Method for producing labeled antibodies, labeled antibodies, and immunoassays

Abstract

To provide a mark antibody in which, an amount of antibodies coupled to a metal-resin composite body is sufficiently large, and by which sensitive immunological measurement can be performed.SOLUTION: There is provided a production method of a mark antibody configured so that, the antibody is adhered to a metal-resin composite body in a coupling step, by mixing, the antibody, and the metal-resin composite body having a structure in which, metal particles are stabilized to resin particles, in the presence of a coupling buffer solution with pH of 6-10 and including a good buffer whose pKa at 20°C is 7.5 or greater, where the good buffer may be preferably one or more kinds selected from: 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid (HEPES); N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS);N-cyclohexyl-3-aminopropanesulfonic acid (CAPS); 2-cyclohexylaminoethanesulfonic (CHES); and N-(tris(hydroxymethyl)methyl)-3-amino-2-hydroxypropanesulfonic acid (TAPSO).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a labeled antibody that can be used, for example, in immunological measurements, a labeled antibody, and an immunological measurement method.

Background Art

[0002] An immunoassay, also called an immunological measurement method, is a method for qualitatively and quantitatively analyzing trace components by utilizing a specific reaction between an antigen and an antibody. Since the antigen-antibody reaction has high sensitivity and reaction selectivity, it is widely used in fields such as pharmaceuticals. Various measurement methods are known for immunoassays depending on their measurement principles. For example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), fluorescence immunoassay (FIA), agglutination methods such as latex (LIA, PA), immunochromatography method (ICA), hemagglutination method (HA), hemagglutination inhibition method (HI), and the like can be mentioned.

[0003] An immunoassay detects an antigen or an antibody qualitatively or quantitatively from changes (concentration changes of an antigen, an antibody, or a complex) when an antigen and an antibody react to form a complex. When detecting these, the detection sensitivity is increased by binding a labeling substance to an antibody, an antigen, or a complex. Therefore, it can be said that the labeling ability of the labeling substance is an important factor that influences the detection ability in immunoassays.

[0004] Therefore, as a labeling substance that enables highly sensitive immunological measurements, a metal-resin composite having a structure in which metal particles are immobilized on resin particles has been proposed (for example, Patent Documents 1 and 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

[0006] For metal-resin complexes to be used as labeling materials in immunoassays, it is necessary to bind them with a sufficient amount of ligand, such as an antibody. If the amount of antibody bound to the metal-resin complex is insufficient, good detection sensitivity cannot be obtained. However, metal-resin complexes dispersed in buffer tend to aggregate, which can reduce the amount of antibody bound and significantly decrease detection sensitivity.

[0007] Therefore, the present invention aims to provide a labeled antibody that has a sufficiently large amount of antibody bound to the metal-resin composite, enabling highly sensitive immunological measurement. [Means for solving the problem]

[0008] As a result of diligent research, the inventors of the present invention have discovered that the above problem can be solved by binding a metal-resin composite having a structure in which metal particles are immobilized on resin particles to an antibody under a specific binding buffer, and have completed the present invention.

[0009] In other words, the present invention provides a method for producing labeled antibodies, comprising a binding step of attaching the antibody to the metal-resin composite, by mixing the antibody and a metal-resin composite having a structure in which metal particles are immobilized on resin particles in the presence of a binding buffer in the pH range of 6 to 10 containing a Good buffer having a pKa of 7.5 or higher at 20°C; It includes.

[0010] The method for producing labeled antibodies of the present invention may also involve the Good buffer being one or more selected from 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 2-cyclohexylaminoethanesulfonic acid (CHES), and N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO).

[0011] In the method for producing labeled antibodies of the present invention, the proportion of the antibody attached to the total amount of antibody used in the binding step may be 50% by weight or more.

[0012] In the method for producing labeled antibodies of the present invention, the metal particles may be silver, nickel, copper, gold, platinum, palladium, or an alloy containing any of these.

[0013] The method for producing labeled antibodies of the present invention may include metal particles having a portion embedded within the resin particles and a portion exposed outside the resin particles.

[0014] In the method for producing labeled antibodies of the present invention, at least some of the metal particles may be distributed three-dimensionally on the surface layer of the resin particles.

[0015] In the method for producing labeled antibodies of the present invention, the resin particles may be polymer particles having substituents in their structure that are capable of adsorbing metal ions.

[0016] In the method for producing the labeled antibody of the present invention, the average particle size of the metal particles may be in the range of 1 to 80 nm.

[0017] In the method for producing labeled antibodies of the present invention, the average particle size of the metal-resin composite may be in the range of 100 to 1000 nm.

[0018] The labeled antibody of the present invention is produced by the method for producing any of the above-labeled antibodies.

[0019] The immunological assay method of the present invention uses the above-labeled antibody. [Effects of the Invention]

[0020] According to the method for producing a labeled antibody of the present invention, by using a specific binding buffer solution, aggregation of the metal-resin complex is suppressed, and a labeled antibody with a large amount of antibody attachment and capable of highly sensitive immunological measurement can be produced. The labeled antibody obtained by the method of the present invention can be advantageously used in various immunological measurements as a material excellent in durability, visibility, visual determination property, and detection sensitivity. [Brief Description of the Drawings]

[0021] [Figure 1] It is a schematic diagram showing the structure of a cross section of a metal-resin complex used in one embodiment of the present invention. [Figure 2] It is a cross-sectional view of an immunochromatographic strip prepared in an example. [Modes for Carrying Out the Invention]

[0022] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate.

[0023] [Method for Producing Labeled Antibody] The method for producing a labeled antibody of the present embodiment includes at least the following step A; Step A) A binding step of attaching an antibody to a metal-resin complex by mixing an antibody and a metal-resin complex having a structure in which metal particles are immobilized on resin particles in the presence of a binding buffer solution in the range of pH 6 to 10 containing a Good buffer (hereinafter sometimes referred to as "specific Good buffer") having a pKa of 7.5 or more at 20°C. can include.

[0024] (Antibody) In this embodiment, there are no particular restrictions on the antibody, and for example, polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments having antigen-binding ability [e.g., H chain, L chain, Fab, F(ab')2, etc.] can be used. Also, any of IgG, IgM, IgA, IgE, or IgD can be used as the immunoglobulin. The animal species that produce the antibody can be humans, as well as animals other than humans (e.g., mice, rats, rabbits, goats, horses, etc.). Specific examples of antibodies include anti-CRP antibody, anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-HCV antibody, anti-IgG antibody, anti-human IgE antibody, anti-human cardiac troponin I antibody, and anti-SARS-CoV-2 (COVID-19) antibody.

[0025] (metal-resin composite) In this embodiment, the metal-resin composite used as a label has a structure in which metal particles are immobilized on resin particles. Details of this metal-resin composite will be described later.

[0026] <Process A: Bonding process> In step A, the antibody is attached to the metal-resin complex by mixing the antibody and the metal-resin complex in the presence of a binding buffer with a pH in the range of 6 to 10. The binding of the metal-resin complex and the antibody in this step is a so-called physical binding, in which the antibody is bound to the surface of the metal particles and / or the surface of the resin particles in the metal-resin complex by physical interaction. Here, binding by physical interaction can include, for example, electrostatic bonding, hydrogen bonding, hydrophilic-hydrophobic interactions, and van der Waals forces.

[0027] (Binding buffer) In this process, a binding buffer containing a specific Good's buffer with a pH in the range of 6 to 10 is used. Good's buffers are biochemically useful buffers with acidic and basic functional groups, and dozens of types have been known to date. Among these, the specific Good's buffer exhibits a remarkable effect in addition to the properties known for general Good's buffers, by effectively suppressing the aggregation of metal-resin complexes in the buffer due to its low ionic strength. In other words, it has been confirmed that the specific Good's buffer exerts an aggregation-inhibiting effect on metal-resin complexes, which is different from its effect on other labeling substances. Therefore, by binding the antibody to the metal-resin complex using a binding buffer containing the specific Good's buffer, it becomes possible to apply the antibody in a state where the aggregation of the metal-resin complex is suppressed, and the amount of antibody adhering to the metal-resin complex can be significantly increased compared to, for example, a binding buffer using an inorganic buffer.

[0028] For specific Good buffering agents, any agent with a pKa of 7.5 or higher at 20°C is acceptable. However, from the viewpoint of ensuring sufficient antibody adhesion, for example, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid [HEPES; pKa (20°C) 7.55], N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid [TAPS; pKa (20°C) 8.40], N-cyclohexyl-3-aminopropanesulfonic acid [CAPS; pKa (20°C) 10.40], 2-cyclohexylaminoethanesulfonic acid [CHES; pKa (20°C) 9.5], N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid [TAPSO; pKa (20°C) 7.7] are preferred. Among the compounds known as Good's buffers, those with a pKa of less than 7.5 at 20°C can suppress the aggregation of metal-resin complexes, but they often fall outside the pH range suitable for antibody binding, making it difficult to ensure sufficient antibody adhesion.

[0029] Furthermore, by using a binding buffer containing a specific Good buffer, clogging of the test strip by the metal-resin complex can be prevented regardless of the type of antibody, thereby suppressing false positives from immunochromatographic negative samples.

[0030] In step A, it is preferable to use a binding buffer with a pH in the range of 6 to 10, and more preferably in the range of 6 to 8. If the pH of the binding buffer is less than 6, the amount of antibody that adheres may decrease, and if the pH exceeds 10, the amount of ionic components added to adjust the pH increases, which increases the ionic strength of the buffer and reduces its ability to suppress the aggregation of the metal-resin complex. If the pH is within the range of 6 to 10, it is possible to bind a sufficient amount of antibody while suppressing the aggregation of the metal-resin complex, and excellent detection sensitivity can be obtained in immunoassays.

[0031] The binding buffer used in step A can be prepared according to conventional methods. For example, the binding buffer can be prepared by mixing an aqueous solution of a specific Good buffer with an alkaline solution of a predetermined concentration so that the pH is within the above range. Preferred alkaline solutions include, for example, sodium hydroxide solution and potassium hydroxide. The pH of the binding buffer can be adjusted using, for example, hydrochloric acid and sodium hydroxide.

[0032] (join operation) In step A, a labeled antibody dispersion can be obtained by mixing the antibody and the metal-resin complex in the presence of a binding buffer and stirring thoroughly. In this step, since the antibody and the metal-resin complex only need to be bound in the binding buffer, the metal-resin complex may be dispersed in the binding buffer beforehand and the antibody may be added thereto, or the antibody may be dispersed in the binding buffer beforehand and the metal-resin complex may be added thereto. Dispersion can be carried out by known methods, but it is preferable to use dispersion means such as sonication, stirring with a rotator, or a vortex mixer. The labeled antibody dispersion thus obtained can be separated as a solid portion, such as the labeled antibody, by solid-liquid separation means such as centrifugation.

[0033] (Antibody binding yield) In this binding process, it is preferable that the ratio of antibodies attached to the metal-resin complex relative to the total amount of antibody used (also called the "antibody binding yield") be 50% by weight or more, and more preferably 70% by weight or more. If the ratio of attached antibodies is less than 50% by weight, it is undesirable because expensive antibodies are not effectively utilized and are wasted. In the present invention, by using a binding buffer containing a specific Good buffer, the antibody binding yield can be increased to 50% by weight or more, thereby enabling excellent detection sensitivity in immunoassays using labeled antibodies while effectively utilizing the antibodies.

[0034] Furthermore, the reagent composition obtained by dispersing the metal-resin composite in a binding buffer preferably has a non-aggregation ratio of 50% or more of the metal-resin composite. Here, the non-aggregation ratio refers to the ratio of metal-resin composites that are dispersed as single particles without aggregating, and can be determined from the measurement results of the particle size distribution based on volume and mass.

[0035] A non-agglutination ratio of 50% or higher increases the antibody binding yield when producing labeled antibodies and increases the amount of antibody attached to the metal-resin complex, resulting in excellent detection sensitivity when performing immunoassays using labeled antibodies. Such a high non-agglutination ratio is achieved by using a binding buffer containing a specific Good buffer when applying it to the metal-resin complex. Conversely, if the non-agglutination ratio is less than 50%, it means that the proportion of aggregated particles in the metal-resin complex is relatively large, resulting in a decrease in both the antibody binding yield when producing labeled antibodies and the amount of antibody attached to the metal-resin complex, as well as inferior detection sensitivity when performing immunoassays using labeled antibodies.

[0036] The method for producing the labeled antibody of this embodiment may further include the following step B.

[0037] <Process B> Step B is a step in which the labeled antibody is dispersed in a blocking buffer within the pH range of 2 to 9. In Step B, blocking is performed to suppress nonspecific adsorption to the labeled antibody by dispersing the labeled antibody obtained in Step A in a blocking buffer within the pH range of 2 to 9. In this case, for example, the labeled antibody that has been separated by a solid-liquid separation means is dispersed in the liquid phase under conditions within the pH range of 2 to 9. From the viewpoint of maintaining the activity of the antibody and suppressing the aggregation of the labeled antibody, the blocking conditions are preferably within the pH range of 4 to 9, and from the viewpoint of suppressing nonspecific adsorption of the labeled antibody, a pH range of 5 to 9 is preferable. If the blocking buffer pH is less than 2, the antibody may be altered and inactivated due to strong acidity, and if the pH exceeds 9, the labeled antibody may aggregate, making dispersion difficult.

[0038] Step B involves, for example, adding a blocking buffer adjusted to the above pH range to a predetermined amount of labeled antibody obtained in Step A, and uniformly dispersing the labeled antibody in the blocking buffer. Preferably, the blocking buffer is a solution of a protein that does not bind to the substance to be detected. Examples of proteins that can be used in the blocking buffer include bovine serum albumin (BSA), ovalbumin, casein, and gelatin. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. Known methods can be used to disperse the labeled antibody, but it is preferable to use dispersion means such as sonication or stirring with a rotator. In this way, a labeled antibody dispersion is obtained in which the labeled antibody is uniformly dispersed. From this labeled antibody dispersion, only the labeled antibody can be separated as a solid portion by solid-liquid separation means such as centrifugation. Furthermore, optional steps such as a washing process and a storage process can be performed as needed. The washing process and storage process will be described below.

[0039] (Cleaning process) The washing process involves adding a washing buffer to the labeled antibody separated by a solid-liquid separation means, and uniformly dispersing the labeled antibody in the washing buffer. For dispersion, it is preferable to use a dispersion method such as sonication. The washing buffer is not particularly limited, but for example, a predetermined concentration of Tris buffer (tris-hydroxymethylaminomethane buffer), glycinamide buffer, or arginine buffer adjusted to a pH range of 8-9 can be used. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. The washing process of the labeled antibody can be repeated multiple times as needed.

[0040] (Preservation process) The preservation process involves adding a preservation buffer to the labeled antibody separated by a solid-liquid separation means, and uniformly dispersing the labeled antibody in the preservation buffer. For dispersion, it is preferable to use a dispersion method such as sonication. As the preservation buffer, for example, a solution obtained by adding a predetermined concentration of an anti-aggregating agent and / or stabilizer to the washing buffer can be used. As anti-aggregating agents, for example, sugars such as sucrose, maltose, lactose, and trehalose, or polyhydric alcohols such as glycerin and polyvinyl alcohol can be used. As stabilizers, there are no particular limitations, but for example, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin can be used. In this way, the preservation process for labeled antibodies can be carried out.

[0041] In each of the above steps, surfactants and preservatives such as sodium azide and parahydroxybenzoic acid esters may be used as needed.

[0042] [Labeled antibody] Labeled antibodies can be produced in the manner described above. Labeled antibodies obtained by the method of the present invention can be used for various immunological measurements, similar to conventional labeled antibodies. For example, by mixing a sample containing analyte with the labeled antibody, allowing it to react, and measuring the resulting color development visually or using an analytical instrument, immunological measurements can be performed.

[0043] <Metal-resin composite> Next, an example of a metal-resin composite used as a label in the method for producing labeled antibodies of this embodiment will be described in detail. The metal-resin composite has a structure in which metal particles are immobilized on resin particles.

[0044] Figure 1 is a schematic cross-sectional view of a metal-resin composite 100 that can be used as a label in this embodiment. The metal-resin composite 100 comprises resin particles 10 and metal particles 20.

[0045] (Structure of metal-resin composites) In the metal-resin composite 100, metal particles 20 are dispersed or immobilized within resin particles 10. Preferably, in the metal-resin composite 100, a portion of the metal particles 20 are distributed three-dimensionally in the surface layer 60 of the resin particles 10, and a portion of the three-dimensionally distributed metal particles 20 are partially exposed outside the resin particles 10, while the remaining portion is enclosed within the resin particles 10. Here, "surface layer" means the area from the surface of the resin particles 10 to 50% of the particle radius in the depth direction. Also, "three-dimensionally distributed" means that the metal particles 20 are dispersed not only in the planar direction of the resin particles 10 but also in the depth direction.

[0046] Here, the metal particles 20 may include one or more selected from metal particles completely enclosed within the resin particles 10 (hereinafter also referred to as "enclosed metal particles 30"), metal particles having a portion embedded within the resin particles 10 and a portion exposed outside the resin particles 10 (hereinafter also referred to as "partially exposed metal particles 40"), and metal particles adsorbed on the surface of the resin particles 10 (hereinafter also referred to as "surface-adsorbed metal particles 50").

[0047] The encapsulated metal particles 30 have their entire surface covered with the resin that makes up the resin particles 10. The partially exposed metal particles 40 have 5% to less than 100% of their surface area covered by the resin constituting the resin particles 10. From the viewpoint of durability as a label for immunological measurement, the lower limit is preferably 20% or more of the surface area, and more preferably 30% or more. The surface-adsorbed metal particles 50 have a surface area that is more than 0% but less than 5% covered by the resin that makes up the resin particles 10.

[0048] For example, when using the metal-resin composite 100 for immunological measurements, antibodies are immobilized on the surface of the resin particles 10, the surface of the partially exposed metal particles 40, or the surface-adsorbed metal particles 50. In this case, antibodies are immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, but not on the encapsulated metal particles 30. However, since all of the metal particles 20, including the encapsulated metal particles 30, exhibit localized surface plasmon absorption, not only the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, but also the encapsulated metal particles 30 contribute to improved visibility as an immunological measurement label. Furthermore, the partially exposed metal particles 40 and the encapsulated metal particles 30 have a larger contact area with the resin particles 10 compared to the surface-adsorbed metal particles 50, and their physical adsorption force, such as the anchoring effect due to the embedding state, is strong, making them less likely to detach from the resin particles 10. Therefore, the durability and stability of the metal-resin composite 100 as an immunological measurement label can be improved.

[0049] Furthermore, the amount of metal particles 20 (total of encapsulated metal particles 30, partially exposed metal particles 40, and surface-adsorbed metal particles 50) loaded onto the metal-resin composite 100 is preferably in the range of 5% to 70% by weight relative to the weight of the metal-resin composite 100. Within this range, the metal-resin composite 100 exhibits excellent visibility, visual determination, and detection sensitivity as a labeling substance. If the amount of metal particles 20 loaded is less than 5% by weight, the amount of immobilized antibody decreases, and the detection sensitivity tends to decline. More preferably, the amount of metal particles 20 loaded is in the range of 15% to 70% by weight.

[0050] Furthermore, it is preferable that 10% to 90% by weight of the metal particles 20 consist of partially exposed metal particles 40 and surface-adsorbed metal particles 50. Within this range, a sufficient amount of antibody can be immobilized on the metal particles 20, resulting in high sensitivity as a labeling substance. It is even more preferable that 20% to 80% by weight of the metal particles 20 consist of partially exposed metal particles 40 and surface-adsorbed metal particles 50.

[0051] Furthermore, it is preferable that 60% to 100% by weight of the metal particles 20 are present in the surface layer 60, and that 5% to 90% by weight of the metal particles 20 present in the surface layer 60 are partially exposed metal particles 40 or surface-adsorbed metal particles 50, as this ensures sufficient immobilization of the antibody on the metal particles 20, thereby increasing the sensitivity as a labeling substance. In other words, it is preferable that 10% to 95% by weight of the metal particles 20 present in the surface layer 60 are encapsulated metal particles 30.

[0052] (Resin particles) The resin particles 10 are preferably polymer particles having substituents in their structure that are capable of adsorbing metal ions. In particular, nitrogen-containing polymer particles are preferred. Nitrogen atoms in nitrogen-containing polymers are preferred because they have excellent visibility and readily chemically adsorb anionic metal ions, which are precursors to metal particles such as silver, nickel, copper, gold, platinum, and palladium, to which antibodies can be easily immobilized. When metal ions adsorbed in the nitrogen-containing polymer are reduced to form metal nanoparticles, some of the generated metal particles 20 become encapsulated metal particles 30 or partially exposed metal particles 40. Furthermore, polymers having functional groups such as carboxylic acids, such as acrylic acid polymers, can adsorb cationic metal ions. Therefore, they readily adsorb cationic metal ions, which are precursors to metal particles such as silver, nickel, copper, gold, platinum, and palladium. This makes it possible to form metal particles 20 such as silver, nickel, copper, gold, platinum, and palladium, and to create alloys with any of the above metals.

[0053] The nitrogen-containing polymers mentioned above are resins having nitrogen atoms in their main chain or side chains, and include, for example, polyamines, polyamides, polypeptides, polyurethanes, polyureas, polyimides, polyimidazoles, polyoxazoles, polypyrroles, and polyanilines. Preferably, they are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. When nitrogen atoms are present in the side chains, a wide range of materials can be used, such as acrylic resins, phenolic resins, epoxy resins, cellulose resins, and melamine resins.

[0054] Furthermore, in the case of resin particles that do not have substituents in their structure that can adsorb metal ions, such as unsubstituted polystyrene particles, metal particles can be immobilized on the particle surface by irradiation of the particle surface with ultrasound or ionizing radiation, or by metal plating of the resin particle surface.

[0055] (metal particles) The structure of the metal particles 20 is not particularly limited, but those that exhibit color development in the state of the metal-resin composite 100 immobilized on the resin particles 10 are preferred because they facilitate visual determination in immunological measurements. Examples of materials for the metal particles 20 include silver, nickel, copper, gold, platinum, and palladium. These metals can be used individually or in composites such as alloys. Preferably, gold, platinum, and palladium are used because they have excellent visibility and allow for easy antibody immobilization, and these metals develop color due to absorption originating from localized surface plasmon resonance. More preferably, gold and platinum have good storage stability. Furthermore, from the viewpoint of obtaining excellent color development when bound to antibodies, gold, platinum, gold alloys, or platinum alloys are the most preferred metal species. Here, a gold alloy means, for example, an alloy consisting of gold and other metal species, containing 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more of gold. Furthermore, a platinum alloy refers to an alloy consisting of, for example, platinum and other metal species, containing 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more, of platinum.

[0056] Furthermore, the average particle diameter of the metal particles 20, as measured by scanning electron microscopy (SEM), is preferably in the range of 1 to 80 nm. If the average particle diameter of the metal particles 20 is less than 1 nm or greater than 80 nm, localized surface plasmons tend to be less likely to appear, resulting in a decrease in sensitivity. The average particle diameter of the metal particles 20 is preferably in the range of 1 nm or more and less than 70 nm, and more preferably in the range of 1 nm or more and less than 50 nm.

[0057] Furthermore, the average particle size of the metal-resin composite 100 is, for example, in the range of 100 to 1000 nm. If the average particle size is less than 100 nm, for example, when gold particles or platinum particles are used as the metal particles 20, the amount of metal particles 20 that can be supported tends to be small, resulting in a tendency for weaker coloration compared to gold particles of the same size. If it exceeds 1000 nm, when used as a labeled antibody or reagent, it tends to clog the pores of chromatographic media such as membrane filters and tends to have reduced dispersibility. The average particle size of the metal-resin composite 100 is preferably in the range of 100 nm or more and less than 700 nm, and more preferably in the range of 100 nm or more and less than 650 nm. Here, the particle size of the metal-resin composite 100 means the particle size of the resin particles 10 plus the length of the protruding portion of the partially exposed metal particles 40 or surface-adsorbed metal particles 50, and can be measured by laser diffraction / scattering, dynamic light scattering, or centrifugal sedimentation.

[0058] The method for producing the metal-resin composite 100 is not particularly limited. For example, a solution containing metal ions can be added to a dispersion of resin particles 10 produced by emulsion polymerization to adsorb the metal ions onto the resin particles 10 (hereinafter referred to as "metal ion-adsorbing resin particles"). Next, the metal ion-adsorbing resin particles can be added to a reducing agent solution to reduce the metal ions and generate metal particles 20, thereby obtaining the metal-resin composite 100. When gold particles are used as the metal particles 20, it is preferable to use an aqueous solution of chloroauric acid (HAuCl4) as the solution containing metal ions. When platinum particles are used as the metal particles 20, it is preferable to use an aqueous solution of chloroplatinic acid (H2PtCl6) as the solution containing metal ions. A metal complex may also be used instead of metal ions.

[0059] Furthermore, instead of water, aqueous alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, and t-butanol, or acids such as hydrochloric acid, sulfuric acid, and nitric acid may be used as the solvent for solutions containing metal ions.

[0060] Furthermore, additives such as water-soluble polymer compounds like polyvinyl alcohol, surfactants, alcohols, ethers like tetrahydrofuran, diethyl ether, and diisopropyl ether, polyols like alkylene glycol, polyalkylene glycol, their monoalkyl or dialkyl ethers, and glycerin, and various water-miscible organic solvents like acetone and methyl ethyl ketone may be added to the metal ion-containing solution as needed. Such additives are effective in accelerating the reduction reaction rate of the metal ions and in controlling the size of the resulting metal particles 20.

[0061] Furthermore, there are no particular restrictions on the reducing agent, but it is preferable to use, for example, sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, hydrazine sulfate, formaldehyde, Rochelle salt, etc. Among these, sodium borohydride, dimethylamine borane, and citric acid are more preferred. Surfactants can be added to the reducing agent solution as needed, or the pH of the solution can be adjusted. pH can be adjusted with buffers such as boric acid or phosphoric acid, acids such as hydrochloric acid or sulfuric acid, or alkalis such as sodium hydroxide or potassium hydroxide. Furthermore, by adjusting the reduction rate of metal ions using the temperature of the reducing agent solution, the particle size of the formed metal particles 20 can be controlled.

[0062] Furthermore, when reducing the metal ions in the metal ion adsorbing resin particles to generate metal particles 20, the metal ion adsorbing resin particles may be added to the reducing agent solution, or the reducing agent may be added to the metal ion adsorbing resin particles. However, the former is preferred from the viewpoint of the ease with which encapsulated metal particles 30 and partially exposed metal particles 40 are generated.

[0063] Furthermore, to maintain the dispersibility of the metal-resin composite 100 in water, dispersants such as citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, and DISPERBYK184 (manufactured by Big Chemie Japan Co., Ltd.) may be added. In addition, the pH can be adjusted with buffers such as boric acid and phosphoric acid, acids such as hydrochloric acid and sulfuric acid, and alkalis such as sodium hydroxide and potassium hydroxide to maintain dispersibility.

[0064] The metal-resin composite 100 having the above configuration can be suitably applied to various immunological assay methods by adsorbing antibodies onto the surfaces of the resin particles 10 and / or metal particles 20. In particular, it can be suitably applied as a material for an immunological labeling substance or immunological reagent that exhibits excellent visual determination in the low-concentration range (high-sensitivity range). Furthermore, there are no particular limitations on the form of the immunological labeling substance or immunological reagent, but for example, the metal-resin composite 100 can be used as a dispersion in water or a pH-adjusted buffer solution. [Examples]

[0065] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited in any way by these examples. Unless otherwise specified in the following examples and comparative examples, various measurements and evaluations are performed as described below.

[0066] <Absorbance measurement of metal-resin composite particles> The absorbance of metal-resin composite particles was measured using a spectrophotometer (Shimadzu Corporation, UV3600) with a 0.01 wt% dispersion of metal-resin composite particles (dispersion medium: water) placed in a quartz glass cell (optical path length 10 mm). The absorbance at 570 nm for gold-resin composites and 400 nm for platinum-resin composites were measured.

[0067] <Measurement of solid content concentration and metal load amount> One g of the dispersion before concentration adjustment was placed in a porcelain crucible and dried at 70°C for 3 hours. The weight was measured before and after drying, and the solid content concentration was calculated using the following formula. Solid content concentration (wt%)= [Weight after drying (g) / Weight before drying (g)] × 100 Furthermore, the samples after the above drying treatment were subjected to further heat treatment at 500°C for 5 hours, and the weights before and after heat treatment were measured. The amount of metal supported was then calculated using the following formula. Metal load (wt%) = [Weight after heat treatment (g) / Weight before heat treatment (g)] × 100

[0068] <Measurement of average particle size and particle size distribution of resin particles and metal-resin composite particles> The measurements were performed using a centrifugal sedimentation particle size distribution analyzer (LUMiSizer610, manufactured by LUM GmbH). The measurements were taken with the particles dispersed in water or a solution.

[0069] <Measurement of average particle size of metal particles> A substrate was prepared by dropping a metal-resin composite particle dispersion onto a metallic mesh with a carbon support film. Images were observed using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000). The area-average diameter of 100 arbitrary metal particles was measured from these images and defined as the average particle diameter.

[0070] [Example 1] <Synthesis of resin particles> Trioctylammonium chloride (1.3 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) were dissolved in 300 g of pure water. Then, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added, and the mixture was stirred under a nitrogen stream at 30°C for 50 minutes, followed by stirring at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.25 g) dissolved in 18.00 g of pure water was added dropwise, and the mixture was stirred at 60°C for 3.5 hours to obtain resin particles A-1 with an average particle size of 349 nm. The particles were precipitated by centrifugation (9000 rpm, 40 minutes), the supernatant was removed, and the mixture was dispersed again in pure water. Impurities were then removed by dialysis. Subsequently, the concentration was adjusted to obtain a 10 wt% resin particle dispersion B-1.

[0071] [Example 2] <Synthesis of resin particles> Trioctylammonium chloride (0.8 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) were dissolved in 300 g of pure water. Then, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added, and the mixture was stirred under a nitrogen stream at 30°C for 50 minutes, followed by stirring at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.25 g) dissolved in 18.00 g of pure water was added dropwise, and the mixture was stirred at 60°C for 3.5 hours to obtain resin particles A-2 with an average particle size of 430 nm. The particles were precipitated by centrifugation (9000 rpm, 40 minutes), the supernatant was removed, and the mixture was dispersed again in pure water. Impurities were then removed by dialysis. Subsequently, the concentration was adjusted to obtain a 10 wt% resin particle dispersion B-2.

[0072] [Example 3] <Synthesis of gold-resin composite particles> 30.0 g of resin particle dispersion B-1 was mixed with 78.0 g of pure water, then 42.9 g of 7 wt% aqueous chloroauric acid solution was added, and the mixture was stirred at room temperature for 3 hours. The resin particles were precipitated from this mixture by centrifugation (3000 rpm, 30 minutes), and excess chloroauric acid was removed by removing the supernatant. Subsequently, the concentration was adjusted to obtain 5 wt% gold ion adsorbed resin particle dispersion C-3.

[0073] Next, 65 g of C-3 was added to 4725 g of pure water, and while stirring at 3°C, 15 g of a 528 mM aqueous dimethylamine borane solution was added dropwise over 2 minutes. The mixture was then stirred at 3°C ​​for 1 hour and at room temperature for 3 hours to obtain gold-resin composite particles D-3 with an average particle size of 357 nm. D-3 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt% gold-resin composite particle dispersion E-3. The absorbance of gold-resin composite particles F-3 in E-3 was 1.38. The average particle size of the gold particles in F-3 was 21 nm, and the gold loading amount was 48.0 wt%.

[0074] [Example 4] <Synthesis of platinum-resin composite particles> 30.0 g of resin particle dispersion B-1 was mixed with 85.0 g of pure water, then 31.0 g of 7 wt% chloroplatinic acid aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. The resin particles were precipitated from this mixture by centrifugation (3000 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. Subsequently, the concentration was adjusted to obtain 5 wt% platinum ion adsorbed resin particle dispersion C-4.

[0075] Next, 70.5 g of C-4 was added to 4725 g of pure water, and 137 g of a 132 mM aqueous dimethylamine borane solution was added dropwise over 20 minutes while stirring at 3°C. The mixture was then stirred at 3°C ​​for 1 hour and at room temperature for 3 hours to obtain platinum-resin composite particles D-4 with an average particle size of 360 nm. D-4 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt% platinum-resin composite particle dispersion E-4. The absorbance of platinum-resin composite particles F-4 in E-4 was 1.75. The average particle size of the platinum particles in F-4 was 3.5 nm, and the platinum load was 39.1 wt%.

[0076] [Example 5] <Synthesis of platinum-resin composite particles> 30.0 g of resin particle dispersion B-2 was mixed with 85.0 g of pure water, then 31.0 g of 7 wt% chloroplatinic acid aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. The resin particles were precipitated from this mixture by centrifugation (3000 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. Subsequently, the concentration was adjusted to obtain 5 wt% platinum ion adsorbed resin particle dispersion C-5.

[0077] Next, 70.5 g of C-5 was added to 4725 g of pure water, and 137 g of a 132 mM aqueous dimethylamine borane solution was added dropwise over 20 minutes while stirring at 3°C. The mixture was then stirred at 3°C ​​for 1 hour and at room temperature for 3 hours to obtain platinum-resin composite particles D-5 with an average particle size of 445 nm. D-5 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt% platinum-resin composite particle dispersion E-5. The absorbance of platinum-resin composite particles F-5 in E-5 was 1.75. The average particle size of the platinum particles in F-5 was 3.7 nm, and the platinum load was 39.4 wt%.

[0078] [Example 6] <Preparation of binding buffer> HEPES buffer, TAPS buffer, CAPS buffer, MES buffer, and MOPS buffer were prepared by preparing 0.1 mol / L free acid solutions of HEPES, TAPS, CAPS, 2-morpholinoethanesulfonic acid [MES; pKa (20℃) 6.15], or 3-morpholinopropanesulfonic acid [MOPS; pKa (20℃) 7.20], and then mixing each with pure water and a 0.1 mol / L sodium hydroxide solution to adjust the buffer concentration and pH as shown in Table 1.

[0079] [Example 1] (Joining process) After mixing 25 μg of anti-CRP antibody with 0.45 mL of 20 mM HEPES buffer (pH 7), 0.05 mL of 1 wt% gold-resin composite particle dispersion E-3 was added, and the mixture was inverted and stirred at room temperature for 2 hours to obtain labeled antibody dispersion J-1 containing anti-CRP antibody labeled with gold-resin composite particles F-3.

[0080] (Blocking process) Next, labeled antibody dispersion J-1 was centrifuged at 3000 rpm for 5 minutes, and after removing the supernatant, 0.5 mL of 5 mM Tris aqueous solution (pH 5) containing 1 wt% BSA was added to the settled sediment, and after ultrasonic dispersion, the mixture was further stirred by inversion at room temperature for 2 hours to obtain labeled antibody dispersion K-1.

[0081] (Cleaning process) Next, the labeled antibody dispersion K-1 was centrifuged at 3000 rpm for 5 minutes. After removing the supernatant, 0.5 mL of a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant was added to the settled sediment and ultrasonically dispersed. This procedure was repeated three times to complete the washing process.

[0082] (Preservation process) Next, after cooling with ice, the mixture was centrifuged at 3000 rpm for 5 minutes. After removing the supernatant, 0.5 mL of a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant and 10 wt% sucrose was added to the settled sediment and ultrasonically dispersed to obtain labeled antibody dispersion L-1.

[0083] (Conjugate pad creation) 0.12 ml of labeled antibody dispersion L-1 was centrifuged at 3000 rpm for 5 minutes, and after removing the supernatant, 0.333 ml of an aqueous solution (pH 8.0) containing 5 wt% sucrose and 2.5 wt% BSA was added to the settled sediment and ultrasonically dispersed to obtain labeled antibody dispersion M-1. Labeled antibody dispersion M-1 was uniformly impregnated into a glass fiber nonwoven fabric and dried at 50°C for 1 hour to prepare conjugate pad N-1. At this time, the volume of labeled antibody dispersion M-1 was adjusted so that the content of gold-resin composite particles F-3 in conjugate pad N-1 was 3 μg per test of evaluation by the immunochromatographic method described later.

[0084] (Preparation of immunochromatographic strips) An immunochromatographic strip with the structure shown in Figure 2 was prepared. First, an anti-CRP antibody was applied to a 25 mm wide nitrocellulose membrane 4 to create a test line 5. An anti-mouse IgG antibody was also applied downstream of test line 5 to create a control line 6. After drying the nitrocellulose membrane 4 at 50°C for 1 hour, a laminate film 1, a conjugate pad N-1 (as a conjugate pad 3), a sample pad 2 (glass fiber nonwoven fabric), and an absorbent pad 7 (cotton nonwoven fabric) were laminated as shown in the cross-sectional view of the immunochromatographic strip in Figure 2. Finally, the strip was cut to a width of 3.5 mm to create the immunochromatographic strip P-1.

[0085] (Preparation of developing solution) An aqueous solution (pH 7.1) containing 50 mM Tris, 150 mM NaCl, 1.0 wt% BSA, and 1.0 wt% PEG cetyl ether was prepared and designated as developing solution Q-1.

[0086] (Evaluation by immunochromatography) Positive control (antigen concentrations 312 ng / ml, 3.12 ng / ml) and negative control (antigen-free) sample solutions were prepared by diluting the CRP antigen with developing solvent Q-1. 50 μl of the sample solution was dropped onto sample pad 2 of immunochromatographic strip P-1. After 30 minutes, the color intensity of test line 5 was measured using an immunochromatographic reader (Hamamatsu Photonics C10066-10). Visual inspection was also performed to determine whether gold-resin composite particles F-3 were clogging the interface between conjugate pad 3 and membrane 4 and downstream of it. The results of the immunochromatographic evaluation are shown in Table 1.

[0087] (Calculation of the amount of bound antibody) The amount of antibody bound to the gold-resin composite particle F-3 was calculated using the supernatant liquid removed by centrifugation in the above blocking process. The amount of bound antibody was calculated by measuring the supernatant liquid using a spectrophotometer, determining the amount of antibody contained in the supernatant liquid from the absorbance at a wavelength of 280 nm, and subtracting the amount of antibody in the supernatant liquid from the amount of antibody used in the binding process (25 μg). The calculated amount of bound antibody and antibody binding yield are shown in Table 1. Note that the binding yield represents the weight percentage (%) of the attached antibody relative to the total amount of antibody used.

[0088] [Examples 2-5, Comparative Examples 1-5] Except for using the metal-resin composite particle dispersion and binding buffer shown in Table 1, the binding process, blocking process, washing process, storage process, conjugate pad preparation, immunochromatographic strip preparation, immunochromatographic evaluation, and calculation of bound antibody amount and antibody binding yield were performed in the same manner as in Example 1. Table 1 shows the calculation results for bound antibody amount and antibody binding yield, and the results of the immunochromatographic evaluation.

[0089] The test results from Examples 1-5 and Comparative Examples 1-5 show that using a binding buffer containing Good's buffer with a pKa of 7.5 or higher at 20°C increases the amount of bound antibody, suppresses clogging of immunochromatographic evaluation results, increases the color intensity of the test line, and improves the sensitivity of the immunochromatography.

[0090] [Table 1]

[0091] [Example 6] (Joining process) After mixing 25 μg of anti-influenza A virus antibody with 0.45 mL of 20 mM HEPES buffer (pH 7), 0.05 mL of 1 wt% gold-resin composite particle dispersion E-3 was added, and the mixture was inverted and stirred at room temperature for 2 hours to obtain labeled antibody dispersion J-6 containing anti-influenza A virus antibody labeled with gold-resin composite particles F-3.

[0092] (Blocking process, washing process, preservation process, conjugate pad preparation) Conjugate pad N-6 was prepared by performing the blocking process, washing process, storage process, and conjugate pad preparation in the same manner as in Example 1, except that labeled antibody dispersion J-6 was used.

[0093] (Preparation of immunochromatographic strips) An immunochromatographic strip with the structure shown in Figure 2 was prepared. First, an anti-influenza A virus antibody was applied to a 25 mm wide nitrocellulose membrane 4 to create a test line 5. An anti-mouse IgG antibody was also applied downstream of test line 5 to create a control line 6. After drying the nitrocellulose membrane 4 at 50°C for 1 hour, a laminate film 1, a conjugate pad N-6 (as a conjugate pad 3), a sample pad 2 (glass fiber nonwoven fabric), and an absorbent pad 7 (cotton nonwoven fabric) were laminated as shown in the cross-sectional view of the immunochromatographic strip in Figure 2. Finally, the strip was cut to a width of 3.5 mm to create the immunochromatographic strip P-6.

[0094] (Preparation of developing solution) An aqueous solution (pH 7.1) containing 50 mM Tris, 150 mM NaCl, 1.0 wt% BSA, and 1.0 wt% Triton X100 was prepared and designated as the developing solution Q-6.

[0095] (Evaluation by immunochromatography) Positive control (antigen concentration 1250 FFU / ml, equivalent to 20 FFU / ml) and negative control (antigen-free) sample solutions were prepared by diluting the influenza A virus inactivated antigen with developing solvent Q-6. 50 μl of the sample solution was dropped onto sample pad 2 of an immunochromatographic strip P-6. After 30 minutes, the color intensity of test line 5 was measured using an immunochromatographic reader (Hamamatsu Photonics C10066-10). Visual inspection was also performed to determine whether gold-resin composite particles F-3 were clogging the interface between conjugate pad 3 and membrane 4 and downstream of it. The results of the immunochromatographic evaluation are shown in Table 2.

[0096] [Comparative Example 6] Except for using 20 mM borate buffer (pH 7) instead of 20 mM HEPES buffer (pH 7) in the conjugation step of Example 6, the conjugation step, blocking step, washing treatment, storage treatment, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 6. Table 2 shows the results of the immunochromatographic evaluation.

[0097] The test results from Example 6 and Comparative Example 6 show that using a binding buffer containing Good's buffer with a pKa of 7.5 or higher at 20°C suppresses clogging in immunochromatographic evaluation results, increases the color intensity of the test lines, suppresses false positives in negative control samples without antigens, and improves the sensitivity of immunochromatography.

[0098] [Table 2]

[0099] [Example Tests 1-4] (Evaluation of the degree of aggregation by mixing binding buffer with metal-resin composite particles) After mixing 0.45 ml of the binding buffer shown in Table 3 with 0.05 ml of 1 wt% gold-resin composite particle dispersion E-3, the mixture was stirred by inversion at room temperature for 2 hours, and the particle size distribution was measured. From the particle size distribution measurement results, the percentage of gold-resin composite particles F-3 that were dispersed as single particles without agglomeration (non-aggregation ratio) was determined. The results are shown in Table 3.

[0100] From the test results of Reference Test Examples 1-4, it can be seen that in Reference Test Examples 1-3, which used a binding buffer containing Good's buffer with a pKa of 7.5 or higher at 20°C, aggregation of gold-resin composite particles F-3 was suppressed compared to Reference Test Example 4, which used a borate buffer. It is presumed that this aggregation suppression effect leads to an increase in antibody adhesion and also suppresses clogging in the immunochromatography strip, contributing to good immunochromatographic evaluation results.

[0101] [Table 3]

[0102] Although embodiments of the present invention have been described in detail above for illustrative purposes, the present invention is not limited to the above embodiments. [Explanation of Symbols]

[0103] 1…Laminate film, 2…Sample pad, 3…Conjugate pad, 4…Nitrocellulose membrane, 5…Test line, 6…Control line, 7…Absorbent pad, 10…Resin particles, 20…Metal particles, 30…Encapsulated metal particles, 40…Partially exposed metal particles, 50…Surface-adsorbed metal particles, 60…Surface layer, 100…Metal-resin composite

Claims

1. A method for producing labeled antibodies, A binding step in which an antibody and a metal-resin composite having a structure in which metal particles are immobilized on resin particles are mixed in the presence of a binding buffer in the pH range of 6 to 10 containing a Good buffer having a pKa of 7.5 or higher at 20°C, thereby attaching the antibody to the metal-resin composite. Includes, The Good buffer is one or more selected from 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 2-cyclohexylaminoethanesulfonic acid (CHES), and N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO). A method for producing labeled antibodies, characterized in that, in the binding step, the proportion of the antibody attached to the total amount of antibody used is 50% by weight or more.

2. The method for producing a labeled antibody according to claim 1, wherein the metal particles are silver, nickel, copper, gold, platinum, palladium, or an alloy containing any of these.

3. A method for producing a labeled antibody according to claim 1 or 2, wherein the metal particles include a portion embedded within the resin particles and a portion exposed outside the resin particles.

4. A method for producing a labeled antibody according to any one of claims 1 to 3, wherein at least a portion of the metal particles are distributed three-dimensionally on the surface layer of the resin particles.

5. A method for producing a labeled antibody according to any one of claims 1 to 4, wherein the resin particles are polymer particles having substituents in their structure that are capable of adsorbing metal ions.

6. A method for producing a labeled antibody according to any one of claims 1 to 5, wherein the average particle size of the metal particles is in the range of 1 to 80 nm.

7. A method for producing a labeled antibody according to claims 1 to 6, wherein the average particle size of the metal-resin composite is in the range of 100 to 1000 nm.

8. An immunoassay method characterized by using a labeled antibody produced by the method for producing a labeled antibody described in any one of claims 1 to 7.

9. An immunochromatographic method characterized by using a labeled antibody produced by the method for producing a labeled antibody described in any one of Claims 1 to 7.

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