JP5991032B2 - Analyte detection method using lectin - Google Patents

Description

  The present invention relates to a method for detecting an analyte using a lectin. More specifically, the present invention relates to a method for detecting an analyte having high sensitivity and low noise using a lectin, including a step of subjecting a sugar chain of an analyte (for example, an antigen) to a predetermined treatment.

  Post-translational modifications such as glycosylation play an extremely important role in order for proteins, which are the main players responsible for vital functions of living organisms, to function in an orderly manner in the cellular society. Most proteins in the body are modified by sugar chains, and the sugar chains added to the proteins are virus infection, protozoan parasitism, infection, toxin binding, hormone binding, fertilization, developmental differentiation, protein stability In recent years, it has become clear that it plays an important role in various life phenomena such as cancer cell metastasis and apoptosis.

  Even for proteins with the same amino acid sequence (proteins with the same name), there are a wide variety of modified sugar chains, and it is known that the sugar chain structure differs depending on the state of the cell producing the protein.

  The relationship between such sugar chain changes and diseases has also been clarified. For example, liver cancer based on α-fetoprotein (AFP) sugar chain fraction measurement as disclosed in Patent Document 1 and Non-Patent Document 1 is known. Differentiation method, prostate cancer differentiation method by prostate specific antigen (PSA) sugar chain fraction measurement as disclosed in Patent Document 2, carcinoembryonic antigen (CEA) as disclosed in Non-Patent Documents 2 and 3 It is applied to a method for differentiating adenocarcinoma by measuring the sugar chain fraction.

  For specific detection of sugar chains on glycoproteins, proteins having the ability to specifically recognize, bind and crosslink sugar chains called lectins are widely used. This is because antibodies against sugar chains are very difficult to produce and difficult to obtain.

  Lectins are inexpensive and can be obtained in large quantities, and are excellent in protein stability and can be stored for a long time. However, lectins have problems such as lower binding activity and lower specificity than antibodies.

  For example, nodafuji lectin (WFA) is known to have N-acetylgalactosamine as the main linking sugar chain, but it binds slightly to galactose, and therefore nonspecifically when galactose residues are present in the reaction system. It will react.

  As a method for simply quantitatively analyzing a sugar chain on a specific protein using a lectin having such characteristics, there is a sandwich assay using an antibody against the protein and a lectin.

  However, this method is effective when the target protein has been purified to some extent, but a large amount of contaminants such as blood proteins and urine used as specimens in normal disease diagnosis, such as glycoproteins and glycolipids other than the measurement target. In the system included in the system, the sensitivity and quantitative performance were remarkably reduced, and the analysis was very difficult. For this reason, the concentration of the target protein in the serum is extremely high (about several μg / mL), and is used only for serum diagnosis and the like.

  As a measure to reduce the influence of contaminants, a blocking agent that suppresses the adsorption of serum contaminants to the surface of the support on which the antibody is immobilized (Patent Document 3), and an absorbent that adsorbs nonspecific substances in advance. Addition (Patent Documents 4 and 5) and investigation of a cleaning solution (Patent Document 3) that can efficiently remove molecules adsorbed on a support have been carried out.

  Here, bovine serum albumin (BSA), casein, and the like are known as blocking agents, and there are others using synthetic polymer materials. Absorbers of non-specific substances are molecules that can be removed from the reaction system by binding molecules that inhibit antigen-antibody reactions and molecules that cause noise, such as glycosaminoglycans and heparins, A sugar chain complex or the like may be used. As the cleaning liquid, the composition of the liquid has been studied, and an example in which the effects of salt strength and various surfactants are examined has been reported (Patent Document 3).

  However, in these background reduction measures, there are very few examples where the quantitative analysis using the detection lectin shows a dramatic effect, and the search for a blocking agent suitable for the target can be extremely time-consuming. It is a problem.

JP-A-61-292062 International Publication WO2010 / 090264 JP 2010-127827 JP 2009-53195 JP 2010-60293 A

Sugar Chains of Human Cord Serum α-Fetoprotein: Characteristics of N-linked Sugar Chains of Glycoproteins Produced in Human Liver and Hepatocellular Carcinomas, K. Yamashita et al., Cancer Res., 53, 1 (1993) Carbohydrate Structures of Nonspecific Cross-reacting Antigen-2, a Glycoprotein Purified from Meconium as an Antigen Cross-reacting with Anticarcinoembryonic Antigen Antibody, K. Yamashita et al., Biol. Chem., 264, 17873 (1989) Structural Studies of the Carbohydrate Moieties of Carcinoembryonic Antigens, K. Yamashita et al., Cancer Res., 47, 3451 (1987)

  In the detection system of an analyte having a sugar chain using a lectin (for example, an antigen), the present invention can detect an analyte that can improve the detection sensitivity and quantitativeness of the lectin by a simple technique (simple configuration). It is an object to provide a method and a detection kit.

  As a result of intensive studies to solve the above problems, the present inventors have found that a high background (noise) factor in a measurement system using lectin is that lectin used for detection has a certain range in sugar chain recognition. Therefore, even if a known treatment method using a blocking agent or the like is used, a certain amount of saccharide other than the detection target bound nonspecifically to a support or a ligand (such as an antibody) for capturing an antigen is generated. It has been found that it may bind to impurities such as proteins and glycolipids. Based on this new knowledge, the present inventors have proposed a glycan recognition molecule (preferably a glycan recognition molecule that binds strongly to the glycan, which is likely to be bound by the labeling lectin and possessed by impurities that are not to be detected. It has been found that a reduction in background and an increase in sensitivity can be obtained by masking with a lectin different from the labeling lectin), and the present invention has been completed. That is, the present invention includes the following matters.

[1] A method for detecting an analyte having a detection target sugar chain in a sample using a labeling lectin that binds to a plurality of types of sugar chains including a detection target sugar chain and a non-detection target sugar chain, Before the detection step of detecting the analyte bound to the labeling lectin,
A labeling treatment in which the labeling lectin is brought into contact with the analyte;
At least one of the non-detection target sugar mask carbohydrate-recognizing molecules that bind to a chain, the pre-detection process lines physician and a mask processing is brought into contact with contaminants having a non-detection target sugar chain (provided that the When the labeling process is performed before the masking process, the mask sugar chain recognition molecule that binds more easily to the non-detection target sugar chain than the labeling lectin is used)
The analyte is a prostate-specific antigen, the labeling lectin is Nodafuji lectin, and the mask sugar chain recognition molecule is a galectin, or
The method for detecting an analyte having a sugar chain to be detected, characterized in that the analyte is α-fetoprotein, the labeling lectin is a lentil lectin, and the sugar chain recognition molecule for the mask is a red bean lectin. .

  [2] The detection pretreatment step further includes a solid phase treatment in which the analyte and the contaminant are brought into contact with a support on which a ligand that binds to the analyte is immobilized. Detection method.

[3] The detection method according to [2], wherein the solid phase treatment is performed before the labeling process and the masking process.
[4] The detection method according to [2], wherein the labeling process and the masking process are performed before the solid-phase treatment.

[5] The detection method according to any one of [1] to [4], wherein the labeling process and the masking process are performed simultaneously.
[6] The detection method according to any one of [1] to [4], wherein the labeling process is performed before the masking process.

[7] The detection method according to any one of [1] to [4], wherein the mask process is performed before the labeling process.
[8] The detection method according to [2], wherein the solid phase treatment is performed after the labeling process and before the masking process.

  [9] The detection method according to [2], wherein the solid-phase treatment is performed after the mask treatment and before the labeling treatment.

[ 10 ] a reagent containing a labeling lectin that binds to a plurality of types of sugar chains including a detection target sugar chain and a non-detection target sugar chain;
A reagent comprising a mask sugar chain recognition molecule that binds to at least one kind of the non-detected sugar chain;
Look contains a reagent comprising a ligand that binds with the analyte having the detection target sugar chain,
The analyte is a prostate-specific antigen, the labeling lectin is Nodafuji lectin, and the mask sugar chain recognition molecule is a galectin, or
The analyte is α-fetoprotein, the labeling lectin is a lentil lectin, and the sugar chain recognition molecule for the mask is a red bean lectin, for detection of an analyte having a sugar chain to be detected kit.

  An example of a conceptual diagram of means for solving the problems of the present invention is shown in FIG. In FIG. 1a showing the conventional embodiment, the labeling lectin (60a) labeled with the labeling body (60b) recognizes a plurality of types of sugar chains including a detection target sugar chain and a non-detection sugar chain. Not only the detection target sugar chain (46c) of the detection target antigen (46) which is a light, but also the non-detection target sugar which is a non-specifically adsorbed glycoprotein (50) or glycolipid (52) which is a contaminant. It also binds to the chain (46d). For example, when a PSA antigen is used as the detection target antigen (46) and a WFA lectin is used as the labeling lectin (60a), N-acetyl-D-galactosamine (GalNAc) corresponds to the detection target sugar chain (46c), and galactose ( Gal) corresponds to the non-detected target sugar chain (46d). On the other hand, in FIG. 1b showing the embodiment of the present invention, the non-detection target sugar chain (46d) of the contaminant is masked by the mask sugar chain recognition molecule (75). ) Can be inhibited from binding to impurities.

  According to the present invention, in a detection system for an antigen using a lectin, an increase in the background of the measurement system is suppressed by a simple technique (simple configuration), and the detection sensitivity and quantification of sugar chains on the target antigen by the lectin are detected. It is possible to provide a method for detecting an antigen and a detection kit capable of improving the sensitivity.

  In addition, since the above-described effect can be obtained only by including a mask sugar chain recognition molecule in the reagent in a sandwich assay system, this is a simple method (simple configuration) and is effective for application in a diagnostic scene.

FIG. 1 shows that a sample is brought into contact with a support on which a ligand is immobilized, and in accordance with a conventional embodiment in the state (a) where an analyte and impurities are present on the support, The mechanism (b) in which the background is generated in the detection of the analyte, and the state (c) in which the effect of reducing the background and increasing the detection sensitivity is achieved according to the embodiment of the present invention are schematically shown. It is a conceptual diagram. FIG. 2 is a conceptual diagram showing an embodiment of the detection method of the present invention in which a solid phase treatment is performed after a labeling process and a mask process are performed simultaneously. FIG. 3 is a conceptual diagram showing an embodiment of the detection method of the present invention, which is performed in the order of mask treatment, solid phase treatment, and labeling treatment. FIG. 4 is a conceptual diagram showing an embodiment of the detection method of the present invention in which a labeling process and a masking process are performed following the solid phase treatment. FIG. 5 is a schematic diagram schematically showing the configuration of an SPFS measuring apparatus suitable for carrying out the analyte detection method of the present invention. 6 is a partially enlarged view of the periphery of the sensor chip of the measuring apparatus of FIG.

Hereinafter, the detection method and detection kit of the present invention will be described.
In the present specification, the “sugar chain to be detected” refers to a sugar chain specifically possessed by an analyte (for example, an antigen serving as a marker that becomes present at a relatively high concentration in blood when a specific disease occurs). In order to detect an analyte, it refers to a sugar chain intended to bind as much detection lectin as possible. On the other hand, the “non-detected sugar chain” is a sugar chain of a contaminant (a glycoprotein, glycolipid, etc. different from the analyte) present in the specimen together with the analyte, Therefore, it refers to a sugar chain that is intended not to bind the detection lectin as much as possible.

The “detection method” in the present specification naturally includes not only a method for qualitatively detecting an analyte but also a method for quantitatively detecting an analyte (analyte quantification method).
≪Labeling lectin used in the present invention, sugar chain recognition molecule for mask, etc.≫
In the method of the present invention, a lectin for labeling, a label, a sugar chain recognition molecule for mask, a specimen containing a ligand, an analyte and impurities, and a support for further immobilizing the ligand as required are used. Hereinafter, each element will be described in detail.

<Lectin>
In the detection method of the present invention, a lectin is used as a labeling lectin for labeling an analyte having a sugar chain to be detected. Moreover, a lectin may be used as a mask sugar chain recognition molecule for masking the detection target sugar chain of a contaminant. In the present invention, the sugar chain recognition molecule used for masking is not limited to a lectin, and includes, for example, an antibody that recognizes a sugar chain to be detected as an epitope and specifically binds to it. From the viewpoint of being inexpensive and available in large quantities, and excellent in protein stability and can be stored for a long period of time, lectins are preferred.

  A lectin binds to a specific sugar chain according to its type with high binding ability, but may bind to another sugar chain with low binding ability. Therefore, in the detection method of the present invention, a lectin that binds to a detection target sugar chain of a detection analyte with a high binding ability while binding to a non-detection target sugar chain of a contaminant with a weak binding ability is used. Applicable when used as A sugar chain recognition molecule that binds with a strong binding ability to a non-detection target sugar chain to which the labeling lectin binds with a weak binding ability is used as a mask sugar chain recognition molecule. Such a sugar chain recognition molecule for a mask is a sugar chain recognition molecule (for example, a label that does not substantially bind to a detection target sugar chain to which a labeling lectin binds with a strong binding ability or binds with a weak binding ability). It is necessary to be a different kind of lectin).

  Lectins are lectins belonging to various molecular families obtained from animals, plants, fungi, bacteria, viruses, etc., that is, ricin B chain-related “R-type lectins” found in all living worlds including bacteria; “Calnexin calreticulin” which exists and is involved in glycoprotein folding, “C-type lectin”, which is widely present in multicellular animals and contains many typical lectins such as “selectin” and “collectin” "Galectins" widely distributed in the animal kingdom and exhibiting specificity for galactose; "Legidae lectins" that form a large family in the plant legumes and "L-type lectins that are structurally similar to this and are involved in animal intracellular transport "Mannose 6-phosphate-binding" P-type lectin "involved in intracellular transport of lysosomal enzymes; including glycosaminoglycans" "Annexin" binds to an acidic sugar chain; belonging to the immunoglobulin super family contain "Siglec" can be classified into such as "I-type lectin."

  Specific examples of the lectin include ACA (sennin clectin), BPL (purple lectin lectin), ConA (Tatami bean lectin), DBA (horsegram lectin), DSA (saturated stag bean lectin), ECA (deigo bean). Lectin), EEL (Spindle Tree lectin), GNA (Yukinohana lectin), GSL I (Glyphonia bean lectin), GSL II (Glyphonia bean lectin), HHL (Amaryllis lectin), Jacarin (Jack fruit lectin), LBA ( Lima bean lectin), LCA (lentil lectin), LEL (tomato lectin), LTL (lotus bean lectin), MPA (American pelvis lectin), NPA (trumpet lectin), PHA- (Bean bean lectin), PHA-L (kidney bean lectin), PNA (peanut lectin), PSA (pea lectin), PTL-I (winged bean lectin), PTL-II (winged bean lectin), PWM (Yodobo lectin), RCA120 (Castor lectin), SBA (soybean lectin), SJA (endurectin), SNA (chicken chick collectin), SSA (Japanese chick lectin), STL (potato lectin), TJA-I (Kikarasuri lectin), TJA-II (Kiara Uri lectin), UDA (Common Stinging Nettle lectin), UEA I (Harienshi lectin), VFA (Fabama lectin), VVA (hairy vetch lectin), WFA (Noda Fuji) Kuching), WGA (bread wheat lectin), AAL (Heero tea Wanda Ke lectin), and the like AOL (Aspergillus Orizerekuchin).

<Labeled body>
In the method of the present invention, a labeled body complexed with a lectin is used to detect the antigen to be detected. For this purpose, a labeled lectin in which a labeled body and a lectin are complexed (bound) is produced.

As the label, a label known to those skilled in the art, such as a fluorescent dye, an enzyme / coenzyme, a chemiluminescent substance, and a radioactive substance, can be used.
Examples of fluorescent dyes include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), and hexachlorofluorescein family fluorescent dyes (Applied Biosystems Japan). ), Coumarin Family Fluorescent Dye (Invitrogen), Rhodamine Family Fluorescent Dye (GE Healthcare Biosciences), Cyanine Family Fluorescent Dye, Indian Carbocyanine Family Fluorescent Dye , Oxazine family fluorescent dyes, thiazine family fluorescent dyes, squaraine family fluorescent dyes, chelated lanthanide family fluorescent dyes, BODIPY® family fluorescent dyes (in vitro ), Naphthalenesulfonic acid family fluorescent dyes, pyrene family fluorescent dyes, triphenylmethane family fluorescent dyes, Alexa Fluor (registered trademark) dye series (Invitrogen Corp.) and other organic fluorescent dyes Is mentioned.

In addition, rare earth complex fluorescent dyes such as Eu and Tb (for example, ATBTA-Eu ³⁺ ), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), Examples of fluorescent dyes include fluorescent proteins typified by red fluorescent protein (DsRed) or Allophycocyanin (APC; LyoFlogen (registered trademark)), and fluorescent fine particles such as latex and silica.

  When using a sample derived from a blood sample for analysis, in order to minimize the effects of light absorption by iron derived from blood cell components in blood, the maximum fluorescence wavelength is set in the near infrared region, such as Cy5 and Alexa Fluor 647. It is desirable to use a fluorescent dye having the same.

The radioactive substance, a radioisotope (such as ^{32 P, 14 C, 125 I} , 3 H, 131 I) can be mentioned.
<Analyte (Substance to be detected)>
In the present invention, a molecule or molecular fragment that has a specific sugar chain recognized by a lectin and can be specifically recognized and bound by an appropriate ligand can be used as an analyte (substance to be detected). In the present specification, when the analyte is an antigen, it may be referred to as “detection target antigen”.

  Examples of such “molecule” or “molecular fragment” include, for example, nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acid), which may be single-stranded or double-stranded), Or nucleosides, nucleotides and their modified molecules); proteins (polypeptides, oligopeptides, etc.); amino acids (including modified amino acids); carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.); lipids; or modifications thereof Examples thereof include molecules and complexes. Among these, proteins (glycoproteins) and lipids (glycolipids) are preferable as the analyte, and proteins (glycoproteins) are more preferable.

  As the analyte of the present invention, for example, proteins such as cancer antigen / tumor marker, signal transduction substance, hormone and the like are suitable. Specific examples of the cancer antigen / tumor marker include PSA, AFP, CEA and the like.

<Samples containing analyte (substance to be detected)>
In the detection method of the present invention, a specimen that may contain an analyte (detection target substance) can be used as a test sample. A specimen that may contain an analyte may be a specimen that actually contains an analyte, or a specimen that does not actually contain an analyte. Examples of such specimens include biological samples such as blood, serum, plasma, urine, spinal fluid, saliva, cells, tissues, or organs, or preparations thereof (eg, biopsy specimens) and biological samples. Can be mentioned. In particular, blood, serum or plasma that may contain a cancer antigen / tumor marker is suitable as a specimen.

  Liquid samples such as blood, serum, plasma, urine, spinal fluid and saliva can be used after diluted with an appropriate buffer. In addition, a solid sample such as a cell, tissue, or organ is homogenized with an appropriate buffer about 2 to 10 times the volume of the solid sample, and the suspension or its supernatant is diluted as it is or further diluted. Can be used.

<Analyte, labeling lectin, mask sugar chain recognition molecule combination>
Specific examples of combinations of analyte (antigen to be detected), labeling lectin, and mask sugar chain recognition molecule (lectin) in the detection method of the present invention are shown in Table 1.

  In addition to those shown in Table 1, detection target antigens such as cancer antigens / tumor markers, contaminants (glycoproteins and glycolipids other than detection target antigens) that are likely to be included in the specimen, and the types of sugar chains each has The lectin for labeling that easily binds to the detection target sugar chain of the former and the mask sugar chain recognition molecule (lectin that easily binds to the non-detection target sugar chain of the latter that the lectin may bind simultaneously. ) Can be appropriately selected and used in combination.

<Ligand (antigen-capturing substance)>
In the present invention, a molecule that specifically recognizes and binds to an analyte (detection target substance) and does not interfere with sugar chain recognition by a lectin can be used as a ligand (antigen capture substance). The ligand is not particularly limited as long as it is an appropriate substance corresponding to the analyte. For example, an antibody, an aptamer, a synthetic peptide, or the like can be used. In particular, a monoclonal antibody against the antigen to be detected is suitable.

  For example, when a cancer antigen / tumor marker or the like is used as an antigen to be detected, it is appropriate to use an antibody (such as a monoclonal antibody) that specifically binds to the antigen as a ligand. For example, when human PSA is used as an antigen, an anti-human PSA antibody may be used.

The above “antibody” is used to mean not only a complete antibody but also any antibody fragment or derivative. In addition to a complete antibody, Fab, Fab ′ ₂ , CDR, humanized antibody, multifunctional antibody, single chain antibody Various antibodies such as (ScFv) are also included.

<Support>
In the detection method of the present invention, a support for immobilizing the ligand (capturing the analyte therein) can be used depending on the measurement system. Examples of the support include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resin, polystyrene resin, polyacrylamide resin, nylon resin and polycarbonate resin, and insoluble supports such as glass. . These supports are used in the form of beads (mainly spherical), plates (mainly planar) and the like. As the beads, a column filled with magnetic beads, resin beads, or the like can be used. In the case of a plate, a multiwell plate (96-well multiwell plate or the like), a biosensor chip, or the like can be used. Note that a planar support such as a plate may be referred to as a substrate.

The binding between the ligand and the support can be performed by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be used commercially.
A ligand that is immobilized on a support and is capable of capturing an analyte in a specimen to be fed and immobilizing the analyte on the support may be referred to as a solid-phased ligand.

<< Detection Method of the Present Invention >>
<Steps included in detection method>
The detection method of the present invention uses a labeling lectin that binds to a plurality of types of sugar chains including a detection target sugar chain and a non-detection target sugar chain, and detects an analyte having the detection target sugar chain in a sample. A detection pretreatment step including the following treatment is included before the detection step of detecting the analyte bound to the labeling lectin:
(1) A labeling treatment in which the labeling lectin is brought into contact with the analyte;
(2) mask treatment in which at least one kind of mask sugar chain recognition molecule that binds to the non-detection target sugar chain is brought into contact with a contaminant having the non-detection target sugar chain; and, if necessary,
(3) Solid phase treatment in which the analyte is brought into contact with a support on which a ligand that binds to the analyte is immobilized.

<Order in which processes and processes are performed>
In the detection method of the present invention, the detection pretreatment process and the detection process are performed in this order. The detection process may be performed after the last process included in the pre-detection process.

  In addition, various embodiments are included in the order of the labeling process, the mask process, and the solid-phase process performed as necessary in the pre-detection process. For example, the labeling process and the masking process can be performed simultaneously. Specifically, the following processing embodiments can be mentioned. Note that “labeling process + masking process” means that the labeling process and the masking process are performed simultaneously, and “→” means that the process on the left side is performed and then the process on the right side is performed. For example, “(labeling process + mask process) → solid phase process” indicates that the solid phase process is performed after the label process and the mask process are performed simultaneously.

(Processing order in the pre-detection process)
(1) Labeling process + masking process (2) Labeling process → masking process (3) Masking process → labeling process (4) (Labeling process + masking process) → Solid phase treatment (5) Labeling process → Masking process → Solid phase (6) Mask treatment → Label treatment → Solidification treatment (7) Label treatment → Solid phase treatment → Mask treatment (8) Mask treatment → Solid phase treatment → Label treatment (9) Solid phase treatment → ( Sign processing + mask processing)
(10) Solid-phase treatment → labeling treatment → mask treatment (11) Solid-phase treatment → mask treatment → labeling treatment FIGS. 2 to 4 show conceptual diagrams of some of these embodiments. 2 is a conceptual diagram corresponding to the above embodiment (4), FIG. 3 is the above embodiment (8), and FIG. 4 is a conceptual diagram corresponding to the above embodiment (11). In any embodiment, the lectin (60a) labeled with the label (60b) is finally contaminated with a glycoprotein (50) having a non-detected target sugar chain (46d), a glycolipid (52), and the like. Binding to the product is suppressed, so that it can bind to the analyte (46) having the sugar chain (46c) to be detected.

  As an embodiment of the present invention that does not include a solid phase treatment, for example, an antibody labeled with a donor fluorophore of a FRET (Fluorescence Resonance Energy Transfer) reagent as a ligand and an FRET reagent labeled as a label for labeling a lectin for labeling are used. A method using an acceptor phosphor is exemplified. In this case, glycoproteins and glycolipids (contaminants) other than analyte are non-specifically bound to the ligand (antibody), causing an increase in background. Therefore, even if the solid phase treatment is not included, the increased background can be reduced by performing the inspection step after the detection pretreatment step including the labeling treatment and the mask treatment as described above. It is possible and the effect of the present invention can be obtained.

As the detection method of the present invention, an embodiment in which the solid phase treatment is included in the detection pretreatment step is preferable from the viewpoint of increasing detection sensitivity.
The order of execution of mask processing in the pre-detection process is as follows: (A) after performing solid-phase treatment, when performing mask treatment on the support, and (B) otherwise (masking prior to solid-phase treatment) The treatment can be broadly divided into cases where the treatment is performed or the solid-phase treatment is not performed.

  In embodiment (A), a support on which an analyte (antigen, etc.) contained in a specimen is immobilized with a ligand (antibody, etc.) together with contaminants such as glycoproteins and glycolipids also contained in the specimen. And contact first. Then, on the support, the analyte is captured by the immobilized ligand, and impurities are adsorbed to the ligand or the support non-specifically. By adding a mask sugar chain-recognizing molecule to the support in such a state, it is possible to mask the non-detection target sugar chains possessed by impurities nonspecifically bound to the support and the ligand. Thereby, the high background at the time of adding a lectin is suppressed.

  In the embodiment (B), the sugar chain recognition molecule for mask is added to an undiluted specimen or a sample prepared by diluting with an appropriate buffer or the like. Thereby, the non-detection object sugar chain which the contaminant contained in a sample has can be masked, and specificity and a sensitivity improve.

  Compared to the embodiment (B) in which the mask treatment is performed in a large amount of impurities, the embodiment (A) in which only the impurities adsorbed on the support and the ligand are masked is more preferable because the noise removal effect is higher. .

In order to quickly prevent the labeling lectin from binding to the non-detection target sugar chain (contaminant), it is preferable to perform the masking process before the labeling process.
The method of the present invention can also be performed in combination with a conventional background suppression method, and a higher background suppression effect can be expected by combining.

<Details of each process and treatment>
Details of each process / process will be described below.
[1. Marking process)
The labeling process is a process in which the labeling lectin is brought into contact with the analyte in order to form an analyte-labeling lectin complex. The labeling lectin used for this treatment is previously complexed with a labeling substance. If the solution containing the labeling lectin is brought into contact with a solution containing the analyte (such as a specimen or a prepared sample) or a support on which the analyte has been captured by solid-phase treatment, the labeling lectin and the analyte are usually naturally. Contact with the light occurs, and both are bonded via the sugar chain to be detected which the analyte has. The reaction conditions such as the amount of labeling lectin used in this treatment, the concentration in the reaction solution, and the reaction time may be appropriately adjusted according to the properties of the labeling lectin or analyte used.

[2. Mask processing)
The mask process is a process in which the mask sugar chain recognition molecule is brought into contact with the contaminant in order to suppress the binding of the labeling lectin to the non-detection target sugar chain of the contaminant. Unlike the labeling lectin, the mask sugar chain recognition molecule (for example, lectin) used in this treatment is not complexed with the labeling substance. Ligand and support on which a non-specifically adsorbed contaminant is obtained by a solution containing a sugar chain-recognizing molecule for a mask (along with an analyte) or a solid phase treatment (with an analyte trapped). In general, the mask sugar chain recognition molecule and the foreign substance naturally come into contact with each other, and both are bonded via the non-detection target sugar chain of the foreign substance. The reaction conditions such as the amount of the sugar chain recognition molecule for mask used in this treatment, the concentration in the reaction solution, and the reaction time may be appropriately adjusted according to the properties of the mask sugar chain recognition molecule to be used and impurities.

[3. Solid phase treatment)
The solid-phase treatment is a treatment performed in a detection system using a substrate-like measurement member (corresponding to a support) or the like, and an analyte and impurities are brought into contact with a support on which a ligand is immobilized. It is processing. A solution containing analyte and contaminants (if labeled and masked, if complexed with labeled lectin and masked sugar recognition molecule, respectively) When contacted with the body, the analyte is usually captured by contact with the ligand, and the contaminants are non-specifically adsorbed by contact with the ligand or support. The reaction conditions such as the reaction time and reaction temperature of this treatment may be appropriately adjusted according to the analyte and ligand used.

[4. Detection process)
The detection step is a step of detecting an analyte bound to the labeling lectin by measuring a signal emitted from the labeling substance complexed with the labeling lectin. Based on the measurement results in this step, data relating to the presence or absence of the analyte in the specimen, the content of the analyte, and the like can be acquired.

(Measuring method)
The measuring method used in the detection step of the present invention is not particularly limited as long as it can measure the signal emitted from the label, and can be performed according to a method known to those skilled in the art suitable for each label. For example, when detecting a lectin labeled with a radioactive substance, it can be detected by liquid scintillation or RIA. When detecting a lectin labeled with a fluorescent dye, it can be detected with a luminometer, an SPFS measuring instrument or the like. When detecting a lectin labeled with an enzyme, add a substrate corresponding to the labeled enzyme, and then detect the chemical change of the substrate by the enzyme, for example, color development, fluorescence, chemiluminescence, etc. Can do.

[Surface Plasmon-field enhanced fluorescence spectroscopy (SPFS) method]
As a measurement method used in the detection method of the present invention, SPFS is preferably used. In SPFS, when the metal thin film formed on the dielectric member is irradiated with excitation light at an angle at which total reflection attenuation (ATR) occurs, the evanescent wave transmitted through the metal thin film is several tens of times due to resonance with the surface plasmon. It is a method of efficiently exciting a phosphor for labeling an analyte (analyte) captured in the vicinity of a metal thin film and measuring the fluorescence signal by utilizing the fact that it is enhanced several hundred times. Since such SPFS is extremely sensitive compared to a general fluorescent labeling method, it can be quantified even when only a very small amount of analyte is present in the sample.

<Measuring member for SPFS>
The measurement member for SPFS is generally used for forming a sandwich-type immune complex, a sensor chip on which a field (measurement region) for performing fluorescence measurement by SPFS is formed and a sandwich-type immune complex is formed. A structure in which various solutions used (analyte-containing samples, labeled ligand solutions, other reaction reagents, etc.) can be held on the measurement area and laminated with members for constructing channels or wells. Take.

  The sensor chip basically includes a transparent support for introducing excitation light into the back surface of the metal thin film, a metal thin film for generating surface plasmon resonance formed on the transparent support, and the metal thin film. Including a reaction layer for capturing the analyte on the sensor surface, and if necessary, the phosphor formed between the metal thin film and the reaction layer is too close to the metal thin film. A spacer layer may be included to prevent the metal quenching of fluorescence caused by.

  The site where the reaction layer is formed corresponds to the measurement region. A reaction layer may be formed over the entire bottom surface of the flow path or the well to be a measurement region, or a reaction layer may be formed only on a part of the bottom surface (with a desired patterning if necessary) to be a measurement region. The area of the measurement region can be adjusted in consideration of the irradiation area of excitation light generally irradiated as laser light. For example, if the spot diameter of excitation light is about 1 mmφ, the assay area is usually designed to have an area of at least several mm square.

  When the SPFS system is a “channel type” that sends various solutions through a sealed channel, a “flow cell” with holes to form the channel is loaded on the sensor chip. Correspondingly, a measurement member is constructed by loading a “top plate” with a liquid feed inlet and a liquid feed outlet at a position corresponding to the hole of the flow cell and fixing them in close contact with each other. To do. The sensor chip surface at a position corresponding to the hole of the flow cell forms the bottom surface of the flow path, and a measurement region is formed there. In the case of the flow channel type, for example, various liquids can be fed into the flow channel from the liquid feed inlet and discharged from the liquid feed outlet using a liquid feeding means including a pump and a tube. Thus, reciprocating and circulating liquid feeding can also be performed. Conditions such as the feeding speed and feeding (circulation) time are as follows: the amount of the sample, the concentration of the analyte in the sample, the size of the flow channel or well, the mode of the reaction layer (the density of the immobilized ligand, etc.), the pump It can be adjusted as appropriate while considering performance and the like.

  On the other hand, when the SPFS system is a “well type” that stores various solutions in a space wider than the flow path, a “well member” having a through-hole for forming a well is formed on the sensor chip. A measuring member is constructed by loading and fixing. In the case of the well type, various liquids can be added to the well and removed using, for example, a pipette-shaped member.

  The flow cell can be made of, for example, a sheet-like polydimethylsiloxane (PDMS). The top plate is made of a material having transparency so that fluorescence emitted from the measurement region can be measured, and can be made of, for example, plate-shaped polymethyl methacrylate (PMMA). Alternatively, the flow cell and the top plate can be made of plastic having a desired shape by molding or photolithography.

  A means for fixing the flow cell or well member in close contact with the sensor chip is not particularly limited. Generally, the pressure may be physically applied from above and below, and if necessary, a transparent support. Adhesives having the same optical refractive index, matching oil, transparent adhesive sheets, and the like may be used.

<SPFS measuring device>
The measuring method according to the present invention can be carried out using a general SPFS measuring apparatus. The SPFS measuring device basically has an SPFS measuring member that can be attached and detached, and a light source and excitation for irradiating excitation light (preferably laser light) of an appropriate wavelength according to the phosphor to be used. A prism for making light incident on the back surface of the metal thin film of the sensor chip at a predetermined angle (when the transparent support uses a sensor chip having a flat substrate shape), receiving the light reflected by the metal thin film and measuring its intensity Light receiver, lens for condensing fluorescence emitted from the phosphor, detector for measuring the intensity of the fluorescence, excitation light and only light having a predetermined wavelength is transmitted from the fluorescence and cut the other light It is equipped with various filters for doing so.

  For more specific modes, for example, various documents such as JP2010-145272, JP2011-80935, JP2008-102117, and Japanese Patent No. 3562912 can be referred to.

Hereinafter, a more detailed configuration example of the SPFS measurement apparatus will be described.
FIG. 5 is a schematic view schematically showing an outline of a quantitative measurement apparatus for explaining the quantitative measurement method for an analyte of the present invention, and FIG. 6 is a partially enlarged view of the quantitative measurement apparatus of FIG.

  As shown in FIGS. 5 and 6, the quantitative measurement apparatus 10 of the present invention includes a prism-shaped dielectric member 12 having a substantially trapezoidal vertical cross-sectional shape, and a metal formed on a horizontal upper surface 12 a of the dielectric member 12. A sensor chip 16 having a film 14 is provided, and the sensor chip 16 is loaded in a sensor chip loading unit 18 of the quantitative measurement apparatus 10.

  Further, as shown in the figure, a light source 20 is disposed on the side of one side surface 12b below the dielectric member 12, and incident light 22 from the light source 20 is transmitted from the lower outside of the dielectric member 12. Then, the light is incident on the side surface 12 b of the dielectric member 12, and is irradiated toward the metal film 14 formed on the upper surface 12 a of the dielectric member 12 through the dielectric member 12.

  Further, a polarizing filter for converting the laser light emitted from the light source 20 into P-polarized light that efficiently generates surface plasmons on the metal film 14 can be provided between the light source 20 and the dielectric member 12. .

  Further, as shown in FIG. 5, a light receiving means 26 that receives the metal film reflected light 24 in which the incident light 22 is reflected by the metal film 14 is provided on the other side surface 12 c below the dielectric member 12. ing.

  The light source 20 is provided with incident angle adjusting means (not shown) that can appropriately change the incident angle α1 of the incident light 22 irradiated from the light source 20 with respect to the metal film 14. On the other hand, the light receiving means 26 is also provided with a movable means (not shown), and even when the reflection angle of the metal film reflected light 24 changes, the metal film reflected light 24 is reliably received in synchronization with the light source 20. Is configured to do.

The sensor chip 16, the light source 20, and the light receiving means 26 constitute an SPR measurement unit 28 that performs SPR measurement of the quantitative measurement device 10 of the present invention.
Above the sensor chip 16, there is provided a light detection means 32 for receiving the fluorescence 30 emitted from a fluorescent material as described later.

For example, a cut filter or a condensing lens can be provided between the sensor chip 16 and the light detection means 32.
The sensor chip 16, the light source 20, and the light detection means 32 constitute an SPFS measurement unit 34 that performs SPFS measurement of the quantitative measurement device 10 of the present invention.

  The light receiving means 26 and the light detecting means 32 are respectively connected to the quantitative calculation means 40, and the light amount of the metal film reflected light 24 received by the light receiving means 26 and the light amount of the fluorescence 30 received by the light detecting means 32. Are transmitted to the quantitative calculation means 40.

  In the sensor chip 16 of the present embodiment, the flow path 36 is formed on the upper surface 14a of the metal film 14. A sensor unit 38 in which a molecule (ligand) that specifically binds to a detection target antigen (analyte) is immobilized is provided in a part of the flow path 36.

≪Detection kit≫
The detection kit of the present invention is used for the detection method of the present invention. The detection kit of the present invention comprises:
(1) a reagent comprising a labeling lectin that binds to a plurality of types of sugar chains including a detection target sugar chain and a non-detection target sugar chain;
(2) a reagent containing a mask sugar chain recognition molecule that binds to at least one non-detection target sugar chain, and (3) a reagent containing a ligand that binds to an analyte having the detection sugar chain,
including.

The detection kit of the present invention further includes:
(4) Instructions for use in which the detection method according to the present invention is described as instructions, and / or (5) a lectin labeling reagent may be included.

  The instruction manual (4) included in the kit is an instruction manual in which any of the detection methods according to the present invention is described as instructions for carrying out the method of the present invention. The specific mode of the instruction manual may be any as long as it can appropriately transmit the above information. For example, it may be printed on a piece of paper, packaging of a kit, a label of a component of the kit, or may be recorded in a computer-readable medium such as a diskette or CD.

The lectin labeling reagent (5) included in the kit is a reagent used for labeling the lectin, and usually contains a label and a reagent that binds the lectin and the label.
≪Application examples≫
The method of the present invention can be used for disease diagnosis. For example, when the detection target antigen (analyte) is PSA, prostate cancer can be diagnosed from the amount of PSA in a patient-derived biological sample quantified by the detection method of the present invention. In addition, when the analyte is AFP, hepatocellular carcinoma can be diagnosed, and when the analyte is CEA, cancer mainly in the digestive tract can be diagnosed.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
(Configuration of quantitative measurement device)
In the following examples, the SPFS measuring device was made and used as a measuring device. The SPFS measurement device has the same configuration as the quantitative measurement device 10.

  In the above configuration, a laser diode (LD) capable of irradiating light with a wavelength of 635 nm is used as the light source 20, and a neutral density filter is used as an optical filter between the light source 20 and the dielectric member 12. (Neutral density filter) was provided so that the photon amount could be adjusted.

  Further, as the dielectric member 12, a 60-degree prism manufactured by Sigma Kogyo Co., Ltd. is used, and a sensor chip 16 is configured by fixing a plasmon excitation sensor, which will be described later, to the upper portion of the dielectric member 12. .

Further, an objective lens is provided as a condensing lens above the sensor chip 16, and a photomultiplier tube (PMT) is used as the light detection means 32.
(Production of plasmon excitation sensor)
A glass transparent flat substrate (S-LAL 10 manufactured by OHARA INC.) Having a refractive index of 1.72 and a thickness of 1 mm was plasma-cleaned, and a chromium thin film was formed on one surface of this substrate by a sputtering method. Thereafter, a gold thin film was further formed on the surface by a sputtering method. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

  Thus, the board | substrate with which the gold thin film was formed was immersed in the ethanol solution which contains 10 mM of 10-carboxy- 1-decanethiol for 24 hours or more, and the SAM film was formed on the surface of the gold thin film. The substrate was removed from this solution, washed with ethanol and isopropanol, and then dried using an air gun.

  A sheet made of polydimethylsiloxane (PDMS) having a groove serving as a flow channel with a height of 0.5 mm and having through holes at both ends of the groove is formed on the groove so that the surface of the SAM film is inside the flow channel. The PDMS sheet (flow path 36) and the plasmon excitation sensor were fixed with screws using pressure from the upper part of the PDMS sheet outside the flow path.

(Solid phase of antibody)
[Preparation Example 1]
[Anti-PSA antibody-immobilized SAM membrane (substrate)]
As described above, ultrapure water was added to the external flow path connected to the plasmon excitation sensor for 10 minutes, and then phosphate buffered saline (PBS) was used for 20 minutes by a peristaltic pump at room temperature (25 ° C.) with a flow rate of 500 μL / min. The liquid was circulated under conditions to equilibrate the surface of the plasmon excitation sensor.

  Subsequently, 5 mL of phosphate buffered saline (PBS) containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM water-soluble carbodiimide (WSC) was fed and circulated for 20 minutes. By circulating and feeding 2.5 mL of a PSA monoclonal antibody (No. 79, 2.5 mg / mL, manufactured by Miku Immune Laboratories) solution for 30 minutes, the antibody was immobilized on the SAM membrane, and the anti-PSA antibody solid phase A modified SAM membrane was prepared.

Thereafter, phosphate buffered saline (PBS) containing 1% by weight bovine serum albumin (BSA) was circulated for 30 minutes to prevent nonspecific adsorption in the flow path.
[Preparation Example 2]
[Anti-AFP antibody-immobilized SAM membrane (substrate)]
An anti-AFP antibody solid phase was prepared in the same manner as in Preparation Example 1, except that an anti-AFP monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Nippon Medical Laboratory) was used instead of the anti-PSA antibody. A modified SAM membrane was prepared.

(Preparation of labeled lectin)
[Production Example 1]
[Fluorescently labeled WFA lectin]
A fluorescently labeled WFA lectin was prepared using a fluorescent substance labeling kit “Alexa Fluor (trade name) 647 protein labeling kit” (manufactured by Invitrogen). WFA lectin (L-1350, Vector) equivalent to 100 μg, 0.1 M sodium bicarbonate and Alexa Fluor 647 reactive dye were mixed and reacted at room temperature for 1 hour, and then subjected to gel filtration chromatography and ultrafiltration. The Alexa Fluor 647 reactive dye, which was not used for labeling, was removed to obtain a fluorescently labeled WFA lectin. Thereafter, the absorbance was measured to quantify the labeled lectin concentration.

[Production Example 2]
[Fluorescently labeled LCA lectin]
Fluorescently labeled LCA lectin was prepared using a fluorescent material labeling kit. A fluorescently labeled LCA lectin was obtained in the same manner as in Preparation Example 1, except that LCA lectin (L-1040, Vector) was used as the lectin.

<Measurement of PSA in serum samples>
Five PSA free pool human sera were prepared as negative control samples. As positive control specimens, serum samples were prepared by adding LNCaP (human prostate cancer cell culture) culture supernatant to each of these five PSA free pool sera to a PSA concentration of 50 pg / mL. A total of 10 samples were each diluted twice with PBS to obtain measurement samples in Examples 1 to 3. As the PSA-free pooled human serum, serum obtained by purchasing normal human pooled serum from Kojin Bio and confirming that the PSA concentration is 0.01 ng / mL or less by ELISA was used.

[Example 1]
(Measurement of PSA in serum samples (1))
Each measurement sample was masked with galectin, then contacted with a fluorescently labeled WFA lectin (Production Example 1), and then reacted with a substrate on which an anti-PSA antibody was immobilized (Preparation Example 1). Details are as follows.

  Galectin was added to 0.1 mL of the measurement sample to a final concentration of 10 μg / mL, and reacted at 37 ° C. for 1 hour. Next, 0.1 mL of a solution of WFA lectin labeled with Alexa Fluor 647 (dissolved in phosphate buffered saline (PBS) to 1 μg / mL) was added and allowed to react at room temperature for 1 hour. 0.1 mL of a measurement sample subjected to mask treatment with galectin and labeling treatment with fluorescently labeled WFA lectin was added to the flow path, and the solution was circulated for 20 minutes at a flow rate of 200 μL / min. Subsequently, TBS containing 0.05% by weight of Tween 20 (TBS-T) was fed and washed for 5 minutes. Thereafter, SPFS measurement was performed with a quantitative measurement apparatus. The measurement sample was also prepared in the process excluding the mask treatment with galectin, and SPFS measurement was performed in the same manner.

  Table 2 shows the measurement results of SPFS. The signal values in the table are actually measured values, and the signal values of the serum with PSA added are shown as PSA-derived signal value + background signal value.

It was confirmed that the value (background) of the PSA free pool serum of the measurement sample that was masked with galectin was reduced to about ¼ compared to the untreated measurement sample.

  In addition, the average value of the S / N ratio (signal value of PSA-added serum / corresponding signal value of PSA-free serum) of 5 specimens was 2.02 for the untreated sample, whereas the masked sample with galectin Then, it increased to 3.62 and it became clear that the detection sensitivity increased.

[Example 2]
(Measurement of PSA in serum samples (2))
Each measurement sample was masked with galectin, reacted with a substrate on which an anti-PSA antibody was immobilized (Preparation Example 1), and then contacted with a fluorescently labeled WFA lectin (Preparation Example 1). Details are as follows.

  Galectin was added to the measurement sample to a final concentration of 10 μg / mL and reacted at 37 ° C. for 1 hour. 0.1 mL of the measurement sample subjected to the mask treatment with galectin was added to the flow path, and the solution was circulated for 20 minutes at a flow rate of 200 μL / min. Subsequently, TBS containing 0.05% by weight of Tween 20 (TBS-T) was fed and washed for 5 minutes. After the reaction, 0.1 mL of WFA lectin solution labeled with Alexa Fluor 647 (dissolved in phosphate buffered saline (PBS) so as to be 1 μg / mL) is added, and sent at a flow rate of 200 μL / min for 5 minutes. Liquid. Again, TBS containing 0.05% by weight of Tween 20 (TBS-T) was fed and washed for 5 minutes. Thereafter, SPFS measurement was performed with a quantitative measurement apparatus. A measurement sample was also prepared in a process excluding the mask treatment with galectin, and SPFS measurement was performed.

  The measurement results of SPFS are shown in Table 3. The signal values in the table are actually measured values, and the signal values of the serum with PSA added are shown as PSA-derived signal value + background signal value.

It was confirmed that the signal value (background) of the PSA free pool serum was reduced to about 1/3 in the measurement sample subjected to the mask treatment with galectin as compared with the untreated measurement sample.

In addition, the average value of the S / N ratio (signal value of PSA-added serum / corresponding PSA-free serum signal) of 5 specimens was 2.64 in the untreated sample, whereas it was 4 in the galactosidase-treated sample. It became clear that the detection sensitivity increased.
[Example 3]
(Measurement of PSA in serum samples (3))
Each measurement sample was reacted with a substrate on which an anti-PSA antibody was immobilized (Preparation Example 1), then masked with galectin, and then contacted with a fluorescently labeled WFA lectin (Preparation Example 1). Details are as follows.

  0.1 mL of the measurement sample was added to the flow path and circulated for 20 minutes at a flow rate of 200 μL / min. Subsequently, 0.1 mL of a PBS solution containing 10 μg / mL of galectin (corresponding to galectin (+)) or PBS not containing galectin (corresponding to galectin (−)) was added and circulated for 20 minutes. Thereafter, TBS (TBS-T) containing 0.05% by weight of Tween 20 was fed and washed for 5 minutes at a flow rate of 200 μL / min. After washing, 0.1 mL of WFA lectin solution labeled with Alexa Fluor 647 (dissolved in phosphate buffered saline (PBS) so as to be 1 μg / mL) is added, and sent at a flow rate of 200 μL / min for 5 minutes. Liquid. Thereafter, TBS (TBS-T) containing 0.05% by weight of Tween 20 was fed and washed for 5 minutes at a flow rate of 200 μL / min. Thereafter, SPFS measurement was performed with a quantitative measurement apparatus. SPFS measurement was also performed in the process excluding the mask treatment with galectin.

  Table 4 shows the measurement results of SPFS. The signal values in the table are actually measured values, and the signal values of the serum with PSA added are shown as PSA-derived signal value + background signal value.

It was confirmed that the measurement sample that had been masked with galectin on the substrate had a value (background) of PSA free pool serum that was reduced to about 1/5 compared to the untreated measurement sample.

  Moreover, the average value of the S / N ratio (signal value of PSA-added serum / corresponding signal value of PSA-free serum) of 5 specimens was 3.50 in the untreated sample, but 13 in the galactosidase-treated sample. It was revealed that the detection sensitivity increased.

  As described above, as shown in Examples 1 to 3, a signal derived from non-specific binding of contaminants (such as glycoproteins) in a serum component having a sugar chain containing galactose, which occurred in the measurement system (back) Ground noise) can be suppressed by masking the galactose with galectin, and it was confirmed that the sensitivity to specifically detect the GalNAc sugar chain structure on PSA was improved.

  In addition, as a result of comparing Example 2 (Table 3) and Example 3 (Table 4), antibody immobilization is targeted to some extent as compared with performing mask treatment in a solution containing a large amount of serum protein. It was confirmed that the effect was higher when performed on the substrate.

<Measurement of AFP in serum samples>
In Examples 1 to 3, the test was performed using PSA as the detection target antigen (analyte), but the same test was performed using AFP as the detection target antigen. Examples 4 to 6 are shown below.

  Five AFP free pool sera were prepared as negative control samples. In addition, as a positive control sample, each of these 5 types of AFP free pool sera was supplied with a control L for Mutasuwako AFP-L3 (trade name, manufactured by Wako Pure Chemical Industries, Ltd., L3 = 20%, AFP concentration 200 ng / mL). Serum samples added to an AFP concentration of 1.0 ng / mL were prepared. A total of 10 samples were used as measurement samples in Examples 4-6. As the AFP free pool human serum, serum obtained by purchasing normal human pool serum from Kojin Bio and confirming that the AFP concentration was 0.01 ng / mL or less by ELISA was used.

[Example 4]
(Measurement of AFP in serum samples (1))
Each measurement sample was masked with concanavalin A lectin and then contacted with a fluorescently labeled LCA lectin (Production Example 2), and then reacted with a substrate on which an anti-AFP antibody was immobilized (Preparation Example 2).

  That is, in Example 1, instead of galectin, concanavalin A lectin, fluorescently labeled WFA lectin (Preparation Example 1) instead of fluorescently labeled LCA lectin (Preparation Example 2), and an anti-PSA antibody-immobilized substrate (preparation) The same procedure as in Example 1 was performed except that instead of Example 1), a substrate on which an anti-AFP antibody was immobilized (preparation example 2) was used.

  Table 5 shows the measurement results of SPFS. The signal values in the table are actually measured values, and the signal values of the AFP-added serum are shown as AFP-derived signal value + background signal value.

It was confirmed that the measurement sample subjected to mask treatment with concanavalin A lectin had a value of AFP free pool serum (background) decreased to about 3/4 compared with the untreated measurement sample. It was also confirmed that there was almost no difference in the value when the AFP-L3-added serum was measured.

  In addition, the average value of the S / N ratio (signal value of the serum with AFP-L3 added / signal value of the corresponding AFP-free serum) of the five specimens was 4.18 in the untreated sample, whereas the sample treated with galactosidase Then, it increased to 4.80, and it became clear that the detection sensitivity increased.

[Example 5]
(Measurement of AFP in serum samples (2))
Each measurement sample was masked with concanavalin A lectin, reacted with a substrate on which an anti-AFP antibody was immobilized (Preparation Example 2), and then contacted with a fluorescently labeled LCA lectin (Preparation Example 2).

  That is, in Example 2, instead of galectin, concanavalin A lectin, fluorescently labeled WFA lectin (Preparation Example 1) instead of fluorescently labeled LCA lectin (Preparation Example 2), and an anti-PSA antibody-immobilized substrate (preparation) The same procedure as in Example 2 was performed except that instead of Example 1), a substrate on which an anti-AFP antibody was immobilized (preparation example 2) was used.

  Table 6 shows the measurement results of SPFS. The signal values in the table are actually measured values, and the signal values of the AFP-added serum are shown as AFP-derived signal value + background signal value.

It was confirmed that the measurement sample subjected to masking with concanavalin A lectin had a value of AFP free pool serum (background) lowered to about 3/2 as compared to the untreated measurement sample. It was also confirmed that there was almost no difference in the value when the AFP-L3-added serum was measured.

  In addition, the average value of the S / N ratio (signal value of AFP-L3-added serum / corresponding AFP-free serum signal value) of the five specimens was 6.43 for the untreated sample, whereas the galactosidase-treated sample was Then, it increased to 8.25, and it became clear that the detection sensitivity increased.

[Example 6]
(Measurement of AFP in serum samples (3))
Each measurement sample was reacted with a substrate on which an anti-AFP antibody was immobilized (Preparation Example 2), then treated with mannosidase, and then contacted with a fluorescently labeled LCA lectin (Preparation Example 2).

  That is, in Example 3, instead of galectin, concanavalin A lectin, fluorescently labeled WFA lectin (Preparation Example 1) instead of fluorescently labeled LCA lectin (Preparation Example 2), and an anti-PSA antibody-immobilized substrate (preparation) The same procedure as in Example 3 was performed except that instead of Example 1), a substrate on which an anti-AFP antibody was immobilized (preparation example 2) was used.

  Table 7 shows the measurement results of SPFS. The signal values in the table are actually measured values, and the signal values of the AFP-added serum are shown as AFP-derived signal value + background signal value.

It was confirmed that the measurement sample on which the mask treatment with concanavalin A lectin was performed on the substrate had a value (background) of the AFP free pool serum lowered to about 12 as compared with the untreated measurement sample.

  In addition, the average value of the S / N ratio (signal value of AFP-L3-added serum / signal value of the corresponding AFP-free serum) of the five specimens was 8.11 in the untreated sample, whereas the sample treated with galactosidase Then, it increased to 15.3, and it became clear that the detection sensitivity increased.

  As described above, as shown in Examples 4 to 6, signals (noise) generated in the measurement system derived from non-specific binding of contaminants (glycoprotein, etc.) in serum components having a sugar chain containing mannose. ) Can be suppressed by masking the mannose with concanavalin A lectin, and it was confirmed that the sensitivity to specifically detect the fucose sugar chain structure on AFP was improved.

  In addition, as a result of comparing Example 5 (Table 6) and Example 6 (Table 7), antibody immobilization is targeted to some extent as compared with performing mask treatment in a solution containing a large amount of serum protein. It was confirmed that the effect was higher when performed on the substrate.

  In the above embodiment, in the SPFS measuring device, the aspect of circulating and feeding various solutions is described as an example, but it is not always necessary to circulate, the aspect of continuing to feed in one direction, the aspect of reciprocating in both directions, Alternatively, various modifications can be made without departing from the object of the present invention, such as a mode in which a predetermined amount of solution is fed and then retained for a predetermined time.

DESCRIPTION OF SYMBOLS 10 Quantitative measurement apparatus 12 Dielectric material 12a Upper surface 12b Side surface 12c Side surface 14 Metal film 14a Upper surface 16 Sensor chip 18 Sensor chip loading part 20 Light source 22 Incident light 24 Metal film reflected light 26 Light receiving means 28 SPR measurement part 30 Fluorescence 32 Light detection means 34 SPFS measurement unit 36 Fine flow path 38 Sensor unit 40 Quantitative calculation means 44 Antigen supplement antibody (ligand)
46 Target antigen (analyte)
46a Protein (antigen)
46b Sugar chain that is neither a detection target sugar chain nor a non-detection target sugar chain 46c Detection target sugar chain 46d Non-detection target sugar chain 50 Non-specifically adsorbed glycoprotein (contaminant)
50a Protein 52 Non-specifically adsorbed glycolipid (contaminant)
52a Lipid 60 Fluorescently labeled lectin (probe)
60a Labeling lectin 60b Labeling body 75 Sugar chain recognition molecule 80 for masking Support

Claims (10)

A method for detecting an analyte having a detection target sugar chain in a sample using a labeling lectin that binds to a plurality of types of sugar chains including a detection target sugar chain and a non-detection target sugar chain. Before the detection step of detecting the analyte bound to the lectin,
A labeling treatment in which the labeling lectin is brought into contact with the analyte;
At least one of the non-detection target sugar mask carbohydrate-recognizing molecules that bind to a chain, the pre-detection process lines physician and a mask processing is brought into contact with contaminants having a non-detection target sugar chain (provided that the When the labeling process is performed before the masking process, the mask sugar chain recognition molecule that is easier to bind to the non-detection target sugar chain than the labeling lectin is used) .
The analyte is a prostate-specific antigen, the labeling lectin is Nodafuji lectin, and the mask sugar chain recognition molecule is a galectin, or
The method for detecting an analyte having a sugar chain to be detected, characterized in that the analyte is α-fetoprotein, the labeling lectin is a lentil lectin, and the sugar chain recognition molecule for the mask is a red bean lectin. .   The detection method according to claim 1, wherein the detection pretreatment step further includes a solid phase treatment in which the analyte and the contaminant are brought into contact with a support on which a ligand that binds to the analyte is immobilized.   The detection method according to claim 2, wherein the solid-phase treatment is performed before the labeling treatment and the mask treatment.   The detection method according to claim 2, wherein the labeling process and the masking process are performed before the solid-phase treatment.   The detection method as described in any one of Claims 1-4 which performs the said label | marker process and the said mask process simultaneously.   The detection method according to claim 1, wherein the labeling process is performed before the masking process.   The detection method according to claim 1, wherein the mask process is performed before the labeling process.   The detection method according to claim 2, wherein the solid-phase treatment is performed after the labeling treatment and before the mask treatment.   The detection method according to claim 2, wherein the solid-phase treatment is performed after the mask treatment and before the labeling treatment. A reagent comprising a labeling lectin that binds to a plurality of types of sugar chains including a detection target sugar chain and a non-detection target sugar chain;
A reagent comprising a mask sugar chain recognition molecule that binds to at least one kind of the non-detected sugar chain;
Look contains a reagent comprising a ligand that binds with the analyte having the detection target sugar chain,
The analyte is a prostate-specific antigen, the labeling lectin is Nodafuji lectin, and the mask sugar chain recognition molecule is a galectin, or
The analyte is α-fetoprotein, the labeling lectin is a lentil lectin, and the sugar chain recognition molecule for the mask is a red bean lectin, for detection of an analyte having a sugar chain to be detected kit.