Antibody or antigen-binding fragment - base material composite

Abstract

To provide an effective immunological measurement method.SOLUTION: An antibody to which a basic amino acid residue is added at the C-terminal or its antigen-binding fragment and a composite comprising a base material are provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a complex comprising an antibody or an antigen-binding fragment and a substrate, a method for producing the complex, a composition comprising the complex, a test method using the complex, and the like. [Background technology]

[0002] Immunological assay methods for detecting antigens in a sample using antibodies bound to insoluble carrier particles have been known for some time (e.g., Patent Document 1). For example, particles with a large surface area are used as the solid carrier, and the efficiency of antigen detection can be increased by binding a large number of antibodies to the particle surface. For example, a method for diagnosing a disease is known that uses specific antibodies against proteins known as disease causative substances or disease markers. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 4413179 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide an efficient immunological measurement method. [Means for solving the problem]

[0005] As a result of extensive research, the present inventors surprisingly found that by adding a basic amino acid to the C-terminus of an antibody, a complex of the antibody and a solid support reacts efficiently with an antigen, thus completing the present invention.

[0006] That is, the present invention provides (1) A complex comprising an antibody or antigen-binding fragment having a basic amino acid added to the C-terminus, and a substrate; (2) The conjugate according to (1), wherein the C-terminus of the antibody or antigen-binding fragment is bound to the surface of the substrate; (3) The complex according to (1) or (2), wherein the surface of the substrate is negatively charged; (4) The complex according to any one of (1) to (3), wherein the basic amino acid is selected from histidine, arginine, lysine, or an artificial amino acid having an amino group in a side chain; (5) The composite according to any one of (1) to (4), wherein the substrate is a particle or a plate-like substrate; (6) The complex according to (5), wherein the particles are latex particles; (7) The conjugate according to any one of (1) to (6), wherein the antigen-binding fragment is F(ab')2, Fab, Fv, scFv, or a diabody; (8) A method for producing a complex comprising an antibody or antigen-binding fragment, the C-terminus of which is oriented so that it binds to the surface of the substrate, and a substrate, the method comprising contacting an antibody or antigen-binding fragment having a basic amino acid added to its C-terminus with a substrate; (9) The method according to (8), wherein the surface of the substrate is negatively charged; (10) A reagent or kit for immunoassay, comprising the complex according to any one of (1) to (7). (11) The immunoassay reagent or kit according to (10), wherein the antibody or antigen-binding fragment in the complex is an antibody or an antigen-binding fragment thereof that specifically binds to a protein selected from the group consisting of H-FABP, PLA2, trypsin, and elastase; (12) A method for detecting a target substance in a sample, comprising contacting the sample with the conjugate according to any one of (1) to (7), wherein the antibody or antigen-binding fragment thereof is an antibody that specifically binds to the target substance, or an antigen-binding fragment thereof; (13) The method according to (12), wherein the target substance is selected from the group consisting of H-FABP, PLA2, trypsin, and elastase; to provide. Effect of the Invention

[0007] According to the present invention, there are provided an antibody-substrate complex for immunoassay, which can detect a target antigen efficiently and with high sensitivity, an immunoassay reagent and kit containing said complex, and an efficient and highly sensitive immunoassay method, in particular an immune agglutination method, which uses said complex. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram comparing the composite of the present invention with a conventional composite. [Diagram 2] 1 shows the results of an antigen binding test using a complex of latex and an anti-H-FABP antibody fragment having a basic amino acid added to the C-terminus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] (Antibody or antigen-binding fragment) The antibody used in the present invention may be an antibody against any antigen. The antibody may be either a polyclonal antibody or a monoclonal antibody, or both may be used. The antibody may be selected to specifically bind to a substance such as a protein, nucleic acid, or sugar that is desired to be detected by the immunological measurement method described below. The antigen may be, for example, a pathogenic substance or a marker of any disease. Examples of proteins include, but are not limited to, H-FABP, PLA2, trypsin, elastase, etc.

[0010] As used herein, an antigen-binding fragment refers to an antibody fragment that retains the antigen-binding ability of a full-length antibody. Examples of antigen-binding fragments include, but are not limited to, F(ab')2, Fab, Fv, scFv, and diabody.

[0011] The antibody or antigen-binding fragment used in the present invention has a basic amino acid added to its C-terminus. Such a basic amino acid may be any basic amino acid, for example, histidine, arginine, or lysine, or an artificial amino acid having an amino group in its side chain. In this specification, an artificial amino acid refers to a non-natural amino acid. An artificial amino acid having an amino group in its side chain may be an amino acid obtained by introducing an amino group into the side chain of a natural or non-natural amino acid. The basic amino acid may be either L-type or D-type.

[0012] Two or more basic amino acids may be added to the C-terminus of the antibody or antigen-binding fragment, for example, but not limited to, 2 to 12 basic amino acids. The two or more basic amino acids may be the same type of basic amino acid or different types of basic amino acids.

[0013] The antibody or antigen-binding fragment can be produced by known methods, for example, from an immunized animal or as a recombinant protein. In the case of a polyclonal antibody, it can generally be prepared by obtaining antiserum from an animal such as a mouse immunized with an antigen. In the case of a monoclonal antibody, for example, it can be obtained by fusing an antibody-producing cell obtained from an animal such as a mouse immunized with an antigen with a myeloma cell to produce a hybridoma that produces a monoclonal antibody, and culturing the hybridoma cell in vivo or in vitro. Furthermore, the obtained antibody may be digested with an enzyme such as pepsin or papain to obtain an antigen-binding fragment such as F(ab')2 or Fab.

[0014] In the case of monoclonal antibodies, the antibody or antigen-binding fragment can be obtained as a recombinant protein by cloning the antibody gene. For example, a nucleic acid containing a sequence encoding the antibody or antigen-binding fragment can be incorporated into an appropriate vector and introduced into a host cell (e.g., mammalian cells, E. coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody or antigen-binding fragment as a recombinant protein. When an antibody or antigen-binding fragment is produced by genetic engineering in this way, a basic amino acid can be easily added to the C-terminus during the production process. For example, a nucleic acid encoding an antibody or antigen-binding fragment to which a basic amino acid has been added to the C-terminus can be obtained by a method of inserting a nucleic acid encoding an antibody or antigen-binding fragment into a vector produced so as to fuse a basic amino acid to the C-terminus of a protein, or by PCR using a primer containing a nucleotide sequence encoding a basic amino acid.

[0015] (base material) The substrate used in the present invention is an insoluble solid carrier, and includes those of various shapes. For example, but not limited to, substrates of spherical (e.g., particles) and plate-like (e.g., plate-like substrate) shapes can be mentioned. The material constituting the substrate is not particularly limited, and various materials such as metal, synthetic polymer, natural polymer, glass, silica, alumina, and ceramic can be mentioned. It may be appropriately selected depending on the shape and application.

[0016] (particle) The particles used in the present invention are insoluble carrier particles, and are not particularly limited, but examples thereof include latex particles, metal colloids (e.g., gold colloids), magnetic beads, liposomes, microcapsules, and particles made of materials such as gelatin, silica, alumina, carbon black, metal compounds, metals, and ceramics. Preferably, particles whose surfaces are negatively charged are used. More preferably, latex particles are used.

[0017] The latex particles may be made of either a synthetic polymer or natural rubber. Preferably, synthetic polymer latex is used. Examples of the synthetic polymer include styrene-based polymers such as polystyrene, acrylic acid-based polymers, and various modified polymers (for example, carboxylic acid-modified polymers in which a carboxyl group is introduced into the above-mentioned polystyrene). Preferably, polystyrene-based particles (polystyrene latex) containing styrene as a main component, which are often used in immunoassays, are used. Two or more types of particles may be used.

[0018] The size (particle diameter) of the particles used in the present invention is not particularly limited. For example, particles having an average particle diameter of about 50 nm to 1000 nm, preferably about 200 nm to 800 nm, and more preferably about 300 nm to 500 nm may be used. Two or more particles having different particle diameters may be used.

[0019] (Plate-shaped substrate) The plate-like substrate used in the present invention is an insoluble substrate. The material of the plate-like substrate is not particularly limited, but examples thereof include synthetic polymers such as styrene-based polymers such as polystyrene, acrylic acid-based polymers, various modified polymers (e.g., carboxylic acid-modified polymers in which carboxyl groups are introduced into the above-mentioned polystyrene), natural polymers such as gelatin, and inorganic substances such as silica, alumina, ceramics, glass, and metals. Preferably, a substrate whose surface is negatively charged is used. Specific examples of the plate-like substrate include plates, multiwell plates, microtiter plates, and dishes.

[0020] (Conjugates Comprising Antibodies or Antigen-Binding Fragments and Substrates) By sensitizing the antibody or antigen-binding fragment to the substrate, a complex (hereinafter also referred to as the "complex of the present invention") containing the antibody or antigen-binding fragment and the substrate can be produced. For example, physical adsorption or chemical binding can be used as the sensitization method, and preferably, chemical binding is used. In the physical adsorption method, the antibody or antigen-binding fragment is hydrophobically adsorbed to the substrate surface by simply contacting (e.g., mixing) the antibody or antigen-binding fragment with the substrate. The physical adsorption method is used, for example, for styrene-based substrates. In the chemical binding method, a condensing agent may be used to promote binding (e.g., dehydration binding, covalent binding) between the antibody or antigen-binding fragment and the functional group on the substrate surface. Preferably, the antibody or antigen-binding fragment is bound to a substrate having a negatively charged functional group (e.g., carboxyl group, sulfo group, etc.) introduced on its surface. The antibody or antigen-binding fragment can be bound to the substrate by contacting the antibody or antigen-binding fragment with the substrate, for example, by mixing the antibody or antigen-binding fragment with the substrate, adding the antibody or antigen-binding fragment to the surface of the substrate, etc. One or more types of antibodies or antigen-binding fragments may be sensitized to the substrate, and the complex of the present invention may contain one or more types of antibodies or antigen-binding fragments.

[0021] In the complex of the present invention thus obtained, the C-terminus of the antibody or antigen-binding fragment is bound to the surface of the substrate. This is believed to be due to the fact that a basic amino acid is added to the C-terminus of the antibody or antigen-binding fragment used in the present invention, and therefore the C-terminus is positively charged. For example, if the surface of the substrate is negatively charged, the positively charged C-terminus of the antibody or antigen-binding fragment is believed to bind to the substrate surface. Thus, according to the present invention, the orientation of the antibody or antigen-binding fragment is efficiently controlled. Thus, an antibody / antigen-binding fragment-substrate complex in which the orientation of the antibody or antigen-binding fragment is controlled is obtained.

[0022] Since a basic amino acid acts as a proton donor at approximately neutral pH, the complex is produced in a buffer solution having a pH of approximately neutral, for example, about pH 5 to pH 8.5.

[0023] (Immunoassay reagents, kits, and immunoassay methods) The complex comprising the antibody or antigen-binding fragment and a substrate can be used in an immunoassay. Accordingly, the present invention provides an immunoassay reagent comprising the complex (hereinafter also referred to as the "immunoassay reagent of the present invention"). Furthermore, the present invention provides an immunoassay method using the complex (hereinafter also referred to as the "immunoassay method of the present invention"). Furthermore, the present invention also provides an immunoassay kit.

[0024] The immunoassay reagent of the present invention may contain additives such as surfactants, stabilizers, and reaction promoters in addition to the conjugate of the present invention. The immunoassay reagent of the present invention preferably contains a conjugate containing an antibody or an antigen-binding fragment and a particle. A reagent containing a conjugate containing an antibody or an antigen-binding fragment and a particle can be used in an immune agglutination assay, as described below. The immunoassay reagent of the present invention may contain one or more types of conjugates of the present invention. The two or more types of conjugates preferably contain different antibodies or antigen-binding fragments against the same antigen.

[0025] The immunoassay kit includes the complex of the present invention, or the antibody or antigen-binding fragment, and the substrate (hereinafter, also referred to as the "immunological assay kit of the present invention"). The immunoassay kit of the present invention may include a buffer solution for use in immunoassay and / or a buffer solution for use in producing the complex of the present invention, in addition to the complex of the present invention, or the antibody or antigen-binding fragment, and the substrate. One example of the immunoassay kit of the present invention includes a complex including an antibody or antigen-binding fragment and a plate-shaped substrate, and may further include a buffer solution for use in immunoassay. Another example of the immunoassay kit of the present invention includes an antibody or antigen-binding fragment and a substrate, and may further include a buffer solution for use in producing the complex of the present invention and / or a buffer solution for use in immunoassay. Yet another example of the immunoassay kit of the present invention includes the immunoassay reagent of the present invention, and may further include a buffer solution for use in immunoassay.

[0026] In immunoassays, a target substance in a sample (also called a specimen) can be detected by using a complex of a substrate and an antibody or antigen-binding fragment that specifically binds to the target substance.

[0027] The target substance may be any substance to be detected, such as a protein, a nucleic acid, a sugar, etc. The target substance may be a pathogenic substance or a marker of any disease, such as, but not limited to, H-FABP, PLA2, trypsin, elastase, etc.

[0028] The sample is not particularly limited as long as it is a sample suspected of containing a target protein or a sample for which the presence or absence of a target protein is to be detected, and examples of the sample include, but are not limited to, blood, serum, plasma, urine, cerebrospinal fluid, saliva, sweat, semen, vaginal secretions, etc.

[0029] The immunoassay of the present invention may be any method for measuring or detecting a target substance by utilizing an antigen-antibody reaction, and examples thereof include, but are not limited to, immunoagglutination assay, enzyme immunoassay, radioimmunoassay, and chemiluminescence assay. In the immunoagglutination assay, preferably, an immunoassay reagent containing a complex containing an antibody or an antigen-binding fragment and a particle is used. For example, when latex particles are used as the particles, it is also called latex immunoturbidimetry. In the immunoassay of the present invention, the antibody or antigen-binding fragment of the complex and the antigen (target substance) are bound by an antigen-antibody reaction, causing agglutination of the complex particles. The agglutination of the particles can be detected by a method known in the art, and examples thereof include, but are not limited to, turbidity measurement by absorbance, particle counting technology by scattered light, and the like. It may be measured by a general-purpose biochemical automatic analyzer. In the case of latex immunoturbidimetry, absorbance at a wavelength of 500 to 900 nm is usually measured. In the enzyme immunoassay, for example, a complex containing an antibody or an antigen-binding fragment and a plate-shaped substrate is used. An antigen (target substance) is bound to an antibody or antigen-binding fragment immobilized on the surface of a plate-like substrate such as a multi-well plate through an antigen-antibody reaction, and then another antibody (secondary antibody) is reacted with the enzyme-labeled antigen. After washing, the enzyme activity remaining on the plate-like substrate is detected.

[0030] The antibody or antigen-binding fragment is bound to the substrate surface in a state where the orientation is controlled so that its C-terminus is bound to the substrate surface, i.e., the antigen-binding site at the N-terminus of the antibody or antigen-binding fragment is free, so that the antigen-antibody reaction is further promoted. Thus, the immunoassay method of the present invention can detect a target substance efficiently and with high sensitivity. Therefore, the immunoassay method of the present invention can detect low concentrations of substances in a sample. When the complex of the present invention contains two or more antibodies or antigen-binding fragments against different epitopes of the same antigen, or when two or more complexes of the present invention are used, each containing an antibody or antigen-binding fragment against a different epitope of the same antigen, the measurement sensitivity for the target substance (antigen) is further amplified.

[0031] The immunoassay method is carried out by contacting the complex of the present invention with a sample. The immunoassay reagent of the present invention may be used as the complex. The contact between the complex and the sample may be carried out by any method, and is not particularly limited. For example, the complex may be mixed with the sample, or the sample may be added to the complex of the present invention. The contact is usually carried out in a buffer solution. The contact is preferably carried out in a neutral buffer solution, for example, about pH 5 to 8.5, preferably about pH 6.0 to 8.0. The buffer solution is not particularly limited, but may be, for example, a phosphate buffer solution such as PBS, a Tris hydrochloride buffer solution, a carbonate buffer solution, a glycine buffer solution, or a Good's buffer solution such as HEPES or MES. The concentration of the buffer solution may be appropriately determined by a person skilled in the art, and may be, for example, 10 mM to 500 mM. The buffer solution may further contain a sensitizer, a stabilizer, etc. Examples of the sensitizer include, but are not limited to, polyethylene glycol (PEG), 2-methacryloyloxyethyl phosphorylcholine (MPC), etc.

[0032] In an immunological measurement method, for example, a buffer solution containing a sensitizer or the like may be mixed with a sample, and then the sample may be mixed with the complex of the present invention. In this way, the reproducibility (stability) of the antigen-antibody reaction can be improved by stabilizing the sample (antigen) and then reacting it with the complex of the present invention (antibody).

[0033] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. EXAMPLES

[0034] Example 1 Preparation of F(ab')2 antibody fragment-latex conjugate 1 Antibody fragment F(ab')2 against phospholipase A2 (PLA2) was prepared and used to prepare an anti-PLA2 antibody-latex conjugate.

[0035] 1. Preparation of antigen-binding fragments (immunogen) One mg of PLA2G1B recombinant protein (Abeomics, Cat. 32-2678) was dissolved in 5 mL of saline solution containing 1 mM TLCK (Tosyl-L-lysyl-chloromethane hydrochloride, Nakarai, Code 34219-94), heated at 37°C for 30 minutes, and then dialyzed against saline solution before use.

[0036] (immunization method) The above immunogen was suspended in an adjuvant (Complete Friends' adjuvant: CFA, GIBCO), and BALB / c mice (5 weeks old, female) were immunized at 25 μg PLA2 / mouse at 2-week intervals. Partial blood was collected during the immunization, and the degree of titer increase at the antiserum level was confirmed as an index of reactivity with the immunogen PLA2. After confirming that the antibody titer had increased sufficiently through multiple immunizations, a saline solution of the above immunogen was administered intraperitoneally or intravenously. Antibody-producing cells were collected from the spleen and fused with myeloma (P3X63Ag8.653, ECACC). Cell fusion was performed using the PEG method, and the fused cells were seeded on a culture plate. The cells were then cultured in a carbon dioxide incubator at 37°C.

[0037] (screening) The culture supernatant of each well into which the fused cells were dispensed was collected, and the antibody titer was confirmed using the reactivity with PLA2 as an index. Then, cells (hybridomas) producing two different antibodies were selected, subcultured, and cloned by limiting dilution. The cells derived from the obtained single colony were used as anti-human PLA2 monoclonal antibody producing hybridomas. Thus, two types of hybridomas were obtained.

[0038] From the above hybridoma, PureLink TMMessenger RNA (mRNA) was obtained using a SMARTer RACE 5' / 3' kit (ThermoFisher). The obtained mRNA was converted to cDNA using a SMARTer RACE 5' / 3' kit (Takara Bio), and sequenced using a gene sequencer. The light chain sequence of the analyzed antibody gene was left as is, and the heavy chain sequence was synthesized by extracting a part of the sequence so as to become F(ab')2, and adding a sequence so that six histidine residues (His) were added to the C-terminus. The fully synthesized sequence was incorporated into the pcDNA3.1(+) vector to make a plasmid so that the antibody could be produced in mammalian cells. The prepared plasmid was introduced into CHO (Chinese hamster ovary) cells using Lipofectamine 3000 (ThermoFisher), and the antibody secreted into the culture supernatant was analyzed using HisTrap. TM Purification was carried out using HP (Cytiva) to obtain the antibody fragments of interest. Hereinafter, the antibody fragments obtained from each hybridoma are referred to as Antibody A and Antibody B, respectively.

[0039] 2. Complex Preparation Antibody A-sensitized latex α liquid and antibody B-sensitized latex β liquid were prepared using the following materials and methods. (material) Latex liquid: Carboxy modified latex (JSR product name: IMMUNTEX) TM ), average particle size 351nm, solid content: 10% ·Antibody A solution: 4.0mg / mL CHO-derived C-terminal His-added antibody A, 40mM NaCl, 20mM HEPES (pH7.4) ·Antibody B solution: 4.0mg / mL CHO-derived C-terminal His-added antibody B, 40mM NaCl, 20mM HEPES (pH7.4) ·MES buffer: 1%BSA, 20mM MES (pH6.0), 0.05%NaN3 ·HEPES buffer: 2%BSA, 20mM HEPES (pH7.0), 150mM NaCl 0.05%NaN3

[0040] (method) 500μL of latex solution (10% w / vol) and 417μL of antibody A solution (4.0mg / mL) were mixed and shaken at 20℃ for 90 minutes. Separately, 347μL of antibody B solution (4.0mg / mL) was added to a mixture of latex solution and antibody A solution prepared in the same manner, and shaken at 20℃ for 90 minutes. The resulting mixture was centrifuged at 10000rpm and 10℃ for 30 minutes using a centrifuge (rotor: R15A (Eppendorf-Himack)). The supernatant was decanted, and 25mL of MES buffer was added to the resulting sediment and dispersed by sonication. The mixture was then shaken at 37℃ for 60 minutes. The resulting dispersion was centrifuged at 10000rpm and 10℃ for 30 minutes using the same centrifuge. The supernatant was decanted, and 50 mL of HEPES buffer was added to the resulting sediment, which was then dispersed by sonication to obtain antibody-sensitized latex α liquid and β liquid.

[0041] Example 2: Preparation of F(ab')2 antibody fragment-latex conjugate 2 We prepared an antibody fragment F(ab')2 against human cardiac fatty acid binding protein (H-FABP) and used it to prepare an anti-H-FABP antibody-latex conjugate.

[0042] 1. Preparation of antigen-binding fragments (immunogen) 1 mg of H-FABP purified from human cardiomyocytes was dissolved in 5 mL of saline solution containing 1 mM TLCK (Tosyl-L-lysyl-chloromethane hydrochloride, Nakarai, Code 34219-94), heated at 37°C for 30 minutes, and then dialyzed against saline solution before use.

[0043] (immunization method) The above immunogen was suspended in an adjuvant (Complete Friends' adjuvant: CFA, manufactured by GIBCO) and immunized with BALB / c mice (5 weeks old, female) at 25 μg H-FABP / mouse at 2-week intervals. Partial blood was collected during the immunization, and the degree of titer increase at the antiserum level was confirmed as an index of reactivity with the immunogen H-FABP. After confirming that the antibody titer had increased sufficiently through multiple immunizations, a saline solution of the above immunogen was administered intraperitoneally or intravenously. Antibody-producing cells were collected from the spleen and fused with myeloma (P3X63Ag8.653, ECACC). Cell fusion was performed using the PEG method, and the fused cells were seeded on a culture plate. The cells were then cultured in a carbon dioxide incubator at 37°C.

[0044] (screening) The culture supernatant of each well into which the fused cells were dispensed was collected, and the antibody titer was confirmed using the reactivity with H-FABP as an index. Then, cells (hybridomas) producing two different antibodies were selected, subcultured, and cloned by limiting dilution. The cells derived from the obtained single colony were used as anti-human PLA2 monoclonal antibody-producing hybridomas. Thus, two types of hybridomas were obtained.

[0045] From the above hybridoma, PureLink TM Messenger RNA (mRNA) was obtained using a SMARTer RACE 5' / 3' kit (ThermoFisher). The obtained mRNA was converted to cDNA using a SMARTer RACE 5' / 3' kit (Takara Bio) and sequenced using a gene sequencer. The light chain sequence of the analyzed antibody gene was left as is, and a part of the heavy chain sequence was extracted so as to become F(ab')2, and a sequence was added to add six His to the C-terminus to produce the antibody by synthesis. The fully synthesized sequence was incorporated into the pcDNA3.1(+) vector to make a plasmid, allowing the antibody to be produced in mammalian cells. The prepared plasmid was introduced into CHO cells using Lipofectamine 3000 (ThermoFisher), and the antibody secreted into the culture supernatant was analyzed using HisTrap.TM Purification was carried out using HP (Cytiva) to obtain the antibody fragments of interest. Hereinafter, the antibody fragments obtained from each hybridoma are referred to as Antibody A and Antibody B, respectively.

[0046] 2. Complex Preparation Antibody A-sensitized latex α liquid and antibody B-sensitized latex β liquid were prepared using the following materials and methods. (material) Latex liquid: Carboxy modified latex (JSR product name: IMMUNTEX) TM ), average particle size 351nm, solid content: 10% ·Antibody A solution: 4.0mg / mL CHO-derived C-terminal His-added antibody A, 40mM NaCl, 20mM HEPES (pH7.4) ·Antibody B solution: 4.0mg / mL CHO-derived C-terminal His-added antibody B, 40mM NaCl, 20mM HEPES (pH7.4) ·MES buffer: 1%BSA, 20mM MES (pH6.0), 0.05%NaN3 ·HEPES buffer: 2%BSA, 20mM HEPES (pH7.0), 150mM NaCl 0.05%NaN3

[0047] (method) 500μL of latex solution (10% w / vol) and 417μL of antibody A solution (4.0mg / mL) were mixed and shaken at 20℃ for 90 minutes. Separately, 347μL of antibody B solution (4.0mg / mL) was added to a mixture of latex solution and antibody A solution prepared in the same manner, and shaken at 20℃ for 90 minutes. The resulting mixture was centrifuged at 10000rpm and 10℃ for 30 minutes using a centrifuge (rotor: R15A (Eppendorf-Himack)). The supernatant was decanted, and 25mL of MES buffer was added to the resulting sediment and dispersed by sonication. The mixture was then shaken at 37℃ for 60 minutes. The resulting dispersion was centrifuged at 10000rpm and 10℃ for 30 minutes using the same centrifuge. The supernatant was decanted, and 50 mL of HEPES buffer was added to the resulting sediment, which was then dispersed by sonication to obtain antibody-sensitized latex α liquid and β liquid.

[0048] Example 3 Binding test using anti-H-FABP antibody fragment-latex complex The following experiment was carried out using the anti-H-FABP antibody fragment-latex complex prepared in Example 2. The compositions of measuring reagent I, reagent II and standard reagent used in the experiment are as follows.

[0049] <Measurement Reagents> (1) Reagent I (R1 Reagent: Buffer Solution) (composition) 0.3% Tween20 0.5% PEG 0.5% bovine serum albumin (BSA) 150mM NaCl ·0.09%NaN3 ·100mM HEPES buffer (pH7.6) (2) Reagent II (R2 Reagent: Latex Reagent) (composition) 0.5% BSA 150mM NaCl ·0.09% NaN3 ·100mM HEPES buffer (pH7.0) Antibody A sensitized latex α liquid (1g / l) Antibody B sensitized latex β solution (1g / l) <Standard Reagents> Standard H-FABP (Sumitomo Bakelite Co., Ltd.) (0, 1.875, 3.75, 7.5, 15, 30, 60, or 120 ng / ml) 20mM phosphate buffer (pH 6.5) 5mM EDTA 2Na 150mM NaCl 0.5% BSA 0.09% Sodium Azide 0.1% Tween20

[0050] The measurement was carried out as follows. 120 μL of reagent I was added to 5 μL of standard reagent of each concentration, and 5 minutes after the addition, 30 μL of reagent II was added, and the absorbance at 700 nm was immediately measured (0 minutes later). The value was designated as ABS1. In addition, 5 minutes after adding reagent II, the absorbance was measured, and the value was designated as ABS2. The value of (ABS2-ABS1) was then calculated as ΔABS. As a control, instead of reagent II, the R2 latex reagent containing full-length anti-human H-FABP monoclonal antibody-sensitized latex contained in Libria (registered trademark) H-FABP (Sumitomo Bakelite Co., Ltd.), a commercially available H-FABP measurement kit, was used, and the experiment was carried out in the same manner. The results are shown in Table 1 and Figure 2.

[0051] [Table 1]

[0052] As is clear from Table 1 and FIG. 2, it was found that the antibody-substrate complex of the present invention has higher measurement sensitivity than the full-length antibody, particularly in the presence of low concentrations of antigen.

[0053] Example 4 Binding test using anti-PLA2 antibody fragment-latex complex The following experiment was carried out using the anti-PLA2 antibody fragment-latex complex prepared in Example 1. The compositions of measuring reagent I, reagent II and standard reagent used in the experiment are as follows.

[0054] <Measurement Reagents> (1) Reagent I (R1 Reagent: Buffer Solution) (composition) 0.3% Tween20 0.5% PEG 0.5% bovine serum albumin (BSA) 150mM NaCl ·0.09%NaN3 ·100mM HEPES buffer (pH7.6) (2) Reagent II (R2 Reagent: Latex Reagent) (composition) 0.5% BSA, 150mM NaCl ·0.09% NaN3 ·100mM HEPES buffer (pH7.0) Antibody A sensitized latex α solution (20μl / 100μl) Antibody B sensitized latex β solution (20μl / 100μl) <Standard Reagents> Recombinant PLA2 [PLA2G1B recombinant protein (Abeomics, Cat. 32-2678): 0, 100, 1000ng / dL] 20mM phosphate buffer (pH 6.5) 5mM EDTA 2Na 150mM NaCl 0.5% BSA 0.09% Sodium Azide 0.1% Tween20

[0055] The measurement was carried out as follows. 120 μL of reagent I was added to 5 μL of the standard reagent of each concentration, and 30 μL of reagent II was added 5 minutes after the addition, and the absorbance at 700 nm was immediately measured (0 minutes later). The value was designated as ABS1. In addition, the absorbance was measured 5 minutes after adding reagent II, and the value was designated as ABS2. The value of (ABS2-ABS1) was then calculated as ΔABS. As a control, in reagent II, anti-PLA2 antibody fragment F(ab')2-sensitized latex prepared by a conventional method was added instead of the antibody-sensitized latex α and β solutions, and the same experiment was carried out. The results are shown in Table 2. The above anti-PLA2 antibody fragment was prepared by digesting the full-length anti-PLA2 antibody with pepsin (Sigma-Aldrich, product number: P7012) in citrate buffer (pH 3.5) at 37° C. for 60 minutes, and a complex with latex was obtained in the same manner as in Example 1.

[0056] [Table 2]

[0057] The antibody-substrate complexes of the invention have been shown to provide greater sensitivity in measurements compared to antibody fragments, particularly in the presence of low concentrations of antigen.

Claims

1. A complex comprising an antibody or antigen-binding fragment having a basic amino acid added to its C-terminus, and a substrate.

2. The complex of claim 1 , wherein the C-terminus of the antibody or antigen-binding fragment is bound to the surface of the substrate.

3. The composite of claim 1 , wherein the surface of the substrate is negatively charged.

4. The complex according to claim 1 , wherein the basic amino acid is selected from histidine, arginine, lysine, or an artificial amino acid having an amino group in its side chain.

5. The composite of claim 1 , wherein the substrate is a particle or platelet-like substrate.

6. The composite of claim 5 , wherein the particle is a latex particle.

7. The complex of claim 1 , wherein the antigen-binding fragment is a F(ab')2, Fab, Fv, scFv, or diabody.

8. A method for producing a complex comprising an antibody or antigen-binding fragment having a basic amino acid added to its C-terminus and a substrate, the method comprising contacting the antibody or antigen-binding fragment with a substrate, the C-terminus of the antibody or antigen-binding fragment being oriented so that it binds to the substrate surface.

9. The method of claim 8 , wherein the surface of the substrate is negatively charged.

10. A reagent or kit for immunoassay, comprising the complex according to any one of claims 1 to 7.

11. The immunoassay reagent or kit according to claim 10, wherein the antibody or antigen-binding fragment in the complex is an antibody or an antigen-binding fragment thereof that specifically binds to a protein selected from the group consisting of H-FABP, PLA2, trypsin, and elastase.

12. A method for detecting a target substance in a sample, comprising contacting the sample with the complex according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is an antibody or an antigen-binding fragment thereof that specifically binds to the target substance.

13. The method of claim 12, wherein the target substance is selected from the group consisting of H-FABP, PLA2, trypsin, and elastase.

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