JP6910699B2 - Lectin target molecule capture method - Google Patents

Description

The present invention relates to a method for capturing a lectin target molecule, a complex containing a lectin target molecule, a reagent and a kit for capturing a lectin target molecule, and the like.

Alpha-fetoprotein (AFP) has three fractions, a non-binding fraction (L1), a weakly binding fraction (L2), and a binding fraction (L3), depending on the binding property with lentil glutinine (Lens culinaris agglutinin: LCA). It is classified into minutes. Of these, AFP-L3 is used as a liver cancer marker. Development of an excellent measurement method for AFP-L3 is required for the diagnosis of liver cancer.

In addition, prostate-specific antigen (PSA) is used as a marker for prostate cancer. However, since the PSA measurement value also increases in benign prostatic hyperplasia and prostatitis, low specificity is a problem. In recent years, changes in PSA sugar chain modification have been reported in prostate cancer, and it is expected that the accuracy of cancer diagnosis will be improved by capturing these changes in sugar chain modification.

Patent Document 1 discloses a method for measuring fucosylated AFP using a lectin.
Patent Document 2 discloses a method for measuring PSA having a sugar chain containing a β-N-acetylgalactosamine residue using a lectin.
Non-Patent Document 1 discloses a method for measuring AFP-L3 using a lectin.
Non-Patent Document 2 discloses that the malignancy of prostate cancer can be determined by measuring the amount of fucosylated PSA.

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

Tamano et al. , Bioscience 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 (eg, AFP-L3 and PSA) using lectins.

As a result of diligent studies, the present inventors have found that the use of lectin in the presence of a lectin-reactive sugar chain-containing entity enhances the binding of the lectin to the lectin target molecule. This finding can be applied to all methods for capturing lectin target molecules, including measurement of lectin target molecules with lectins. The present inventors have completed the present invention based on such findings. The above patented and non-patented documents do not describe or suggest a method of enhancing the binding of lectins to lectin target molecules (AFP-L3 and PSA).

That is, the present invention is as follows.
[1] A method for capturing a lectin target molecule, which comprises binding the lectin to the lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.
[2] The method of [1], wherein the lectin is a legume lectin or a mushroom lectin.
[3] The method of [1] or [2], wherein the lectin is a fucose-specific lectin.
[4] The method according to any one of [1] to [3], wherein the lectin is lentil glutinin (LCA), concanavalin A (ConA) or Heero Chawantake lectin (AAL).
[5] The method according to any one of [1] to [4], wherein the lectin target molecule is a glycoprotein.
[6] The method of [5], wherein the glycoprotein is AFP-L3 or PSA.
[7] The method of [5], wherein the glycoprotein is hemopexin (HPX) or transferrin (TF).
[8] The method according to any one of [1] to [7], wherein the entity is a glycoprotein having an N-type sugar chain binding consensus sequence.
[9] The method of [8], wherein the glycoprotein having an N-type sugar chain-binding consensus sequence is an immunoglobulin having an N-type sugar chain-binding consensus sequence in a variable region.
[10] The method according to any one of [1] to [8], wherein the entity is hepatitis B virus surface antigen (HBs antigen), human chorionic gonadotropin α chain (hCGα) or luteinizing hormone (LH).
[11] The method according to any one of [1] to [10], which is a method for measuring a lectin target molecule, which comprises measuring the lectin target molecule after binding of the lectin to the lectin target molecule.
[12] The method of [11], which further uses an affinity substance specific to the lectin target molecule.
[13] Any method of [1] to [12], which is a method including the following:
(1) The lectin target molecule is bound to an affinity substance specific to the lectin target molecule to obtain a first complex containing the lectin target molecule and the affinity substance specific to the lectin target molecule;
(2) In the presence of a lectin-reactive sugar chain-containing substance, lectin is bound to the first complex to obtain a lectin target molecule, an affinity substance specific to the lectin target molecule, a lectin, and a lectin-reactive sugar chain-containing substance. Obtaining a second complex containing; and (3) measuring the amount of lectin target molecule contained in the second complex.
[14] A complex containing a lectin target molecule, a lectin, and a lectin-reactive sugar chain-containing entity.
[15] A reagent for capturing a lectin target molecule, which comprises a lectin and a lectin-reactive sugar chain-containing entity in a mixed form.
[16] A kit for capturing a lectin target molecule, which comprises a lectin and a lectin-reactive sugar chain-containing entity.

The method of the present invention can enhance the binding of a lectin to a lectin target molecule by using the lectin in the presence of a lectin-reactive sugar chain-containing entity. Therefore, the method of the present invention is useful, for example, for measuring, extracting, concentrating, purifying, detecting, and quantifying a lectin target molecule. More specifically, when the method of the present invention using a lectin in the presence of a lectin-reactive sugar chain-containing substance is used for measuring a lectin target molecule, it is more sensitive and stable than the use of a lectin alone. Lectin target molecules can be measured.
The complex of the present invention is useful, for example, in practicing the capture method of the present invention.
The reagents and kits of the present invention are useful, for example, for the convenient implementation of the capture method of the present invention.

FIG. 1 is a diagram showing confirmation of molecular weight by SDS-PAGE and LCA reactivity by lectin blot for purified mouse IgG F (ab') _{2 fragment antibodies A, B, C and D.} FIG. 2 is a diagram showing confirmation of LCA reactivity by ELISA for purified mouse IgG F (ab') _{2 fragment antibodies A, B, C and D.} FIG. 3 shows the specific detection of AFP-L3 by LCA in the presence of LCA-reactive F (ab') _{2 fragment antibody A.} FIG. 4 shows the specific detection of AFP-L3 by LCA in the presence of LCA-reactive F (ab') _{2 fragment antibody B.} FIG. 5 is a diagram showing the results of detection of AFP-L3 and AFP-L1 by LCA in the presence of LCA non-reactive F (ab') _{2 fragment antibody C.} FIG. 6 is a diagram showing the results of detection of AFP-L3 and AFP-L1 by LCA in the presence of LCA non-reactive F (ab') _{2 fragment antibody D.} FIG. 7 is a diagram showing confirmation of molecular weight by SDS-PAGE and LCA reactivity by lectin blot for a sugar chain-depleted mouse IgG F (ab') _{2 fragment.} FIG. 8 is a diagram showing confirmation of LCA reactivity by ELISA for a sugar chain-containing mouse IgG F (ab') ₂ fragment and a sugar chain-depleted mouse IgG F (ab') _{2 fragment.} FIG. 9 shows AFP by LCA in the presence of sugar chain-containing mouse IgG F (ab') _2- HRP showing LCA reactivity or sugar chain-depleted mouse IgG F (ab') _{2-HRP not showing LCA reactivity.} It is a figure which shows the detection result of -L3 and AFP-L1. FIG. 10 is a diagram showing the detection results of AFP-L3 and AFP-L1 by LCA in the presence of an antibody having an N-type sugar chain binding consensus sequence. Anti-TNFα antibody (P): Anti-TNFα antibody having an N-type sugar chain binding consensus sequence in the variable region; Anti-TNFα antibody (N): Anti-TNFα antibody having no N-type sugar chain binding consensus sequence in the variable region; Anti-IL- 10 antibody (P'): anti-IL-10 antibody having an N-type sugar chain binding consensus sequence in the variable region; anti-IL-10 antibody (N'): anti-IL having no N-type sugar chain binding consensus sequence in the variable region -10 antibody. FIG. 11 is a diagram showing specific detection of AFP-L3 by LCA in the presence of a lectin-reactive antigen (HBs antigen). FIG. 12 is a diagram showing confirmation of molecular weight by SDS-PAGE and LCA reactivity by lectin blot for sugar chain-removed HBs antigen. FIG. 13 is a diagram showing confirmation of LCA reactivity by ELISA with respect to the sugar chain-containing HBs antigen and the sugar chain-removed HBs antigen. FIG. 14 shows the detection results of AFP-L3 and AFP-L1 by LCA in the presence of sugar chain-containing HBsAg-HRP showing LCA reactivity or sugar chain-removed HBsAg-HRP not showing LCA reactivity. Is. FIG. 15 shows the detection of AFP-bound sugar chains by Hiirochawantakerectin (AAL) in the presence of _{mouse IgG F (ab') 2-} HRP or HBsAg-HRP having an N-type sugar chain-binding consensus sequence. It is a figure which shows. FIG. 16 is a diagram showing the detection of AFP-bound sugar chains by concanavalin A (ConA) in the presence of _{mouse IgG F (ab') 2-} HRP or HBsAg-HRP having an N-type sugar chain-binding consensus sequence. FIG. 17 is a diagram showing the detection of PSA-bound sugar chains by AAL in the presence of _{mouse IgG F (ab') 2-} HRP or HBsAg-HRP having an N-type sugar chain-binding consensus sequence. FIG. 18 shows the specific detection of AFP-L3 by LCA in the presence of _{mouse IgG F (ab') 2-} HRP having an N-type sugar chain consensus sequence. Co-addition conditions: A solution containing LCA and mouse IgG F (ab') ₂ antibody A-HRP was incubated in the AFP capture well; wash conditions: the solution containing LCA was incubated in the AFP capture well and then washed and unreacted LCA. Was removed. _{The mouse IgG F (ab') 2} antibody A-HRP was then added to the AFP capture wells from which the unreacted LCA had been removed and then incubated. FIG. 19 shows the specific detection of AFP-L3 by LCA in the presence of HBsAg-HRP. Simultaneous addition conditions: Incubate the solution containing LCA and HBsAg-HRP in the AFP capture well; Wash condition: Incubate the solution containing LCA in the AFP capture well and then wash to remove unreacted LCA from the AFP capture well. .. Then, in the AFP capture well from which the unreacted LCA was removed, a solution containing HBsAg-HRP was added and then incubated. FIG. 20 is a diagram showing specific detection of AFP-L3 by AAL in the presence of HBsAg-HRP. Simultaneous addition conditions: AAL and HBsAg-HRP-containing solution was incubated in the AFP capture well; wash condition: AAL-containing solution was incubated in the AFP capture well and then washed to remove unreacted AAL from the AFP capture well. .. HBsAg-HRP was then added to the AFP capture wells from which unreacted AAL had been removed and then incubated. FIG. 21 is a diagram showing specific detection of AFP-L3 by LCA under the condition that LCA-reactive antibody fragment A or B is a solid phase. (+): LCA reactivity; (-): LCA non-reactivity FIG. 22 shows the detection of hemopexin (HPX) -linked AAL-reactive fucosylated sugar chains in the serum of liver cancer patients by _{mouse IgG F (ab') 2-} HRP having an N-type sugar chain-binding consensus sequence in the presence of AAL. It is a figure which shows. 1% BSA-PBS: Control; HCC-1, HCC-2: Serum from liver cancer patients; NHS-1, NHS-2: Serum from healthy subjects. FIG. 23 shows the detection of transferrin (TF) -linked AAL-reactive fucosylated sugar chains in the serum of liver cancer patients by _{mouse IgG F (ab') 2-} HRP having an N-type sugar chain-binding consensus sequence in the presence of AAL. It is a figure which shows. 1% BSA-PBS: Control; HCC-1, HCC-2: Serum from liver cancer patients; NHS-1, NHS-2: Serum from healthy subjects.

The present invention provides a method for capturing a lectin target molecule, which comprises binding the lectin to the lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity.

Lectin is a protein that recognizes a specific sugar chain structure and exhibits binding activity. As the lectin, for example, those derived from animals (eg, vertebrates, invertebrates), plants (eg, legumes, grasses), and fungi (eg, mushrooms, koji) are known. Examples of legume lectins include concanavalin A (ConA), lentil glutinin (LCA), and pea lectin (Pisum sativum). Examples of the mushroom lectin include Heero Chawantake Lectin (AAL) and the like.

The lectin used in the present invention can also be appropriately selected depending on the sugar chain structure of the lectin target molecule. Examples of the lectin used in the present invention include fucose-specific lectins having an affinity for fucose, galectin having an affinity for galactose, sialic acid-reactive lectins, and the like, but fucose-specific lectins are preferable. Examples of fucose-specific lectins include lectins that recognize a sugar chain structure (core fucose structure) in which fucose is added by α1,6 to GlcNAc at the base of an N-type sugar chain [eg, LCA, pea methivum (PSA)). ], And a lectin that recognizes the sugar chain structure in which fucose is added to GlcNAc of N-type sugar chain, O-type sugar chain, and glycolipid sugar chain by any of α1, 2, α1,3, α1,4, and α1,6. [Example, AAL, Aspergillus oryzae Lectin (AOL)].

A lectin target molecule is a target molecule to be captured in the present invention that has the ability to bind to a lectin. The target molecule is a molecule consisting of or containing a specific sugar chain to which the lectin binds.

Preferably, the lectin target molecule is a glycoprotein. Glycoproteins as lectin target molecules include, for example, glycoproteins derived from mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms, or viruses. The glycoprotein as a lectin target molecule may be a glycoprotein that is desired to be measured, extracted, concentrated or purified. Specifically, the glycoprotein is a protein having an N-type sugar chain binding consensus sequence. The N-type sugar chain binding consensus sequence is an amino acid sequence composed of amino acid residues of Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline). N-type sugar chains are known to bind to asparagine (Asn) residues in the N-type sugar chain binding consensus sequence. More specifically, the lectin target molecule includes, but is not limited to, for example, AFP-L3, PSA, hemopexin (HPX), and transferrin (TF). PSA is known as a modified protein for sugar chains such as N-type sugar chains.

The lectin-reactive sugar chain-containing substance refers to a molecule containing a lectin-reactive sugar chain or a solid phase to which a lectin-reactive sugar chain is added. The lectin-reactive sugar chain-containing entity is different from the lectin target molecule to be captured by the methods of the invention. A lectin-reactive sugar chain-containing entity is not an antibody that uses a lectin or a lectin target molecule as an antigen, because the sugar chain reacts with the lectin. Examples of the molecule containing a lectin-reactive sugar chain include a biomolecule and an artificial synthetic product (eg, an artificial synthetic molecule such as an artificial synthetic polymer). Examples of the solid phase include particles (eg, magnetic particles); membranes (eg, nitrocellulose membrane, filter paper), supports such as columns; and containers such as plates (eg, multi-well plates) and tubes. Solid phase materials include, for example, glass, plastics, polysaccharide matrices, and metals. The lectin-reactive sugar chain-containing entity can be appropriately selected depending on the type of lectin used. For example, such selection can be made by assessing the ability of the sugar chain or sugar chain-containing entity to bind to the lectin (see Example, Example 2).

Preferably, the lectin-reactive sugar chain-containing entity is a biomolecule containing a lectin-reactive sugar chain. Biomolecules include, for example, polypeptides and nucleic acids. More preferably, the lectin-reactive sugar chain-containing entity is a glycoprotein. Glycoproteins as lectin-reactive sugar chain-containing entities include, for example, glycoproteins derived from mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms, or viruses. Glycoproteins as lectin-reactive sugar chain-containing entities also include, for example, immunoglobulins (eg, antibodies), secretory proteins, membrane-bound proteins, hormones, cytokines, chemocaines, virus-derived antigens, enzymes, extracellular matrices, etc. Examples include extracellular vesicular membrane proteins, tumor-specific antigens, and their partial peptides.

Preferably, the glycoprotein as a lectin-reactive sugar chain-containing entity is a protein having an N-type sugar chain binding consensus sequence. The N-type sugar chain binding consensus sequence is an amino acid sequence composed of amino acid residues of Asn-X-Ser or Asn-X-Thr (X is an amino acid residue other than proline). N-type sugar chains are known to bind to asparagine (Asn) residues in the N-type sugar chain binding consensus sequence. More specifically, as a protein having an N-type sugar chain binding consensus sequence, for example, hepatitis B virus surface antigen, human chorionic gonadotropin α chain, luteinizing hormone, PSA antigen, HE4 antigen, MUC1 antigen, thyroglobulin, etc. In addition, an antibody having an N-type sugar chain binding consensus sequence in a variable region can be mentioned.

More preferably, the glycoprotein as a lectin-reactive sugar chain-containing entity is an antibody having an N-type sugar chain binding consensus sequence in a variable region. Antibodies having an N-type sugar chain binding consensus sequence in the variable region include, for example, natural full-length antibodies and modified antibodies. Modified antibodies include, for example, antibody fragments having variable regions (eg, Fab, F (ab') ₂ ), and single chain antibodies. Further, examples of the antibody having the N-type sugar chain binding consensus sequence in the variable region include IgG, IgM, IgA, IgD, IgE, and IgY. The antibody having the N-type sugar chain binding consensus sequence in the variable region may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

In the method of the present invention, a lectin is bound to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing entity. In other words, the method of the present invention is carried out in a system containing at least three components of a lectin target molecule and a lectin and a lectin-reactive sugar chain-containing substance, and contains a lectin and a lectin-reactive sugar chain as a means for capturing the lectin target molecule. The entity is used together. By using the lectin in the presence of a lectin-reactive sugar chain-containing entity, the binding of the lectin to the lectin target molecule can be enhanced. Therefore, the method of the present invention is useful, for example, for the measurement, extraction, concentration, purification, quantification, and detection of a lectin target molecule.

As the lectin target molecule, those contained in the liquid sample are used. In other words, the method of the present invention is carried out in a system in which a liquid sample containing a lectin target molecule is added with a lectin and a lectin-reactive sugar chain-containing entity. The origin of the liquid sample is not particularly limited, and it may be a biological sample derived from an organism, an environmental sample, or the like. Organisms from which biological samples are derived include, for example, mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens) and other animals and insects. , Microorganisms, plants, fungi, and fish, preferably mammals, fungi, and fish, more preferably mammals, and even more preferably humans. Biological samples can also be blood-related samples (eg, whole blood, serum, plasma), saliva, urine, milk, tissue or cell extracts, or mixtures thereof, which are blood itself or samples derived from blood. May be good. Examples of environmental samples include samples derived from soil, seawater, and freshwater. As the liquid sample, those subjected to pretreatment may be used. Such pretreatments include, for example, centrifugation, fractionation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.

The amount of the lectin used in the method of the present invention is not particularly limited as long as the target lectin can be captured in the presence of the lectin-reactive sugar chain-containing entity, and is, for example, 0.1 μg / mL to 100 mg / mL. .. The amount of lectin is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 10 μg / mL or more. The amount of lectin is also preferably 20 mg / mL or less, more preferably 10 mg / mL or less, even more preferably 2 mg / mL or less. Such an amount of lectin may be added to the liquid sample.

The amount of the lectin-reactive sugar chain-containing entity used in the method of the present invention is not particularly limited as long as it can support the capture of the lectin target molecule by the lectin, and is, for example, 0.001 μg / mL to 100 mg / mL. .. The amount of the lectin-reactive sugar chain-containing entity is preferably 0.005 μg / mL or more, more preferably 0.02 μg / mL or more, and even more preferably 0.1 μg / mL or more. The amount of the lectin-reactive sugar chain-containing entity is also preferably 20 mg / mL or less, more preferably 5 mg / mL or less, and even more preferably 1 mg / mL or less. Such an amount of lectin-reactive sugar chain-containing entity may be added to the liquid sample.

In a preferred embodiment, the method of the invention is a method of measuring a lectin target molecule. In the measuring method of the present invention, the lectin target molecule can be measured after the lectin is bound to the lectin target molecule. The measurement method of the present invention can be carried out according to an immunological method, and can be carried out by the same method as the immunological method by using a lectin-reactive sugar chain-containing entity and a lectin in combination instead of an antibody. can. Immunoassays to which the measurement method of the present invention conforms include, for example, enzyme-linked immunosorbent assay (EIA) (eg, chemiluminescent EIA (CLEIA), enzyme-linked immunosorbent ELISA (ELISA)), fluorescence immunoassay, chemiluminescent immunoassay. Methods, electrochemical luminescence immunoassays, aggregation methods, immunostaining, flowmetry methods, biolayer interference methods, In Situ PLA methods, chemically amplified luminescent proximity homogenius assays, line blot methods, Western blotting. Be done. The measuring method of the present invention is carried out qualitatively or quantitatively, and the presence or absence or amount of a lectin target molecule can be evaluated.

In the method of the present invention, an affinity substance specific to the lectin target molecule may be further used. The affinity substance specific to the lectin target molecule used in the method of the present invention binds to the lectin target molecule at a site (eg, peptide region) different from the lectin binding site (sugar chain) in the lectin target molecule. Examples of the affinity substance specific to the lectin target molecule include antibodies and aptamers, with antibodies being preferred.

The amount of the affinity substance specific to the lectin target molecule used in the method of the present invention is not particularly limited as long as it can sufficiently bind the lectin target molecule, and is, for example, 0.0001 μg / mL to 10 mg / mL. be. The amount of the affinity substance is preferably 0.001 μg / mL or more, more preferably 0.005 μg / mL or more, and even more preferably 0.01 μg / mL or more. The amount of the affinity substance is also preferably 2 mg / mL or less, more preferably 0.5 mg / mL or less, still more preferably 0.1 mg / mL or less. Such an amount of affinity material may be added to the liquid sample.

If an affinity substance specific for the lectin target molecule is further used, the measurement method of the present invention may be carried out in a sandwich manner. For example, an affinity substance specific to the lectin target molecule may be used as the immobilization means, and a lectin and a lectin-reactive sugar chain-containing entity may be used in combination as the detection means. Alternatively, a lectin and a lectin-reactive sugar chain-containing entity may be used in combination as the immobilization means, and an affinity substance specific to the lectin target molecule may be used as the detection means.

In the measuring method of the present invention, a lectin-reactive sugar chain-containing entity or a lectin, or an affinity substance specific to a lectin target molecule may be labeled with a labeling substance. Labeling substances include, for example, enzymes (eg, peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (eg, streptavidin and biotin, one of which is complementary to the sense strand and antisense strand nucleic acid. One of them), fluorescent substance or protein (eg, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substance (eg, luciferase, equolin, acridinium ester, tris (2,2'-) Bipyridyl) lutenium, luminol), radioactive substances (eg, ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²⁵ I). Preferably, a lectin-reactive sugar chain-containing entity or an affinity substance specific to the lectin-reactive sugar chain-containing entity is labeled.

More specifically, when the measuring method of the present invention is carried out in a sandwich style, it can be carried out by the following steps:
(1) The lectin target molecule is bound to an affinity substance specific to the lectin target molecule to obtain a first complex containing the lectin target molecule and the affinity substance specific to the lectin target molecule;
(2) In the presence of a lectin-reactive sugar chain-containing substance, lectin is bound to the first complex to obtain a lectin target molecule, an affinity substance specific to the lectin target molecule, a lectin, and a lectin-reactive sugar chain-containing substance. Obtaining a second complex containing; and (3) measuring the amount of lectin target molecule contained in the second complex.

In step (1), as the lectin target molecule, those contained in the liquid sample as described above can be used. Therefore, as a specific embodiment of the step (1), the step (1) may be performed by adding an affinity substance specific to the lectin target molecule to the liquid sample containing the lectin target molecule. In step (1), an affinity substance specific to the amount of the lectin target molecule as described above can be used, but preferably, an excess amount of the lectin target molecule is specific to the expected lectin target molecule. Affinity substance may be used. As the affinity substance specific to the lectin target molecule, it is preferable to use a substance immobilized on a solid phase as described above. Binding of the lectin target molecule and the affinity substance specific to the lectin target molecule can be achieved by incubating both in a solution (eg, buffer), thereby resulting in a lectin target molecule and a lectin target molecule. A first complex containing a specific affinity substance is obtained.

In step (2), the second complex is obtained. As a specific embodiment of the step (1), by adding a lectin and a lectin-reactive sugar chain-containing entity to the solution containing the first complex obtained in the step (1), the step (2) May be done. In step (2), the above-mentioned amount of lectin and the above-mentioned amount of lectin-reactive sugar chain-containing substance can be used, but preferably, an excess equivalent with respect to the expected lectin target molecule. Lectin and an excess equivalent of the lectin-reactive sugar chain-containing substance may be used. Either the lectin or the lectin-reactive sugar chain-containing entity may be labeled with the labeling substance, but preferably the lectin-reactive sugar chain-containing entity may be labeled with the labeling substance. Binding of lectins to the first complex in the presence of lectin-reactive sugar chain-containing entities can be achieved by incubating the three in a solution (eg, buffer), thereby the lectin target molecule, A second complex containing an affinity substance specific for the lectin target molecule, a lectin, and a lectin-reactive sugar chain-containing substance is obtained. When a substance immobilized on a solid phase is used as an affinity substance specific for the lectin target molecule, the second complex is immobilized on the solid phase. In this case, a cleaning step consisting of removal and addition of the liquid phase may be provided after the step (2).

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

The present invention also provides a complex comprising a lectin target molecule, a lectin and a lectin-reactive sugar chain-containing entity. The complex of the present invention may further contain an affinity substance specific for the lectin target molecule. The complex of the present invention can be made by incubating and binding these constituents in solution.

Definitions, examples and preferred examples of lectin target molecules, lectins, lectin-reactive sugar chain-containing entities, and affinity substances specific for lectin target molecules in the complex of the present invention are as described above.

The complex of the present invention is useful, for example, in practicing the capture method of the present invention.

The present invention further provides a reagent for capturing a lectin target molecule, which comprises a lectin and a lectin-reactive sugar chain-containing entity in a mixed form. In other words, the capture reagent of the present invention comprises a composition of a lectin and a lectin-reactive sugar chain-containing entity. Since the lectin and the lectin-reactive sugar chain-containing substance contained in the reagent of the present invention are in a mixed form, a complex is formed between the lectin and the lectin-reactive sugar chain-containing substance in the capture reagent of the present invention. You may.

When the reagent of the present invention is used for measuring a lectin target molecule, either the lectin or the lectin-reactive sugar chain-containing substance may be labeled with a labeling substance, but the lectin-reactive sugar chain-containing substance is preferably labeled. It may be labeled with a substance. Alternatively, if neither the lectin nor the lectin-reactive sugar chain-containing entity is labeled with the labeling substance, the reagent of the present invention may contain the labeling substance in an unmixed form with the lectin and the lectin-reactive sugar chain-containing entity. good.

The reagent of the present invention may further contain an affinity substance specific for the lectin target molecule. The affinity substance specific to the lectin target molecule may be labeled with a labeling substance. Further, the affinity substance specific to the lectin target molecule may be immobilized on the solid phase as described above. In this case, the reagents of the present invention can be suitably used for measuring lectin target molecules in a sandwich mode. The reagent of the present invention contains an affinity substance specific to the lectin target molecule in either a mixed form or a non-mixed form with the lectin and the lectin-reactive sugar chain-containing entity, but is preferably contained in the non-mixed form.

Definitions, examples and preferred examples of lectin target molecules, lectins, lectin-reactive sugar chain-containing entities, and affinity substances specific for lectin target molecules in the reagents of the present invention are as described above.

The reagent of the present invention is useful, for example, for simple implementation of the capture method of the present invention, and can be suitably used, for example, for the measurement method of the present invention.

The present invention also provides a kit for capturing a lectin target molecule, which comprises a lectin and a lectin-reactive sugar chain-containing entity. The lectin and lectin-reactive sugar chain-containing entities contained in the kits of the present invention are in a mixed form (ie, in the form of a composition) or in a non-mixed form (ie, in a form isolated from each other). Therefore, the components contained in the kit of the present invention are provided in a form contained in different containers (eg, tubes, plates) or in different compartments of the same container.

When the kit of the present invention is used for measuring a lectin target molecule, either the lectin or the lectin-reactive sugar chain-containing substance may be labeled with a labeling substance, but preferably the lectin-reactive sugar chain-containing substance is labeled. It may be labeled with a substance. Alternatively, if neither the lectin nor the lectin-reactive sugar chain-containing substance is labeled with a labeling substance, the kit of the present invention is labeled with a labeling substance and / or an affinity substance (labeled with a labeling substance) specific for the lectin target molecule. May be included).

The kit of the present invention may further contain an affinity substance specific for the lectin target molecule. In this case, the kit of the present invention can be suitably used for measuring a lectin target molecule in a sandwich mode. The affinity substance specific to the lectin target molecule may be immobilized on the solid phase as described above.

In the kit of the present invention, the definitions, examples and preferable examples of the lectin target molecule, the lectin, the lectin-reactive sugar chain-containing entity, and the affinity substance specific to the lectin target molecule are as described above.

The kit of the present invention is useful for simple implementation of the capture method of the present invention, for example, and can be suitably used for the measurement method of the present invention, for example.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

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

Specifically, the preparation was carried out as follows.
(1) Mouse IgG1 antibodies A, B, C and D, which are not anti-LCA antibodies, were digested with pepsin. Digestion was carried out by adding pepsin (SIGMA) to mouse IgG1 antibodies A, B, C and D buffered in citrate buffer, reacting at 37 ° C. for 1 hour, and stopping the reaction by adding Tris buffer. ..
(2) Gel filtration was performed with Superdex 200 increase 10/300 GL equilibrated with PBS to obtain _{4 kinds of purified mouse IgG F (ab') 2 fragments.}
(3) Purified mouse IgG F (ab') ₂ fragment antibodies A, B, C and D were labeled with horseradish peroxidase (HRP). Labeling of HRP was carried out using Peroxidase Labeling Kit-NH2 (Dojin Kagaku Kenkyusho) according to the attached protocol, and four purified antibody fragments-HRP (mouse IgG F (ab') _2- HRP) were obtained.

Example 2: Confirmation of purification degree, molecular weight and lectin reactivity of antibody fragment The purification degree, molecular weight and lectin reactivity of the four antibody fragments prepared in Example 1 were confirmed.

Specifically, the confirmation was performed as follows.
(1) The molecular weights of purified mouse IgG F (ab') ₂ fragment antibodies A, B, C and D were confirmed by SDS-PAGE (Fig. 1, left).
(2) The LCA reactivity of purified mouse IgG F (ab') ₂ fragment antibodies A, B, C and D was confirmed by a lectin blot (Fig. 1, right).
(3) The LCA reactivity of purified mouse IgG F (ab') ₂ fragment antibodies A, B, C and D was confirmed by ELISA. Purified mouse IgG F (ab') ₂ fragment antibodies A, B, C and D were diluted with PBS to 5 μg / mL each. 50 μL of the diluted solution was incubated in U96 MaxiSorp Nunc-Immuno Plate (Thermo) at 4 ° C. for 24 hours to solidify the antibody. The antibody-immobilized wells were washed 3 times with 250 μL of PBST and then blocked with 150 μL of 10% blocking reagent (Carbofree blocking solution; Vector) at 37 ° C. for 1 hour. The antibody-immobilized wells were washed 3 times with 250 μL PBST. Next, 50 μL of a solution diluted with LCA-HRP (J-Oil Mills) to 5 μg / mL with a 10% blocking reagent was added, and the solution was incubated at 37 ° C. for 1 hour in an antibody-immobilized well. Wells were washed 3 times with 250 μL PBST. 50 μL of TMB substrate solution (Dako) was added to the wells after washing, and the reaction was carried out at room temperature for 5 minutes. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm (Fig. 2).

As a result, it was confirmed that antibody fragments A and B are LCA-reactive, but antibody fragments C and D are LCA-non-reactive (Table 1, FIGS. 1 and 2).

The antibody used was analyzed.
Antibody A had an N-type sugar chain binding consensus sequence in the heavy chain of the antibody variable region. This indicates that the F (ab') ₂ fragment is modified with an N-type sugar chain. Therefore, it is considered that the F (ab') ₂ fragment antibody A exhibits lectin reactivity via an N-type sugar chain.
Antibody B is similar to antibody A.
Antibody C did not have an N-type sugar chain binding consensus sequence in either the heavy chain or the light chain of the antibody variable region. This indicates that the F (ab') ₂ fragment is not modified with an N-type sugar chain. Therefore, it is considered that the F (ab') ₂ fragment antibody C does not show lectin reactivity.
Antibody D is similar to antibody C.

Example 3: Construction of an AFP-L3 measurement system using a lectin in the presence of a lectin-reactive antibody fragment Immunological measurement of AFP-L3 using _{the mouse IgG F (ab') 2-HRP prepared in Example 1.} I built a system.

Specifically, the construction was carried out as follows.
(1) anti-AFP F (ab') ₂ was diluted with PBS to 10 μg / mL. 50 μL of the diluted solution was incubated in U96 MaxiSorp Nunc-Immuno Plate at 4 ° C. for 24 hours to solidify the antibody.
(2) The antibody-immobilized well was washed 3 times with 250 μL of PBST and then blocked with 150 μL of 10% blocking reagent at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Next, 50 μL of AFP-L3 solution and AFP-L1 solution, whose concentrations were converted from the measured values of Mutasuwako AFP-L3 (Wako Pure Chemical Industries, Ltd.), diluted 1000 ng / mL to 2 n with 10% blocking reagents, respectively. The solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Next, the mouse IgG F (ab') _2- HRP prepared in Example 1 was diluted with a 10% blocking reagent to 5 μg / mL, and LCA (Vector) was further added to 100 μg / mL. A mixture was prepared, the mixture was added to antibody-immobilized wells, and incubated at 37 ° C. for 1 hour.
(5) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Then, 50 μL of TMB substrate solution (Dako) was added and reacted at room temperature for 5 minutes. After terminating the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.

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

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

Example 4: Confirmation of involvement of lectin-reactive sugar chain in lectin-reactive antibody fragment in AFP-L3 detection N-glycosidase F was used for the sugar chain of _{mouse IgG F (ab') 2 antibody B prepared in Example 1.} It was confirmed whether or not the sugar chain-removed mouse IgG F (ab') ₂ antibody B maintained the AFP-L3 reactivity in the coexistence of LCA.

Specifically, the confirmation was performed as follows.
(1) The mouse IgG F (ab') ₂ antibody B prepared in Example 1 was treated with N-glycosidase F. For N-glycosidase F treatment, N-glycosidase F (ROCHE) was added to _{mouse IgG F (ab') 2} antibody B buffer exchanged with Tris-hydrochloric acid buffer, and the mixture was reacted at 37 ° C. for 24 hours. N-glycosidase F is an enzyme that specifically cleaves the GlcNAc-Asn bond between an N-linked sugar chain and a protein.
(2) Gel filtration was performed with Superdex 200 increase 10/300 GL equilibrated with PBS to obtain a _{sugar chain-removed mouse IgG F (ab') 2 fragment.}
(3) Sugar chain-removed mouse IgG F (ab') _2- HRP was prepared in the same manner as in Example 1.
(4) The degree of purification, molecular weight and LCA reactivity were confirmed in the same manner as in Example 2 (FIGS. 7 and 8).
(5) Similar to Example 3, the AFP-L3 reactivity of LCA in the presence of the labeled antibody was confirmed (FIGS. 9, Table 3). The AFP concentration was examined at 1 μg / mL.

As a result, LCA could not detect AFP-L3 in the presence of the sugar chain-removing labeled antibody fragment (FIGS. 7 and 8) that lost LCA reactivity (Tables 3 and 9).

From the above, it was shown that the sugar chain in the variable region of the antibody is important for the detection of AFP-L3 by the lectin.

Example 5: AFP-L3 detection by lectin in the presence of mouse IgG having an N-type sugar chain consensus sequence in the variable region The presence or absence of the N-type sugar chain consensus sequence in the variable region of the antibody determines AFP-L3 detection. We examined whether it would affect it.

Specifically, the examination was conducted as follows.
(1) An anti-TNFα antibody (P) having an N-type sugar chain binding consensus sequence (Asn-Ser-Ser) in the variable region and an anti-TNFα antibody (N) not having it, and an N-type sugar chain binding consensus sequence in the variable region (1) The anti-IL-10 antibody (P') having Asn-Asp-Thr) and the anti-IL-10 antibody (N') not having Asn-Asp-Thr) were HRP-labeled in the same manner as in Example 1, respectively.
(2) Similar to Example 3, the AFP-L3 reactivity in the coexistence of LCA was confirmed. The AFP concentration was examined at 5 μg / mL.

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

From the above, it was shown that the lectin can specifically detect AFP-L3 in the presence of an antibody having an N-type sugar chain binding consensus sequence in a variable region (regardless of the type of antigen to which this antibody binds).

Example 6: Construction of AFP-L3 measurement system by lectin in the presence of lectin-reactive antigen HBs antigen (hepatitis B virus surface antigen), which is a glycoprotein having an N-type sugar chain binding consensus sequence other than mouse IgG, is used. Therefore, an immunological measurement system for AFP-L3 was constructed.

Specifically, the construction was carried out as follows.
(1) HBs antigens having an N-type sugar chain binding consensus sequence were HRP-labeled in the same manner as in Example 1.
(2) Similar to Example 3, the AFP-L3 reactivity in the coexistence of LCA was confirmed. The AFP concentration was examined by diluting from 1000 ng / mL to 2 n. HBsAg-HRP was reacted at 2 μg / mL.

As a result, LCA was able to specifically detect AFP-L3 in the presence of HBsAg having an N-type sugar chain binding consensus sequence (Tables 5 and 11).

From the above, it was shown that the lectin can specifically detect AFP-L3 not only in the presence of an antibody but also in the presence of a lectin-reactive antigen.

Example 7: Confirmation of involvement of lectin-reactive sugar chains in the antigen in AFP-L3 detection By removing the sugar chains of the HBs antigen with N-glycosidase F, the sugar chain-removed HBs antigen is AFP in the coexistence of LCA. It was confirmed whether the −L3 reactivity was maintained.

Specifically, the confirmation was performed as follows.
(1) N-glycosidase F treatment of HBs antigen was carried out. The N-glycosidase F treatment was carried out using the reaction solution attached to N-glycosidase F and PNGase F (New England Biolabs) according to the protocol attached to PNGase F, and sugar chains were removed.
(2) The sugar chain-removed HBs antigen was labeled with HRP in the same manner as in Example 1.
(3) Similar to Example 2, the degree of purification, molecular weight and LCA reactivity were confirmed.
(4) Similar to Example 6, the AFP-L3 reactivity in the coexistence of LCA was confirmed. The AFP concentration was examined at 1 μg / mL. HBsAg-HRP was added at 1 μg / mL.

As a result, LCA could not detect AFP-L3 in the presence of the sugar chain-removing labeled antigen (FIGS. 12 and 13) that lost LCA reactivity (Tables 6 and 14).

From the above, it was shown that the sugar chain in the glycoprotein antigen is important for the detection of AFP-L3 by the lectin.

Example 8: Detection of AFP-L3 by lectin in the presence of lectin-reactive glycoprotein LCA was able to detect AFP-L3 not only in the presence of an antibody but also in the presence of an antigen (Example 7). Therefore, another glycoprotein having an N-type sugar chain binding consensus sequence was HRP-labeled on the amino acid sequence, and it was confirmed whether LCA could detect AFP-L3 in the presence of the other glycoprotein.

Specifically, the confirmation was performed as follows.
(1) HCGα (human chorionic gonadotropin α chain) and LH (luteinizing hormone) having an N-type sugar chain binding consensus sequence in the amino acid sequence were labeled with HRP in the same manner as in Example 1, respectively.
(2) Similar to Example 3, the AFP-L3 reactivity of LCA was confirmed. The AFP concentration was examined at 1 μg / mL.

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

数値は、ＯＤ４５０の測定値を示す。

The numerical value shows the measured value of OD450.

From the above, it was shown that the lectin can specifically detect AFP-L3 in the presence of the lectin-reactive glycoprotein.

Example 9: Construction of a detection system for AFP-linked sugar chains by various lectins in the presence of a lectin-reactive labeled antibody or a lectin-reactive labeled antigen Mouse IgG F (ab') _2- HRP having an N-type sugar chain-binding consensus sequence. Alternatively, in the presence of HBs antigen-HRP, a detection system for AFP-binding sugar chains having the following structural formula was constructed by using hirochawantake lectin (AAL) and concanavalin A (ConA) as lectins.

Regarding the above structural formula, it is reported that 60% or more is contained in the cultured cells (Huh7) and the hepatocellular carcinoma sample according to the previously reported (Nakagawa et al., J Proteome Res. 2008 Jun; 7 (6): 2222-33). The structural formula used is shown as a representative example.

Specifically, the construction was carried out as follows.
(1) In the same manner as in Example 3, AFP-L3 and AFP-L1 were diluted with a 10% blocking reagent to 1 μg / mL, respectively, and the reaction was carried out using the diluted solution.
(2) The antibody-immobilized well was washed 3 times with 250 μL of PBST. _{Next, the mouse IgG F (ab') 2-} HRP having the N-type sugar chain binding consensus sequence prepared in Example 1 and the HBs antigen-HRP prepared in Example 6 were each adjusted to 1 μg / mL with a 10% blocking reagent. To prepare a mixture prepared by diluting the blocking reagent as described above, and further adding either AAL (J-Oil Mills) to 10 μg / mL or ConA (Vector) to 10 μg / mL, and the mixed solution to solidify the antibody. It was added to the phased well and incubated at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 250 μL of PBST. 50 μL of TMB substrate solution was added to the washed wells, and the reaction was carried out at room temperature for 5 minutes. After terminating the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.

As a result, AAL and ConA can detect AFP-bound sugar chains in the presence of _{mouse IgG F (ab') 2-} HRP or HBsAg-HRP having an N-type sugar chain consensus sequence, similar to LCA. It was completed (Table 9, FIGS. 15 and 16).

From the above, it was shown that AFP-bound sugar chains can be detected not only in LCA but also in other lectins in the presence of glycoprotein.

Example 10: Construction of a detection system for PSA-linked sugar chains by lectin in the presence of a lectin-reactive labeled antibody or lectin-reactive labeled antigen AAL-reactive sugar chains are bound to PSA (prostate-specific antigen). It has been reported. PSA-linked AAL-reactive fucosylated sugar chain having the following structural formula using AAL as a lectin in the presence of _{mouse IgG F (ab') 2-} HRP and HBs antigen-HRP having an N-type sugar chain-binding consensus sequence. The detection system of was constructed.

Regarding the above structural formula, according to a previous report (Tajiri et al., J Glycobiology. 2008 Jan; 18 (1): 2-8), three cases of prostate cancer examination were analyzed, and it was reported that the relief abundance was the highest in common. The structural formula is shown as a typical example.

Specifically, the construction was carried out as follows.
(1) anti-PSA F (ab') ₂ was diluted with PBS to 10 μg / mL. 50 μL of the diluted solution was incubated in U96 MaxiSorp Nunc-Immuno Plate at 4 ° C. for 24 hours to solidify the antibody.
(2) The antibody-immobilized well was washed 3 times with 250 μL of PBST and then blocked with 150 μL of 10% blocking reagent at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 250 μL of PBST of PBST, and Lumipulse PSA-N standard PSA solution (Fujirebio) 100 ng / mL, 40 ng / mL, 5 ng / mL, 0.5 ng / mL, 0 ng. 50 μL of / mL was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 250 μL of PBST. _{The mouse IgG F (ab') 2-} HRP having the N-type sugar chain consensus sequence prepared in Example 1 and the HBs antigen-HRP prepared in Example 6 were each adjusted to 1 μg / mL with a 10% blocking reagent. And AAL was added to 10 μg / mL respectively. The solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(5) The antibody-immobilized well was washed 3 times with 250 μL of PBST. 50 μL of TMB substrate solution was added to the washed wells, and the reaction was carried out at room temperature for 5 minutes. After terminating the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.

As a result, AAL detects PSA-bound AAL-reactive sugar chains in a concentration-dependent manner in the presence of _{mouse IgG F (ab') 2-} HRP or HBsAg-HRP having an N-type sugar chain-binding consensus sequence. (Table 10, FIG. 17).

From the above, it was shown that the lectin can detect not only AFP-L3 but also other lectin-reactive glycoproteins such as PSA in the presence of a lectin-reactive labeled antibody or a lectin-reactive labeled antigen.

Example 11: Enhancement of binding force between lectin and target molecule by glycoprotein (1)
This example was carried out to prove the enhancement of the binding force between the lectin and the target molecule by glycoprotein. Conditions detected after the reaction of the target molecule (AFP-L3) and the lectin (LCA) with a glycoprotein (mouse IgG F (ab') _2- HRP or HBs antigen-HRP having an N-type sugar chain binding consensus sequence). (Washing conditions) and target molecule (AFP-L3) lectin (LCA) in the presence _{of glycoprotein (mouse IgG F (ab') 2-HRP or HBs antigen-HRP with N-type sugar chain binding consensus sequence)} It was examined whether there was a difference in the detectability of the target molecule under the conditions of reaction with (simultaneous addition condition). The number of washes was the same between the conditions.

Specifically, the examination was conducted as follows.
(1) anti-AFP F (ab') ₂ was diluted with PBS to 10 μg / mL. 100 μL of the diluted solution was incubated in F16 MaxiSorp Nunc-Immuno Plate (Thermo) at 4 ° C. for 24 hours to solidify the antibody.
(2) The antibody-immobilized well was washed 3 times with 300 μL of PBST and then blocked with 200 μL of 10% blocking reagent at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Then, the AFP-L3 solution and the AFP-L1 solution, each of which had their concentrations converted from the measured values of Mutaswaco AFP-L3, were diluted with a 10% blocking reagent to 500 ng / mL. 100 μL of the diluted solution was added and incubated at 37 ° C. for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Under washing conditions, LCA was diluted with 10% blocking reagent to 100 μg / mL, 100 μL of the diluted solution was added to the antibody-immobilized well, and the mixture was incubated at 37 ° C. for 1 hour. Under the co-addition condition, 100 μL of only 10% blocking reagent was added to the antibody-immobilized well, and the mixture was incubated at 37 ° C. for 1 hour.
(5) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Under washing conditions, the mouse IgG F (ab') ₂ antibody A-HRP 5 μg / mL prepared in Example 1 or the HBsAg-HRP prepared in Example 6 was diluted with a 10% blocking reagent to 2 μg / mL. Then, 100 μL of the diluted solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour. Under the co-addition conditions, the mouse IgG F (ab') ₂ antibody A-HRP 5 μg / mL or HBsAg-HRP prepared in Example 1 was diluted with a 10% blocking reagent to 2 μg / mL, and LCA was further added. Each was added so as to be 100 μg / mL. 100 μL of the diluted solution was added to the antibody-immobilized wells and incubated at 37 ° C. for 1 hour.
(6) The antibody-immobilized well was washed 3 times with 300 μL of PBST. 100 μL of TMB substrate solution was added to the wells after washing, and the reaction was carried out at room temperature for 5 minutes. After terminating the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.

As a result, the reactivity to AFP-L3 was significantly attenuated by adding a washing step after the reaction between AFP-L3 and lectin (LCA) (FIGS. 18 and 19). AFP-L3 was reacted with a lectin (LCA) in the presence of a labeled glycoprotein (mouse IgG F (ab') _{2-HRP or HBsAg-HRP having an N-type sugar chain consensus sequence) under the simultaneous addition conditions.} Only AFP-L3 could be detected specifically (FIGS. 18 and 19).

From the above, it was shown that the binding force between the lectin and the target molecule is enhanced by reacting the lectin (LCA) with the target molecule (AFP-L3) in the presence of glycoprotein.

Example 12: Enhancement of binding force between lectin and target molecule by glycoprotein (2)
This example was carried out to prove the enhancement of the binding force between the lectin and the target molecule by glycoprotein. After the reaction between the target molecule (AFP-bound fucosylated sugar chain) and the lectin (AAL), it was washed, and the conditions (washing conditions) detected by glycoprotein (HBsAg-HRP) and the target molecule (AFP-bound fucosylated sugar chain) were determined. It was examined whether there is a difference in the detectability of the target molecule under the condition of reacting with the lectin (AAL) in the presence of glycoprotein (HBs antigen-HRP) (concurrent addition condition). The number of washes was the same between the conditions.

Specifically, the examination was conducted as follows.
(1) In the same manner as in Example 11, 100 μL of AFP-L3 and AFP-L1 diluted to 500 ng / mL were added to the wells on which _{anti-AFP F (ab') 2 was immobilized, and the temperature was 37 ° C.} Incubated for hours.
(2) The antibody-immobilized well was washed 3 times with 300 μL PBST. Under washing conditions, AAL was diluted with 10% blocking reagent to 10 μg / mL, 100 μL of diluted AAL solution was added to antibody-immobilized wells, and the resulting solution was incubated at 37 ° C. for 1 hour. Under the co-addition conditions, 100 μL of only 10% blocking reagent was added to the antibody immobilization well, and the obtained solution was incubated at 37 ° C. for 1 hour.
(3) The antibody-immobilized well was washed 3 times with 300 μL of PBST. Under washing conditions, the HBsAg-HRP prepared in Example 6 was diluted with a 10% blocking reagent to a concentration of 2 μg / mL, and 100 μL of the diluted solution was incubated at 37 ° C. for 1 hour. Under the simultaneous addition conditions, the HBsAg-HRP prepared in Example 6 was diluted with a 10% blocking reagent to a concentration of 2 μg / mL, and the diluted HBsAg-HRP solution was further diluted with AAL to a concentration of 10 μg / mL. 100 μL of the diluted solution was added to the antibody-immobilized wells, and the mixture was incubated at 37 ° C. for 1 hour.
(4) The solution obtained in (3) was washed 3 times with 300 μL PBST. 100 μL of TMB substrate solution was added to the washed wells, and the mixture was reacted at room temperature for 5 minutes. After terminating the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.

As a result, the reactivity to the AFP-bound fucosylated sugar chain was significantly attenuated by adding a washing step after the reaction between the AFP-linked fucosylated sugar chain and the lectin (AAL) (FIG. 20). The AFP-bound fucosylated sugar chain could be detected only under the simultaneous addition condition of reacting the AFP-linked fucosylated sugar chain with the lectin (AAL) in the presence of the labeled glycoprotein (HBsAg-HRP) (Fig.). 20).

From the above, it was shown that the binding force between the lectin and the target molecule is enhanced by reacting the lectin (AAL) with the target molecule (AFP-bound fucosylated sugar chain) in the presence of glycoprotein.

Example 13: Construction of a detection system for AFP-L3 using _{mouse IgG F (ab') 2} having an N-type sugar chain consensus sequence as a solid phase In this experiment, mouse IgG F (ab') prepared in Example 1 ( ab _{') 2} was carried out to prove whether capture AFP-L3 on the solid phase. Solid phase of mouse IgG F (ab') ₂ fragment antibodies A and B having an N-type sugar chain consensus sequence and mouse IgG F (ab') ₂ fragment antibodies C and D having no N-type sugar chain consensus sequence. _{Then, it was examined whether mouse IgG F (ab') 2} could specifically capture AFP-L3 by reacting AFP-L3 and AFP-L1 with these immobilized antibodies in the coexistence of LCA.

Specifically, the examination was conducted as follows.
(1) The purified mouse IgG F (ab') ₂ fragment antibodies A, B, C and D prepared in Example 1 were diluted with PBS to 10 μg / mL. In a U96 MaxiSorp Nunc-Immuno Plate, 50 μL of the diluted solution was incubated at 37 ° C. for 1 hour to solidify.
(2) The antibody-immobilized well was washed 3 times with 250 μL of PBST and blocked with 150 μL of 10% blocking reagent at 37 ° C. for 24 hours.
(3) The antibody-immobilized well was washed 3 times with 250 μL of PBST, and the AFP-L3 solution and the AFP-L1 solution whose concentrations were converted from the measured values of Mutaswaco AFP-L3 were each 5 μg / mL with 10% blocking reagent. The solution was prepared by further diluting to 100 μg / mL of LCA and 1 μg / mL of anti-AFP F (ab') _2-HRP. 50 μL of the prepared solution was added to the antibody-immobilized well and incubated at 37 ° C. for 1 hour.
(4) The antibody-immobilized well was washed 3 times with 250 μL of PBST. Then, 50 μL of TMB substrate solution was added and reacted at room temperature for 5 minutes. After terminating the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.

As a result, LCA was able to specifically detect AFP-L3 under the condition that the LCA-reactive antibody fragment A or B was used as a solid phase (FIG. 21).

From the above, it was shown that the lectin target molecule can be captured by using the immobilized lectin-reactive sugar chain-containing entity in combination with the lectin.

Example 14: Construction of a detection system for hemopexin (HPX) -linked sugar chain and transferase (TF) -linked sugar chain using mouse IgG-HRP having an N-type sugar chain-binding consensus sequence in the variable region N-type sugar in the variable region Whether mouse IgG having a chain-binding consensus sequence can be reacted in coexistence with AAL to detect HPX-linked AAL-reactive fucosylated sugar chains and TF-linked AAL-reactive fucosylated sugar chains in the serum of liver cancer patients. investigated.

Specifically, the examination was conducted as follows.
(1) Mouse IgG having an N-type sugar chain binding consensus sequence in the amino acid sequence of the variable region was labeled with HRP in the same manner as in Example 1.
(2) Anti-HPX antibody (R & D SYSTEMS Clone # 698813) and anti-TF antibody (abcam Clone # 2A2) were treated with N glycosidase. In the N glycosidase treatment, N glycosidase F (ROCHE) was added to the anti-HPX antibody buffer exchanged with Trisic acid buffer, and the mixture was reacted at 37 ° C. for 24 hours. Then, limit filtration of 100 kDa was carried out to remove N glycosidase.
(3) The anti-HPX antibody and anti-TF antibody treated with N glycosidase were diluted with PBS to 2 μg / mL, dispensed into C96 MaxiSup Nunc-Immuno Plate (Thermo) at 50 μL / well, and dispensed at 4 ° C. It was immobilized for 24 hours.
(4) 200 μL of 250 μL of 1% BSA-PBS was added, and blocking was performed at 4 ° C. for 24 hours.
(5) Two hepatocellular carcinoma patient sera (HCC-1, HCC-2) and two healthy person sera (NHS-1, NHS-) washed three times with 300 μL PBST and diluted 500-fold with 1% BSA-PBS. 2) was added in 50 μL and incubated at 37 ° C. for 1 hour.
(6) Wash three times with 300 μL PBST, dilute mouse IgG-HRP having an N-type sugar chain consensus sequence in the variable region with a diluent (manufactured in-house) so as to be 2 μg / mL, and further AAL (manufactured in-house). Vector) was added to 20 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour.
(7) Washing was performed 3 times with 300 μL PBST, 50 μL of TMB substrate solution (Thermo) was added, the reaction was carried out at room temperature for 2 minutes, the reaction was stopped with 1N sulfuric acid, and the absorbance was measured at 450 nm.

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

From the above, it was shown that the lectin target molecule can be captured by using the immobilized lectin-reactive sugar chain-containing entity in combination with the lectin.

Claims (14)

Includes binding of a lectin to a lectin target molecule in the presence of a lectin-reactive sugar chain-containing substance using a system in which a lectin and a lectin-reactive sugar chain-containing substance are added to a liquid sample containing the lectin target molecule, respectively. , A method for capturing a lectin target molecule. The method according to claim 1, wherein the lectin is a legume lectin or a mushroom lectin. The method according to claim 1 or 2, wherein the lectin is a fucose-specific lectin. The method according to any one of claims 1 to 3, wherein the lectin is lentil glutinin (LCA), concanavalin A (ConA) or Heero Chawantake lectin (AAL). The method according to any one of claims 1 to 4, wherein the lectin target molecule is a glycoprotein. The method of claim 5, wherein the glycoprotein is AFP-L3 or PSA. The method of claim 5, wherein the glycoprotein is hemopexin (HPX) or transferrin (TF). The method according to any one of claims 1 to 7, wherein the entity is a glycoprotein having an N-type sugar chain binding consensus sequence. The method according to claim 8, wherein the glycoprotein having an N-type sugar chain-binding consensus sequence is an immunoglobulin having an N-type sugar chain-binding consensus sequence in a variable region. The method according to any one of claims 1 to 8, wherein the entity is a hepatitis B virus surface antigen (HBs antigen), human chorionic gonadotropin α chain (hCGα) or luteinizing hormone (LH). The method according to any one of claims 1 to 10, which is a method for measuring a lectin target molecule, which comprises measuring the lectin target molecule after binding of the lectin to the lectin target molecule. The method of claim 11, further using an affinity substance specific for the lectin target molecule. The method according to any one of claims 1 to 12, which is a method including the following:
(1) The lectin target molecule is bound to an affinity substance specific to the lectin target molecule to obtain a first complex containing the lectin target molecule and the affinity substance specific to the lectin target molecule;
(2) Lectin reactivity using a system in which a lectin and a lectin-reactive sugar chain-containing substance are added to a liquid sample containing a lectin target molecule and a first complex containing an affinity substance specific to the lectin target molecule . In the presence of a sugar chain-containing substance, the lectin is bound to the first complex to form a second complex containing a lectin target molecule, an affinity substance specific to the lectin target molecule, a lectin, and a lectin-reactive sugar chain-containing substance. To obtain; and (3) to measure the amount of lectin target molecule contained in the second complex. A kit for capturing a lectin target molecule, which comprises a lectin and a lectin-reactive sugar chain-containing entity in a non-mixed form.

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