JP2017120242A - Resin-metal complex, labeling substance, immunological measurement method, reagent for immunological measurement, method for measuring analyte, kit for analyte measurement, and test strip for lateral flow type chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a resin-metal composite which has less adhesion to a container wall surface and can be measured with high accuracy even in quantitative measurement when used as a labeling substance for immunological measurement. A resin-metal composite 100 includes resin particles 10 and metal particles 20. The resin particles 10 are copolymers of a nitrogen-containing monomer and a polyvalent vinyl compound, and the weight composition ratio (nitrogen-containing monomer: polyvalent vinyl compound) is in the range of 49: 1 to 30:20. The metal particles 20 are dispersed or immobilized on the resin particles 10, and a part of the metal particles 20 is three-dimensionally distributed on the surface layer portion 60 of the resin particles 10. The metal particles 20 include an encapsulated metal particle 30 completely contained in the resin particle 10, a partially exposed metal particle 40 having a portion embedded in the resin particle 10 and a portion exposed outside the resin particle 10, and a resin. It contains surface-adsorbed metal particles 50 adsorbed on the surface of the particles 10. [Selection diagram] Fig. 1

Description

  The present invention includes, for example, a resin-metal complex that can be used as a labeling substance in immunological measurement, a labeling substance using the same, an immunological measurement method, an immunological measurement reagent, an analyte measurement method, an analyte, and the like. The present invention relates to a light measurement kit and a test strip for lateral flow chromatography.

  There are various methods of immunological measurement (immunoassay) depending on the measurement principle. For example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), latex agglutination (LIA, PA), immunochromatography ( ICA), hemagglutination method (HA), hemagglutination inhibition method (HI) and the like.

  In the immunoassay, an antigen or antibody is detected qualitatively or quantitatively from a change (a change in the concentration of the antigen, antibody or complex) when the antigen reacts with the antibody to form a complex. When these are detected, the detection sensitivity is increased by binding a labeling substance to the antibody, antigen or complex. Therefore, it can be said that the labeling ability of the labeling substance is an important factor affecting the detection ability in the immunoassay. Also in the immunoassay exemplified above, as labeling substances, red blood cells (in the case of HA), latex particles (in the case of LIA), fluorescent dyes (in the case of FIA), radioactive elements (in the case of RIA), enzymes (in the case of EIA), Chemiluminescent materials (in the case of CLIA) are used.

By the way, when colored fine particles are used as the labeling substance, detection can be confirmed by visual observation without using a special analyzer, so that simpler measurement is expected. Examples of such colored fine particles include colloidal particles of metals and metal oxides, latex particles colored with a pigment, and the like (Patent Document 1, Patent Document 4 and the like). However, since the color tone of the colloidal particles is determined depending on the particle diameter and preparation conditions, it is difficult to obtain a desired vivid dark color tone, that is, there is a problem that visibility is insufficient.
Further, the colored latex particles have a problem that the coloring effect by the coloring matter is low and the visual judgment is insufficient. In order to solve this problem, if the coloring amount of the dye is increased, the dye covers the surface of the latex, and the original surface state of the latex particles is damaged, so that it is difficult to bind the antigen or antibody. was there. In addition, it is not always possible to improve the performance by clogging the pores of a chromatographic medium such as a membrane filter or causing non-specific aggregation of latex particles and increasing the coloring of the dye to increase the color. There was also a problem of not sticking.

  In order to improve the visibility of the above-mentioned labeling substance, after the antibody (labeled antibody) bound with the labeling substance reacts with the antigen to form a complex, the labeling substance is further modified with other metals. An immunochromatographic method for amplifying a substance detection sensitivity is disclosed (Patent Documents 2 and 5).

  Patent Document 3 proposes a colored latex composed of gold nanoparticles bonded to the surface of a polymer-based latex particle as a labeling substance.

  Patent Document 4 discloses polymer latex particles coated with metallic gold, suggesting application to reagents that can be used in microscopy and immunoassay methods.

  Non-Patent Document 1 discloses a microgel in which gold nanoparticles are supported on poly-2-vinylpyridine latex particles, and the pH responsiveness of the particle size of the microgel is determined based on the localized surface plasmon of the gold nanoparticles. Confirmed from changes in absorption behavior.

JP-A-5-10950 JP 2011-117906 A JP 2009-168495 A Japanese Patent Laid-Open No. 3-206959 JP 2009-192270 A

K. Akamatsu, M. Shimada, T .; Tsuruoka, H .; Nawafune, S .; Fujii and Y.J. Nakamura Langmuir 2010, 26, 1254-1259.

  In the immunological measurement, when the amount of the labeling substance with respect to the antibody, antigen or substance to be measured is not constant, it becomes difficult to quantitatively grasp the substance to be measured. Therefore, it is very important to apply a labeling substance in a reagent in an accurate amount to an antibody, an antigen or a substance to be measured for quantitative evaluation of the substance to be measured. However, when fine particles having an average particle size of about several hundreds of nanometers are used as the labeling substance, since the surface free energy is large, aggregation tends to occur or the particles tend to adhere to the inner wall surface of the reagent container. In particular, if the labeling substance remains attached to the inner wall surface in the reagent container, the amount of the labeling substance contained in the reagent to be tested is not constant. As a result, an error occurs in the quantitative evaluation. Even when qualitative evaluation is performed, the evaluation result may vary. In particular, when the substance to be measured is a minute amount, the influence is great.

  The object of the present invention is that the adhesion to the container wall surface is small, and when used as a labeling substance for immunological measurement, high-precision measurement is possible even in quantitative measurement and qualitative measurement when the substance to be measured is a minute amount. It is to provide a resin-metal composite that can be used.

  As a result of intensive studies, the present inventors have found that the above problems can be solved by setting the copolymerization ratio of the resin component constituting the resin-metal composite to a specific range, and have completed the present invention.

  That is, the resin-metal composite of the present invention comprises resin particles and a plurality of metal particles fixed to the resin particles, and the resin particles are co-polymerized with a nitrogen-containing monomer and a polyvalent vinyl compound. The weight composition ratio (nitrogen-containing monomer: polyvalent vinyl compound) is in the range of 49: 1 to 30:20.

  In the resin-metal composite of the present invention, the supported amount of the metal particles may be in the range of 5% by mass to 70% by mass with respect to the weight of the resin-metal composite.

  In the resin-metal composite of the present invention, 60% by mass to 100% by mass of the metal particles may be present in the surface layer portion of the resin particles.

  In the resin-metal composite of the present invention, the average particle size of the metal particles may be in the range of 1 to 80 nm.

  The resin-metal composite of the present invention may have an average particle size in the range of 50 to 1000 nm.

  In the resin-metal composite of the present invention, the metal particles may be gold particles or platinum particles.

  The labeling substance of the present invention includes any one of the above resin-metal complexes.

  The labeling substance of the present invention may be used by adsorbing an antigen or antibody on the surface of the resin-metal complex.

  The immunological measurement method of the present invention uses any of the above-mentioned labeling substances.

  The reagent for immunological measurement of the present invention comprises any one of the above resin-metal complexes.

The analyte measurement method of the present invention is an analyte measurement method for detecting or quantifying an analyte contained in a sample.
The method for measuring an analyte of the present invention uses a lateral flow type chromatographic test strip including a membrane and a determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane.
And the measuring method of the analyte of the present invention comprises the following steps (I) to (III);
Step (I): contacting the analyte contained in the sample with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with any one of the resin-metal complexes,
Step (II): a step of bringing the complex containing the analyte and the labeled antibody formed in Step (I) into contact with a capture ligand in the determination unit;
Step (III): a step of measuring the color development intensity derived from localized surface plasmon resonance of the resin-metal complex and light energy absorption by electronic transition,
The process including this is performed.

The analyte measurement kit of the present invention is an analyte measurement kit for detecting or quantifying an analyte contained in a sample using a lateral flow type chromatographic test strip.
The analyte measurement kit of the present invention includes a membrane, and a test strip for lateral flow type chromatography including a determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane;
An antibody that specifically binds to the analyte, a detection reagent comprising a labeled antibody labeled with any one of the resin-metal complexes, and
Is included.

The test strip for lateral flow chromatography of the present invention is
It is a test strip for lateral flow type chromatography for detecting or quantifying an analyte contained in a sample.
The test strip for lateral flow chromatography of the present invention is
A membrane,
A determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane in a direction in which the sample is developed,
On the upstream side of the determination unit, a reaction unit including a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with any one of the resin-metal complexes, and
Is included.

  In the resin-metal composite of the present invention, since the copolymerization ratio of the nitrogen-containing monomer and the polyvalent vinyl compound constituting the resin particles is controlled within a specific range, the occurrence of aggregation and the inner wall surface of the reagent container Adhesion to is effectively suppressed. Therefore, by using the resin-metal complex of the present invention as a labeling substance, the labeling substance remains in the container, and the measurement error caused by the amount of the labeling substance with respect to the antibody, antigen or substance to be measured varies. Without causing it, it is possible to perform quantitative immunological measurement with high accuracy. Therefore, the resin-metal complex of the present invention is used for, for example, a labeling substance for immunological measurement such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, HI, and a reagent for immunological measurement. Can be preferably applied.

It is a schematic diagram which shows the structure of the cross section of the resin-metal composite_body | complex which concerns on one embodiment of this invention. It is a schematic diagram which shows the structure of the cross section of the one aspect | mode of a resin-metal composite. It is a schematic diagram which shows the structure of the cross section of another aspect of a resin-metal composite body. It is explanatory drawing which shows the outline | summary of the measuring method of the analyte using the test strip for lateral flow type | mold chromatography which concerns on one embodiment of this invention. It is a photograph which shows the adhesion test result with respect to PP container. It is a photograph which shows the adhesion test result with respect to PC container. It is a photograph which shows the adhesion test result with respect to PC container. It is a photograph which shows the adhesion test result with respect to a PET container.

[Resin-metal composite]
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. FIG. 1 is a schematic cross-sectional view of a resin-metal composite according to an embodiment of the present invention. The resin-metal composite 100 includes resin particles 10 and metal particles 20. In the resin-metal composite 100, the metal particles 20 are fixed to the resin particles 10. The resin particles 10 are relatively larger particles than the metal particles 20. That is, in the resin-metal composite 100, a relatively large number of relatively small metal particles 20 are fixed to the large resin particles 10. As shown in FIG. 1, the relationship between the particle diameter D1 of the entire resin-metal composite 100, the particle diameter D2 of the resin particle 10, and the particle diameter D3 of the metal particle 20 is D1>D2> D3.

  Further, in the resin-metal composite 100, a part of the metal particles 20 may be distributed three-dimensionally in the surface layer portion 60 of the resin particle 10. In this case, a part of the three-dimensionally distributed metal particles 20 may be partially exposed outside the resin particles 10, and the remaining part may be included in the resin particles 10. Specifically, as shown in FIG. 1, metal particles 20 that are completely encapsulated in the resin particles 10 (hereinafter, also referred to as “encapsulated particles 30”) are embedded in the resin particles 10. Metal particles (hereinafter also referred to as “partially exposed particles 40”) having exposed portions and portions exposed to the outside of the resin particles 10, and metal particles adsorbed on the surface of the resin particles 10 (hereinafter referred to as “surface adsorbed particles 50”). Is also present.) Is preferably present.

  For example, when the resin-metal complex 100 is used as a labeling substance for immunological measurement or a reagent for immunological measurement, an antibody or a surface of the resin particle 10 or the surface of the partially exposed particle 40 or the surface adsorbed particle 50 Antigen is immobilized and used. At this time, the antibody or antigen is immobilized on the partially exposed particles 40 and the surface adsorbed particles 50, but not immobilized on the encapsulated particles 30. However, all of the partially exposed particles 40, the surface adsorbed particles 50, and the encapsulated particles 30 exhibit light energy absorption by electronic transition in addition to the localized surface plasmon resonance. In addition, the encapsulated particles 30 also contribute to improving the visibility of the immunological measurement labeling substance and the immunological measurement reagent. Furthermore, since the partially exposed particles 40 and the encapsulated particles 30 have a larger contact area with the resin particles 10 than the surface adsorbed particles 50, an anchor effect or the like due to the embedded state is exhibited, so that physical adsorption The force is strong and it is difficult to detach from the resin particles 10. Therefore, the durability and stability of the immunological measurement labeling substance and the immunological measurement reagent using the resin-metal complex 100 can be improved.

  Hereinafter, the resin-metal complex 100 is applied to a labeling substance for immunological measurement (hereinafter also simply referred to as “labeling substance”) or a reagent for immunological measurement (hereinafter also simply referred to as “reagent”). Will be described as an example.

  The inner particles 30 are all covered with the resin constituting the resin particles 10 on the entire surface. Further, the partially exposed particles 40 are such that 5% or more and less than 100% of the surface area is covered with the resin constituting the resin particles 10. From the viewpoint of durability of the immunological measurement labeling substance and the immunological measurement reagent, the lower limit thereof is preferably 20% or more, more preferably 30% or more of the surface area. Further, it is preferable that the surface adsorbing particles 50 are covered with a resin constituting the resin particles 10 in a range of more than 0% and less than 5% of the surface area.

  In addition, the loading amount of the metal particles 20 (the total of the encapsulated particles 30, the partially exposed particles 40, and the surface adsorption particles 50) on the resin-metal composite 100 is 5 mass with respect to the weight of the resin-metal composite 100. It is preferable that it is% -70 mass%. If it is this range, the resin-metal composite body 100 is excellent in the visibility as a labeling substance, visual judgment property, and detection sensitivity. When the loading amount of the metal particles 20 is less than 5% by mass, the amount of antibody or antigen immobilized is decreased, and the detection sensitivity tends to decrease. The supported amount of the metal particles 20 is more preferably 15% by mass to 70% by mass, and further preferably 15% by mass to 60% by mass. On the other hand, if it exceeds 70% by mass, the specific gravity of the resin-metal composite is increased, and the dispersibility in a dispersion medium such as water tends to deteriorate.

  Further, 10 mass% to 90 mass% of the metal particles 20 are preferably the partially exposed particles 40 and the surface adsorbed particles 50. If it is this range, since the fixed quantity of the antibody or antigen on the metal particle 20 can be ensured sufficiently, the sensitivity as a labeling substance is high. It is more preferable that 20% by mass to 80% by mass of the metal particles 20 are partly exposed particles 40 and surface adsorbed particles 50. From the viewpoint of durability of the immunological measurement labeling substance and the immunological measurement reagent, The surface adsorbed particles 50 are more preferably 20% by mass or less.

  In addition, when the resin-metal complex 100 is used for immunological measurement, in order to obtain excellent detection sensitivity, 60% by mass to 100% by mass, preferably 75% by mass to 100% by mass of the metal particles 20 is obtained. %, More preferably 85% by mass to 100% by mass is present in the surface layer portion 60, more preferably in the range of 40% of the particle radius in the depth direction from the surface of the resin particle 10. Further, 5 mass% to 90 mass% of the metal particles 20 present in the surface layer portion 60 are partially exposed particles 40 or surface adsorbed particles 50, indicating that the amount of antibody or antigen immobilized on the metal particles 20 is fixed. Since it can be secured sufficiently, the sensitivity as a labeling substance is increased, which is preferable. In other words, it is preferable that 10% by mass to 95% by mass of the metal particles 20 existing in the surface layer part 60 is the inclusion particles 30.

  Here, the “surface layer portion” refers to the surface of the resin particle 10 on the basis of the outermost position of the resin-metal composite 100 (that is, the protruding end portion of the partially exposed particle 40 or the surface adsorbed particle 50). Means a range of 50% of the particle radius in the depth direction. The “three-dimensional distribution” means that the metal particles 20 are dispersed not only in the surface direction of the resin particles 10 but also in the depth direction.

  As described above, since the encapsulated particle 30 also expresses light energy absorption by electronic transition in addition to the localized surface plasmon resonance, not only the partially exposed particle 40 and the surface adsorbed particle 50 but also the encapsulated particle 30 is immunological. This contributes to the improvement of the visibility of the labeling substance for measuring and the reagent for immunological measurement. From such a viewpoint of improving visibility, the resin-metal composite 100 is distributed in such a manner that the encapsulated particles 30 are concentrated in a certain range in the depth direction from the surface of the resin particles 10 as shown in FIG. 2A, for example. However, it is preferable that the encapsulated particles 30 are not present near the center of the resin particles 10. More specifically, in order to effectively express light energy absorption due to electronic transition in addition to localized surface plasmon resonance by the encapsulated particles 30, for example, 0 to 200 nm in the depth direction from the surface of the resin particles 10. 70% by mass or more, preferably 80% by mass or more, and more preferably 90 to 100% by mass of the encapsulated particles 30 may be present in the range. In particular, when the region (encapsulated particle distribution region) in which all (100% by mass) of the encapsulated particles 30 are distributed is within a range of, for example, 0 to 100 nm from the surface of the resin particle 10, the localized surface by the encapsulated particles 30 is used. In addition to plasmon resonance, light energy absorption due to electronic transition can be maximized, which is preferable.

  Further, the resin-metal composite 100 may not include the encapsulated particles 30. For example, as shown in FIG. 2B, in the resin-metal composite 100, all of the metal particles 20 may be fixed to the surface of the resin particle 10 without overlapping in the radial direction of the resin particle 10. In this case, the metal particles 20 are composed of partially exposed particles 40 and surface adsorbed particles 50.

[Resin particles]
The resin particle 10 is a copolymer of a nitrogen-containing monomer and a polyvalent vinyl compound. Here, examples of the nitrogen-containing monomer include aromatic amines and aliphatic amines. When this nitrogen-containing monomer and polyvalent vinyl compound are polymerized, polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, polyaniline, acrylic resin having a nitrogen atom in the side chain, phenol In a resin, epoxy resin, or the like, a copolymer having monomer units derived from a polyvalent vinyl compound is obtained. A preferred nitrogen-containing monomer is vinylpyridine. As vinylpyridine, for example, 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine and the like can be used. Therefore, the main chain of the polymer constituting the resin particle 10 is preferably poly-2-vinylpyridine, poly-3-vinylpyridine, or poly-4-vinylpyridine.

  On the other hand, the polyvalent vinyl compound functions as a crosslinking agent, and examples thereof include divinylbenzene, trivinylbenzene, divinylnaphthalene, and divinylbiphenyl. Of these, o-divinylbenzene, m-divinylbenzene, and p-divinylbenzene, which are excellent in the reactivity of the crosslinking reaction, are preferable.

  The copolymer of a nitrogen-containing monomer and a polyvalent vinyl compound is a polymer particle having a substituent in the structure capable of adsorbing metal ions. The nitrogen atom in the copolymer of the nitrogen-containing monomer and the polyvalent vinyl compound is an anionic metal that is a precursor of metal particles such as gold, platinum, and palladium, which has excellent visibility and can easily immobilize antigens or antibodies. Easy to chemisorb ions. In the present embodiment, in order to reduce metal ions adsorbed in the copolymer of the nitrogen-containing monomer and the polyvalent vinyl compound to form metal nanoparticles, some of the generated metal particles 20 are encapsulated metal particles. 30 or partially exposed metal particles 40.

  The weight composition ratio of the monomer-derived component in the copolymer of the nitrogen-containing monomer and the polyvalent vinyl compound constituting the resin particle 10 (nitrogen-containing monomer: polyvalent vinyl compound) is the resin-metal composite on the inner wall of the reagent container. From the viewpoint of achieving both suppression of adhesion of 100 and improvement in detection sensitivity when immunological measurement is performed using the resin-metal complex 100 as a labeling substance, the range is 49: 1 to 30:20, and 48: It is preferably within the range of 2 to 35:15, more preferably 48: 2 to 40:10, and most preferably within the range of 48: 2 to 45: 5.

  In the weight composition ratio (nitrogen-containing monomer: polyvalent vinyl compound), if the ratio of nitrogen-containing monomer exceeds 49: 1, the resin-metal complex 100 tends to adhere to the inner wall of the reagent container. When performing an immunological measurement using the metal complex 100 as a labeling substance, it is difficult to obtain the accuracy and reproducibility of quantitative evaluation. Further, when the ratio of the nitrogen-containing monomer is increased beyond 49: 1, the crosslink density due to the polyvalent vinyl compound is relatively lowered, so that the resin particles 10 are softened and the metal ions easily penetrate into the interior. As a result, metal particles are generated inside the resin particles 10, the inclusion metal particles 30 increase, the ratio of the partially exposed metal particles 40 and the surface adsorbed metal particles 50 is relatively reduced, and the particle diameter is There is a tendency to become smaller.

  On the other hand, in the weight composition ratio (nitrogen-containing monomer: polyvalent vinyl compound), when the ratio of the polyvalent vinyl compound exceeds 30:20, the crosslinking density by the polyvalent vinyl compound becomes relatively high, and the resin particles When the immunological measurement is performed using the resin-metal complex 100 as a labeling substance, the detection sensitivity tends to decrease.

[Metal particles]
For example, silver, nickel, copper, gold, platinum, palladium or the like can be applied to the metal particles 20. Gold, platinum, and palladium are preferable because they are excellent in visibility and can easily immobilize antigens or antibodies. These are preferable because they express localized surface plasmon resonance and absorption derived from light energy absorption by electronic transition. More preferably, it is gold with good storage stability. These metals can be used as a simple substance or a composite such as an alloy. Here, for example, a gold alloy means an alloy composed of gold and a metal species other than gold and containing 10% by weight or more of gold.

[Average particle size]
The average particle size of the metal particles 20 measured by observation with a scanning electron microscope (SEM) (that is, the average of the particle size D3 in FIG. 1) is preferably 1 to 80 nm, for example. When the average particle diameter of the metal particles 20 is less than 1 nm or exceeds 80 nm, the sensitivity tends to decrease because localized surface plasmon resonance and light energy absorption due to electronic transitions are less likely to occur. When the metal particle 20 is a gold particle, the average particle diameter of the metal particle 20 is preferably 20 nm or more and less than 70 nm, and more preferably 22 nm or more and less than 50 nm. When the metal particles 20 are platinum particles, the average particle diameter is preferably 1 nm or more and 50 nm or less from the viewpoint of obtaining high detection sensitivity when the resin-metal complex 100 is used for immunological measurement. More preferably, they are 1 nm or more and 30 nm or less, More preferably, they are 1 nm or more and 20 nm or less, Most preferably, they are 1 nm or more and 15 nm or less. When the average particle diameter of the platinum particles is 15 nm or less, particularly excellent detection sensitivity can be obtained when the resin-metal complex 100 is used as a labeling substance for immunochromatography.

Moreover, it is preferable that the average particle diameter (namely, average of the particle diameter D1 in FIG. 1) of the resin-metal composite body 100 is 50-1000 nm, for example. If the average particle size of the resin-metal composite 100 is less than 50 nm, for example, the amount of metal particles supported tends to be reduced, so that coloring tends to be weaker than that of metal particles of the same size. When a substance or reagent is used, it tends to be clogged in the pores of a chromatographic medium such as a membrane filter, and the dispersibility tends to decrease. From the viewpoint of improving the dispersibility when the resin-metal complex 100 is used as a labeling substance or reagent, and obtaining high detection sensitivity when the resin-metal complex 100 is used for immunological measurement, When the metal particles 20 are gold particles,
Preferably, they are 100 nm or more and less than 700 nm, More preferably, they are 340 nm or more and less than 650 nm. When the metal particle 20 is a platinum particle, it is preferably 100 nm or more and 600 nm or less, more preferably 250 nm or more and 600 nm or less, and most preferably 300 nm or more and 600 nm or less. In particular, when the average particle diameter of the resin-metal composite 100 is 300 nm or more, excellent detection sensitivity can be obtained stably when the resin-metal composite 100 is used as a labeling substance for immunochromatography.
Here, the particle diameter D1 of the resin-metal composite 100 means a value obtained by adding the length of the protruding portion of the partially exposed particle 40 or the surface adsorbed particle 50 to the particle diameter of the resin particle 10, and the laser diffraction / It can be measured by a scattering method, a dynamic light scattering method, or a centrifugal sedimentation method.

[Method for producing resin-metal composite]
Although the manufacturing method of the resin-metal complex 100 is not particularly limited, for example, the resin particles 10 can be manufactured by an emulsion polymerization method. In the production of the resin particles 10, both suppression of the adhesion of the resin-metal complex 100 to the inner wall of the reagent container and improvement of detection sensitivity when performing immunological measurement using the resin-metal complex 100 as a labeling substance are achieved. In view of the above, the feed weight ratio (nitrogen-containing monomer: polyvalent vinyl compound), which is a raw material monomer of a copolymer of a nitrogen-containing monomer and a polyvalent vinyl compound, is in the range of 49: 1 to 30:20, preferably 48. : Within the range of 2 to 35:15, more preferably within the range of 48: 2 to 40:10, and most preferably within the range of 48: 2 to 45: 5.

  The emulsion polymerization is preferably performed using a polymerization initiator in the presence of a surfactant and a stabilizer, for example. Here, preferable surfactants include, for example, Aliquat 336 (manufactured by Aldrich), trioctylmethylammonium chloride, tetrabutylammonium hydrogen sulfate, tetramethylammonium chloride, tetramethylammonium bromide, benzylbutylammonium chloride, benzyltrimethyl Examples include ammonium chloride, benzyltriethylammonium chloride, n-octyltrimethylammonium chloride, methyltri-n-octylammonium chloride, trimethylphenylammonium chloride, tripropylammonium chloride, and trimethylstearylammonium chloride. Preferred stabilizers include, for example, polyethylene glycol methyl ethyl ether methacrylate, lauryl acrylate, stearyl acrylate, isostearyl acrylate, polyethylene glycol methyl ethyl acrylate, stearyl methacrylate, isostearyl acrylate, polyethylene glycol modified stearyl methacrylate, methoxy polyethylene glycol methacrylate. Is mentioned. Preferable polymerization initiators include, for example, 2,2-azobis (2-methylpropionamidine) dihydrochloride, 2,2′-azobis (isobutyronitrile), 2,2′-azobis [2- (2- Imidazolin-2-yl) propane] dihydrochloride, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] disulfate dihydrate, 2,2′-azobis [N- (2- Carboxyethyl) -2-methylpropionamidine] tetrahydrate, 2,2′-azobis [2- (2-imidazolin-2-yl) propane], 2,2′-azobis (1-imino-1-pyridino- 2-methylpropane) dihydrochloride, 2,2′-azobis [2-methyl-N- (2-hydroxyethyl) propionamide], 4,4′-azobis (4-cyanovaleric acid) And the like. The pH in the polymerization reaction of the resin particles 10 is preferably adjusted, for example, within the range of pH 5-10 from the viewpoint of emulsification of the organic compound, and the reaction temperature is, for example, 30-95 from the viewpoint of the activity of the polymerization initiator. Within the range of ° C is preferred.

  Next, a solution containing metal ions is added to the obtained dispersion of resin particles 10 to adsorb the metal ions to the resin particles 10 (hereinafter referred to as “metal ion adsorption resin particles”). Furthermore, the metal ion adsorption resin particles are added to the reducing agent solution to reduce the metal ions to generate the metal particles 20, thereby obtaining the resin-metal composite 100.

Further, for example, when gold particles are used as the metal particles 20, examples of the solution containing metal ions include chloroauric acid (HAuCl ₄ ) aqueous solution. Further, a gold complex may be used in place of the gold ion. Examples of the solution containing platinum ions include chloroplatinic acid (H ₂ PtCl ₆ ) aqueous solution, platinum chloride (PtCl ₂ ) solution, and the like. A platinum complex may be used instead of platinum ions.

  Further, as a solvent for a solution containing metal ions, instead of water, water-containing alcohol or alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, hydrochloric acid, sulfuric acid, nitric acid An acid such as the above may be used.

  Further, in the solution, if necessary, for example, water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, diisopropyl ether; alkylene glycol, polyalkylene glycol, these Additives such as various water-miscible organic solvents such as polyols such as monoalkyl ether or dialkyl ether, glycerol, etc .; ketones such as acetone and methyl ethyl ketone may be added. Such an additive is effective for accelerating the reduction reaction rate of metal ions and controlling the size of the metal particles 20 to be produced.

  Moreover, a well-known thing can be used for a reducing agent. For example, sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, erythorbic acid, sodium phosphinate, hydroquinone, Rochelle Examples include salts. Of these, sodium borohydride, dimethylamine borane, and citric acid are preferable. A surfactant can be added to the reducing agent solution as necessary, and the pH of the solution can be adjusted. For pH adjustment, buffers such as boric acid and phosphoric acid, acids such as hydrochloric acid and sulfuric acid, alkalis such as sodium hydroxide and potassium hydroxide, and the like can be used. Furthermore, the particle size of the metal particles to be formed can be controlled by adjusting the reduction rate of the metal ions depending on the temperature of the reducing agent solution.

  Further, when the metal ions in the metal ion adsorption resin particles are reduced to produce the metal particles 20, the metal ion adsorption resin particles may be added to the reducing agent solution, or the reducing agent may be added to the metal ion adsorption resin. Although it may be added to the particles, the former is preferable from the viewpoint of easy generation of the encapsulated metal particles 30 and the partially exposed metal particles 40.

In order to maintain the dispersibility of the resin-metal composite 100 in water, for example, citric acid, poly-L-lysine, polyvinyl pyrrolidone, polyvinyl pyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 (Big Chemy Japan) A dispersant may be added.
Furthermore, the dispersibility can be maintained by adjusting the pH with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.

  The resin-metal complex 100 having the above configuration is used as a labeling substance, for example, by adsorbing an antigen or antibody on the surface of the metal particle 20, for example, EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA. It is preferably applicable to immunological measurement methods such as HI. In particular, it can be preferably applied as a labeling substance for immunological measurement or a reagent for immunological measurement excellent in visual determinability in a low concentration region (high sensitivity region). The form of the immunological measurement labeling substance or immunological measurement reagent is not particularly limited. For example, a dispersion in which the resin-metal complex 100 is dispersed in water or a pH-adjusted buffer. Can be used as

  A method for adsorbing an antigen or antibody on the surface of the metal particle 20 is not particularly limited, and a known physical adsorption or chemical adsorption method can be used. For example, the resin-metal complex 100 is immersed in a buffer solution containing an antigen or antibody, and physical adsorption such as incubation, or SH groups are introduced into the antigen or antibody and reacted with the resin-metal complex 100 to cause Au. Examples include chemisorption such as -SH bond formation. Of these, chemisorption is preferable because the bond between the metal particles 20 and the antigen or antibody becomes strong.

  As uses other than the labeling substance for immunological measurement of the resin-metal complex 100 or the reagent for immunological measurement, it can be preferably applied as a solid catalyst, pigment, paint, conductive material, electrode, sensor element.

  Next, an analyte measurement method using the resin-metal complex 100 as a labeling substance, a lateral flow chromatography test strip, and an analyte detection / quantification kit will be described.

[Test strip for lateral flow chromatography]
First, a lateral flow type chromatographic test strip (test strip) according to an embodiment of the present invention will be described with reference to FIG. As will be described later, the test strip 200 can be preferably used in the analyte measuring method of one embodiment of the present invention.

  The test strip 200 includes a membrane 110. The membrane 110 is provided with a sample addition unit 120, a determination unit 130, and a liquid absorption unit 140 in order in the sample development direction.

<Membrane>
As the membrane 110 used in the test strip 200, a membrane used as a membrane material in a general test strip can be applied. The membrane 110 is formed of an inert substance (a substance that does not react with the analyte 160, various ligands, etc.) made of a microporous material that exhibits a capillary phenomenon and develops the sample at the same time as the sample is added. It is. Specific examples of the membrane 110 include a fibrous or non-woven fibrous matrix composed of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc., membrane, filter paper, glass fiber filter paper, cloth, Cotton etc. are mentioned. Among these, membranes composed of cellulose derivatives and nylon, filter paper, glass fiber filter paper, etc. are preferably used, more preferably nitrocellulose membrane, mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membrane, nylon membrane Filter paper is used.

  The test strip 200 is preferably provided with a support that supports the membrane 110 for easier operation. As the support, for example, plastic can be used.

<Sample addition part>
The test strip 200 may have a sample adding unit 120 for adding a sample including the analyte 160. The sample addition unit 120 is a part for receiving a sample including the analyte 160 in the test strip 200. The sample addition unit 120 may be formed on the membrane 110 upstream of the determination unit 130 in the direction in which the sample is developed, or, for example, cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon A sample addition pad made of a material such as cotton cloth may be provided on the membrane 110 to constitute the sample addition unit 120.

<Determining unit>
A capture ligand 131 that specifically binds to the analyte 160 is fixed to the determination unit 130. The capture ligand 131 can be used without particular limitation as long as it forms a specific bond with the analyte 160. For example, an antibody against the analyte 160 can be preferably used. The capture ligand 131 is immobilized so as not to move from the determination unit 130 even when a sample is provided to the test strip 200. The capture ligand 131 may be fixed directly or indirectly to the membrane 110 by physical or chemical bonding or adsorption.

  The determination unit 130 is not particularly limited as long as the complex 170 including the labeled antibody 150 and the analyte 160 is in contact with the capture ligand 131 that specifically binds to the analyte 160. For example, the capture ligand 131 may be directly fixed to the membrane 110, or the capture ligand 131 may be fixed to a pad made of cellulose filter paper, glass fiber, nonwoven fabric, or the like fixed to the membrane 110. .

<Liquid absorption part>
The liquid-absorbing part 140 is formed by a pad of a water-absorbing material such as cellulose filter paper, non-woven fabric, cloth or cellulose acetate, for example. The moving speed of the sample after the development front (front line) of the added sample reaches the liquid absorption part 140 varies depending on the material, size, and the like of the liquid absorption part 140. Therefore, the optimum speed for detection / quantification of the analyte 160 can be set by selecting the material, size, etc. of the liquid absorption part 140. In addition, the liquid absorption part 140 is an arbitrary configuration and may be omitted.

  The test strip 200 may further include arbitrary parts such as a reaction part and a control part as necessary.

<Reaction part>
Although not shown, the test strip 200 may be formed with a reaction part including the labeled antibody 150 on the membrane 110. The reaction unit can be provided upstream of the determination unit 130 in the direction in which the sample flows. In addition, you may utilize the sample addition part 120 in FIG. 3 as a reaction part. When the test strip 200 has a reaction part, if the sample containing the analyte 160 is supplied to the reaction part or the sample addition part 120, the analyte 160 contained in the sample and the labeled antibody 150 can be brought into contact with each other in the reaction part. it can. In this case, since the complex 170 containing the analyte 160 and the labeled antibody 150 can be formed by simply supplying the sample to the reaction part or the sample addition part 120, so-called one-step type immunochromatography is possible. Become.

  The reaction part is not particularly limited as long as it includes the labeled antibody 150 that specifically binds to the analyte 160, but may be one in which the labeled antibody 150 is directly applied to the membrane 110. Alternatively, the reaction part may be formed by, for example, immobilizing the membrane 110 with a pad (conjugate pad) made of cellulose filter paper, glass fiber, nonwoven fabric, or the like impregnated with the labeled antibody 150.

<Control part>
Although illustration is omitted, the test strip 200 may be formed with a control unit in which a capture ligand that specifically binds to the labeled antibody 150 is fixed to the membrane 110 in the direction in which the sample is developed. By measuring the color intensity at the control unit together with the determination unit 130, the sample provided for the test strip 200 is developed and reaches the reaction unit and the determination unit 130 to confirm that the test has been performed normally. be able to. The control unit is prepared in the same manner as the determination unit 130 described above except that another type of capture ligand that specifically binds to the labeled antibody 150 is used instead of the capture ligand 131. The configuration can be taken.

[Analyte measurement method]
Next, a method for measuring the analyte 160 according to an embodiment of the present invention performed using the test strip 200 will be described.

The measuring method of the analyte 160 of the present embodiment is a measuring method of the analyte 160 that detects or quantifies the analyte 160 contained in the sample. The method for measuring the analyte 160 according to the embodiment uses the test strip 200 including the membrane 110 and the determination unit 130 in which the capture ligand 131 that specifically binds to the analyte 160 is fixed to the membrane 110. (I) to (III);
Step (I): a resin-metal complex having a structure in which a plurality of metal particles 20 are immobilized on resin particles 10 with the analyte 160 contained in the sample and an antibody that specifically binds to the analyte 160 Contacting the labeled antibody 150 labeled with 100,
Step (II): a step of bringing the complex containing the analyte 160 and the labeled antibody 150 formed in Step (I) into contact with the capture ligand 131 in the determination unit 130;
Step (III): a step of measuring the color development intensity derived from the localized surface plasmon resonance of the resin-metal composite 100 and light energy absorption by electronic transition,
Can be included.

Process (I):
Step (I) is a step of bringing the analyte 160 contained in the sample into contact with the labeled antibody 150. As long as the complex 170 containing the analyte 160 and the labeled antibody 150 is formed, the mode of contact is not particularly limited. For example, the sample may be provided to the sample addition unit 120 or the reaction unit (not shown) of the test strip 200, and the analyte 160 may be brought into contact with the labeled antibody 150 in the reaction unit, or before the sample is applied to the test strip 200. The analyte 160 in the sample may be brought into contact with the labeled antibody 150.

  The composite 170 formed in the step (I) expands and moves on the test strip 200 and reaches the determination unit 130.

Process (II):
In step (II), in the determination unit 130 of the test strip 200, the complex 170 formed in step (I) and including the analyte 160 and the labeled antibody 150 is brought into contact with the capture ligand 131. When the complex 170 is brought into contact with the capture ligand 131, the capture ligand 131 specifically binds to the analyte 160 of the complex 170. As a result, the complex 170 is captured by the determination unit 130.

  Since the capture ligand 131 does not specifically bind to the labeled antibody 150, when the analyte 160 and the unbound labeled antibody 150 reach the determination unit 130, the analyte 160 and the unbound labeled antibody 150 are unbound. Passes through the determination unit 130. Here, when a control unit (not shown) to which another capture ligand that specifically binds to the labeled antibody 150 is fixed is formed on the test strip 200, the labeled antibody 150 that has passed through the determination unit 130 is developed. Then, bind to the other capture ligand at the control unit. As a result, the labeled antibody 150 that does not form the complex 170 with the analyte 160 is captured by the control unit.

  After step (II), if necessary, before step (III), for example, the test strip 200 is washed with a buffer solution commonly used in biochemical tests such as water, physiological saline, and phosphate buffer solution. A cleaning step may be performed. Through the washing step, the labeled antibody 150 (the labeled antibody 150 that is not bound to the analyte 160 and does not form the complex 170) that has not been captured by the determination unit 130 or the determination unit 130 and the control unit is removed. be able to.

  By performing the cleaning process, in step (III), the determination unit 130 or the color development due to the absorption of light energy due to the localized surface plasmon resonance and electron transition of the resin-metal composite 100 in the determination unit 130 and the control unit , The background color intensity can be reduced, the signal / background ratio can be increased, and the detection sensitivity and quantitativeness can be further improved.

Process (III):
Step (III) is a step of measuring the color intensity derived from localized surface plasmon resonance of the resin-metal composite 100 and light energy absorption due to electronic transition. After performing the above-mentioned step (II) or the cleaning step as required, the test strip 200 measures the color development intensity derived from the localized surface plasmon resonance of the resin-metal composite 100 and light energy absorption due to electronic transition. To do.

  When the control part is formed on the test strip 200, the labeled antibody 150 is captured by another capture ligand in the control part in the step (II) to form a complex. Therefore, in the step (III), in the test strip 200, not only the determination unit 130 but also the control unit can generate a color due to localized surface plasmon resonance and light energy absorption due to electron transition. In this way, by measuring the color intensity in the control unit as well as the determination unit 130, it is possible to confirm whether the sample provided for the test strip 200 has developed normally and reached the reaction unit and the determination unit 130.

<Sample and analyte>
The sample in the analyte measurement method of the present embodiment is not particularly limited as long as it includes a substance that can be an antigen such as a protein as the analyte 160. For example, a biological sample containing the analyte 160 of interest (ie, whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swab, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin exudate , Extracts from tissues, cells and stool, etc.) and food extracts. If necessary, in order to facilitate the specific binding reaction between the labeled antibody 150 and the capture ligand 131 and the analyte 160, the analyte 160 contained in the sample is pretreated prior to the step (I). May be. Here, examples of the pretreatment include chemical treatment using various chemicals such as acid, base, and surfactant, and physical treatment using heating, stirring, ultrasonic waves, and the like. In particular, when the analyte 160 is a substance that is not normally exposed on the surface, such as an influenza virus NP antigen, it is preferable to perform treatment with a surfactant or the like. As the surfactant used for this purpose, a nonionic surfactant can be used in consideration of specific binding reaction, for example, binding reactivity between the ligand 160 and the analyte 160 such as antigen-antibody reaction. .

  In addition, the sample may be appropriately diluted with a solvent (water, physiological saline, buffer, or the like) used in a normal immunological analysis method or a water-miscible organic solvent.

  Examples of the analyte 160 include tumor markers, signaling substances, proteins such as hormones (including polypeptides and oligopeptides), nucleic acids (single-stranded or double-stranded DNA, RNA, polynucleotides, oligos) Other molecules such as nucleotides, PNA (including peptide nucleic acids)) or nucleic acid-containing substances, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or sugar chains, lipids, and the like. And as long as it specifically binds to the capture ligand 131, for example, carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, α-fetoprotein (AFP), Immunosuppressive acidic protein (IAP), CA15-3, CA125, estrogen receptor, progesterone Putter, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, influenza virus, human hemoglobin, chlamydia antigen , Group A β-streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin and the like. Among these, antigens solubilized by nonionic surfactants are preferable, and antigens that form self-assemblies such as viral nucleoproteins are more preferable.

<Labeled antibody>
In the step (I), the labeled antibody 150 is used to contact the analyte 160 contained in the sample to form a complex 170 including the analyte 160 and the labeled antibody 150. The labeled antibody 150 is obtained by labeling an antibody that specifically binds to the analyte 160 with the resin-metal complex 100 having a structure in which a plurality of metal particles 20 are immobilized on the resin particle 10. Here, “labeling” means that the resin-metal complex 100 is directly attached to the antibody to the extent that the resin-metal complex 100 is not detached from the labeled antibody 150 in steps (I) to (III). It means that it is fixed indirectly by chemical or physical bonding or adsorption. For example, the labeled antibody 150 may be one in which the resin-metal complex 100 is directly bonded to the antibody, or the antibody and the resin-metal complex 100 are bonded via an arbitrary linker molecule. Or those fixed to insoluble particles.

In the present embodiment, the “antibody” is not particularly limited. For example, in addition to a polyclonal antibody, a monoclonal antibody, an antibody obtained by genetic recombination, an antibody fragment [for example, an H chain , L chain, Fab, F (ab ′) ₂ etc.] can be used. Further, IgG, IgM, IgA, IgE, or IgD may be used as the immunoglobulin. The animal species that produce the antibody may be humans or animals other than humans (eg, mice, rats, rabbits, goats, horses, etc.). Specific examples of the antibody include 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.

<Preferred production method of labeled antibody>
Next, a preferred method for producing the labeled antibody 150 will be described. The production of the labeled antibody 150 is at least the following step A;
Step A) including the step of obtaining labeled antibody 150 by mixing and binding the resin-metal complex 100 with the antibody under the first pH condition, preferably further comprising Step B;
Step B) A step of treating the labeled antibody 150 under a second pH condition can be included.

[Step A]
In step A, the resin-metal complex 100 is mixed with an antibody under a first pH condition to obtain a labeled antibody 150. In step A, the solid resin-metal complex 100 is preferably brought into contact with the antibody in a state of being dispersed in the liquid phase. The first pH condition varies depending on the metal species of the metal particles 20 in the resin-metal composite 100.

  When the metal particles 20 of the resin-metal composite 100 are gold particles (including gold alloy particles; the same applies hereinafter), the first pH condition is that the binding with the antibody is the resin-metal composite 100. From the viewpoint of uniformly contacting the resin-metal complex 100 and the antibody while maintaining the dispersion of the antibody and the activity of the antibody, conditions within the range of pH 2 to 7 are preferred, and acidic conditions such as pH 2.5 to 5.5 are preferred. Within the range is more preferable. When the metal particles 20 are gold particles, when the resin-metal complex 100 and the antibody are bound under conditions where the pH is less than 2, the antibody may be denatured and deactivated due to strong acidity. When the metal complex 100 and the antibody are mixed, they aggregate and become difficult to disperse. However, if the antibody is not inactivated by strong acidity, the treatment can be performed even at a pH of less than 2.

  Further, when the metal particles 20 of the resin-metal composite 100 are particles other than gold, such as platinum, palladium, or alloys thereof, the first pH condition is that the binding with the antibody is the resin-metal composite. From the viewpoint of uniformly contacting the resin-metal complex 100 and the antibody while maintaining the dispersion of the body 100 and the activity of the antibody, conditions within the range of pH 2 to 10 are preferable, and for example, the range of pH 5 to 9 is more preferable. . When the metal particles 20 are particles other than gold, if the conditions for bonding the resin-metal complex 100 and the antibody are less than pH 2, the antibody may be denatured and deactivated due to strong acidity. When the resin-metal complex 100 and the antibody are mixed, they aggregate and become difficult to disperse. However, if the antibody is not inactivated by strong acidity, the treatment can be performed even at a pH of less than 2.

  Step A is preferably performed in a binding buffer adjusted to the first pH condition. For example, a predetermined amount of the resin-metal complex 100 is mixed with the binding buffer adjusted to the above pH and mixed thoroughly. As the binding buffer, for example, a boric acid solution adjusted to a predetermined concentration can be used. The pH of the binding buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide.

  Next, a labeled antibody-containing liquid can be obtained by adding a predetermined amount of the antibody to the obtained mixed liquid, sufficiently stirring and mixing. The labeled antibody-containing liquid thus obtained can be fractionated only with the labeled antibody 150 as a solid part by solid-liquid separation means such as centrifugation.

[Step B]
In the process B, the labeled antibody 150 obtained in the process A is treated under the second pH condition to perform blocking that suppresses nonspecific adsorption to the labeled antibody 150. In this case, the labeled antibody 150 separated by the solid-liquid separation means is dispersed in the liquid phase under the second pH condition. This blocking condition varies depending on the metal species of the metal particles 20 in the resin-metal composite 100.

  When the metal particles 20 of the resin-metal composite 100 are gold particles, the second pH condition is, for example, within a range of pH 2 to 9 from the viewpoint of maintaining the activity of the antibody and suppressing aggregation of the labeled antibody 150. From the viewpoint of suppressing non-specific adsorption of the labeled antibody 150, acidic conditions, for example, in the range of pH 2 to 6, are more preferable. If the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if it exceeds pH 9, the labeled antibody 150 aggregates, making dispersion difficult.

  Further, when the metal particles 20 of the resin-metal complex 100 are particles other than gold, the second pH condition is, for example, from pH 2 to pH 2 from the viewpoint of maintaining the activity of the antibody and suppressing aggregation of the labeled antibody 150. The range of 10 is preferable, and from the viewpoint of suppressing nonspecific adsorption of the labeled antibody 150, the range of pH 5 to 9 is more preferable. If the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if it exceeds pH 10, the labeled antibody 150 aggregates, making dispersion difficult.

  The step B is preferably performed using a blocking buffer adjusted to the second pH condition. For example, the blocking buffer adjusted to the above pH is added to a predetermined amount of labeled antibody 150, and the labeled antibody 150 is uniformly dispersed in the blocking buffer. As the blocking buffer, for example, a protein solution that does not bind to the detection target is preferably used. Examples of proteins that can be used in the blocking buffer include bovine serum albumin, ovalbumin, casein, and gelatin. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. For dispersing the labeled antibody 150, it is preferable to use a dispersing means such as ultrasonic treatment. In this way, a dispersion in which the labeled antibody 150 is uniformly dispersed is obtained.

  A dispersion of labeled antibody 150 is obtained as described above. From this dispersion, only the labeled antibody 150 can be fractionated as a solid part by solid-liquid separation means such as centrifugation. Moreover, a cleaning process, a preservation | save process, etc. can be implemented as needed. Hereinafter, the cleaning process and the storage process will be described.

(Cleaning process)
In the washing treatment, a washing buffer solution is added to the labeled antibody 150 sorted by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the washing buffer solution. For dispersion, for example, a dispersion means such as ultrasonic treatment is preferably used. The washing buffer is not particularly limited, but for example, a Tris buffer solution, a glycinamide buffer solution, an arginine buffer solution, or the like having a predetermined concentration adjusted within a pH range of 8 to 9 may be used. it can. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. The washing treatment of the labeled antibody 150 can be repeated a plurality of times as necessary.

(Save process)
In the storage process, a storage buffer is added to the labeled antibody 150 collected by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the storage buffer. For dispersion, for example, a dispersion means such as ultrasonic treatment is preferably used. As the storage buffer, for example, a solution obtained by adding a predetermined concentration of an anti-aggregation agent and / or stabilizer to the washing buffer can be used. As the aggregation inhibitor, for example, saccharides typified by sucrose, maltose, lactose, trehalose, polyhydric alcohols typified by glycerin, polyvinyl alcohol, and the like can be used. The stabilizer is not particularly limited. For example, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin can be used. Thus, the preservation | save process of the labeled antibody 150 can be performed.

  In each of the above steps, a surfactant or a preservative such as sodium azide or paraoxybenzoate can be used as necessary.

[Analyte detection / quantification kit]
An analyte measurement kit according to an embodiment of the present invention uses, for example, a lateral flow type chromatographic test strip 200 and an analyte 160 contained in a sample based on the analyte measurement method of the present embodiment. It is a kit for detecting or quantifying.

The kit of this embodiment is
A membrane 110;
A lateral flow type chromatographic test strip 200 including a determination unit 130 in which a capture ligand that specifically binds to the analyte 160 is fixed to the membrane 110;
A detection reagent comprising a labeled antibody 150 in which an antibody that specifically binds to the analyte 160 is labeled with a resin-metal complex 100 having a structure in which a plurality of metal particles 20 are immobilized on resin particles 10;
Is included. The kit of the present embodiment may further include other components as necessary.

  In using the kit according to the present invention, after the step (I) is performed by bringing the analyte 160 in the sample into contact with the labeled antibody 150 in the detection reagent, the reaction section or the sample addition section 120 of the test strip 200 is performed. The step (II) and the step (III) may be sequentially performed by supplying a sample. Alternatively, after a detection reagent is applied to the upstream side of the determination unit 130 of the test strip 200 and appropriately dried to form a reaction part, the formed reaction part or a position upstream of the reaction part (for example, The sample may be added to the sample addition unit 120) and the steps (I) to (III) may be sequentially performed.

  EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples. Unless otherwise specified in the following examples and comparative examples, various measurements and evaluations are as follows.

<Measurement of absorbance of resin-metal composite>
Absorbance of the resin-metal composite was measured by placing a resin-metal composite dispersion (dispersion medium: water) prepared at 0.01 wt% into an optical white glass cell (optical path length 10 mm), and an instantaneous multi-photometry system (Otsuka The absorbance at 570 nm for gold and 400 nm for platinum was measured using MCPD-3700) manufactured by Denki Co., Ltd.

<Measurement of solid content concentration and metal loading>
A magnetic crucible was charged with 1 g of a resin-metal composite dispersion before concentration adjustment, and heat treatment was performed at 70 ° C. for 3 hours. The weight before and after the heat treatment was measured, and the solid content concentration was calculated by the following formula.

  Solid content concentration (wt%) = [weight after drying (g) / weight before drying (g)] × 100

The sample after the heat treatment was further heat-treated at 500 ° C. for 5 hours, the weight before and after the heat treatment was measured, and the metal loading was calculated from the following formula.
Metal loading (wt%) =
[Weight after heat treatment at 500 ° C. (g) / Weight before heat treatment at 500 ° C. (g)] × 100

<Measurement of average particle diameter of resin particles and resin-metal composite>
The measurement was performed using a disk centrifugal particle size distribution analyzer (CPS Disc Centrifuge DC24000 UHR, manufactured by CPS instruments, Inc.). The measurement was performed in a state where the resin-metal composite was dispersed in water.

<Measurement of average particle diameter of metal particles>
From a substrate observed by dropping a resin-metal composite dispersion onto a metallic mesh with a carbon support film, observed with a field emission scanning electron microscope (FE-SEM; manufactured by Hitachi High-Technologies Corporation, SU-9000). The area average diameter of 100 arbitrary metal particles was measured.
<Evaluation by immunochromatograph>
Using the resin-metal complex-labeled antibody dispersion prepared in each example, the following immunochromatographic measurement was performed to evaluate the performance of the resin-metal complex.
(Evaluation method)
The evaluation was performed using a monochrome screen for influenza A evaluation (manufactured by Adtech), and the coloring levels after 5 minutes, 10 minutes and 15 minutes were compared. In the performance evaluation, the antigen used was a 2-fold dilution series (1-fold to 1024-fold) of influenza A positive control (APC) (the virus concentration before dilution of APC was 5000 FFU / ml).
(Evaluation procedure)
3 μl of the resin-metal complex labeled antibody dispersion was added to each well of a 96-well plate, and 100 μl each of the APC 2-fold dilution series (1-fold to 1024-fold) and the negative control were mixed. Next, 50 μl of this mixed dispersion was added to a monochrome screen for evaluation of influenza A, and the color development level after 5 minutes, 10 minutes and 15 minutes was evaluated. A color development level of 0.5 or more after 15 minutes was judged as “good”. The color development level was determined using a color sample for gold colloid determination (manufactured by Adtec).

[Example 1]
<Synthesis of resin particles>
Trioctyl ammonium chloride (1.20 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) are dissolved in 300 g of pure water, and then 2-vinylpyridine (40.00 g) and divinylbenzene (10.00 g) are added. The mixture was stirred at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (0.250 g) dissolved in 18.00 g of pure water was added dropwise over about 2 minutes, and the mixture was stirred at 150 rpm and 60 ° C. for 3.5 hours. By stirring, resin particles A-1 having an average particle diameter of 307 nm were obtained. After sedimentation by centrifugation (9000 rpm, 40 minutes) and removal of the supernatant, the operation of dispersing again in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-1.

<Synthesis of resin-gold composite>
After adding 11.25 g of pure water to 10 wt% resin particle dispersion B-1 (4.57 g) prepared in Example 1, 400 mM chloroauric acid aqueous solution (7.34 g) was added and stirred at room temperature for 3 hours. . The mixture was allowed to stand for 24 hours, and then the resin particles A-1 were precipitated by centrifugation (8000 rpm, 20 minutes), and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare 2.5 wt% gold ion adsorption resin particle dispersion C-1.

  Next, the above-mentioned 2.5 wt% gold ion adsorption resin particle dispersion C-1 (4.33 g) was added to 158 g of pure water, and stirred at 150 rpm at 3 ° C., a 528 mM dimethylamine borane aqueous solution (1.0 g). Was added dropwise over 2 minutes, followed by stirring at 3 ° C. for 3 hours and at room temperature for 2 hours to obtain a resin-gold composite D-1 having an average particle diameter of 310 nm. The resin-gold complex D-1 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of redispersing in 140 g of pure water was repeated three times, followed by purification by dialysis, By adjusting, 1 wt% of resin-gold composite dispersion E-1 was obtained. The absorbance of the resin-gold complex F-1 in the resin-gold complex dispersion E-1 was 1.07 as a result of measurement according to the above method. Moreover, the average particle diameter of the gold particles in F-1 was 22.7 nm, and the amount of gold supported was 49.5 wt%.

<Evaluation of immunochromatography>
100 μg of influenza antibody was mixed with 1 ml (0.1 wt%) of the obtained resin-gold complex dispersion E-1 and stirred at room temperature for about 3 hours to bind the antibody to the resin-gold complex F-1. It was. The bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the surface of the resin-gold complex F-1. The resin-gold complex labeled antibody dispersion G-1 was prepared by centrifuging at 12000 rpm for 5 minutes at 4 ° C. and then suspended in a buffer containing 0.2% bovine serum albumin.

  Using the produced resin-gold complex labeled antibody dispersion G-1, measurements were made by immunochromatography to evaluate the performance of the resin-gold complex dispersion E-1. The results are shown in Table 1.

  From Table 1 above, it was confirmed that the resin-gold complex-labeled antibody dispersion G-1 exhibited a good color development with a color development level of 0.5 or more after 15 minutes for a 64-fold diluted antigen. It was.

[Example 2]
<Synthesis of resin-platinum composite>
After adding 14 g of pure water to 10 wt% resin particle dispersion B-1 (4.57 g) prepared in Example 1, a 400 mM chloroplatinic acid aqueous solution (5.00 g) was added, and the mixture was stirred at room temperature for 3 hours. Then, after leaving still for 24 hours, the resin particle A-1 was precipitated by centrifugation (8000 rpm, 20 minutes), and the operation | work which removes a supernatant liquid was repeated 3 times, and the excess chloroplatinic acid was removed. Thereafter, the concentration was adjusted to prepare 5 wt% platinum ion adsorption resin particle dispersion C-2.

  Next, 5 wt% platinum ion adsorption resin particle dispersion C-2 (2.06 g) was added to 139 g of pure water, and 132 mM dimethylamine borane aqueous solution (4 g) was added over 20 minutes while stirring at 150 rpm and 3 ° C. After dripping, the resin-platinum composite D-2 with an average particle diameter of 310 nm was obtained by stirring at 3 degreeC for 3 hours, and at room temperature for 2 hours. The resin-platinum complex D-2 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of redispersing in 140 g of pure water was repeated three times, followed by purification by dialysis, By adjusting, a 1 wt% resin-platinum composite dispersion E-2 was obtained. The absorbance of the resin-platinum complex F-2 in the resin-platinum complex dispersion E-2 was 1.62 as a result of measurement according to the above method. Moreover, the average particle diameter of the platinum particles in the resin-platinum composite F-2 was less than 5.0 nm, and the supported amount of platinum was 33.5 wt%.

<Evaluation of immunochromatography>
100 μg of influenza antibody was mixed with 1 ml (0.1 wt%) of the obtained resin-platinum complex dispersion E-2 and stirred at room temperature for about 3 hours to bind the antibody to the resin-platinum complex F-2. It was. A bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the surface of the resin-platinum complex F-2. The resin-platinum complex-labeled antibody dispersion G-2 was prepared by centrifuging at 12000 rpm and 4 ° C. for 5 minutes, and suspending in a buffer containing 0.2% bovine serum albumin.

  Using the produced resin-platinum complex labeled antibody dispersion G-2, measurement by immunochromatography was performed to evaluate the performance of the resin-platinum complex dispersion E-2. The results are shown in Table 2.

  From Table 2 above, it was confirmed that the resin-platinum complex-labeled antibody dispersion G-2 exhibited a good color development with a color development level of 0.5 or more after 15 minutes for a 128-fold diluted antigen. It was.

[Example 3]
Trioctyl ammonium chloride (1.20 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) are dissolved in 300 g of pure water, and then 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) are added. The mixture was stirred at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (0.250 g) dissolved in 18.00 g of pure water was added dropwise over about 2 minutes, and the mixture was stirred at 150 rpm and 60 ° C. for 3.5 hours. By stirring, resin particles A-3 having an average particle diameter of 361 nm were obtained. After sedimentation by centrifugation (9000 rpm, 40 minutes) and removal of the supernatant, the operation of dispersing again in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-3.
<Synthesis of resin-gold composite>
After adding 255 g of pure water to 10 wt% resin particle dispersion B-3 (92.0 g) prepared in Example 3, 400 mM chloroauric acid aqueous solution (147 g) was added, and the mixture was stirred at room temperature for 3 hours. The mixture was allowed to stand for 24 hours, and then the resin particles A-3 were precipitated by centrifugation (3000 rpm, 30 minutes), and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare 2.5 wt% gold ion adsorption resin particle dispersion C-3.

  Next, the 2.5 wt% gold ion adsorbing resin particle dispersion C-3 (43.3 g) is added to 1580 g of pure water, and stirred at 150 rpm at 3 ° C., a 528 mM dimethylamine borane aqueous solution (10.0 g). Was added dropwise over 2 minutes, and the mixture was stirred at 3 ° C. for 3 hours and at room temperature for 2 hours to obtain a resin-gold composite D-3 having an average particle size of 365 nm. After the resin-gold complex D-3 was precipitated by centrifugation (3000 rpm, 30 minutes), the supernatant was removed, and the work of redispersing in 1620 g of pure water was repeated three times, followed by purification by dialysis. By adjusting, a 1 wt% resin-gold composite dispersion E-3 was obtained. The absorbance of the resin-gold complex F-3 in the resin-gold complex dispersion E-3 was 0.96 as a result of measurement according to the above method. In addition, the average particle size of gold particles in the resin-gold composite F-3 was 23.1 nm, and the amount of gold supported was 54.1 wt%. In this resin-gold composite F-3, the gold particles are partly exposed having encapsulated particles completely encapsulated in the resin particles, a part embedded in the resin particle, and a part exposed outside the resin particle. It contained gold particles and surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles. In addition, 100% of gold particles were present in the surface layer portion.

<Evaluation of immunochromatography>
100 μg of influenza antibody was mixed with 1 ml (0.1 wt%) of the obtained resin-gold complex dispersion E-3 and stirred at room temperature for about 3 hours to bind the antibody to the resin-gold complex F-3. It was. The bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the surface of the resin-gold complex F-3. The resin-gold complex labeled antibody dispersion G-3 was prepared by centrifuging at 12000 rpm and 4 ° C. for 5 minutes and then suspending in a buffer containing 0.2% bovine serum albumin.

  Using the prepared resin-gold complex labeled antibody dispersion G-3, measurement by immunochromatography was performed to evaluate the performance of the resin-gold complex dispersion E-3. The results are shown in Table 3.

  From Table 3 above, it was confirmed that the resin-gold complex-labeled antibody dispersion G-3 showed good color development with a color development level of 0.5 or more after 15 minutes for a 256-fold diluted antigen. It was.

[Example 4]
<Synthesis of resin-platinum composite>
After adding 54 g of pure water to 10 wt% resin particle dispersion B-3 (18.2 g) prepared in Example 3, 400 mM chloroplatinic acid aqueous solution (20 g) was added, and the mixture was stirred at 30 ° C. for 3 hours. The mixture was allowed to stand for 24 hours, and then the resin particles A-3 were precipitated by centrifugation (3000 rpm, 30 minutes), and the operation of removing the supernatant was repeated three times to remove excess chloroplatinic acid. Thereafter, the concentration was adjusted to prepare 5 wt% platinum ion adsorbing resin particle dispersion C-4.

  Next, 5 wt% platinum ion adsorption resin particle dispersion C-4 (20.6 g) was added to 1392 g of pure water, and 132 mM dimethylamine borane aqueous solution (40 g) was added over 20 minutes while stirring at 150 rpm and 3 ° C. After the dropwise addition, the mixture was stirred at 3 ° C. for 3 hours and at room temperature for 2 hours to obtain a resin-platinum composite D-4 having an average particle diameter of 369 nm. The resin-platinum complex D-4 was precipitated by centrifugation (3000 rpm, 10 minutes), the supernatant was removed, and then the work of redispersing in 1400 g of pure water was repeated three times, followed by purification by dialysis, By adjusting, 1 wt% of resin-platinum composite dispersion E-4 was obtained. The absorbance of the resin-platinum complex F-4 in the resin-platinum complex dispersion E-4 was 1.61 as a result of measurement according to the above method. Moreover, the average particle diameter of the platinum particles in the resin-platinum composite F-4 was less than 5.0 nm, and the supported amount of platinum was 40.0 wt%.

<Evaluation of immunochromatography>
The same operation as in Example 2 was performed to prepare a resin-platinum complex labeled antibody dispersion G-4.

  Using the prepared resin-platinum complex labeled antibody dispersion G-4, measurement by immunochromatography was performed to evaluate the performance of the resin-platinum complex dispersion E-4. The results are shown in Table 4.

  From Table 4 above, it was confirmed that the resin-platinum complex-labeled antibody dispersion G-4 exhibited a good color development with a color development level of 0.5 or more after 15 minutes for a 512-fold diluted antigen. It was.

[Example 5]
Trioctyl ammonium chloride (0.40 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) are dissolved in 300 g of pure water, and then 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) are added. The mixture was stirred at 150 rpm at 30 ° C. for 50 minutes and then at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (0.250 g) dissolved in 18.00 g of pure water was added dropwise over about 2 minutes, and the mixture was stirred at 150 rpm and 60 ° C. for 3.5 hours. By stirring, resin particles A-5 having an average particle diameter of 428 nm were obtained. After sedimentation by centrifugation (9000 rpm, 40 minutes) and removal of the supernatant, the operation of dispersing again in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-5.
<Synthesis of resin-gold composite>
After adding 11.25 g of pure water to 10 wt% resin particle dispersion B-5 (4.57 g) produced in Example 5, 400 mM chloroauric acid aqueous solution (7.34 g) was added and stirred at room temperature for 3 hours. . After this mixture was allowed to stand for 24 hours, resin particles A-5 were precipitated by centrifugation (8000 rpm, 20 minutes), and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare 2.5 wt% gold ion adsorption resin particle dispersion C-5.
Next, the above-mentioned 2.5 wt% gold ion adsorption resin particle dispersion C-5 (4.33 g) was added to 158 g of pure water, and stirred at 150 rpm at 3 ° C., a 528 mM dimethylamine borane aqueous solution (1.0 g). Was added dropwise over 2 minutes, followed by stirring at 3 ° C. for 3 hours and at room temperature for 2 hours to obtain a resin-gold composite D-5 having an average particle size of 438 nm. The resin-gold complex D-5 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of redispersing in 140 g of pure water was repeated three times, followed by purification by dialysis, By adjusting, 1 wt% of resin-gold composite dispersion E-5 was obtained. The absorbance of the resin-gold complex F-5 in the resin-gold complex dispersion E-5 was 1.02 as a result of measurement according to the above method. In addition, the average particle diameter of the gold particles in the resin-gold composite F-5 was 22.1 nm, and the amount of gold supported was 51.1 wt%.

<Evaluation of immunochromatography>
100 μg of influenza antibody was mixed with 1 ml (0.1 wt%) of the obtained resin-gold complex dispersion E-5 and stirred at room temperature for about 3 hours to bind the antibody to resin-gold complex F-5. It was. The bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the surface of the resin-gold complex F-5. The resin-gold complex labeled antibody dispersion G-5 was prepared by centrifuging at 12000 rpm and 4 ° C. for 5 minutes and then suspending in a buffer containing 0.2% bovine serum albumin.

  Using the prepared resin-gold complex labeled antibody dispersion G-5, measurement by immunochromatography was performed to evaluate the performance of the resin-gold complex dispersion E-5. The results are shown in Table 5.

  From Table 5 above, it was confirmed that the resin-gold complex labeled antibody dispersion G-5 showed good color development with a color development level of 0.5 or more after 15 minutes for a 256-fold diluted antigen. It was.

[Example 6]
<Synthesis of resin-platinum composite>
After adding 14 g of pure water to 10 wt% resin particle dispersion B-5 (4.57 g) prepared in Example 5, 400 mM chloroplatinic acid aqueous solution (5.00 g) was added, and the mixture was stirred at room temperature for 3 hours. Then, after leaving still for 24 hours, the resin particle A-5 was precipitated by centrifugation (8000 rpm, 20 minutes), and the operation | work which removes a supernatant was repeated 3 times, and the excess chloroplatinic acid was removed. Thereafter, the concentration was adjusted to prepare 5 wt% platinum ion adsorbing resin particle dispersion C-6. Next, 5 wt% platinum ion adsorption resin particle dispersion C-6 (2.06 g) was added to 139 g of pure water, and 132 mM dimethylamine borane aqueous solution (4 g) was added over 20 minutes while stirring at 150 rpm at 3 ° C. After the dropwise addition, the mixture was stirred at 3 ° C. for 3 hours and at room temperature for 2 hours to obtain a resin-platinum complex D-6 having an average particle diameter of 459 nm. The resin-platinum complex D-6 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of redispersing in 140 g of pure water was repeated three times, followed by purification by dialysis. By adjusting, 1 wt% of a resin-platinum composite dispersion E-6 was obtained. The absorbance of the resin-platinum complex F-6 in the resin-platinum complex dispersion E-6 was 1.66 as a result of measurement according to the above method. Moreover, the average particle diameter of the platinum particles in the resin-platinum composite F-6 was less than 5.0 nm, and the amount of platinum supported was 37.8 wt%.

<Evaluation of immunochromatography>
The same operation as in Example 2 was performed to prepare a resin-platinum complex labeled antibody dispersion G-6.

  Using the produced resin-platinum complex labeled antibody dispersion G-6, measurement by immunochromatography was performed to evaluate the performance of the resin-platinum complex dispersion E-6. The results are shown in Table 6.

  From Table 6 above, it was confirmed that the resin-platinum complex-labeled antibody dispersion G-6 showed good color development with a color development level of 0.5 or more after 15 minutes for a 1024-fold diluted antigen. It was.

[Comparative Example 1]
Trioctyl ammonium chloride (3.00 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) are dissolved in 300 g of pure water, and then 2-vinylpyridine (49.50 g) and divinylbenzene (0.50 g) are added. The mixture was stirred at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (0.250 g) dissolved in 18.00 g of pure water was added dropwise over about 2 minutes, and the mixture was stirred at 150 rpm and 60 ° C. for 3.5 hours. By stirring, resin particles A-7 having an average particle diameter of 370 nm were obtained. After sedimentation by centrifugation (9000 rpm, 40 minutes) and removal of the supernatant, the operation of dispersing again in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-7.

<Synthesis of resin-gold composite>
After adding 11.25 g of pure water to 10 wt% resin particle dispersion B-7 (4.57 g) prepared in Comparative Example 1, 400 mM chloroauric acid aqueous solution (7.34 g) was added and stirred at room temperature for 3 hours. . The mixture was allowed to stand for 24 hours, and then the resin particles A-7 were precipitated by centrifugation (8000 rpm, 20 minutes), and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare 2.5 wt% gold ion adsorption resin particle dispersion C-7.

  Next, the above-mentioned 2.5 wt% gold ion adsorption resin particle dispersion C-7 (4.33 g) was added to 158 g of pure water, and stirred at 150 rpm at 3 ° C., a 528 mM dimethylamine borane aqueous solution (1.0 g). Was added dropwise over 2 minutes, followed by stirring at 3 ° C. for 3 hours and at room temperature for 2 hours to obtain a resin-gold composite D-7 having an average particle diameter of 393 nm. The resin-gold complex D-7 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of re-dispersing in 140 g of pure water was repeated three times, followed by purification by dialysis treatment. By adjusting, a 1 wt% resin-gold composite dispersion E-7 was obtained. The absorbance of the resin-gold complex F-7 in the resin-gold complex dispersion E-7 was 0.92 as a result of measurement according to the above method. In addition, the average particle diameter of the gold particles in the resin-gold composite F-7 was 12.3 nm, and the amount of gold supported was 55.9 wt%.

<Evaluation of immunochromatography>
100 μg of influenza antibody was mixed with 1 ml (0.1 wt%) of the obtained resin-gold complex dispersion E-7 and stirred at room temperature for about 3 hours to bind the antibody to resin-gold complex F-7. It was. A bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the surface of the resin-gold complex F-7. The resin was collected by centrifuging at 12000 rpm and 4 ° C. for 5 minutes, and suspended in a buffer containing 0.2% bovine serum albumin to prepare a resin-gold complex labeled antibody dispersion G-7.

  Using the produced resin-gold complex labeled antibody dispersion G-7, measurement by immunochromatography was performed to evaluate the performance of the resin-gold complex dispersion E-7. The results are shown in Table 7.

  From Table 7 above, it was confirmed that the resin-gold complex-labeled antibody dispersion G-7 showed good color development with a color development level of 0.5 or more after 15 minutes for a 64-fold diluted antigen. It was.

[Comparative Example 2]
<Synthesis of resin-platinum composite>
After adding 14 g of pure water to 10 wt% resin particle dispersion B-7 (4.57 g) prepared in Comparative Example 1, a 400 mM chloroplatinic acid aqueous solution (5.00 g) was added, and the mixture was stirred at room temperature for 3 hours. Then, after leaving still for 24 hours, the resin particle A-7 was precipitated by centrifugation (8000 rpm, 20 minutes), and the operation | work which removes a supernatant was repeated 3 times, and the excess chloroplatinic acid was removed. Thereafter, the concentration was adjusted to prepare 5 wt% platinum ion adsorbing resin particle dispersion C-8.
Next, 5 wt% platinum ion adsorption resin particle dispersion C-8 (2.06 g) was added to 139 g of pure water, and 132 mM dimethylamine borane aqueous solution (4 g) was added over 20 minutes while stirring at 150 rpm and 3 ° C. After dripping, the resin-platinum composite D-8 with an average particle diameter of 382 nm was obtained by stirring at 3 degreeC for 3 hours, and at room temperature for 2 hours. The resin-platinum complex D-8 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of redispersing in 140 g of pure water was repeated three times, followed by purification by dialysis. By adjusting, 1 wt% of resin-platinum composite dispersion E-8 was obtained. The absorbance of the resin-platinum complex F-8 in the resin-platinum complex dispersion E-8 was 1.50 as a result of measurement according to the above method. Moreover, the average particle diameter of the platinum particles in the resin-platinum composite F-8 was less than 5.0 nm, and the supported amount of platinum was 38.4 wt%.

<Evaluation of immunochromatography>
The same operation as in Example 2 was performed to prepare a resin-platinum complex labeled antibody dispersion G-8.

  Using the produced resin-platinum complex labeled antibody dispersion G-8, measurement by immunochromatography was performed to evaluate the performance of the resin-platinum complex dispersion E-8. The results are shown in Table 8.

  From Table 8 above, it was confirmed that the resin-platinum complex-labeled antibody showed a good color development with a color development level of 0.5 or more after 15 minutes for 512-fold diluted antigen.

[Comparative Example 3]
Trioctyl ammonium chloride (1.50 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) are dissolved in 300 g of pure water, and then 2-vinylpyridine (49.50 g) and divinylbenzene (0.50 g) are added. The mixture was stirred at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (0.50 g) dissolved in 18.00 g of pure water was added dropwise over about 2 minutes, and the mixture was stirred at 150 rpm and 60 ° C. for 3.5 hours. By stirring, resin particles A-9 having an average particle diameter of 430 nm were obtained. After sedimentation by centrifugation (9000 rpm, 40 minutes) and removal of the supernatant, the operation of dispersing again in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-9.

<Synthesis of resin-gold composite>
After adding 11.25 g of pure water to 10 wt% resin particle dispersion B-9 (4.57 g) prepared in Comparative Example 3, a 400 mM chloroauric acid aqueous solution (7.34 g) was added, and the mixture was stirred at room temperature for 3 hours. . The mixture was allowed to stand for 24 hours, and then the resin particles A-9 were precipitated by centrifugation (8000 rpm, 20 minutes), and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare 2.5 wt% gold ion adsorption resin particle dispersion C-9.

  Next, the above-mentioned 2.5 wt% gold ion adsorption resin particle dispersion C-9 (4.33 g) was added to 158 g of pure water, and stirred at 150 rpm at 3 ° C., a 528 mM dimethylamine borane aqueous solution (1.0 g). Was added dropwise over 2 minutes, and the mixture was stirred at 3 ° C. for 3 hours and at room temperature for 2 hours to obtain a resin-gold composite D-9 having an average particle diameter of 442 nm. The resin-gold complex D-9 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of redispersing in 140 g of pure water was repeated three times, followed by purification by dialysis, By adjusting, a 1 wt% resin-gold composite dispersion E-9 was obtained. The absorbance of the resin-gold complex F-9 in the resin-gold complex dispersion E-9 was 1.26 as a result of measurement according to the above method. In addition, the average particle diameter of the gold particles in the resin-gold composite F-9 was 16.1 nm, and the amount of gold supported was 53.9 wt%.

<Evaluation of immunochromatography>
100 μg of influenza antibody was mixed with 1 ml (0.1 wt%) of the obtained resin-gold complex dispersion E-9 and stirred at room temperature for about 3 hours to bind the antibody to resin-gold complex F-9. It was. A bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the surface of the resin-gold complex F-9. The resin-gold complex labeled antibody dispersion G-9 was prepared by centrifuging at 12000 rpm for 5 minutes at 4 ° C. and then suspending in a buffer containing 0.2% bovine serum albumin.

  Using the produced resin-gold complex labeled antibody dispersion G-9, measurement by immunochromatography was performed to evaluate the performance of the resin-gold complex dispersion E-9. The results are shown in Table 9.

  From Table 9 above, it was confirmed that the resin-gold complex-labeled antibody dispersion G-9 showed good color development with a color development level of 0.5 or more after 15 minutes for a 256-fold diluted antigen. It was.

[Comparative Example 4]
<Synthesis of resin-platinum composite>
After adding 14 g of pure water to 10 wt% resin particle dispersion B-9 (4.57 g) prepared in Comparative Example 3, 400 mM chloroplatinic acid aqueous solution (5.00 g) was added, and the mixture was stirred at room temperature for 3 hours. Then, after leaving still for 24 hours, the resin particle A-9 was precipitated by centrifugation (8000 rpm, 20 minutes), and the operation | work which removes a supernatant liquid was repeated 3 times, and the excess chloroplatinic acid was removed. Thereafter, the concentration was adjusted to prepare 5 wt% platinum ion adsorbing resin particle dispersion C-10.

  Next, 5 wt% platinum ion adsorption resin particle dispersion C-10 (2.06 g) was added to 139 g of pure water, and 132 mM dimethylamine borane aqueous solution (4 g) was added over 20 minutes while stirring at 150 rpm and 3 ° C. After dripping, the resin-platinum composite D-10 with an average particle diameter of 454 nm was obtained by stirring at 3 degreeC for 3 hours, and room temperature for 2 hours. The resin-platinum complex D-10 was precipitated by centrifugation (8000 rpm, 10 minutes), the supernatant was removed, and the work of redispersing in 140 g of pure water was repeated three times, followed by purification by dialysis, By adjusting, 1 wt% of a resin-platinum composite dispersion E-10 was obtained. The absorbance of the resin-platinum complex F-10 in the resin-platinum complex dispersion E-10 was 1.45 as a result of measurement according to the above method. Moreover, the average particle diameter of the platinum particles in the resin-platinum composite F-10 was less than 5.0 nm, and the supported amount of platinum was 37.7 wt%.

<Evaluation of immunochromatography>
The same operation as in Example 2 was performed to prepare a resin-platinum complex labeled antibody dispersion G-10.

  Using the produced resin-platinum complex labeled antibody dispersion G-10, measurement by immunochromatography was performed to evaluate the performance of the resin-platinum complex dispersion E-10. The results are shown in Table 10.

  From Table 10 above, it was confirmed that the resin-platinum complex-labeled antibody dispersion G-10 exhibited a good color development with a color development level of 0.5 or more after 15 minutes for a 1024-fold diluted antigen. It was.

[Adhesion test]
The resin-metal composite dispersions obtained in Examples 1 to 3 and Comparative Examples 1 and 2 (concentration: 1% by mass; dispersion medium water) are placed in a container of the following material, and 24 hours later on the inner wall of the container The adhesion of the metal-resin composite was visually observed, and it was judged that there was no adhesion as “◯” and that there was no adhesion as “x”. The results are shown in Table 11. The weight composition ratio in Table 11 means the weight composition ratio (vinylpyridine: divinylbenzene) in the copolymer of vinylpyridine and divinylbenzene.
<Container material>
PP: Polypropylene PC: Polycarbonate PET: Polyethylene terephthalate

  In addition, photographs showing the results of adhesion tests on PP containers, PC containers, and PET containers are shown in FIGS. 4, 5A, 5B, and 6, respectively. 4, 5 </ b> A and 5 </ b> B, and FIG. 6, A is Comparative Example 1, B is Example 3, C is Example 1, D is Comparative Example 2, E is Example 4, and F is Example 2. Is shown.

  From Table 11 and FIGS. 4 to 6, the weight composition ratio in the copolymer of vinylpyridine and divinylbenzene was controlled in PP containers, PC containers, and PET containers that are widely used as reagent containers for immunological measurement. In the example, there was no adhesion of the resin-metal composite to the inner wall of the container. On the other hand, in Comparative Examples 1 and 2 in which the weight composition ratio in the copolymer of vinylpyridine and divinylbenzene is 49.5: 0.5, any of the PP container, the PC container, and the PET container is attached to the inner wall of the container. Adhesion of the resin-metal composite was observed.

  As mentioned above, although embodiment of this invention was described in detail for the purpose of illustration, this invention is not restrict | limited to the said embodiment.

DESCRIPTION OF SYMBOLS 10 ... Resin particle, 20 ... Metal particle, 30 ... Encapsulated metal particle, 40 ... Partially exposed metal particle, 50 ... Surface adsorbed metal particle, 60 ... Surface layer part, 100 ... Resin-metal composite, 110 ... Membrane, 120 ... Sample addition unit, 130 ... determination unit, 131 ... capture ligand, 140 ... liquid absorption part, 150 ... labeled antibody, 160 ... analyte, 170 ... complex, 200 ... test strip

Claims (13)

Resin particles,
A plurality of metal particles fixed to the resin particles;
With
Resin in which the resin particles are a copolymer of a nitrogen-containing monomer and a polyvalent vinyl compound, and the weight composition ratio (nitrogen-containing monomer: polyvalent vinyl compound) is in the range of 49: 1 to 30: 20- Metal composite.   2. The resin-metal composite according to claim 1, wherein the amount of the metal particles supported is in the range of 5 mass% to 70 mass% with respect to the weight of the resin-metal composite.   The resin-metal composite according to claim 1 or 2, wherein 60% by mass to 100% by mass of the metal particles are present in a surface layer portion of the resin particles.   The resin-metal composite according to any one of claims 1 to 3, wherein the average particle diameter of the metal particles is in the range of 1 to 80 nm.   The resin-metal composite according to any one of claims 1 to 4, wherein an average particle diameter is in a range of 50 to 1000 nm.   The resin-metal composite according to claim 1, wherein the metal particles are gold particles or platinum particles.   A labeling substance comprising the resin-metal complex according to any one of claims 1 to 6.   The labeling substance according to claim 7, which is used by adsorbing an antigen or antibody on the surface of the resin-metal complex.   An immunological assay using the labeling substance according to claim 7 or 8.   A reagent for immunological measurement comprising the resin-metal complex according to any one of claims 1 to 6. A method for measuring an analyte for detecting or quantifying an analyte contained in a sample, comprising:
Using a lateral flow type chromatographic test strip including a membrane and a determination part in which a capture ligand that specifically binds to the analyte is immobilized on the membrane, the following steps (I) to (III);
Step (I): a labeled antibody obtained by labeling the analyte contained in the sample and an antibody that specifically binds to the analyte with the resin-metal complex according to any one of claims 1 to 6. Contacting,
Step (II): a step of bringing the complex containing the analyte and the labeled antibody formed in Step (I) into contact with a capture ligand in the determination unit;
Step (III): a step of measuring the color development intensity derived from localized surface plasmon resonance of the resin-metal complex and light energy absorption by electronic transition,
A method for measuring an analyte, comprising performing a step comprising: An analyte measurement kit for detecting or quantifying an analyte contained in a sample using a lateral flow type chromatographic test strip,
A lateral flow type chromatographic test strip including a membrane and a determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane;
An antibody that specifically binds to the analyte, a detection reagent comprising a labeled antibody labeled with the resin-metal complex according to any one of claims 1 to 6,
An analyte measurement kit for detecting or quantifying an analyte containing A test strip for lateral flow chromatography for detecting or quantifying an analyte contained in a sample,
A membrane,
A determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane in a direction in which the sample is developed,
A reaction part containing a labeled antibody labeled with the resin-metal complex according to any one of claims 1 to 6, wherein the antibody that specifically binds to the analyte is upstream of the determination part.
Lateral flow chromatographic test strip.