after adding HBs antigen-HRP. Figure 21 is a diagram showing a specific detection of AFP-L3 by LCA under conditions where the LCA reactive antibody fragment A or B as the solid phase. (+): LCA reactivity; (-): LCA nonreactive Figure 22 is a mouse IgG F (ab ') hemopexin liver cancer patient serum by 2-HRP (HPX) binding AAL detection of reactive fucosylated sugar chain having N-type sugar chain binding consensus sequence in the presence of AAL It illustrates. 1% BSA-PBS: control; HCC-1, HCC-2: liver cancer patient sera 2 cases; NHS-1, NHS-2: healthy human serum 2 cases. Figure 23 is a mouse IgG F (ab ') transferrin liver cancer patient serum by 2-HRP (TF) binding AAL detection of reactive fucosylated sugar chain having N-type sugar chain binding consensus sequence in the presence of AAL It illustrates. 1% BSA-PBS: control; HCC-1, HCC-2: liver cancer patient sera 2 cases; NHS-1, NHS-2: healthy human serum 2 cases.

The present invention, in the presence of lectin-reactive carbohydrate-containing substance comprises binding the lectin in the lectin target molecule, provides a method for capturing a lectin target molecule.

Lectins are proteins that exhibit binding activity by recognizing a specific sugar chain structure. The lectin, for example, an animal (e.g., vertebrates, invertebrates), plants (e.g., Leguminosae, Gramineae), fungi (e.g., mushroom, koji) those derived from is known. The legume lectins, for example, jack bean lectin (Concanavalin A: ConA), Lens culinaris agglutinin (Lens culinaris agglutinin: LCA), pea lectin (Pisum sativum) and the like. The mushroom lectin, for example, Aleuria aurantia lectin (Aleuria aurantia Lectin: AAL), and the like.

Lectins used in the present invention may also be appropriately selected depending on the sugar chain structure of lectin target molecule. The lectins used in the present invention, for example, fucose-specific lectins having affinity for fucose, galectin with affinity for galactose, but sialic acid reactive lectins include, fucose-specific lectins are preferred. Fucose-specific lectins, e.g., a lectin that recognizes the root of GlcNAc to fucose α1,6 the added sugar chain structure of N-glycans (core fucose structure) Example, LCA, pea lectin (Pisum sativum: PSA) ], and N-type sugar chains, O-type sugar chains and α1,2 fucose GlcNAc of glycolipid sugar chain, alpha1,3, alpha] l, 4, which recognizes a sugar chain structure that is added in either α1,6 lectin [example, AAL, Aspergillus oryzae lectin (Aspergillus oryzae lectin: AOL)] and the like.

The lectin target molecule, having the ability to bind to lectin refers to a target molecule to be trapped in the present invention. Target molecule, comprising a specific carbohydrate lectin binding, or molecules containing the specific sugar chains.

Preferably, the lectin target molecule is a glycoprotein. The glycoproteins of the lectin target molecule, for example, mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms or virus-derived glycoproteins, and the like. Glycoprotein as lectin target molecule, measurement, extraction, concentration or purification may be desired glycoprotein. Specifically, the glycoprotein is a protein having a N-type sugar chain binding consensus sequence. N-type sugar chain binding consensus sequence, the amino acid residues of the Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline) is the amino acid sequence composed of. N-type sugar chains, to bind to asparagine (Asn) residues of N-type sugar chain binding consensus sequence is known. More specifically, the lectin target molecule, for example, AFP-L3, PSA, hemopexin (HPX), and although transferrin (TF) and the like, but is not limited thereto. PSA is known as the modified protein of sugar chains such as N-type sugar chains.

The lectin-reactive carbohydrate-containing substance refers to a solid phase molecule containing lectin reactive sugar chain or lectin reactive sugar chain is added. Lectin-reactive carbohydrate-containing entity, different from the lectin target molecules to be captured in the process of the present invention. Lectin-reactive carbohydrate-containing substance is a than the sugar chains are reacted with the lectin, no lectin or lectin target molecule with an antibody to the antigen. The molecule containing a lectin reactive sugar chains, for example, biomolecules, and artificial synthetic (e.g., artificially synthesized molecules, such as artificial synthetic polymer) and the like. The solid phase, for example, particles (e.g., magnetic particles); membrane (e.g., nitrocellulose membrane, filter paper), a support such as a column; and plates (e.g., multi-well plates), and a container such as a tube. The solid phase material, e.g., glass, plastic, a matrix of polysaccharide, and metals. Lectin-reactive carbohydrate-containing substance may be appropriately selected depending on the kind of the lectin used. For example, such selection can be performed by evaluating the binding ability of the sugar chains or sugar chain-containing substance of the lectin (e.g., see Example 2).

Preferably, the lectin-reactive carbohydrate-containing substance is a biological molecule containing the lectin reactive sugar chains. The biological molecules such as polypeptides and nucleic acid. More preferably, the lectin-reactive carbohydrate-containing substance is a glycoprotein. The glycoproteins of the lectin-reactive carbohydrate-containing substance, for example, mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms or virus-derived glycoproteins, and the like. Examples of glycoproteins as lectins sugar chain-containing substance, e.g., an immunoglobulin (eg, antibody) secreted proteins, membrane-bound proteins, hormones, cytokines, chemokines, virus-derived antigens, enzymes, extracellular matrix, extracellular vesicle membrane proteins, tumor-specific antigen, and a partial peptide thereof and the like.

Preferably, the glycoprotein of the lectin-reactive carbohydrate-containing substance is a protein having a N-type sugar chain binding consensus sequence. N-type sugar chain binding consensus sequence, the amino acid residues of the Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline) is the amino acid sequence composed of. N-type sugar chains, to bind to asparagine (Asn) residues of N-type sugar chain binding consensus sequence is known. More specifically, examples of proteins with N-type sugar chain binding consensus sequence, for example, B-type hepatitis virus surface antigen, human chorionic gonadotropin α chain, luteinizing hormone, PSA antigen, HE4 antigen, MUC1 antigen, thyroglobulin, and antibodies with N-type sugar chain binding consensus sequence the variable region thereof.

More preferably, the glycoprotein of the lectin-reactive carbohydrate-containing substance is an antibody having an N-type sugar chain binding consensus sequence the variable region. The antibodies with N-type sugar chain binding consensus sequence the variable region, for example, native full-length antibodies and modified antibodies of. The modified antibodies, for example, antibody fragments with variable regions (e.g., Fab, F (ab ') 2), and single chain antibodies. As the antibody with an N-type sugar chain binding consensus sequence the variable region, e.g., IgG, IgM, IgA, IgD, IgE, and IgY, and the like. Antibodies with N-type sugar chain binding consensus sequence to the variable region may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

The method of the present invention, in the presence of lectin-reactive carbohydrate-containing substance, the lectin is bound to a lectin target molecule. In other words, the method of the present invention, lectin target molecule, as well as performed in a system comprising at least three components that lectins and lectin-reactive carbohydrate-containing substance, lectins and lectin-reactive carbohydrate content as a capture means lectin target molecule entity is used in combination. By using lectins in the presence of lectin-reactive carbohydrate-containing substance, capable of enhancing binding to lectin target molecule lectin. Thus, the method of the invention, for example, measurement of the lectin target molecule, extraction, concentration, purification, quantification, useful for the detection.

Lectin target molecule, those included in the liquid sample is used. In other words, the method of the present invention, a liquid sample containing a lectin target molecule, carried out in lectins and lectin-reactive carbohydrate containing entity the system was added. Origin of the liquid sample is not particularly limited, it may be a a well be or environmental sample, a biological sample from the organism. The organism from which the biological sample, e.g., mammals (e.g., human, monkey, mouse, rat, rabbit, cow, pig, horse, goat, sheep), birds (e.g., chicken) or the like of animals, insects , microorganisms, plants, fungi, although fish and the like, preferably a mammal, fungi, fish, more preferably a mammal, even more preferably a human. Biological samples also include blood-related samples is a sample derived from blood itself or blood (eg, whole blood, serum, plasma), saliva, urine, milk, tissue or cell extract, or a mixture thereof it may be. The environmental samples, soil, sea water, and a sample from the fresh water. Liquid sample may be used after subjected to pretreatment. Examples of such pre-treatment, for example, centrifugation, fractionation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring the like.

The amount of 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 lectin-reactive carbohydrate-containing substance, for example, at 0.1μg / mL ~ 100mg / mL . The amount of lectin is preferably 1 [mu] g / mL or more, more preferably 5 [mu] g / mL or more, still more preferably at least 10 [mu] g / mL. The amount of lectin also preferably not more than 20 mg / mL, more preferably 10 mg / mL or less, still more preferably not more than 2 mg / mL. Such amount of lectin, may be added to the liquid sample.

The amount of lectin reactivity sugar chain-containing substance used in the method of the present invention is not particularly limited as long as it is an amount capable of supporting the capture of lectin target molecule by lectin, for example, at 0.001μg / mL ~ 100mg / mL . The amount of lectin reactivity sugar chain-containing substance is preferably 0.005 / mL or more, more preferably 0.02 .mu.g / mL or more, still more preferably at least 0.1 [mu] g / mL. The amount of lectin reactivity sugar chain-containing substance is also preferably not more than 20 mg / mL, more preferably 5 mg / mL or less, still more preferably not more than 1 mg / mL. Such an amount of lectin reactivity sugar chain-containing substance may be added to the liquid sample.

In a preferred embodiment, the method of the present invention is a method of measuring the lectin target molecule. In the measurement method of the present invention, it is possible to measure lectin target molecule after binding to the lectin target molecule lectin. Measuring method of the present invention can be carried out according to an immunological technique, the combined use of lectin reactivity sugar chain-containing substance and lectin in place of the antibody, be carried out by the same method as immunological techniques it can. The immunological technique which measuring method is analogous to the present invention, for example, enzyme immunoassay (EIA) (eg, chemiluminescent EIA (CLEIA), enzymes adsorbed EIA (ELISA)), fluorescence immunoassay, chemiluminescence immunoassay mentioned method, electrochemiluminescence immunoassay, agglutination method, immunostaining, flow cytometry method, Bio-layer interferometry, an In Situ PLA method, the chemically amplified luminescence proximity homogeneous assay, line blot, Western blotting It is. Measuring method of the present invention, qualitative or quantitatively performed, it is possible to test for the presence or amount of lectin target molecule.

In the method of the present invention may be further used is specific affinity substance lectin target molecule. Affinity substance specific for lectin target molecule used in the methods of the present invention bind to lectin target molecule at different sites (e.g., peptide region) and the lectin binding sites of the lectin in the target molecule (sugar). The affinity substance specific for lectin target molecule, for example, there may be mentioned antibodies and aptamers, antibodies are preferred.

The amount of specific affinity substances lectin target molecule used in the method of the present invention is not particularly limited as long as it is an amount capable of sufficiently binding lectin target molecule, for example, at 0.0001μg / mL ~ 10mg / mL is there. The amount of affinity substance is preferably 0.001 [mu] g / mL or more, more preferably 0.005 / mL or more, even more preferably at least 0.01 [mu] g / mL. The amount of affinity substance is also preferably not more than 2 mg / mL, more preferably 0.5 mg / mL or less, still more preferably not more than 0.1 mg / mL. Affinity substance of such an amount may be added to the liquid sample.

If specific affinity substance is further used to lectin target molecule, the measuring method of the present invention may be performed in a sandwich fashion. For example, the immobilization means, specific affinity substance is used in the lectin target molecule, as detection means, lectins and lectin-reactive carbohydrate-containing substance may be used in combination. Alternatively, the solid phase means, in combination lectins and lectin-reactive carbohydrate-containing substance, as the detection means may be used specific affinity substance is a lectin target molecule.

In the measurement method of the present invention, the lectin-reactive carbohydrate-containing entity or lectin or specific affinity substance lectin target molecule, it may be labeled with a labeling substance. As the labeling substance, for example, an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase, beta-galactosidase), affinity substance (e.g., one of streptavidin and biotin, complementary sense strand and antisense strand of a nucleic acid from each other one), a fluorescent substance or protein (example out, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent materials (e.g., luciferin, aequorin, acridinium ester, tris (2,2 bipyridyl) ruthenium, luminol), radioactive materials (e.g., 3 H, 14 C, 32 P, 35 S, 125 I) can be mentioned. Preferably, the lectin-reactive carbohydrate-containing entity or specific affinity substance lectins sugar chain-containing substance, is labeled.

More specifically, the measuring method of the present invention, when carried out in a sandwich manner, can be carried out by the following steps:
(1) a lectin target molecule by binding to the specific affinity substance lectin target molecule to obtain a first complex comprising a specific affinity substance lectin target molecule and a lectin target molecule;
(2) the lectin in the presence of lectin-reactive carbohydrate-containing substance is bound to the first complex, lectin target molecule, a lectin target molecule specific affinity substance, lectins and lectin-reactive carbohydrate-containing substance obtaining a second complex comprising; and (3) measuring the amount of lectin target molecules contained in the second mixture.

In step (1), lectin target molecule can be used which are contained in the liquid sample as described above. Accordingly, as a specific embodiment of step (1), a liquid sample containing a lectin target molecule, by adding a specific affinity substance lectin target molecule, step (1) may be performed. In step (1), it can be used specific affinity substance lectin target molecule in an amount as described above, preferably, the expected relative lectins target molecule, specific lectin target molecule excess equivalents it may be used affinity substance. The affinity substance specific for lectin target molecule, it is preferred to use a fixed to a solid phase as described above. Binding of the lectin target molecule and a lectin target molecule specific affinity substance, both solutions (eg, buffer) can be accomplished by incubation in which the lectin target molecule and a lectin target molecule first complex comprising specific affinity substance can be obtained.

In step (2), the second complex is obtained. Step As a specific embodiment of (1), the solution containing the first complex obtained in step (1), by adding lectins, and lectin-reactive carbohydrate-containing entities, step (2) it may be performed. In step (2), the amount of lectin, as described above, and the amount of lectin reactivity sugar chain-containing substance as described above can be used for preferably lectin target molecules which are expected, excess equivalent it may be used lectins and excess equivalents of lectins sugar chain-containing substance. While either lectin or lectin-reactive carbohydrate-containing substance may be labeled with a labeling substance, preferably lectins sugar chain-containing substance may be labeled with a labeling substance. Binding to the first complex of the lectin in the presence of a lectin-reactive carbohydrate-containing entity, a tripartite solution (e.g., buffer) can be accomplished by incubation in which the lectin target molecule, specific affinity substance lectin target molecule, a second complex comprising lectin and lectins sugar chain-containing substance is obtained. If used as immobilized on a solid phase as a specific affinity substance lectin target molecule, the second complex is immobilized on a solid phase. In this case, after the step (2), a washing step may be provided consists of removal and addition of the liquid phase.

In step (3), an index signal from the labeling substance in the second complex, can measure the amount of lectin target molecule.

The present invention also provides a complex comprising lectin target molecules, lectins and lectin-reactive carbohydrate-containing entities. Complexes of the present invention may further contain a specific affinity substance lectin target molecule. Complexes of the present invention, by binding by incubating these configuration factors in solution, can be produced.

In conjugates of the present invention, lectin target molecules, lectins, lectin-reactive carbohydrate-containing substance, and definitions of specific affinity substances lectin target molecule, examples and preferred examples are as mentioned above.

Complexes of the present invention, for example, useful in the practice of the capture method of the present invention.

The present invention further includes lectins and lectin-reactive carbohydrate-containing substance in a mixed form, to provide a capture reagent lectin target molecule. In other words, the capture reagent of the present invention comprises a composition of lectins and lectin-reactive carbohydrate-containing entities. Since lectins and lectin-reactive carbohydrate-containing substance contained in the reagent of the present invention is in the mixed form, in the capture reagent of the present invention have complex is formed between the lectin and lectin-reactive carbohydrate-containing substance it may be.

When reagents of the present invention is used to measure lectin target molecule, but either the lectin or lectins sugar chain-containing substance may be labeled with a labeling substance, it is preferably lectins sugar chain-containing substance labeled it may be labeled with a substance. Alternatively, if neither of the lectin or lectin-reactive carbohydrate-containing substance not labeled with a labeling substance, the reagent of the present invention may contain a labeling substance lectins and lectin-reactive carbohydrate-containing substance and a non-mixed form good.

Reagents of the invention may further comprise a specific affinity substance lectin target molecule. Specific affinity substance lectin target molecule, may be labeled with a labeling substance. Moreover, the affinity substance specific for the lectin target molecule may be immobilized on a solid phase as described above. In this case, the reagent of the present invention can be suitably used to measure lectin target molecules in a sandwich manner. Reagent of the present invention, a specific affinity substance lectin target molecule, including at either the mixed form or unmixed form with lectin and lectins sugar chain-containing substance, preferably contains a non-mixed form.

In the reagent of the present invention, lectin target molecules, lectins, lectin-reactive carbohydrate-containing substance, and definitions of specific affinity substances lectin target molecule, examples and preferred examples are as mentioned above.

Reagent of the present invention is useful, for example, a simple implementation of the acquisition method of the present invention, for example, can be suitably used in the measuring method of the present invention.

The present invention also includes lectins and lectin-reactive carbohydrate-containing entities, provides capture kit lectin target molecule. Lectins and lectin-reactive carbohydrate-containing substance contained in the kit of the present invention is mixed form (i.e., the form of the composition) or in, or to the non-mixed form (i.e., forms which are isolated from each other). Thus, the components contained in the kit of the present invention, different containers are provided (e.g., tube, plate) form housed or accommodated form in different compartments of the same container.

If the kit of the present invention is used to measure lectin target molecule, but either the lectin or lectins sugar chain-containing substance may be labeled with a labeling substance, it is preferably lectins sugar chain-containing substance labeled it may be labeled with a substance. Alternatively, if neither of the lectin or lectin-reactive carbohydrate-containing substance not labeled with a labeling substance, the kit of the present invention is labeled with a labeling substance and / or lectin target molecule specific affinity substance (labeling substance may be) may be contained.

Kits of the present invention may further contain a specific affinity substance lectin target molecule. In this case, the kit of the present invention can be suitably used to measure lectin target molecules in a sandwich manner. Affinity substance specific for the lectin target molecule may be immobilized on a solid phase as described above.

In the kit of the present invention, lectin target molecules, lectins, lectin-reactive carbohydrate-containing substance, and definitions of specific affinity substances lectin target molecule, examples and preferred examples are as mentioned above.

Kits of the present invention is useful, for example, a simple implementation of the acquisition method of the present invention, for example, can be suitably used in the measuring method of the present invention.

Hereinafter, detailed explanation of the present invention embodiment, the present invention is not limited to these examples.

Example 1: Preparation of antibody fragment, and a mouse IgG1 not prepared anti LCA antibody of antibody fragment -HRP was digested with pepsin, LCA reactive mouse IgG F (ab ') 2 fragments and LCA non-reactive mouse IgG F ( ab ') were prepared 2 fragments.

Specifically, the preparation was carried out as follows.
(1) anti-LCA not an antibody mouse IgG1 antibodies A, B, C and D was digested with pepsin. Digestion, was added murine IgG1 antibody A was buffer exchanged into citrate buffer, B, pepsin (SIGMA) in C and D, 37 ° C. for 1 hour the reaction was carried out by stopping the reaction by the addition of Tris buffer .
(2) subjected to gel filtration in Superdex200 increase 10 / 300GL equilibrated with PBS, to obtain a four purified mouse IgG F (ab ') 2 fragments.
(3) purified mouse IgG F (ab ') 2 fragment antibodies A, B, and C and D, were labeled with horseradish peroxidase (HRP). Labeling of HRP, using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories), performed according to the attached protocol, obtain the four purified antibody fragment-HRP (mouse IgG F (ab ') 2 -HRP ).

Example 2: Purification of the antibody fragment, the molecular weight and four antibodies fragments prepared in lectin reactivity confirmed in Example 1 to confirm the purity, molecular weight and lectin reactivity.

More specifically, the confirmation was carried out as follows.
(1) purified mouse IgG F (ab ') 2 fragment antibodies A, B, the molecular weight of C and D was confirmed by SDS-PAGE (Fig. 1 left).
(2) purified mouse IgG F (ab ') 2 fragment antibodies A, B, and LCA reactive C and D were confirmed by lectin blotting (Figure 1 right).
(3) purified mouse IgG F (ab ') 2 fragment antibodies A, B, and LCA reactive C and D were confirmed by ELISA. Diluted in PBS purified mouse IgG F (ab ') 2 fragment antibodies A, B, C and D so that each 5 [mu] g / mL. In U96 MaxiSorp Nunc-Immuno Plate (Thermo), and incubated 4 ° C. 24 hours diluted solution 50 [mu] L, was immobilized the antibody. The antibody-immobilized well, washed 3 times with PBST 250 [mu] L, 10% blocking reagent; and 37 ° C. 1 hour at (Carbofree blocking solution Vector) 150μL. The antibody-coated wells were washed three times with PBST 250μL. Then, the diluted solution 50μL as a 10% blocking reagent LCA-HRP to (J-Oil Mills) becomes 5 [mu] g / mL was added, the solution was incubated 37 ° C. 1 hour in antibody-immobilized wells. The wells were washed 3 times with PBST 250μL. 50μL of TMB substrate solution (Dako) was added to the wells after washing were 5 minutes of reaction at room temperature. After the reaction was stopped with 1N sulfuric acid and the absorbance was measured at 450 nm (Figure 2).

As a result, antibody fragments A and B are LCA reactive, antibody fragments C and D was confirmed to be LCA non-reactive (Table 1, Figure 2).

Figure JPOXMLDOC01-appb-T000001

The analysis of the antibodies used.
Antibodies A had a N-type sugar chain binding consensus sequence to the heavy chain of the antibody variable regions. This indicates that the F (ab ') 2 fragment is modified by N-type sugar chains. Therefore, F (ab ') 2 fragment antibodies in A is considered to indicate a lectin reactivity through the N-type sugar chains.
Antibody B is the same as antibody A.
Antibody C is a N-type sugar chain binding consensus sequence did not also have a light chain in heavy chain antibody variable regions. This is, F (ab ') 2 fragments show that it is not modified by N-type sugar chains. Therefore, F (ab ') 2 fragment antibodies in C is considered to show no lectin reactivity.
Antibodies D is the same as antibody C.

Example 3: Mice were prepared in Construction Example 1 of AFP-L3 measurement system by lectin in the presence of a lectin-reactive antibody fragment IgG F (ab ') with 2-HRP, immunological determination of AFP-L3 It was constructed system.

Specifically, the construction was carried out as follows.
(1) the anti-AFP F (ab ') 2 was diluted with PBS to a 10 [mu] g / mL. In U96 MaxiSorp Nunc-Immuno Plate, and incubated 4 ° C. 24 hours diluted solution 50 [mu] L, was immobilized the antibody.
(2) the antibody-immobilized well, washed 3 times with PBST 250 [mu] L, and 37 ° C. 1 hour at 10% blocking reagent 150 [mu] L.
(3) an antibody immobilized wells were washed 3 times with PBST 250 [mu] L. Then, the AFP-L3 solution and AFP-L1 solution obtained by converting the concentration from the measured values ​​of mu task Wako AFP-L3 (Wako Pure Chemical Industries, Ltd.), 10% blocking reagent, respectively, the solution 50μL were 2n dilution from 1000 ng / mL was 37 ° C. 1 hour addition to antibody-immobilized wells.
(4) an antibody immobilized wells were washed 3 times with PBST 250 [mu] L. Then diluted to 5 [mu] g / mL in mouse IgG F (ab ') 2 -HRP 10% blocking reagent each prepared in Example 1, further LCA the (Vector) was added to a 100 [mu] g / mL the mixture was prepared, the mixture was incubated 37 ° C. 1 hour addition to antibody-immobilized wells.
(5) an antibody immobilized wells were washed 3 times with PBST 250 [mu] L. Then allowed to react for 5 minutes at room temperature was added a 50μL of TMB substrate solution (Dako). After the reaction was stopped with 1N sulfuric acid and the absorbance was measured at 450nm.

As a result, LCA were able to specifically detect AFP-L3 in the presence of LCA-reactive antibody fragment A or B (Table 2, Fig. 3-6).

Figure JPOXMLDOC01-appb-T000002

From the above, lectins, it was shown that can specifically detect AFP-L3 in the presence of a labeled lectin-reactive antibody fragment.

Example 4: the AFP-L3 detection mouse IgG was prepared in the confirmation Example 1 of the involvement of lectin reactivity sugar chain lectin-reactive antibody fragments in F (ab ') 2 sugar chain N- glycosidase F antibody B by removing in, deglycosylation mouse IgG F (ab ') 2 antibody B was confirmed whether maintaining the AFP-L3 reactivity in the coexistence LCA.

More specifically, the confirmation was carried out as follows.
(1) mouse IgG F prepared in Example 1 (ab ') was performed N- glycosidase F treatment 2 antibody B. N- glycosidase F treatment, the addition of mouse IgG F was buffer exchanged into Tris-HCl buffer (ab ') 2 antibody B N- glycosidase F (ROCHE), was reacted 37 ° C. 24 hours. N- glycosidase F is an enzyme which specifically cleaves GlcNAc-Asn bond between N-linked sugar chains and proteins.
(2) subjected to gel filtration in Superdex200 increase 10 / 300GL equilibrated with PBS, and deglycosylation-mouse IgG F (ab ') was obtained 2 fragment.
(3) a sugar chain removal mouse IgG F (ab ') 2 -HRP , was prepared in the same manner as in Example 1.
(4) In the same manner as in Example 2, it was confirmed purity, molecular weight and LCA reactivity (Fig. 7,8).
(5) in the same manner as in Example 3, it was confirmed AFP-L3 reactive LCA in the presence of labeled antibody (Fig. 9, Table 3). AFP concentration was examined at 1 [mu] g / mL.

As a result, in the presence of deglycosylation labeled antibody fragment which has lost LCA reactivity (Fig. 7, 8), LCA could not detect AFP-L3 (Table 3, Figure 9).

Figure JPOXMLDOC01-appb-T000003

From the above, the sugar chain of the variable region of the antibody has been shown to be important for the detection of AFP-L3 by lectin.

Example 5: Non N-type sugar chain binding consensus sequence in the variable region of AFP-L3 detection antibody by lectin in the presence of mouse IgG having the N-type sugar chain binding consensus sequence to the AFP-L3 detected in the variable region It was investigated whether or not the impact.

Specifically, the study was carried out as follows.
(1) N-type sugar chain binding consensus sequence in the variable region (Asn-Ser-Ser) anti TNFα antibody without the anti-TNFα antibody (P) having a (N), N-type sugar chain binding consensus sequence in the variable region ( the Asn-Asp-Thr) 'anti-IL-10 antibodies without a) (N' anti-IL-10 antibody (P with) were HRP-labeled similarly to each example 1.
(2) in the same manner as in Example 3, it was confirmed AFP-L3 reactive under LCA coexist. AFP concentration was examined in 5μg / mL.

As a result, LCA in the presence of an antibody with an N-type sugar chain binding consensus sequence was able to specifically detect AFP-L3 (Table 4, Figure 10).

Figure JPOXMLDOC01-appb-T000004

From the above, lectin was shown to be capable of specifically detecting the AFP-L3 in the presence of the antibody (the antibody is any kind of antigen binding) with N-type sugar chain binding consensus sequence the variable region.

Example 6: Using the HBs antigen is a glycoprotein having an N-type sugar chain binding consensus sequence other than the construction mouse IgG AFP-L3 measurement system by lectin in the presence of a lectin-reactive antigen (B hepatitis virus surface antigen) Te, it was constructed immunoassay system of AFP-L3.

Specifically, the construction was carried out as follows.
(1) The HBs antigen with N-type sugar chain binding consensus sequence, was HRP labeled similarly to each Example 1.
(2) in the same manner as in Example 3, it was confirmed AFP-L3 reactive under LCA coexist. AFP concentration was examined by 2n dilution from 1000 ng / mL. HBs antigen -HRP was reacted with 2 [mu] g / mL.

As a result, LCA is in the presence of HBs antigen with N-type sugar chain binding consensus sequence was able to specifically detect AFP-L3 (Table 5, Figure 11).

Figure JPOXMLDOC01-appb-T000005

From the above, lectins, not only antibody, even in the presence of lectin-reactive antigen, was shown to be specifically detected AFP-L3.

Example 7: in AFP-L3 detected by removing sugar chains confirmation HBs antigen involved lectin reactivity sugar chain of the antigen in the N- Glycosidase F, deglycosylation HBs antigen under coexistence LCA AFP -L3 was sure to maintain the reactivity.

More specifically, the confirmation was carried out as follows.
(1) it was performed N- glycosidase F treatment of HBs antigen. N- glycosidase F treatment, using N- glycosidase F and PNGaseF (New England Biolabs) reaction solution came, performed according to the protocol PNGaseF attached were deglycosylation.
(2) a sugar chain removal HBs antigen was labeled with HRP in the same manner as in Example 1.
(3) In the same manner as in Example 2, it was confirmed purity, molecular weight and LCA reactivity.
(4) In the same manner as in Example 6, it was confirmed AFP-L3 reactive under LCA coexist. AFP concentration was examined at 1 [mu] g / mL. HBs antigen -HRP was added at 1 [mu] g / mL.

As a result, LCA, in the presence of deglycosylation labeled antigen which has lost LCA reactivity (Fig. 12 and 13) failed to detect AFP-L3 (Table 6, Figure 14).

Figure JPOXMLDOC01-appb-T000006

Thus, sugar chains of glycoprotein in the antigen have been shown to be important for the detection of AFP-L3 by lectin.

Example 8: even in the presence of an antigen not only AFP-L3 detection antibody by lectin in the presence of a lectin-reactive glycoprotein, LCA was possible to detect AFP-L3 (Example 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。

More specifically, the confirmation was carried out as follows.
(1) a hCGα with N-type sugar chain binding consensus sequence in the amino acid sequence (human chorionic gonadotropin α chain) and LH (luteinizing hormone), it was HRP labeled similarly to each Example 1.
(2) in the same manner as in Example 3, it was confirmed AFP-L3 reactive LCA. AFP concentration was examined at 1 [mu] g / mL.

As a result, LCA in the presence of hCGα or LH, it was possible to specifically detect AFP-L3 (Table 7,8).

Figure JPOXMLDOC01-appb-T000007
Numbers indicate the measured values ​​of OD450.

Figure JPOXMLDOC01-appb-T000008

From the above, the lectin in the presence of lectin-reactive glycoprotein, was shown to be specifically detected AFP-L3.

Example 9 Mice with lectin reactivity labeled antibody or lectin reactivity detection system constructed N-type sugar chain binding consensus sequence of AFP-linked sugar chains by various lectins in the presence of a labeled antigen IgG F (ab ') 2 -HRP or in the presence of HBs antigen-HRP, Aleuria aurantia lectin (Aleuria aurantia lectin: AAL) and by using concanavalin a and (ConA) as lectin, it was constructed detection system AFP linked sugar chain having the following structural formula.

Figure JPOXMLDOC01-appb-C000009

The above structural formula, previously described (Nakagawa et al, J Proteome Res 2008 Jun; 7 (6):.. 2222-33), the reported cell culture (Huh7) and contained 60% hepatocellular carcinoma in the specimen the structural formula is shown as a representative example.

Specifically, the construction was carried out as follows.
Similarly to (1) Example 3, the AFP-L3 and AFP-L1 diluted to respectively 1 [mu] g / mL in 10% blocking reagent, the reaction was carried out using the dilute solution.
(2) an antibody-immobilized wells were washed 3 times with PBST 250 [mu] L. 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 blocking reagent diluted to further AAL (J-oil Mills) was 10 [mu] g / mL or ConA a (Vector) preparing a mixture by adding one to a 10 [mu] g / mL,, antibody solid the mixture in addition to the phasing wells and incubated for 37 ° C. 1 hour.
(3) an antibody immobilized wells were washed 3 times with PBST 250 [mu] L. Adding TMB substrate solution 50μL to wells after washing was conducted 5 minutes reaction at room temperature. After the reaction was stopped with 1N sulfuric acid and the absorbance was measured at 450nm.

As a result, AAL and ConA in the presence of mouse IgG F (ab ') 2 -HRP or HBs antigen -HRP having N-type sugar chain binding consensus sequence, to detect the AFP-linked sugar chains like the LCA You can have (Table 9, Figure 15 and 16).

Figure JPOXMLDOC01-appb-T000010

Thus, for the other lectin it is not limited to the LCA, in the presence of the glycoprotein, was shown to be capable of detecting the AFP-linked sugar chains.

Example 10: it is constructed AAL reactive glycan detection system of PSA-linked sugar chain by a lectin is bound to a PSA (prostate specific antigen) in the presence of lectin-reactive labeled antibody or lectin reactivity labeled antigen It has been reported. In the presence of mouse IgG F (ab ') 2 -HRP and HBs antigen -HRP having N-type sugar chain binding consensus sequence, was used AAL as lectin, PSA binding AAL reactive fucosylated sugar chain having the following structural formula It was constructed of the detection system.

Figure JPOXMLDOC01-appb-C000011

The above structural formula, previously described (Tajiri et al, J Glycobiology 2008 Jan; 18 (1):.. 2-8) by analyzes prostate Ganken 3 example, it is reported that the highest common is Relative abundance and has the structural formula shown as a representative example.

Specifically, the construction was carried out as follows.
(1) the anti-PSA F (ab ') 2 was diluted with PBS to a 10 [mu] g / mL. In U96 MaxiSorp Nunc-Immuno Plate, and incubated 4 ° C. 24 hours diluted solution 50 [mu] L, was immobilized the antibody.
(2) the antibody-immobilized well, washed 3 times with PBST 250 [mu] L, and 37 ° C. 1 hour at 10% blocking reagent 150 [mu] L.
(3) Antibody immobilized wells were washed three times with PBST 250 [mu] L of PBST, Lumipulse PSA-N standard PSA solution (Fujirebio) 100ng / mL, 40ng / mL, 5ng / mL, 0.5ng / mL, 0ng / mL was added 50μL to antibody-immobilized well, and incubated for 37 ° C. 1 hour.
(4) an antibody immobilized wells were washed 3 times with PBST 250 [mu] L. As the HBs antigen -HRP prepared with mouse IgG F (ab ') 2 -HRP in Example 6 with an N-type sugar chain binding consensus sequence prepared in Example 1, the respective 1 [mu] g / mL in 10% blocking reagent diluted, were added respectively to further AAL so that 10 [mu] g / mL. The solution was incubated 37 ° C. 1 hour addition to antibody-immobilized wells.
(5) an antibody immobilized wells were washed 3 times with PBST 250 [mu] L. Adding TMB substrate solution 50μL to wells after washing was conducted 5 minutes reaction at room temperature. After the reaction was stopped with 1N sulfuric acid and the absorbance was measured at 450nm.

As a result, AAL is mouse IgG F (ab ') having an N-type sugar chain binding consensus sequence in the presence of 2-HRP or HBs antigen-HRP, detecting the PSA binding AAL reactive carbohydrate concentration-dependent manner could be (Table 10, Figure 17).

Figure JPOXMLDOC01-appb-T000012

From the above, the lectin in the presence of lectin-reactive labeled antibody or lectin reactivity labeled antigen, not only AFP-L3, was shown to be capable of detecting the other lectin-reactive glycoproteins such as PSA.

Example 11: Enhancement of binding force between the lectin and the target molecules by glycoprotein (1)
This example was conducted to demonstrate the enhanced binding force between the lectin and the target molecule by glycoproteins. Were washed after the reaction of the target molecule (AFP-L3) and lectin (LCA), were detected in the glycoprotein (mouse IgG F having N-type sugar chain binding consensus sequence (ab ') 2-HRP or HBs antigen-HRP) Conditions and (wash conditions), lectin in the presence of the target molecule (mouse IgG F having N-type sugar chain binding consensus sequence (ab ') 2-HRP or HBs antigen-HRP) (AFP-L3) glycoproteins (LCA) in conditions reacted with (simultaneous addition condition) was examined whether there is a difference between the detectability of the target molecule. The number of washing, aligned between the conditions.

Specifically, the study was carried out as follows.
(1) the anti-AFP F (ab ') 2 was diluted with PBS to a 10 [mu] g / mL. In F16 MaxiSorp Nunc-Immuno Plate (Thermo), and incubated 4 ° C. 24 hours diluted solution 100 [mu] L, was immobilized the antibody.
(2) the antibody-immobilized well, washed 3 times with PBST 300 [mu] L, and 37 ° C. 1 hour at 10% blocking reagent 200 [mu] L.
(3) an antibody immobilized wells were washed 3 times with PBST 300 [mu] L. Then, was diluted to 500ng / mL AFP-L3 solution was converted density from the measured values ​​of mu task Wako AFP-L3 and AFP-L1 solution with 10% blocking reagent, respectively. As the diluted solution 100μL was 37 ° C. 1 hour added.
(4) an antibody immobilized wells were washed 3 times with PBST 300 [mu] L. The wash conditions, the LCA was diluted with 10% blocking reagent such that 100 [mu] g / mL, were the diluted solutions 100μL was 37 ° C. 1 hour addition to antibody-immobilized wells. The simultaneous addition conditions, only 10% blocking reagent was added 100μL to antibody-immobilized well, and incubated for 37 ° C. 1 hour.
(5) an antibody immobilized wells were washed 3 times with PBST 300 [mu] L. The wash conditions, diluted with 10% blocking reagent to the HBs antigen -HRP prepared with mouse IgG F (ab ') 2 antibody A-HRP 5μg / mL or Example 6 prepared in Example 1 becomes 2 [mu] g / mL and, thereof a dilute solution 100μL was 37 ° C. 1 hour addition to antibody-immobilized wells. The simultaneous addition conditions, mouse IgG F (ab ') prepared in Example 1 and 2 antibodies A-HRP 5μg / mL or HBs antigen -HRP diluted with 10% blocking reagent such that the 2 [mu] g / mL, a more LCA They were added respectively as a 100 [mu] g / mL. Adding the diluted solution 100μL antibody immobilized wells and incubated for 37 ° C. 1 hour.
(6) an antibody immobilized wells were washed 3 times with PBST 300 [mu] L. Adding TMB substrate solution 100μL to wells after washing was conducted 5 minutes reaction at room temperature. After the reaction was stopped with 1N sulfuric acid and the absorbance was measured at 450nm.

As a result, by inserting the cleaning step after the reaction with AFP-L3 and lectin (LCA), reactivity to AFP-L3 was significantly attenuated (Fig. 18 and 19). The AFP-L3, simultaneous addition conditions reacted with lectin (LCA) in the presence of labeled glycoprotein (mouse IgG F having N-type sugar chain binding consensus sequence (ab ') 2-HRP or HBs antigen-HRP) only, it was possible to specifically detect AFP-L3 (Figure 18, 19).

As described above, by reacting lectin (LCA) and target molecule (AFP-L3) in the presence of the glycoprotein, it was shown to bind strength between lectin and the target molecule is enhanced.

Example 12: Enhancement of binding force between the lectin and the target molecules by glycoprotein (2)
This example was conducted to demonstrate the enhanced binding force between the lectin and the target molecule by glycoproteins. Were washed after the reaction of the target molecule (AFP binds fucosylated sugar chain) and lectin (AAL), and glycoprotein (HBs antigen-HRP) was detected with conditions (washing conditions), the target molecule (AFP binding fucosylated sugar chain) lectin (AAL) and reacted condition (simultaneous addition conditions) in the presence of the glycoprotein (HBs antigen-HRP), and examined whether there is a difference between the detectability of the target molecule. The number of washing, aligned between the conditions.

Specifically, the study was carried out as follows.
(1) As in Example 11, was added 100μL of anti-AFP F (ab ') AFP-L3 and AFP-L1, respectively 2 to immobilized wells was diluted to 500 ng / mL, 37 ° C. 1 time was incubated.
(2) an antibody immobilized wells were washed 3 times with 300μL of PBST. The wash conditions, the AAL was diluted with 10% blocking reagent such that 10 [mu] g / mL, added AAL solution 100μL diluted in antibody-immobilized well, the resulting solution was incubated 37 ° C. 1 hour. The simultaneous addition conditions, only the addition 100 [mu] L 10% blocking reagent in the antibody-immobilized well, the resulting solution was incubated 37 ° C. 1 hour.
(3) an antibody immobilized wells were washed 3 times with PBST 300 [mu] L. The wash conditions, the HBs antigen -HRP prepared in Example 6 was diluted with 10% blocking reagent such that the 2 [mu] g / mL, and the diluted solution 100μL incubated 37 ° C. 1 hour. The simultaneous addition conditions, the HBs antigen -HRP prepared in Example 6 was diluted with 10% blocking reagent such that the 2 [mu] g / mL, the HBs antigen -HRP solution diluted, such an amount as to result in the AAL and 10 [mu] g / mL each was added to and incubated antibody immobilized diluted wells solution 100μL was added 37 ° C. 1 hour.
(4) The resulting solution was washed three times with 300μL of PBST with (3). The wells after washing adding 100μL of TMB substrate solution was reacted at room temperature for 5 minutes. After the reaction was stopped with 1N sulfuric acid and the absorbance was measured at 450nm.

As a result, by inserting the cleaning step after the reaction with the AFP binding fucosylated sugar chains and lectin (AAL), reactivity to AFP binding fucosylated sugar chains was significantly attenuated (Figure 20). The AFP binding fucosylated sugar chains, only the simultaneous adding conditions reacted with lectin (AAL) in the presence of labeled glycoprotein (HBs antigen-HRP), was able to detect the AFP binding fucosylated sugar chain (Fig. 20).

As described above, by lectin (AAL) is reacted with a target molecule in the presence of the glycoprotein (AFP binding fucosylated sugar chain), it was shown that lectin binding force between the target molecule is enhanced.

Example 13: Using mouse IgG F (ab ') 2 with N-type sugar chain binding consensus sequence to the solid phase, building the experimental detection systems AFP-L3 is a mouse IgG F prepared in Example 1 ( ab ') 2 was carried out to prove whether capture AFP-L3 on the solid phase. Mouse IgG F having N-type sugar chain binding consensus sequence (ab ') 2 fragment antibodies in A and B, and murine IgG without the N-type sugar chain binding consensus sequence F (ab') solid 2 fragment antibodies C and D However, followed by allowing the AFP-L3 and AFP-L1 reacting with these immobilized antibody under LCA coexistence, mouse IgG F (ab ') 2 was examined whether specifically capture AFP-L3.

Specifically, the study was carried out as follows.
(1) purified mouse IgG F prepared in Example 1 (ab ') 2 fragment antibodies A, B, was diluted with PBS to C and D so that the 10 [mu] g / mL. In U96 MaxiSorp Nunc-Immuno Plate, for immobilization and incubated 37 ° C. 1 h the diluted solution 50 [mu] L.
(2) an antibody-immobilized wells were washed 3 times with PBST 250 [mu] L, and 37 ° C. 24 hour at 10% blocking reagent 150 [mu] L.
(3) Antibody-a phased wells were washed 3 times with PBST 250 [mu] L, mu task Wako AFP-L3 AFP-L3 was converted to concentration from the measured value of the solution and AFP-L1 solution 5 [mu] g / mL at 10% blocking reagent, respectively diluted to, solution was prepared further by adding LCA of 100 [mu] g / mL and anti-AFP F (ab ') 2 -HRP so that 1 [mu] g / mL. The prepared solution 50μL was incubated 37 ° C. 1 hour addition to antibody-immobilized wells.
(4) an antibody immobilized wells were washed 3 times with PBST 250 [mu] L. Then allowed to react for 5 minutes at room temperature was added a TMB substrate solution 50 [mu] L. After the reaction was stopped with 1N sulfuric acid and the absorbance was measured at 450nm.

As a result, LCA were able to specifically detect AFP-L3 under conditions and solid phase LCA reactive antibody fragment A or B (Figure 21).

From the above, it can be captured lectin target molecule is indicated by a combination of the immobilized lectin reactive sugar chain-containing substance with lectin.

Example 14: Using mouse IgG-HRP with N-type sugar chain binding consensus sequence to the variable region, hemopexin (HPX) linked sugar chains and transferrin (TF) N-type sugar build the variable region of the detection system of linked sugar chains using a mouse IgG having a chain binding consensus sequence, whether reacted in the coexistence with AAL, it can detect HPX binding AAL reactive fucosylated sugar chains and TF binding AAL reactive fucosylated sugar chains of liver cancer patients serum investigated.

Specifically, the study was carried out as follows.
(1) mouse IgG with N-type sugar chain binding consensus sequence in the amino acid sequence of the variable regions was performed HRP labeled in the same manner as in Example 1.
(2) it was carried out N-glycosidase treatment of anti-HPX antibody (R & D SYSTEMS Clone # 698813) and anti-TF antibody (abcam Clone # 2A2). N-glycosidase treatment, anti-HPX antibodies buffer exchanged into Tris-buffered saline, was added N-glycosidase F (ROCHE Inc.) was reacted 37 ° C. 24 hours. Then, conduct ultrafiltration of 100 KDa, to remove N-glycosidase.
(3) the N-glycosidase treated anti HPX antibody and anti-TF antibody was performed was diluted with PBS to a 2μg / mL, C96 MaxiSoup Nunc-Immuno Plate (Thermo Co.) dispensed at 50μL / well, 4 ℃ 24 hours immobilized.
(4) a 1% BSA-PBS for 250μL was added 200 [mu] L, and 4 ° C. 24 h blocking.
(5) was washed three times with 300μL of PBST, 1% BSA-PBS at 500-fold diluted liver cancer patient sera 2 cases (HCC-1, HCC-2) to healthy human serum 2 patients (NHS-1, NHS- 2), were incubated 50μL added 37 ° C. 1 hour.
(6) washing three times with 300μL of PBST, mouse IgG-HRP with N-type sugar chain binding consensus sequence the variable region such that the 2 [mu] g / mL, diluted in diluent (home-grown), further AAL ( added respectively Vector Co.) as a 20 [mu] g / mL, and incubated 37 ° C. 1 hour.
(7) was washed three times with 300μL of PBST, and reacted at room temperature for 2 minutes was added to 50μL of TMB substrate solution (Thermo Co.), after the reaction is stopped with 1N sulfuric acid and the absorbance was measured at 450nm.

As a result, by using the mouse IgG with N-type sugar chain binding consensus sequence the variable region, the coexistence with AAL, HPX binding AAL reactive fucosylated sugar chains of liver cancer patients serum (Fig. 22) and TF binding AAL reaction It could be detected sexual fucosylated sugar chain (Figure 23).

From the above, it can be captured lectin target molecule is indicated by a combination of the immobilized lectin reactive sugar chain-containing substance with lectin.

Claims (16)

  1. In the presence of lectin-reactive carbohydrate-containing substance comprises binding the lectin in the lectin target molecule, the capture method of lectin target molecule.
  2. Lectins are legume lectins or mushroom lectin The method of claim 1, wherein.
  3. Lectins are fucose-specific lectins, Method according to claim 1 or 2.
  4. Lectin, lens culinaris agglutinin (LCA), a concanavalin A (ConA) or Aleuria aurantia lectin (AAL), any one method according to claim 1-3.
  5. Lectin target molecule is a glycoprotein, any one method according to claim 1-4.
  6. Glycoprotein is AFP-L3 or PSA, method of claim 5, wherein.
  7. Glycoprotein is hemopexin (HPX) or transferrin (TF), The method of claim 5, wherein.
  8. The entity is a glycoprotein having an N-type sugar chain binding consensus sequence, any one method according to claim 1-7.
  9. Glycoproteins with N-type sugar chain binding consensus sequence is an immunoglobulin having an N-type sugar chain binding consensus sequence the variable region The method of claim 8.
  10. It said entity, B type hepatitis virus surface antigen (HBs antigen), human chorionic gonadotropin α chain (HCGarufa) or luteinizing hormone (LH), any one method according to claim 1-8.
  11. Is a method of measuring the lectin target molecule comprising measuring lectin target molecule after binding to the lectin target molecule lectin, any one method according to claim 1 to 10.
  12. Using more specific affinity substance lectin target molecule The method of claim 11.
  13. A method comprising the following method of any one of claims 1 to 12:
    (1) a lectin target molecule by binding to the specific affinity substance lectin target molecule to obtain a first complex comprising a specific affinity substance lectin target molecule and a lectin target molecule;
    (2) the lectin in the presence of lectin-reactive carbohydrate-containing substance is bound to the first complex, lectin target molecule, a lectin target molecule specific affinity substance, lectins and lectin-reactive carbohydrate-containing substance obtaining a second complex comprising; and (3) measuring the amount of lectin target molecules contained in the second mixture.
  14. Lectin target molecules, lectins and lectin-reactive carbohydrate complex containing containing entity.
  15. Lectin and lectin reactive sugar chain-containing substance in mixed form, the capture reagent lectin target molecule.
  16. Lectin and lectin reactive sugar chain-containing substance, capture kit lectin target molecule.
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