Patent Description:
Innumerable chemical substances exist in an organism, so the qualitative and quantitative analysis of specific trace components in the organism is an extremely important technique. In fields, such as medical care, pharmacy, health food, biotechnology and environment, drugs and food having effects only on specific parts (chemical substances) in organisms, analysis devices and diagnostic drugs and the like detecting slight changes in organisms develop along with the technique.

One of the analysis techniques is immunoassay. This technique is also called immunological assay, which is a method that qualitatively and quantitatively analyzes trace components by utilizing antigen-antibody specific reaction as one of immunoreactions. Because of high sensitivity or reaction selectivity, the antigen-antibody reaction is widely applied in the fields. Various assay methods exist in immunoassay according to assay principles. Examples are listed as follows: enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), fluorescence immunoassay (FIA), agglutination of latex and so on (latex immunoassay (LIA), particle agglutination (PA)), immunochromatography (ICA), hemagglutination (HA) and hemagglutination inhibition (HI). Moreover, besides immunoassay, there are physical and chemical assays, biological assay and so on.

Immunoassay qualitatively or quantitatively assays an antigen or an antibody according to change in the reaction between the antigen and the antibody for forming the composite (concentration change of the antigen, the antibody or a composite). When these are assayed, the antibody, the antigen or the composite are bound with a marker, and thereby assay sensitivity is increased.

Therefore, it can be said that the marking capability of the marker is a significant factor affecting assay capability in immunoassay. In the foregoing illustrated immunoassays, erythrocytes (the case of HA), latex particles (the case of LIA), fluorochromes (the case of FIA), radioactive elements (the case of RIA), enzymes (the case of EIA) and chemiluminescent substances (the case of CLIA) and so on can be adopted as markers.

However, when colored microparticles are used as a marker, assay can be determined visually without using a special analysis device, so it can be expected that simpler assay may be realized. As such colored microparticles, examples are listed as follows: colloidal particles of metals and metal oxides and latex particles colored by utilizing a pigment (patent document <NUM>, patent document <NUM>, etc.).

However, because the color of the colloidal particles is determined according to particle sizes and preparation conditions, there exists a problem that it is hard to obtain a desired bright rich color, that is, visual recognizability is insufficient.

In addition, the colored latex particles have the problem of low coloring effect of the utilized pigment and insufficient visual determinability. Furthermore, if the coloring amount of the pigment is increased in order to solve the problem, then the pigment covers the surface of the latex, the original surface state of the latex particles is impaired, and as a result, there exists a problem that it is hard to bind the antigen or the antibody. In addition, there also exists the following problems: blockage in pores of a chromatographic medium such as a membrane filter, or nonspecific agglutination produced by the latex particles or uncertain association between rich coloration resulting from the increase in the coloring amount of the pigment and an increase in performance.

In order to improve the visual recognizability of the marker, following immunochromatography is disclosed: after an antibody (marked antibody) bound with a marker react with an antigen to form a composite, other metals modify the marker, and thereby the assay sensitivity of the marker is increased (patent document <NUM> and patent document <NUM>). However, in the method, a special device is needed in order to modify metal silver. As a result, operation is complex, and it is hard to achieve a stable increase. In addition, it is considered that assay cost is consumed due to the fact that a special device is required, so applicable purposes and application environment are limited.

In addition, a coloring latex containing gold nanoparticles bound with the surfaces of polymeric latex particles (patent document <NUM>) is disclosed. By binding the surfaces of the polymeric latex particles with the gold nanoparticles, the gold nanoparticles as colorant itself can help to increase visual determinability or assay sensitivity. On the other hand, the gold nanoparticles itself are excellent for the binding of an antigen or an antibody, so, even if the gold nanoparticles are bound until a degree of sufficiently rich color, an enough amount of antigen or antibody can be bound.

γ rays irradiate a dispersion of styrene-acrylic acid copolymer latex and a precursor of gold nanoparticles (i.e. HAuCl), so that the surface of the latex is bound with the gold nanoparticles, and thereby the coloring latex is formed. However, since the gold nanoparticles are only bound with the surface of the latex, not only is the quantity of carried gold particles manifesting surface plasmon absorption limited, but also the gold nanoparticles can easily come off. As a result, the visual recognizability or sensitivity of it as an immunoassay reagent may not be sufficient. In addition, because of the irradiation of electromagnetic radioactive rays such as γ rays, the latex may be injured. Further, although the description of patent document <NUM> discloses preferred ranges of latex size or gold nanoparticle size, whether it has been verified in these preferred ranges in the embodiments is not clear, so a specified basis of preferred ranges does not exist.

In addition, patent document <NUM> discloses a metal gold-coated polymer latex particle, and suggests the application of a reagent applicable to microscopic examination and immunoassay.

However, the material or particle size of the metal gold-coated polymer latex particle is not disclosed. Further, the effect of it as a reagent applicable to immunoassay has not been verified. Therefore, the effect of it as a reagent in metal gold and polymer latex particles is not clear.

In addition, patent document <NUM> discloses a nanocomposite comprising a matrix layer and metal microparticles. The matrix layer comprises solid backbone parts and voids formed by the solid backbone parts, and the metal microparticles are fixed on the solid backbone parts. Patent document <NUM> discloses compositions of a multimodal detection agent comprising a plurality of metallic nanoparticles attached to a surface of a polymeric carrier, together with a method of fabricating the same. Patent document <NUM> discloses an immunoassay test device comprising a plastic enclosure having a top section and a bottom section. The top section of the enclosure has an opening for receiving sample and an opening for visualizing test results and for visualizing a control result. Bottom section comprises a tray into which fits a capture membrane. Patent document <NUM> discloses a hapten-linker-large group conjugate for use in a kinetic-based not approaching equilibrium assay. The hapten-linker-large group conjugate is of the general formula: X - W - Y - Z, wherein: X is a hapten; W is an optional thioether or ether group; Y is a linker of <NUM> or more atoms in length; and Z is a large group of sufficient size to provide steric hindrance with respect to the binding of X to a ligand in the absence of Y.

According to what is mentioned above, although the latex particle bound or coated with the gold nanoparticle is expected as an immunoassay reagent, in the prior art, durability or visual recognizability is not sufficient. In addition, even if visual recognizability is high, applicable purposes and application environment are limited.

The present invention is directed to provide a marker which is applicable to immunoassay, has excellent sensitivity, durability and visual recognizability and can realize high-sensitivity determination without requiring the addition of a special device or operation steps.

As the result of the research effort of the inventor and the others, it is discovered that a resin-metal composite with a specific structure can be utilized to solve the problem, and thereby the present invention is achieved.

That is, the marker of the present invention is as defined in the appended claim <NUM>. Dependent claims are directed to some beneficial embodiments. The marker can be used in an immunoassay method. An immunoassay reagent is as defined in appended claim <NUM>. A measuring method is as defined in appended claim <NUM>. An analyte measurement kit is as defined in appended claim <NUM>. A lateral-flow chromatographic test strip is as defined in appended claim <NUM>.

The marker of the present invention is provided with the resin-metal composite with the structure formed by immobilizing the metal particles on the resin particle. Therefore, the quantity of the carried metal particles manifesting localized surface plasmon absorption on the resin particle is high. Therefore, the marker of the present invention as an excellent material having excellent durability and visual recognizability and capable of realizing high-sensitivity determination without requiring the addition of a special device or operation steps can be preferably applied in immunoassay, such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA and HI.

In reference to the drawings, the embodiments of the present invention are elaborated hereinafter.

A marker of the first embodiment of the present invention is provided with a resin-metal composite with a structure formed by immobilizing metal particles on a resin particle, and the average particle size of the resin-metal composite exceeds <NUM>. <FIG> is a schematic diagram of the section of the resin-metal composite composing the marker of the present embodiment. The resin-metal composite <NUM> is provided with the resin particles <NUM> and the metal particles <NUM>. For example, the marker of the present embodiment which is provided with the resin-metal composite <NUM> can be preferably used as an immunoassay reagent or a material therefor.

In the resin-metal composite <NUM>, the metal particles <NUM> are dispersed or immobilized on the resin particle <NUM>. In addition, portions of the metal particles <NUM> of the resin-metal composite <NUM> are three-dimensionally distributed in a surface layer part <NUM> of the resin particle <NUM>, moreover, portions of the three-dimensionally distributed metal particles <NUM> are partially exposed from the resin particle <NUM>, and the rest portions are encased in the resin particle <NUM>.

Here, metal particles completely encased in the resin particle <NUM> (also called "encased metal particles <NUM>" hereinafter), metal particles having a portion embedded in the resin particle <NUM> and a portion exposed from the resin particle <NUM> (also called "partially exposed metal particles <NUM>" hereinafter) and metal particles adsorbed on the surface of the resin particle <NUM> (also called "surface-adsorbed metal particles <NUM>" hereinafter) exist among the metal particles <NUM>.

When the resin-metal composite <NUM> is used as the marker, an antibody or an antigen is immobilized on the partially exposed metal particles <NUM> or the surface-adsorbed metal particles <NUM> for use. At this point, the antibody or the antigen is immobilized on the partially exposed metal particles <NUM> and the surface-adsorbed metal particles <NUM>, but, on the other hand, is not immobilized on the encased metal particles <NUM>. However, because the metal particles <NUM> including the encased metal particles <NUM> all manifest localized surface plasmon absorption, not only can the partially exposed metal particles <NUM> and the surface-adsorbed metal particles <NUM> help to increase the visual recognizability of the marker, but also the encased metal particles <NUM> can help to increase the visual recognizability of the marker. Further, compared with the surface-adsorbed metal particles <NUM>, the partially exposed metal particles <NUM> and the encased metal particles <NUM> have large contact area with the resin particle <NUM>, and, besides, can hardly come off from the resin particle <NUM> due to the high adsorption force of physics such as anchoring effect produced by the embedded state. Therefore, the durability and stability of the marker using the resin-metal composite <NUM> can become excellent.

The whole surface of each encased metal particle <NUM> is covered by resin forming the resin particle <NUM>. In addition, <NUM> percent to less than <NUM> percent of the surface area of each partially exposed metal particle <NUM> is covered by the resin forming the resin particle <NUM>. From the point of durability, the lower limit is preferably <NUM> percent or more of the surface area, more preferably <NUM> percent or more. In addition, more than <NUM> percent but less than <NUM> percent of the surface area of each surface-adsorbed metal particle <NUM> is covered by the resin forming the resin particle <NUM>.

In addition, relative to the weight of the resin-metal composite <NUM>, the quantity of the carried metal particles <NUM> (the total of the encased metal particles <NUM>, the partially exposed metal particles <NUM> and the surface-adsorbed metal particles <NUM>) of the resin-metal composite <NUM> is preferably 5wt% (percentage by weight) to 70wt%. If it is in the range, then the visual recognizability, visual determinability and assay sensitivity of the resin-metal composite <NUM> as the marker are excellent. If the quantity of the carried metal particles <NUM> is less than 5wt%, then there exists a tendency of decrease in the amount of the immobilized antibody or antigen and decrease in assay sensitivity. The quantity of the carried metal particles <NUM> is more preferably 15wt% to 70wt%.

In addition, preferably, 10wt% to 90wt% of the metal particles <NUM> are the partially exposed metal particles <NUM> and the surface-adsorbed metal particles <NUM>. If it is in the range, then the amount of the antibody or the antigen immobilized onto the metal particles <NUM> can be sufficiently guaranteed, so the sensitivity of it as the marker is high. More preferably, 20wt% to 80wt% of the metal particles <NUM> are the partially exposed metal particles <NUM> and the surface-adsorbed metal particles <NUM>, and from the point of durability, more preferably, the surface-adsorbed metal particles <NUM> are 20wt% or less.

In addition, in order to obtain excellent assay sensitivity in immunoassay, preferably 60wt% to 100wt%, preferably 75wt% to 100wt% or more preferably 85wt% to 100wt% of the metal particles <NUM> exist in the surface layer part <NUM>, and, more preferably, exist in a range of <NUM>% of particle radius in a depth direction from the surface of the resin particle <NUM>. In addition, <NUM> wt% to <NUM> wt% of the metal particles <NUM> existing in the surface layer part <NUM> are the partially exposed metal particles <NUM> or the surface-adsorbed metal particles <NUM>, sufficiently guaranteeing the amount of the antibody or the antigen immobilized onto the metal particle <NUM>, so the sensitivity of it as the marker is high as preferred. In other words, 10wt% to 95wt% of the metal particles <NUM> existing in the surface layer part <NUM> can be the encased metal particles <NUM>.

Here, the "surface layer part" means a range of <NUM>% of the particle radius in the depth direction from the surface of the resin particle <NUM> with the outermost position (i.e. the protruding ends of the partially exposed metal particles <NUM> or the surface-adsorbed metal particles <NUM>) of the resin-metal composite <NUM> as a datum. In addition, "two-dimensionally distributed" means that the metal particles <NUM> are distributed in the surface direction of the resin particle <NUM>. "three-dimensionally distributed" means that the metal particles <NUM> are distributed not only in the surface direction of the resin particle <NUM> but also in the depth direction. From the point of the metal particles <NUM> hard to come off from the resin particle <NUM> and the point of the amount of the carried metal particles <NUM> becoming larger, it is preferred that the metal particles <NUM> are "three-dimensionally distributed".

In addition, in the present embodiment, the average particle size of the resin-metal composite <NUM> exceeds <NUM>. If the average particle size of the resin-metal composite <NUM> is <NUM> or less, then there exists a tendency of decrease in the visual recognizability or sensitivity of the marker. The average particle size of the resin-metal composite <NUM> is preferably of more than <NUM> to <NUM>, more preferably of <NUM> to less than <NUM>. Here, the particle size of the resin-metal composite <NUM> means a value which is obtained by adding the particle size of the resin particle <NUM> with the length of the protruding portions of the partially exposed metal particles <NUM> or the surface-adsorbed metal particles <NUM>, and can be determined by a laser diffraction/scattering method, a dynamic light scattering method or a centrifugal precipitation method.

Preferably the resin particle <NUM> is a polymer particle which is provided with a substituent group capable of adsorbing metal ions in the structure. In particular, a nitrogenous polymer particle is preferred. Nitrogen atoms in a nitrogenous polymer can easily chemically adsorb the precursor (i.e. anionic metal ions) of a metal particle, such as gold or palladium, which has excellent visual recognizability and can easily immobilize an antigen or an antibody, so it is preferred. In the present embodiment, the metal ions adsorbed in the nitrogenous polymer are reduced, so that metal nanoparticles are formed, so portions of the produced metal particles <NUM> become the encased metal particles <NUM> or the partially exposed metal particles <NUM>. In addition, because carboxylic acid and the like can adsorb cationic metal ions like a crylic acid polymer, the precursor of a metal particle, such as silver, nickel or copper (i.e. cationic metal ions), can be easily adsorbed, so that the metal particles <NUM> of silver, nickel, copper or the like can be formed, and an alloy of a metal, such as gold or palladium, can be made.

On the other hand, in the case that it is a resin particle rather than the nitrogenous polymer having the substituent group capable of adsorbing metal ions in the structure, for example, in the case of polystyrene, the metal ions can hardly be adsorbed in resin. As a result, the majority of the produced metal particles <NUM> are the surface-adsorbed metal particles <NUM>. As mentioned above, the contact area between the surface-adsorbed metal particles <NUM> and the resin particle <NUM> is small, so there exists a tendency of low bonding force between the resin and the metal and great affection of the metal particles <NUM> coming off from the resin particle <NUM>.

The nitrogenous polymer is resin which has nitrogen ions on the main chain or the side chain, for example, polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole or polyaniline. Among these, polyamine, such as poly-<NUM>-vinylpyridine, poly-<NUM>-vinylpyridine or poly-<NUM>-vinylpyridine, is preferred. In addition, for example, when there are nitrogen atoms on the side chain, acrylic resin, phenolic resin and epoxy resin can be widely utilized.

The metal particles <NUM> can apply silver, nickel, copper, gold and palladium. Gold and palladium which have excellent visual recognizability and can easily immobilize an antigen or an antibody are preferred. These manifest absorption stemming from localized surface plasmon resonance, and therefore are preferred. Gold with good stability in storage is more preferred. These metals can be used as monomers or composites such as alloys. Here, for example, gold alloy means an alloy which contains gold and metal varieties except gold, and contains 10wt% or more of gold.

In addition, for example, the average particle size of the metal particles <NUM> relaying on scanning electron microscope (SEM) observation to determine length is preferably <NUM> to <NUM>. In the case that the average particle size of the metal particles <NUM> is less than <NUM> or exceeds <NUM>, a localized surface plasmon can hardly manifest, so there exists a tendency of decrease in sensitivity. When the metal particles <NUM> are gold particles, the average particle size of the metal particles <NUM> in the first embodiment is preferably of <NUM> to less than <NUM>, more preferably of <NUM> to less than <NUM>.

A marker of the second embodiment of the present invention is provided with a resin-metal composite with a structure formed by immobilizing metal particles on a resin particle, and the average particle size of the metal particles is in a range of more than <NUM> and less than <NUM>. Except the range of the average particle size of the metal particles and the range of the average particle size of the resin-metal composite that are different from that of the resin-metal composite <NUM> of the first embodiment (<FIG>), the resin-metal composite composing a marker of the present embodiment is the same as the resin-metal composite <NUM> of the first embodiment (<FIG>). In reference to <FIG>, the differences from the first embodiment are described as central points hereinafter.

In the resin-metal composite <NUM> used in the second embodiment, for example, the average particle size of the metal particles <NUM> depending on scanning electron microscope (SEM) observation to determine length is more than <NUM> and less than <NUM>. If the average particle size of the metal particles <NUM> is <NUM> or less, then there exists a tendency of decrease in sensitivity; and if it is <NUM> or more, then there exists a tendency of decrease in visual recognizability. More preferably, the average particle size of the metal particles <NUM> is <NUM> to less than <NUM>.

In addition, for example, the average particle size of the resin-metal composite <NUM> is preferably <NUM> to <NUM>. If the average particle size of the resin-metal composite <NUM> is less than <NUM>, then, for example, when the gold particles are used as the metal particles <NUM>, there exists a tendency that the quantity of the carried gold particles becomes less, and therefore there exists a tendency that coloring becomes weak in comparison with gold particles of the same size; and if the average particle size of the resin-metal composite <NUM> exceeds <NUM>, then there exists a tendency of easy blockage in pores of a chromatographic medium, such as a membrane filter, or a tendency of decrease in dispersibility when the resin-metal composite <NUM> is prepared into a reagent. The average particle size of the resin-metal composite <NUM> is preferably <NUM> to less than <NUM>, more preferably <NUM> to less than <NUM>. Here, the particle size of the resin-metal composite <NUM> means a value which is obtained by adding the particle size of the resin particle <NUM> with the length of the protruding portions of the partially exposed metal particles <NUM> or the surface-adsorbed metal particles <NUM>, and can be determined by a laser diffraction/scattering method, a dynamic light scattering method or a centrifugal precipitation method.

In the resin-metal composite <NUM> used in the marker of the second embodiment, the other constitution is the same as that of the resin-metal composite <NUM> used in the first embodiment, and therefore description is omitted.

Preparation methods for the resin-metal composites <NUM> used in the markers of the first embodiment and the second embodiment are not specially limited. For example, solution containing metal ions is added into dispersion of resin particles <NUM> prepared by the emulsion polymerization method, so that the metal ions are adsorbed on the resin particles <NUM> (called "metal ion-adsorbing resin particles hereinafter"). Further, the metal ion-adsorbing resin particles are added into a reducing agent solution, so that the metal ions are reduced to form metal particles <NUM>, and thereby the resin-metal composite <NUM> is obtained.

In addition, for example, when gold particles are used as the metal particles <NUM>, aqueous chloroauric acid (HAuCl4) solution can be used as the solution containing the metal ions. In addition, a metal complex can be used to substitute for the metal ions.

In addition, as a solvent for the solution containing the metal ions, aqueous alcohol or alcohol, such as methanol, ethanol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol or tert-butyl alcohol, or acid, such as hydrochloric acid, sulfuric acid or nitric acid, can be used to substitute for water.

In addition, if needed, an additive, such as various water-miscible organic solvents (such as water-soluble macromolecular compound (such as polyvinyl alcohol), surfactant, alcohols, ethers (such as tetrahydrofuran, diethyl ether and diisopropyl ether), polyhydric alcohols (such as alkylene glycol, polyalkylene glycol, their monoalkyl ether or dialkyl ether and glycerol) and ketones (such as acetone and methyl ethyl ketone)), can be added into the solution. Such additive accelerates the speed of the reduction reaction of the metal ions, and is also effective in controlling the size of the produced metal particles <NUM>.

In addition, a well-known reducing agent can be used. Examples are listed as follows: sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium hypophosphite, hydroquinone and Rochelle salt. Sodium borohydride, dimethylamine borane or citric acid is preferred. If needed, surfactant can be added into the reducing agent solution, or the pH of the solution is regulated. The pH can be regulated by a buffering agent (such as boric acid or phosphoric acid), acid (such as hydrochloric acid or sulfuric acid) or alkali (such as sodium hydroxide or potassium hydroxide).

Further, the reduction speed of the metal ions is regulated by the temperature of the reducing agent solution, so that the particle size of the formed metal particles can be controlled.

In addition, when the metal ions in the metal ion-adsorbing resin particles are reduced to produce the metal particles <NUM>, the metal ion-adsorbing resin particles can be added into the reducing agent solution, or the reducing agent can be added into the metal ion-adsorbing resin particles, nevertheless, from the point of the easiness of the production of encased metal particles <NUM> and partially exposed metal particles <NUM>, the former is preferred.

In addition, in order to keep the dispersibility of the resin-metal composite <NUM> in water, for example, a dispersing agent, such as citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, disperbyk <NUM>, disperbyk <NUM> or disperbyk <NUM> (produced by BYK-Chemie Japan), can be added.

Further, pH can be regulated by a buffering agent (such as boric acid or phosphoric acid), acid (such as hydrochloric acid or sulfuric acid) or alkali (such as sodium hydroxide or potassium hydroxide), and dispersibility is kept.

Particularly, by adsorbing the antigen or antibody on the surfaces of the metal particles <NUM>, the resin-metal composite <NUM> with the above-mentioned composition is preferably applicable as a marker to immunoassay, such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA and HI. In addition, it can be particularly used as a marker with excellent visual determinability in low-concentration regions (high-sensitivity regions). In addition, the form of the marker is not specially limited, for example, it can be used as dispersion which is formed by dispersing the resin-metal composite <NUM> into water or a pH-regulated buffer.

Methods for adsorbing the antigen or antibody onto the surface of the metal particles <NUM> are not specially limited, and the well-known physical adsorption and chemical adsorption methods can be utilized. Examples are listed as follows: physical adsorption, such as immersing the resin-metal composite <NUM> into a buffer containing the antigen or antibody for incubation, or chemical adsorption, such as introducing an SH group into the antigen or antibody to react with the resin-metal composite <NUM> to form an Au-SH bond. In order to make the bonding between the metal particles <NUM> and the antigen or antibody become firm, chemical adsorption is preferred.

A method for measuring analyte, a lateral-flow chromatographic test strip and an analyte assay and quantification kit which adopt the resin-metal composite <NUM> as a marker are then described.

Firstly, in reference to <FIG>, a lateral-flow chromatographic test strip (test strip) in an embodiment of the present invention is described. As mentioned hereinafter, the test strip <NUM> can be preferably used in a method for measuring analyte in an embodiment of the present invention.

The test strip <NUM> is provided with a membrane <NUM>. In the membrane <NUM>, a sample addition portion <NUM>, a determination portion <NUM> and a liquid-absorbing portion <NUM> are sequentially arranged in the spreading direction of a sample.

A material which is used as a membrane in ordinary test strips can be applied as the membrane <NUM> used in the test strip <NUM>. For example, the membrane <NUM> is formed by an insert material including the following microporous materials (substances not reacting with an analyte <NUM> and various ligands, etc), the microporous substances show capillarity, and when added, the sample spreads. Specific examples as the membrane <NUM> are listed as follows: fibrous or non-woven fibrous matrices and films containing polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon and cellulose derivative, etc., filter paper, glass fiber filter paper, cloth and cotton. Among these, preferably, a film containing cellulose derivatives or nylon, filter paper or glass fiber filter paper can be used, or more preferably, a cellulose nitrate film, a mixed nitrocellulose ester (a mixture of cellulose nitrate and cellulose acetate) film, a nylon film or filter paper can be used.

In order to make operation easier, the test strip <NUM> is preferably a supporting body with the supporting membrane <NUM>. For example, as the supporting body, plastic can be used.

The test strip <NUM> can also be provided with the sample addition portion <NUM> for the addition of a sample containing the analyte <NUM>. In the test strip <NUM>, the sample addition portion <NUM> is a portion for receiving the sample containing the analyte <NUM>. In the spreading direction of the sample, the sample addition portion <NUM> can be formed on the membrane <NUM> further upstream than the determination portion <NUM>, or a sample addition pad containing a material, such as cellulose filter paper, glass fibers, polyurethane, polyacetate, cellulose acetate, nylon or cotton, can be arranged on the membrane <NUM> to form the sample addition portion <NUM>.

Capturing ligands <NUM> which are specifically bound with the analyte <NUM> are immobilized in the determination portion <NUM>. As long as the capturing ligands <NUM> are specifically bound with the analyte <NUM>, there is no special limitation in use, for example, an antibody for the analyte <NUM> can be preferably used. Even if the sample is supplied to the test strip <NUM>, the capturing ligands <NUM> are immobilized in a manner of not moving from the determination portion <NUM>. The capturing ligands <NUM> only need to be directly or indirectly immobilized on the membrane <NUM> by physical bonding or chemical bonding or adsorption.

In addition, as long as the determination portion <NUM> is composed like a composite <NUM> containing a marked antibody <NUM> and the analyte <NUM> in contact with the capturing ligands <NUM> specifically bound with the analyte <NUM>, there is no special limitation. For example, the capturing ligands <NUM> can be directly immobilized on the membrane <NUM>, or the capturing ligands <NUM> can be immobilized on a pad containing cellulose filter paper, glass fibers or non-woven cloth fixed on the membrane <NUM>.

For example, the liquid-absorbing portion <NUM> can be formed by a pad of water-absorbing material such as cellulose filter paper, non-woven cloth, cloth and cellulose acetate. The moving speed of the sample after the front line of spreading of the added sample arrives at the liquid-absorbing portion <NUM> is different according to the materials and sizes of the liquid-absorbing portion <NUM>. Therefore, by choosing the material and size of the liquid-absorbing portion <NUM>, the most suitable speed can be set for the assay and quantification of the analyte <NUM>. Moreover, the composition of the liquid-absorbing portion <NUM> is optional, and therefore can be omitted.

If needed, the test strip <NUM> can also comprise any portions, such as a reaction portion and a control portion.

Although graphical representation is omitted, in the test strip <NUM>, the reaction portion containing the marked antibody <NUM> can be formed on the membrane <NUM>. In the flowing direction of the sample, the reaction portion can be arranged further upstream than the determination portion <NUM>. Furthermore, the sample addition portion <NUM> in <FIG> can be used as the reaction portion. When the test strip <NUM> has the reaction portion, if the sample containing the analyte <NUM> is supplied to the reaction portion or the sample addition portion <NUM>, then, in the reaction portion, the analyte <NUM> contained in the sample can be in contact with the marked antibody <NUM>. In this case, the sample is only supplied to the reaction portion or the sample addition portion <NUM>, so that the composite <NUM> containing the analyte <NUM> and the marked antibody <NUM> can be formed, so a so-called one-step immunochromatography can be implemented.

As long as the reaction portion contains the marked antibody <NUM> specifically bound with the analyte <NUM>, there is no special limitation, and the reaction portion can be formed by directly applying the marked antibody <NUM> on the membrane <NUM>. Or the reaction portion can also be formed by immobilizing a pad (conjugate pad) on the membrane <NUM>, wherein the pad containing cellulose filter paper, glass fibers or non-woven cloth and the marked antibody <NUM> is impregnated in the pad.

Although graphical representation is omitted, the control portion can also be formed on the test strip <NUM> in the spreading direction of the sample by immobilizing the capturing ligands specifically bound with the marked antibody <NUM> on the membrane <NUM>. By determining colored intensity in the determination portion <NUM> and the control portion together, it can be determined that the sample supplied to the test strip <NUM> arrives at the reaction portion and the determination portion <NUM> after spreading, and examination can be normally carried out. Moreover, except using other types of capturing ligands specifically bound with the marked antibody <NUM> to replace the capturing ligands <NUM>, the control portion can be made in the same way as the determination portion <NUM>, and can adopt the same composition.

A method for assaying the analyte <NUM> using the test strip <NUM> in an embodiment of the present invention is then described.

The method for assaying the analyte <NUM> in the present invention is a method for assaying the analyte <NUM> which can assay or quantify the analyte <NUM> contained in the sample. The method for assaying the analyte <NUM> in the present invention can use the test strip <NUM> comprising the membrane <NUM> and the determination portion <NUM> formed by immobilizing the capturing ligands <NUM> specifically bound with the analyte <NUM> on the membrane <NUM>, and comprises Step (I) to Step (III) below:.

Step (I):
Step (I) is a step of making the analyte <NUM> contained in the sample contact with the marked antibody <NUM>. As long as the composite <NUM> containing the analyte <NUM> and the marked antibody <NUM> is formed, there is no special limitation in the form of contact. For example, the sample can be supplied to the sample addition portion <NUM> or reaction portion (graphical representation omitted) of the test strip <NUM> and the analyte <NUM> is made contact with the marked antibody <NUM> in the reaction portion, or, before the sample is supplied to the test strip <NUM>, the analyte <NUM> in the sample can be made contact with the marked antibody <NUM>.

The composite <NUM> formed in Step (I) is moved after spreading on the test strip <NUM>, and arrives at the determination portion <NUM>.

Step (II):
Step (II) is a step of making the composite <NUM> containing the analyte <NUM> and the marked antibody <NUM> formed in Step (I) contact with the capturing ligands <NUM> in the determination portion <NUM> of the test strip <NUM>. If the composite <NUM> is made contact with the capturing ligands <NUM>, then the capturing ligands <NUM> are specifically bound with the analyte <NUM> of the composite <NUM>. As a result, the composite <NUM> is captured in the determination portion <NUM>.

Furthermore, because the capturing ligands <NUM> are not specifically bound with the marked antibody <NUM>, when the marked antibody <NUM> not bound with the analyte <NUM> arrives at the determination portion <NUM>, the marked antibody <NUM> not bound with the analyte <NUM> passes through the determination portion <NUM>. Here, when the control portion (graphical representation omitted) on which other capturing ligands specifically bound with the marked antibody <NUM> are immobilized is formed in the test strip <NUM>, the marked antibody <NUM> which has passed through the determination portion <NUM> continues to spread, and is bound with the other capturing ligands in the control portion. As a result, the marked antibody <NUM> which does not form the composite <NUM> along with the analyte <NUM> is captured in the control portion.

After Step (II), if needed, before Step (III), for example, a cleaning step of cleaning the test strip <NUM> by utilizing a buffer commonly used in biochemical examination, such as water, normal saline or phosphate buffer, can be implemented. By means of the cleaning step, the marked antibody <NUM> (marked antibody <NUM> not bound with the analyte <NUM> to form the composite <NUM>) which is not captured in the determination portion <NUM> or the determination portion <NUM> and the control portion can be removed.

By implementing the cleaning step, when coloration caused by the localized surface plasmon resonance of the resin-metal composite <NUM> in the determination portion <NUM> or the determination portion <NUM> and the control portion is determined in Step (III), the colored intensity of the background can be decreased, the signal/background ratio can be increased, and assay sensitivity or quantifiability can be further increased.

Step (III):
Step (III) is a step of determining colored intensity derived from the localized surface plasmon resonance of the resin-metal composite <NUM>. After Step (II) or the cleaning step, if needed, is implemented, in the test strip <NUM>, colored intensity derived from the localized surface plasmon resonance of the resin-metal composite <NUM> is determined.

Moreover, when the control portion is formed in the test strip <NUM>, by means of Step (II), in the control portion, the marked antibody <NUM> is captured by the other capturing ligands to form the composite. Therefore, in Step (III), in the test strip <NUM>, not only can coloration caused by localized surface plasmon resonance be generated in the determination portion <NUM>, but also coloration caused by localized surface plasmon resonance be generated in the control portion. Thus, by determining colored intensity in the determination portion <NUM> and the control portion together, whether the sample supplied to the test strip <NUM> arrives at the reaction portion and the determination portion <NUM> after spreading normally can be determined.

As long as the sample in the method for measuring analyte in the present embodiment contains a substance capable of becoming an antigen, such as protein, as the analyte <NUM>, there is no special limitation. Examples are listed as follows: an organism sample (i.e. whole blood, serum, plasma, urine, saliva, phlegm, rhinal swab fluid or pharyngeal swab fluid, spinal fluid, amniotic fluid, nipple secretion, tear, sweat, extract coming from skin, extract coming from tissues or cells and feces, etc.) containing the target analyte <NUM> or extract of food. If needed, in order to easily cause the specific binding reaction between both the marked antibody <NUM> and the capturing ligands <NUM> and the analyte <NUM>, before Step (I), the analyte <NUM> contained in the sample is pretreated. Here, as pretreatment, chemical treatment utilizing various chemicals (such as acid, alkali and surfactant) or physical treatment utilizing heating, agitation and ultrasonic waves can be adopted. In particular, when the analyte <NUM> is a substance normally not exposed to the surface, such as an influenza virus NP antigen, surfactant is preferably utilized for treatment. As the surfactant for this purpose, non-ionic surfactant can be used in consideration of specific binding reaction, such as binding reactivity between ligands for antigen-antibody reaction and the analyte <NUM>.

In addition, the sample can be properly diluted by a solvent (such as water, normal saline or buffer) or a water-miscible organic solvent normally used in immunological analysis.

As the analyte <NUM>, examples are listed as follows: proteins such as tumor pointers, signal transmission substances and hormone (including polypeptide, oligopeptide and the like), nucleic acids (including single-stranded or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and polynucleotide, oligonucleotide, peptide nucleic acid (PNA) and the like) or substances with nucleic acid, saccharides (including oligosaccharide, polysaccharides, carbohydrate chain and the like) or substances with carbohydrate chains, and other molecules such as lipid. As long as it can be specifically bound with the marked antibody <NUM> and the capturing ligands <NUM>, there is no special limitation. Examples are listed as follows: carcino-embryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-<NUM>, α-fetoprotein (AFP), immunosuppressive acidic protein (IAP), CA15-<NUM>, CA125, estrogen receptor, luteal hormone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotrophin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, influenza virus, human hemoglobin, chlamydia antigen, group A β hemolytic streptococcus antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin and glycated albumin. Among these, antigen which can be dissolved by non-ionic surfactant is preferred, and antigen which is formed from self-aggregate like the nucleoprotein of virus is more preferred.

The marked antibody <NUM> is used in Step (I) to contact with the analyte <NUM> contained in the sample, so that the composite <NUM> containing the analyte <NUM> and the marked antibody <NUM> is formed. The marked antibody <NUM> is formed by utilizing the resin-metal composite <NUM> having the structure formed by immobilizing the plurality of metal particles <NUM> on the resin particle <NUM> to mark an antibody specifically bound with the analyte <NUM>. Here, the so-called "marking" means directly or indirectly immobilizing the resin-metal composite <NUM> onto the antibody by chemical binding or physical binding or adsorption on the basis of the degree of the resin-metal composite <NUM> not coming off from the marked antibody <NUM> in Step (I) to Step (III). For example, the marked antibody <NUM> can be formed by directly binding the antibody with the resin-metal composite <NUM>, or can be formed by binding the antibody with the resin-metal composite <NUM> through any tie molecules or respectively immobilizing them on insoluble particles.

In addition, in the present embodiment, there is no special limitation on "antibody", for example, besides polyclonal antibodies, monoclonal antibodies and antibodies obtained by genetic recombination, antibody fragments (such as H chain, L chain, Fab and F(ab')<NUM>) capable of being bound with antigens can be used. In addition, immune globulin can be any one of IgG, IgM, IgA, IgE and IgD. Animal species producing antibodies can be the human being and animals (e.g. mouse, rat, rabbit, goat and horse) except the human being. Specific examples as the antibody are listed as follows: anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-HCV antibody, anti-IgG antibody and anti-human IgE antibody.

A preferred preparation method for the marked antibody <NUM> is then described. The preparation of the marked antibody <NUM> can at least comprise Step A below:.

In Step A, under the first pH condition, the resin-metal composite <NUM> and the antibody are mixed, so that the marked antibody <NUM> is obtained. Preferably, in Step A, the solid resin-metal composite <NUM> contacts with the antibody in the state of being dispersed in the liquid phase. The first pH condition is different according to the metal varieties of the metal particles <NUM> in the resin-metal composite <NUM>.

When the metal particles <NUM> of the resin-metal composite <NUM> are gold particles (including gold alloy particles; the same is true hereinafter), from the point of making the resin-metal composite <NUM> uniformly contact with the antibody under the state of maintaining the dispersion of the resin-metal composite <NUM> and the activity of the antibody in order to be bound with the antibody, the first pH condition is preferably a condition of pH in a range from <NUM> to <NUM>, and further, an acidic condition is more preferred, for example, pH is in a range from <NUM> to <NUM>. When the metal particles <NUM> are gold particles, if the condition in binding the resin-metal composite <NUM> with the antibody is pH less than <NUM>, then there will exist a situation that the antibody is deteriorated and inactivated due to strong acidity; and if pH exceeds <NUM>, then the resin-metal composite <NUM> will be aggregated and hardly dispersed when mixed with the antibody. However, when the antibody is not inactivated due to strong acidity, even if pH is less than <NUM>, treatment can still be carried out.

In addition, when the metal particles <NUM> of the resin-metal composite <NUM> are particles rather than gold particles, e.g. palladium particles or their alloys, from the point of making the resin-metal composite <NUM> uniformly contact with the antibody under the state of maintaining the dispersion of the resin-metal composite <NUM> and the activity of the antibody in order to be bound with the antibody, the first pH condition is preferably a condition of pH in a range from <NUM> to <NUM>, and more preferably, for example, pH is in a range from <NUM> to <NUM>. When the metal particles <NUM> are particles rather than gold particles, if the condition in binding the resin-metal composite <NUM> with the antibody is pH less than <NUM>, then there will exist a situation that the antibody is deteriorated and inactivated due to strong acidity; and if pH exceeds <NUM>, then the resin-metal composite <NUM> will be aggregated and hardly dispersed when mixed with the antibody. However, when the antibody is not inactivated due to strong acidity, even if pH is less than <NUM>, treatment can still be carried out.

Preferably, Step A is carried out in binding buffer regulated to the first pH condition. For example, a specified amount of resin-metal composite <NUM> is mixed in the binding buffer regulated to the pH, and is sufficiently mixed. For example, as the binding buffer, boric acid solution regulated to specified concentration can be used. For example, the pH of the binding buffer can be regulated by using hydrochloric acid, sodium hydroxide, etc..

Afterwards, a specified amount of antibody is added into the obtained mixture, and is sufficiently agitated and mixed, and thereby a marked antibody-containing solution can be obtained. For example, only the marked antibody <NUM> as solid part is separated out from the marked antibody-containing solution obtained in this way by a solid-liquid separation method, such as centrifugal separation.

In Step B, under the second pH condition, the marked antibody <NUM> obtained in Step A is treated in order to carry out blockage of inhibiting the non-specific adsorption of the marked antibody <NUM>. In this case, under the second pH condition, the marked antibody <NUM> which is separated out by the solid-liquid separation method is dispersed in the liquid phase. The condition of blockage is different according to the metal varieties of the metal particles <NUM> in the resin-metal composite <NUM>.

When the metal particles <NUM> of the resin-metal composite <NUM> are gold particles, from the point of keeping the activity of the antibody and inhibiting the aggregation of the marked antibody <NUM>, for example, the second pH condition is preferably pH in a range from <NUM> to <NUM>, and further, from the point of inhibiting the non-specific adsorption of the marked antibody <NUM>, an acidic condition is more preferred, for example, pH is in a range from <NUM> to <NUM>. If the condition of blockage is pH less than <NUM>, then there will exist a situation that the antibody is deteriorated and inactivated due to strong acidity; and if pH exceeds <NUM>, then the marked antibody <NUM> will be aggregated and hardly dispersed.

In addition, when the metal particles <NUM> of the resin-metal composite <NUM> are particles rather than gold particles, from the point of keeping the activity of the antibody and inhibiting the aggregation of the marked antibody <NUM>, for example, the second pH condition is preferably pH in a range from <NUM> to <NUM>, and from the point of inhibiting the non-specific adsorption of the marked antibody <NUM>, more preferably, pH is in a range from <NUM> to <NUM>. If the condition of blockage is pH less than <NUM>, then there will exist a situation that the antibody is deteriorated and inactivated due to strong acidity; and if pH exceeds <NUM>, then the marked antibody <NUM> will be aggregated and hardly dispersed.

Preferably, Step B is carried out by using blocking buffer regulated to the second pH condition. For example, the blocking buffer regulated to the pH is added into a specified amount of marked antibody <NUM>, and the marked antibody <NUM> is uniformly dispersed into the blocking buffer. For example, preferably, as the blocking buffer, a solution of protein not bound with an assayed substance is used. As the protein capable of being used in the blocking buffer, examples are listed as follows: bovine serum albumin, ovalbumin, casein and gelatin. More specifically, preferably, a bovine serum albumin solution regulated to specified concentration is used. For example, the pH of the blocking buffer can be regulated by using hydrochloric acid, sodium hydroxide, etc. Preferably, the marked antibody <NUM> can be dispersed by using a dispersion method, such as ultrasonic treatment. Dispersion in which the marked antibody <NUM> is uniformly dispersed is obtained in this way.

Dispersion of the marked antibody <NUM> can be obtained in this way. For example, only the marked antibody <NUM> as solid part is separated out from the dispersion by a solid-liquid separation method, such as centrifugal separation. In addition, if needed, cleaning treatment, storage treatment and the like can be implemented. Cleaning treatment and storage treatment are described hereinafter.

Cleaning treatment adds cleaning buffer into the marked antibody <NUM> separated out by the solid-liquid separation method, and uniformly disperses the marked antibody <NUM> into the cleaning buffer. For example, preferably, a dispersion method, such as ultrasonic treatment, is used for dispersion. There is no limitation on the cleaning buffer, for example, Tris(hydroxymethyl)aminomethane buffer, glycin amide buffer or arginine buffer of specified concentration regulated to pH in a range from <NUM> to <NUM> can be used. For example, the pH of the cleaning buffer can be regulated by using hydrochloric acid, sodium hydroxide, etc. If needed, the cleaning treatment of the marked antibody <NUM> can be repeated multiple times.

During storage treatment, storage buffer is added into the marked antibody <NUM> separated out by the solid-liquid separation method, and the marked antibody <NUM> is uniformly dispersed into the storage buffer. For example, preferably, a dispersion method, such as ultrasonic treatment, is used for dispersion. For example, as the storage buffer, a solution which is prepared by adding anticoagulant and/or stabilizer of specified concentration into the cleaning buffer can be used. For example, as the anticoagulant, saccharides represented by sucrose, maltose, lactose and trehalose or polyhydric alcohols represented by glycerol and polyvinyl alcohol can be used. There is no limitation on the stabilizer, for example, protein, such as bovine serum albumin, ovalbumin, casein or gelatin, can be used. The storage treatment of the marked antibody <NUM> can be carried out in this way.

In each above-mentioned step, if needed, surfactant or preservative, such as sodium azide or paraben, can be used,.

The analyte measurement kit in an embodiment of the present invention is a kit which is used to assay or quantify the analyte <NUM> contained in the sample by using the test strip <NUM> for lateral flow chromatography according to the method for measuring analyte in the present embodiment.

The kit of the present embodiment comprises:.

When the kit of the present invention is in use, after Step (I) is implemented by making the analyte <NUM> in the sample contact with the marked antibody <NUM> in the assay reagent, the sample is supplied to the reaction portion or sample addition portion <NUM> of the test strip <NUM>, and Step (II) and Step (III) are then sequentially implemented. Or the assay reagent can be applied further upstream than the determination portion <NUM> of the test strip <NUM>, it is suitable to add the sample on the formed reaction portion or place further upstream than the reaction portion (e.g. the sample addition portion <NUM>) after the reaction portion is formed by drying, and Step (I) to Step (III) are then sequentially implemented.

The present invention is then described in detail in reference to embodiments, however, the present invention is not limited by these embodiments. In the following embodiments and comparative examples, various determinations and evaluations are based on the following way, unless otherwise indicated.

With regard to the absorbance of the resin-metal composite, resin-metal composite dispersion (dispersion medium: water) prepared to <NUM>. 01wt% is added into an optical whiteboard glass unit (the optical path length is <NUM>), and an instant multi-metering system (produced by Otsuka Electronics Co. , MCPD-<NUM>) is used to determine absorbance of <NUM> in the case of gold. In the case of gold, absorbance in <NUM> which is <NUM> or more is set as ∘ (Good), absorbance in <NUM> which is <NUM> to less than <NUM> is set as △ (Acceptable), and absorbance in <NUM> which is less than <NUM> is set as × (Unacceptable).

<NUM> of dispersion before concentration regulation is added into a magnetic crucible, and is treated by heat under <NUM> for <NUM> hours. Weights before and after heat treatment are determined, and solid component concentration is worked out by the following formula.

In addition, under <NUM>, the sample treated by heat is further treated by heat for <NUM> hours, weights before and after heat treatment are determined, and the carried metal amount is worked out by the following formula.

A disc centrifuge type particle size distribution determination device (CPS Disc Centrifuge DC24000 UHR, produced by CPS instruments, Inc. ) is used for determination. Determination is carried out in the state of the resin-metal composite dispersed in water.

Determination utilizing immunochromatography shown hereinafter is carried out by using resin-metal composite-marked antibody dispersion prepared in each embodiment, and the performance of the resin-metal composite dispersion is evaluated.

Evaluation is carried out by using a monochrome screen for influenza A evaluation (produced by Adtec company), and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are compared. In performance evaluation, two-fold dilution (<NUM> to <NUM> folds) of an influenza A positive control (APC) (the concentration of virus before APC dilution is 5000FFU/ml) is used as an antigen.

3µl of resin-metal composite-marked antibody dispersion is added into each well of a <NUM>-well plate, and 100µl of two-fold dilution (<NUM> to <NUM> folds) of the APC and 100µl of negative control are mixed. Afterwards, 50µl is added into the monochrome screen for influenza A evaluation, and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are evaluated. A color sample for colloidal gold determination (produced by Adtec company) is used to determine the coloration levels.

The determination of the average particle size of the metal particles means the determination of the surface mean diameter of the metal particles according to an image of a substrate produced by dripping the resin-metal composite dispersion into metallic meshes with carbon supporting membranes observed by a field emission scanning electron microscope (FE-SEM; produced by Hitachi High-Technologies, SU-<NUM>).

After Aliquat <NUM> [produced by Aldrich company] (<NUM>) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, <NUM>) are dissolved into <NUM> of pure water, <NUM>-vinylpyridine (<NUM>-VP, <NUM>) and divinyl benzene (DVB, <NUM>) are added, and under nitrogen flow, agitation is performed at 250rpm under <NUM> for <NUM> minutes. After agitation, <NUM>,<NUM>'-azobis(<NUM>-methylpropionamidine) dihydrochloride (AIBA, <NUM>) dissolved in <NUM> of pure water is dripped for <NUM> minutes, and is agitated at 250rpm under <NUM> for <NUM> hours, and thereby resin particles, the average particle size of which is <NUM>, are obtained. The resin particles are precipitated by centrifugal separation (9000rpm, <NUM> minutes), supernatant is removed, the resin particles are then dispersed in pure water again, and thereby 10wt% resin particle dispersion is obtained.

<NUM> aqueous chloroauric acid solution (<NUM>) is added into the resin particle dispersion (<NUM>), and is left alone under room temperature for <NUM> hours. Afterwards, the resin particles are precipitated by centrifugal separation (3000rpm, <NUM> minutes), supernatant is removed, so that redundant chloroauric acid is removed, the resin particles are then dispersed into <NUM> of pure water again, and thereby gold ion-adsorbing resin particle dispersion is prepared. After the gold ion-adsorbing resin particle dispersion (<NUM>) is dripped into <NUM> aqueous dimethylamine borane solution (<NUM>) for <NUM> minutes, agitation is performed under room temperature for <NUM> hours, and thereby a resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3000rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is then dispersed into <NUM> of pure water again, an ultrafiltration membrane is used for refining, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the resin-metal composite in the resin-gold composite dispersion determined according to the method is <NUM>. In addition, the average particle size of the gold particles in the resin-metal composite is <NUM>, and the amount of carried gold is <NUM>. The scanning electron microscope (SEM) picture of the prepared resin-gold composite is shown in <FIG>.

Besides adding <NUM> aqueous chloroauric acid solution (<NUM>) into the resin particle dispersion (<NUM>) obtained in embodiment <NUM>, by the same method as embodiment <NUM>, gold ion-adsorbing resin particle dispersion, a resin-gold composite (average particle size: <NUM>) and 1wt% resin-gold composite dispersion are obtained. The absorbance of the resin-gold composite in the resin-gold composite dispersion is <NUM>. In addition, the average particle size of the gold particles in the resin-metal composite is <NUM>, and the amount of carried gold is <NUM>.

<NUM>-VP (<NUM>) and DVB (<NUM>) are added into <NUM> of pure water, and under nitrogen flow, agitation is performed at 250rpm under <NUM> for <NUM> minutes. After <NUM> minutes of agitation, AIBA (<NUM>) dissolved in <NUM> of pure water is dripped for <NUM> minutes, agitation is performed at 250rpm for <NUM> hours, and thereby resin particles, the average particle size of which is <NUM>, are obtained. The resin particles are precipitated by centrifugal separation (9000rpm, <NUM> minutes), supernatant is removed, the resin particles are then dispersed in pure water again, and thereby 10wt% resin particle dispersion is obtained.

<NUM> aqueous chloroauric acid solution (<NUM>) is added into the resin particle dispersion (<NUM>), and is left alone under room temperature for <NUM> hours. Afterwards, the resin particles are precipitated by centrifugal separation (3000rpm, <NUM> minutes), supernatant is removed, so that redundant chloroauric acid is removed, the resin particles are then dispersed into <NUM> of pure water again, and thereby gold ion-adsorbing resin particle dispersion is prepared. After the gold ion-adsorbing resin particle dispersion (<NUM>) is dripped into <NUM> aqueous dimethylamine borane solution (<NUM>) for <NUM> minutes, agitation is performed under room temperature for <NUM> hours, and thereby a resin-gold composite, the average particle size of which is <NUM>, is obtained. After 10wt% dispersion (BYK194) (600µl) is added into the resin-gold composite and agitated for <NUM> hour, precipitation is performed by centrifugal separation (9000rpm, <NUM> minutes), and supernatant is removed. Afterwards, an appropriate amount of pure water is added for dispersion again, an ultrafiltration membrane is used for refining, and thereby 1wt% resin-gold composite dispersion is obtained. The absorbance of the resin-gold composite in the resin-gold composite dispersion is <NUM>. In addition, the average particle size of the gold particles in the resin-metal composite is <NUM>, and the amount of carried gold is <NUM>.

100µg of influenza antibody is mixed into <NUM> (<NUM>. 1wt%) of coloring latex (produced by Merck Millipore company, coloring Estapor functional particle, K1030, average particle size: <NUM>, absorbance in <NUM>: <NUM>, absorbance in <NUM>: <NUM>), and is agitated under room temperature for about <NUM> hours, so that the coloring latex and the antibody are bound. Bovine serum albumin solution is added until final concentration is changed into <NUM>%, and is agitated under room temperature for <NUM> hours to block the coloring latex. A coloring latex-marked antibody is prepared by recovery after <NUM> minutes of centrifugal separation at 12000rpm under <NUM> and suspension in buffer containing <NUM>% of bovine serum albumin.

The prepared coloring latex-marked antibody is used to carry out determination utilizing immunochromatography shown below, and the performance of the coloring latex is evaluated.

Evaluation is carried out by using the monochrome screen for influenza A evaluation (produced by Adtec company), and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are compared. In performance evaluation, two-fold dilution (<NUM> to <NUM> folds) of an influenza A positive control (APC) (the concentration of virus before APC dilution is 5000FFU/ml) is used as an antigen.

3µl of coloring latex-marked antibody is added into each well of the <NUM>-well plate, and 100µl of two-fold dilution (<NUM> to <NUM> folds) of the APC and 100µl of negative control are mixed. Afterwards, 50µl is added into the monochrome screen for influenza A evaluation, and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are evaluated. The result is represented below.

According to Table 1A, it can be determined that the coloring latex-marked antibody shows good coloration for the antigen diluted <NUM> folds.

The results of absorbance of the above-mentioned embodiments and comparative examples are gathered and shown in Table 1B.

<NUM> of <NUM> aqueous chloroauric acid solution is added into a <NUM> three-neck round-bottomed flask, a heating reflux device is used to boil the aqueous chloroauric acid solution as the aqueous chloroauric acid solution is violently agitated, <NUM> of <NUM> aqueous sodium citrate solution is added after boiling, and whether the solution is changed from light yellow to deep red is determined. After continuing to be heated for <NUM> minutes as the solution is agitated, the solution is agitated under room temperature for about <NUM> minutes and left alone to be cooled. A membrane filter, the pore diameter of which is <NUM>, is used to filter the solution, and the solution is transferred into a conical flask and stored in the shade. The average particle size of the prepared particles is <NUM>.

100µg of influenza antibody is mixed into <NUM> of obtained colloidal gold (OD=<NUM>), and is agitated under room temperature for about <NUM> hours, so that the colloidal gold is bound with the antibody. Bovine serum albumin solution is added until final concentration is changed into <NUM>%, and is agitated under room temperature for <NUM> hours to block the surface of the colloidal gold. A colloidal gold-marked antibody is prepared by recovery after <NUM> minutes of centrifugal separation at 12000rpm under <NUM> and suspension in buffer containing <NUM>% of bovine serum albumin.

The prepared colloidal gold-marked antibody is used to carry out determination utilizing immunochromatography shown below, and the performance of the colloidal gold is evaluated.

3µl of colloidal gold-marked antibody is added into each well of the <NUM>-well plate, and 100µl of two-fold dilution (<NUM> to <NUM> folds) of the APC and 100µl of negative control are mixed. Afterwards, 50µl is added into the monochrome screen for influenza A evaluation, and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are evaluated. The result is represented below.

According to Table <NUM>, it can be determined that the colloidal gold-marked antibody shows good coloration for the antigen diluted <NUM> folds.

After Aliquat <NUM> [produced by Aldrich company] (<NUM>) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, <NUM>) are dissolved into <NUM> of pure water, <NUM>-vinylpyridine (<NUM>-VP, <NUM>) and divinyl benzene (DVB, <NUM>) are added, and under nitrogen flow, agitation is performed at 150rpm under <NUM> for <NUM> minutes, and is then performed under <NUM> for <NUM> minutes. After agitation, <NUM>,<NUM>'-azobis(<NUM>-methylpropionamidine) dihydrochloride (AIBA, <NUM>) dissolved in <NUM> of pure water is dripped for <NUM> minutes, and is agitated at 150rpm under <NUM> for <NUM> hours, and thereby resin particles, the average particle size of which is <NUM>, are obtained. The resin particles are precipitated by centrifugal separation (9000rpm, <NUM> minutes), supernatant is removed, the resin particles are dispersed into pure water again, and after this operation is performed three times, impurities are removed by dialysis. Afterwards, concentration is regulated, and thereby 10wt% resin particle dispersion is obtained.

After <NUM> of pure water is added into the resin beads (<NUM>), <NUM> aqueous chloroauric acid solution (<NUM>) is added, and the resin beads are kept under room temperature for <NUM> hours. Afterwards, the resin particles are precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, this operation is repeated three times, and thereby redundant chloroauric acid is removed. Afterwards, concentration is regulated, and thereby <NUM>. 5wt% gold ion-adsorbing resin particle dispersion is prepared.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, the mixed solution of <NUM> aqueous dimethylamine borane solution (<NUM>) and <NUM> aqueous boric acid solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby a resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>.

100µg of influenza antibody is mixed into <NUM> of obtained resin-gold composite dispersion (<NUM>. 1wt%), and agitation is performed under room temperature for about <NUM> hours, so that the resin-gold composite is bound with the antibody. Bovine serum albumin solution is added until final concentration is changed into <NUM>%, and agitation is performed under room temperature for <NUM> hours to block the surface of the resin-gold composite. Resin-gold composite-marked antibody dispersion is prepared by recovery after <NUM> minutes of centrifugal separation at 12000rpm under <NUM> and suspension in buffer containing <NUM>% of bovine serum albumin.

Determination utilizing immunochromatography is carried out by using the prepared resin-gold composite-marked antibody dispersion, and the performance of the resin-gold composite dispersion is evaluated. The result is represented below.

According to Table <NUM>, it can be determined that the resin-gold composite-marked antibody shows good coloration for the antigen diluted <NUM> folds.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, <NUM> aqueous dimethylamine borane solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>. In the resin-gold composite, the gold particles include encased gold particles completely encased in the resin particle, partially exposed gold particles having a portion embedded in the resin particle and a portion exposed from the resin particle, and surface-adsorbed gold particles absorbed on the surface of the resin particle, and at least portions of the gold particles are three-dimensionally distributed on the surface section of the resin particle.

After Aliquat <NUM> [produced by Aldrich company] (<NUM>) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, <NUM>) are dissolved into <NUM> of pure water, <NUM>-vinylpyridine (<NUM>-VP, <NUM>) and divinyl benzene (DVB, <NUM>) are added, and under nitrogen flow, agitation is performed at 150rpm under <NUM> for <NUM> minutes, and is then performed under <NUM> for <NUM> minutes. After agitation, <NUM>,<NUM>'-azobis(<NUM>-methylpropionamidine) dihydrochloride (AIBA, <NUM>) dissolved in <NUM> of pure water is dripped for <NUM> minutes, and agitation is performed at 150rpm under <NUM> for <NUM> hours, and thereby resin particles, the average particle size of which is <NUM>, are obtained. The resin particles are precipitated by centrifugal separation (9000rpm, <NUM> minutes), supernatant is removed, the resin particles are dispersed into pure water again, and after this operation is performed three times, impurities are removed by dialysis. Afterwards, concentration is regulated, and thereby 10wt% resin particle dispersion is obtained.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, <NUM> aqueous dimethylamine borane solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite dispersion determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>. In the resin-gold composite, the gold particles include encased gold particles completely encased in the resin particle, partially exposed gold particles having a portion embedded in the resin particle and a portion exposed from the resin particle, and surface-adsorbed gold particles absorbed on the surface of the resin particle, and at least portions of the gold particles are three-dimensionally distributed on the surface section of the resin particle.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, <NUM> aqueous dimethylamine borane solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite dispersion determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>. In the resin-gold composite, the gold particles include encased gold particles completely encased in the resin particle, partially exposed gold particles having a portion embedded in the resin particle and a portion exposed from the resin particle, and surface-adsorbed gold particles absorbed on the surface of the resin particle, and at least portions of the gold particles are three-dimensionally distributed on the surface section of the resin particle.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, <NUM> aqueous dimethylamine borane solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite dispersion determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>. In the resin-gold composite, the gold particles include encased gold particles completely encased in the resin particle, partially exposed gold particles having a portion embedded in the resin particle and a portion exposed from the resin particle, and surface-adsorbed gold particles absorbed on the surface of the resin particle, and at least portions of the gold particles are three-dimensionally distributed on the surface section of the resin particle.

After <NUM>-(Diisopropylamino)ethyl methacrylate (DPA, <NUM>), poly(propylene glycol)diacrylate (<NUM>) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, <NUM>) are dissolved into <NUM> of pure water, and under nitrogen flow, agitation is performed at 150rpm under <NUM> for <NUM> minutes, and is then carried out under <NUM> for <NUM> minutes. After agitation, ammonium persulphate (APS, <NUM>) dissolved in <NUM> of pure water is dripped for <NUM> minutes, and is agitated at 150rpm under <NUM> for <NUM> hours, and thereby resin particles, the average particle size of which is <NUM>, are obtained. The resin particles are precipitated by centrifugal separation (9000rpm, <NUM> minutes), supernatant is removed, the resin particles are dispersed into pure water again, and after this operation is performed three times, impurities are removed by dialysis. Afterwards, concentration is regulated, and thereby 10wt% resin particle dispersion is obtained.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, <NUM> aqueous dimethylamine borane solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby a resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite dispersion determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>. In the resin-gold composite, the gold particles include encased gold particles completely encased in the resin particle, partially exposed gold particles having a portion embedded in the resin particle and a portion exposed from the resin particle, and surface-adsorbed gold particles absorbed on the surface of the resin particle, and at least portions of the gold particles are three-dimensionally distributed on the surface section of the resin particle.

100µg of influenza antibody is mixed into <NUM> of obtained resin-gold composite dispersion (<NUM>. 1wt%), and agitation is performed under room temperature for about <NUM> hours, so that the resin-gold composite is bound with the antibody. Bovine serum albumin solution is added until final concentration is changed into <NUM>%, and is agitated under room temperature for <NUM> hours to block the surface of the resin-gold composite. Resin-gold composite-marked antibody dispersion is prepared by recovery after <NUM> minutes of centrifugal separation at 12000rpm under <NUM> and suspension in buffer containing <NUM>% of bovine serum albumin.

After Aliquat <NUM> [produced by Aldrich company] (<NUM>) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, <NUM>) are dissolved into <NUM> of pure water, <NUM>-vinylpyridine (<NUM>-VP, <NUM>) and divinyl benzene (DVB, <NUM>) are added, and under nitrogen flow, agitation is performed at 150rpm under <NUM> for <NUM> minutes, and is then performed under <NUM> for <NUM> minutes. After agitation, <NUM>,<NUM>'-azobis(<NUM>-methylpropionamidine) dihydrochloride (AlBA, <NUM>) dissolved in <NUM> of pure water is dripped for <NUM> minutes, agitation is performed at 150rpm under <NUM> for <NUM> hours, and thereby resin particles, the average particle size of which is <NUM>, are obtained. The resin particles are precipitated by centrifugal separation (9000rpm, <NUM> minutes), supernatant is removed, the resin particles are dispersed into pure water again, and after this operation is performed three times, impurities are removed by dialysis. Afterwards, concentration is regulated, and thereby 10wt% resin particle dispersion is obtained.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, <NUM> aqueous dimethylamine borane solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite dispersion determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>. The scanning electron microscope (SEM) picture of the surface of the obtained resin-gold composite is shown in <FIG>, and the scanning transmission electron microscope (STEM) picture of its section is shown in <FIG> In the resin-gold composite, the gold particles include encased gold particles completely encased in the resin particle, partially exposed gold particles having a portion embedded in the resin particle and a portion exposed from the resin particle, and surface-adsorbed gold particles absorbed on the surface of the resin particle, and at least portions of the gold particles are three-dimensionally distributed on the surface section of the resin particle.

5wt% gold ion-adsorbing resin particle dispersion (<NUM>) is then added into <NUM> of pure water, <NUM> aqueous dimethylamine borane solution (<NUM>) is dripped for <NUM> minutes as agitation is performed at 160rpm under <NUM>, agitation is then performed under room temperature for <NUM> hours, and thereby resin-gold composite, the average particle size of which is <NUM>, is obtained. The resin-gold composite is precipitated by centrifugal separation (3100rpm, <NUM> minutes), supernatant is removed, the resin-gold composite is dispersed into pure water again, this operation is repeated three times, refining and concentration regulation are performed by dialysis, and thereby 1wt% resin-gold composite dispersion is obtained. The result of absorbance of the prepared resin-gold composite dispersion determined according to the method is <NUM>. In addition, the average particle size of the formed gold particles is <NUM>, and the amount of carried gold is <NUM>. The scanning electron microscope (SEM) picture of the surface of the obtained resin-gold composite is shown in <FIG>, and the scanning transmission electron microscope (STEM) picture of its section is shown in <FIG> In the resin-gold composite, the gold particles include encased gold particles completely encased in the resin particle, partially exposed gold particles having a portion embedded in the resin particle and a portion exposed from the resin particle, and surface-adsorbed gold particles absorbed on the surface of the resin particle, and at least portions of the gold particles are three-dimensionally distributed on the surface section of the resin particle.

If the section image of <FIG> of embodiment <NUM> and the section image of <FIG> of embodiment <NUM> are compared, then the assay sensitivity of immunochromatography of embodiment <NUM> in which <NUM>% to <NUM>%, preferably <NUM>% to <NUM>%, of the gold particles exist in a range of <NUM>% of particle radius in a depth direction from the surface of the resin particle is more excellent.

After Aliquat <NUM> [produced by Aldrich company] (<NUM>) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, <NUM>) are dissolved into <NUM> of pure water, <NUM>-vinylpyridine (<NUM>-VP, <NUM>) and divinyl benzene (DVB, <NUM>) are added, and under nitrogen flow, agitation is performed at 250rpm under <NUM> for <NUM> minutes. After agitation, <NUM>,<NUM>'-azobis(<NUM>-methylpropionamidine) dihydrochloride (AIBA, <NUM>) dissolved in <NUM> of pure water is dripped for <NUM> minutes, and is agitated at 250rpm under <NUM> for <NUM> hours, and thereby resin particles A-<NUM>, the average particle size of which is <NUM>, are obtained. A-<NUM> is precipitated by centrifugal separation (9000rpm, <NUM> minutes), supernatant is removed, the resin particles are then dispersed in pure water again, and thereby <NUM>. 1wt% resin particle dispersion B-<NUM> is obtained.

<NUM> aqueous chloroauric acid solution (<NUM>) is added into B-<NUM> (<NUM>), and is left alone under room temperature for <NUM> hours. Afterwards, the resin particles are precipitated by centrifugal separation (3000rpm, <NUM> minutes), supernatant is removed, so that redundant chloroauric acid is removed, the resin particles are then dispersed into <NUM> of pure water again, and thereby gold ion-adsorbing resin particle dispersion C-<NUM> is prepared. After C-<NUM> (<NUM>) is dripped into <NUM> aqueous dimethylamine borane solution (<NUM>) for <NUM> minutes, agitation is performed under <NUM> for <NUM> hour, and is then performed under room temperature for <NUM> hours, and thereby resin-gold composite D-<NUM>, the average particle size of which is <NUM>, is obtained. D-<NUM> is precipitated by centrifugal separation (3000rpm, <NUM> minutes), supernatant is removed, an appropriate amount of pure water is then added for dispersion again, an ultrafiltration membrane is then used for refining, and thereby 1wt% resin-gold composite dispersion E-<NUM> is obtained. The result of absorbance of the resin-gold composite F-<NUM> in E-<NUM> determined according to the method is <NUM>. In addition, the average particle size of the gold particles in F-<NUM> is <NUM>, and the amount of carried gold is <NUM>.

In the experimental examples and reference experimental examples, the following reagents and so on are used.

Anti-influenza A monoclonal antibody (<NUM>/mL/PBS): Produced by Adtec Co.

Influenza A positive control (APC): Prepared by using sample treatment solution (produced by Adtec Co. ) to dilute an influenza A virus passivation antigen (produced by Adtec Co. ) <NUM> folds. The antigen concentration of the APC is equivalent to 5000FFU/ml.

Negative control: Sample treatment solution (produced by Adtec Co.

AuNCP beads: Resin-gold composite (1wt%; average particle size: <NUM>) obtained in preparation example <NUM>.

<NUM> of AuNCP beads as resin-metal composite are put into microtubes [IBIS (registered trademark; produced by AS ONE company) <NUM>], and <NUM> of binding buffer a is added. After upside-down mixing for sufficient mixing, 100µg of anti-influenza A monoclonal antibody <NUM> is added, moreover, upside-down agitation is performed under room temperature for <NUM> hours, and thereby marked antibody-containing solution A-<NUM> which contains the anti-influenza A monoclonal antibody marked by utilizing the resin-metal composite is obtained.

Afterwards, after the marked antibody-containing solution A-<NUM> is cooled by ice bath, centrifugal separation is performed at 12000rpm for <NUM> minutes, supernatant is removed, <NUM> of blocking buffer a is then added into solid component residue, ultrasonic dispersion treatment is performed for <NUM> to <NUM> seconds, further, upside-down agitation is performed under room temperature for <NUM> hours, and thereby marked antibody-containing solution B-<NUM> is obtained.

Afterwards, after the marked antibody-containing solution B-<NUM> is cooled by ice bath, centrifugal separation is performed at 12000rpm for <NUM> minutes, supernatant is removed, <NUM> of cleaning buffer is then added into solid component residue, and ultrasonic dispersion treatment is performed for <NUM> to <NUM> seconds. This operation is repeated three times as cleaning treatment.

Afterwards, after ice bath cooling, centrifugal separation is performed at 12000rpm for <NUM> minutes, supernatant is removed, <NUM> of storage buffer is then added into solid component residue, ultrasonic dispersion treatment is performed for <NUM> to <NUM> seconds, and thereby marked antibody-containing solution C-<NUM> is obtained.

Binding buffer b is used to substitute for binding buffer a in the binding step of experimental example <NUM>, and besides, marked antibody-containing solution A-<NUM>, marked antibody-containing solution B-<NUM> and marked antibody-containing solution C-<NUM> are obtained in the same way as experimental example <NUM>.

Binding buffer c is used to substitute for binding buffer a in the binding step of experimental example <NUM>, and besides, marked antibody-containing solution A-<NUM>, marked antibody-containing solution B-<NUM> and marked antibody-containing solution C-<NUM> are obtained in the same way as experimental example <NUM>.

Binding buffer d is used to substitute for binding buffer a in the binding step of experimental example <NUM>, and besides, marked antibody-containing solution A-<NUM>, marked antibody-containing solution B-<NUM> and marked antibody-containing solution C-<NUM> are obtained in the same way as experimental example <NUM>.

When binding buffer e is used to substitute for binding buffer a in the binding step of experimental example <NUM>, resin-metal composite is aggregated, so it is hard to obtain marked antibody-containing solution.

When binding buffer f is used to substitute for binding buffer a in the binding step of experimental example <NUM>, a resin-metal composite is aggregated, so it is hard to obtain marked antibody-containing solution.

Blocking buffer b is used to substitute for the blocking buffer a in the blocking step of experimental example <NUM>, and besides, marked antibody-containing solution B-<NUM> and marked antibody-containing solution C-<NUM> are obtained in the same way as experimental example <NUM>.

Blocking buffer c is used to substitute for the blocking buffer a in the blocking step of experimental example <NUM>, and besides, marked antibody-containing solution B-<NUM> and marked antibody-containing solution C-<NUM> are obtained in the same way as experimental example <NUM>.

As the result of using blocking buffer d to substitute for the blocking buffer a in the blocking step of experimental example <NUM>, the marked antibody after the binding step shows good dispersibility, however, after the blocking step, marked antibody is aggregated, so it is hard to obtain marked antibody-containing solution.

Binding buffer g is used to substitute for binding buffer a in the binding step of experimental example <NUM>, and besides, marked antibody-containing solution A-<NUM>, marked antibody-containing solution B-<NUM> and marked antibody-containing solution C-<NUM> are obtained in the same way as experimental example <NUM>.

Evaluation is carried out by using the monochrome screen for influenza A evaluation (produced by Adtec company), and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are compared. A color sample for colloidal gold determination (produced by Adtec company) is used to determine the coloration levels. In screening and evaluation, antigen uses an influenza A positive control (APC). In performance evaluation, antigen uses <NUM>-fold dilution (<NUM>- to <NUM>-fold dilution) of the APC.

3µL of marked antibody-containing solution C-<NUM>, 3µL of marked antibody-containing solution C-<NUM>, 3µL of marked antibody-containing solution C-<NUM>, 3µL of marked antibody-containing solution C-<NUM>, 3µL of marked antibody-containing solution C-<NUM>, 3µL of marked antibody-containing solution C-<NUM> and 3µL of marked antibody-containing solution C-<NUM> which are obtained in experimental example <NUM> to experimental example <NUM> are respectively added into seven wells of the <NUM>-well plate, and 100µL of APC is mixed into each well. Afterwards, 50µL is added into the monochrome screen for influenza A evaluation, and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are evaluated. The result is shown in Table <NUM>. Moreover, the higher the values in Table <NUM> are, the higher coloration levels are (intenser coloration).

According to Table <NUM>, it can be determined that the marked antibody-containing solution C-<NUM> obtained in experimental example <NUM> shows the strongest coloration, having excellent marking performance.

3µL of marked antibody-containing solution C-<NUM> obtained in experimental example <NUM> is added into each of <NUM> wells of the <NUM>-well plate, and 100µL of <NUM>-fold dilution (<NUM>- to <NUM>-fold dilution, respectively represented as APC×<NUM> to APC×<NUM>) of the APC and 100µL of negative control are mixed. Afterwards, 50µL is added into the monochrome screen for influenza A evaluation, and coloration levels after <NUM> minutes, <NUM> minutes and <NUM> minutes are evaluated. The result is shown in Table <NUM>. Moreover, the higher the values in Table <NUM> are, the higher coloration levels are (intenser coloration).

According to Table <NUM>, it can be determined that the marked antibody-containing solution C-<NUM> obtained in experimental example <NUM> shows good coloration for the antigen diluted <NUM> folds, having excellent marking performance.

Priority is claimed of <CIT>, and <CIT>.

Claim 1:
A marker, characterized by comprising a resin-metal composite (<NUM>) with a structure formed by immobilizing metal particles (<NUM>) on a resin particle (<NUM>),
having any one of constitution (A) and constitution (B):
(A) the average particle size of the resin-metal composite (<NUM>) exceeding <NUM>; or
(B) the average particle size of the metal particles (<NUM>) being in a range of more than <NUM> and less than <NUM>;
wherein the metal particles (<NUM>) comprise metal particles (<NUM>) completely encased in the resin particle (<NUM>) and metal particles (<NUM>) having a portion embedded in the resin particle (<NUM>) and a portion exposed from the resin particle (<NUM>), and
the material of the metal particles (<NUM>) is selected from a group consisting of silver, nickel, copper, gold, palladium, and alloys thereof.