JP7218052B2 - Specimen treatment liquid and immunochromatographic kit using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a sample treatment liquid that can be used in an immunochromatography kit that includes metal-resin composite particles as a labeling substance.

Conventionally, metal colloids and resin particles colored with pigments have been used as labeling substances for immunochromatography. In recent years, resin microparticles to which metal nanoparticles are adsorbed have been proposed as new labeling substances to replace them. For example, Patent Document 1 proposes a colored latex composed of gold nanoparticles bound to the surface of polymer-based latex particles. By binding gold nanoparticles to the surface of polymer-based latex particles, the gold nanoparticles themselves serve as a coloring agent to improve visual determination and detection sensitivity, while the gold nanoparticles themselves also have binding properties to antigens or antibodies. Therefore, it is considered that a sufficient amount of antigen or antibody can be bound even if gold nanoparticles are bound to such an extent that the color becomes sufficiently dark.

In addition, the present inventors previously proposed metal-resin composite particles having a specific structure as such colored fine particles as labeling substances that enable highly sensitive immunological measurements (Patent Document 3 and 4). Since the metal-resin composite particles have a structure in which a plurality of metal particles are three-dimensionally immobilized on the surface layer of the resin particles, the metal particles are supported in a large amount, and the metal particles are detached from the resin particles. It has the characteristic that it is difficult to Furthermore, metal particles have the property of exhibiting optical energy absorption due to electronic transition in addition to localized surface plasmon resonance.

However, although immunochromatographic kits using these metal-resin composite particles as labeling substances have high sensitivity, they have problems such as background coloring and non-specific adsorption, depending on the type of antibody. Therefore, when the metal-resin composite particles are used as labeling substances for immunochromatography kits, they cannot be applied depending on the type of antibody.

A typical sample treatment liquid used in an immunochromatography kit is an aqueous solution consisting of a buffer, salt, water-soluble protein, and surfactant. The optimum composition of the sample treatment solution varies greatly depending on the measurement conditions, sample collection method, labeling substance, antibody, and the like. For example, a sample treatment solution using a metal colloid as a labeling substance (Patent Documents 5 to 7) and a case using resin particles colored with a dye (Patent Documents 8 and 9) are disclosed.
However, until now, no investigation has been made on a specimen treatment liquid in the case of using metal-resin composite particles as a labeling substance.

JP 2009-168495 A JP-A-3-206959 WO2016/002742 WO2016/002743 JP 2014-210761 A Japanese Patent No. 5597190 Japanese Patent Application No. 2010-200793 Japanese Patent Application No. 10-268180 Japanese Patent Application No. 2000-152974

For example, when an evaluation is made using a specimen treatment liquid having a composition suitable for resin particles colored with a conventional dye, there is a problem of background coloring. Therefore, the background can be reduced by changing the surfactant, but there is a problem that non-specific adsorption occurs.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a sample treatment liquid that does not cause background coloring and non-specific adsorption.

As a result of intensive studies, the inventors of the present invention have found that the above-described problems can be solved by a sample treatment liquid having a specific composition, and completed the present invention.

The sample processing solution of the present invention is a sample processing solution for an immunochromatographic kit using a labeling substance comprising metal-resin composite particles, and is an aqueous solution containing 0.3 to 5 wt% of a polysorbate surfactant. characterized by

The sample treatment solution of the present invention preferably further contains 0.01 to 5 wt % of a milk-derived blocking agent.

The specimen treatment solution of the present invention preferably further contains 0.3 to 1.0 wt% buffer and 0.5 to 2.0 wt% salt component.

The sample treatment solution of the present invention preferably further contains 0.1 to 3.0 wt % of serum-derived protein.

The sample treatment liquid of the present invention preferably has a pH of 7-9.

The sample treatment solution of the present invention preferably further contains a polyoxyethylene (alkylphenyl) ether surfactant at 0.20 wt % or less.

An immunochromatography kit of the present invention is characterized by comprising the sample treatment liquid described above.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a sample treatment liquid that does not cause background coloring and non-specific adsorption.

1 is a schematic cross-sectional view of a "metal-resin composite particle " according to an embodiment of the present invention; FIG. 1 is a cross- sectional view showing the structure of an immunochromatography kit to which the sample treatment liquid of the present invention is applied ; FIG.

<Metal-resin composite particles>
Next, the metal-resin composite particles used as a labeling substance in the method for producing a labeled antibody of the present embodiment will be described in detail.
The metal-resin composite particles have a structure in which metal particles are immobilized on resin particles. Further, an organic dye may be immobilized on the metal-resin composite particles. The structure of the organic dye or metal particles is not limited, but as long as the resin particles are colored by being immobilized on the resin particles, it can be easily visually determined for immunological measurement applications such as immunochromatography. It is preferable because

Examples of metal-resin composite particles in which metal particles are immobilized on resin particles will be described below.
FIG. 1 is a schematic cross-sectional view of a metal-resin composite particle having a structure in which a plurality of metal particles are immobilized on a resin particle, which can be preferably used as a labeling substance in the present embodiment. The metal-resin composite 100 includes resin particles 10 and metal particles 20 .

Metal-resin composite 100 has metal particles 20 dispersed or immobilized in resin particles 10 . In addition, the metal-resin composite 100 has a part of the metal particles 20 distributed two-dimensionally or three-dimensionally in the surface layer portion 60 of the resin particles 10, and the metal particles distributed two-dimensionally or three-dimensionally. A part of 20 is partially exposed outside the resin particles 10 , and the remaining part is included in the resin particles 10 .

Here, the metal particles 20 include metal particles completely enclosed in the resin particles 10 (hereinafter also referred to as “encapsulated metal particles 30”), portions embedded in the resin particles 10, and particles outside the resin particles 10. Metal particles having exposed portions (hereinafter also referred to as “partially exposed metal particles 40”) and metal particles adsorbed to the surface of the resin particles 10 (hereinafter also referred to as “surface-adsorbed metal particles 50”) are exist. The partially exposed metal particles 40 and the surface-adsorbed metal particles 50 correspond to the "first particles" in the present invention, and the included metal particles 30 correspond to the "second particles" in the present invention.

For example, when the metal-resin composite 100 is used for immunological measurements, an antibody is immobilized on the partially exposed metal particles 40 or the surface-adsorbed metal particles 50 for use. At that time, the antibodies are immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50 , but not on the encapsulated metal particles 30 . However, since all of the metal particles 20 including the encapsulated metal particles 30 exhibit localized surface plasmon absorption, not only the partially exposed metal particles 40 and the surface-adsorbed metal particles 50 but also the encapsulated metal particles 30 are immunologically Contributes to improved visibility as a mark for target measurement. Furthermore, the partially exposed metal particles 40 and the included metal particles 30 have a larger contact area with the resin particles 10 than the surface-adsorbed metal particles 50, and also have a physical adsorption force such as an anchor effect due to the embedded state. It is strong and does not easily detach from the resin particles 10 . Therefore, excellent durability and stability as an immunoassay label using the metal-resin composite 100 can be achieved.

The entire surface of the encapsulated metal particles 30 is covered with the resin that constitutes the resin particles 10 . Moreover, 5% or more and less than 100% of the surface area of the partially exposed metal particles 40 is covered with the resin forming the resin particles 10 . More than 0% and less than 5% of the surface area of the surface-adsorbed metal particles 50 is covered with the resin forming the resin particles 10 .

In addition, the amount of metal particles 20 supported on the metal-resin composite 100 (total of the encapsulated metal particles 30, the partially exposed metal particles 40, and the surface-adsorbed metal particles 50) is relative to the weight of the metal-resin composite 100. , 5 wt % to 70 wt %. Within this range, the metal-resin composite 100 is excellent in visibility, visual determination and detection sensitivity as a labeling substance. If the supported amount of the metal particles 20 is less than 5 wt %, the immobilized amount of the antibody tends to decrease and the detection sensitivity tends to decrease. The supported amount of the metal particles 20 is more preferably 15 wt % to 70 wt %.

Also, 10 wt % to 90 wt % of the metal particles 20 are preferably partially exposed metal particles 40 and surface-adsorbed metal particles 50 . Within this range, a sufficient amount of antibody immobilized on the metal particles 20 can be ensured, resulting in high sensitivity as a labeling substance. More preferably, 20 wt % to 80 wt % of the metal particles 20 are the partially exposed metal particles 40 and the surface-adsorbed metal particles 50 , and from the viewpoint of durability, the surface-adsorbed metal particles 50 are more preferably 20 wt % or less. .

60 wt % to 100 wt % of the metal particles 20 are present in the surface layer portion 60, and 5 wt % to 90 wt % of the metal particles 20 present in the surface layer portion 60 are the partially exposed metal particles 40 or the surface adsorbed metal particles 50. Since a sufficient amount of antibody immobilized on the metal particles 20 can be ensured, it is preferable that there is a high sensitivity as a labeling substance. In other words, 10 wt % to 95 wt % of the metal particles 20 present in the surface layer portion 60 are preferably the included metal particles 30 .

Here, the “surface portion” means a range of 50% of the particle radius in the depth direction from the surface of the resin particle 10 . The phrase "three-dimensionally distributed" 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. On the other hand, the phrase “two-dimensionally distributed” means that the metal particles 20 are dispersed equidistantly in the depth direction from the surface of the resin particles 10 . Since the resin particles 10 have curved surfaces, the metal particles 20 cannot actually stay within the two-dimensional plane. Therefore, the metal particles 20 are distributed two-dimensionally on the surface of the resin particles 10 . The projection distribution of the metal particles 20 in the plane on which the resin particles 10 and the metal particles 20 are projected is not intended. An example of the “two-dimensional distribution” is the case where all of the metal particles 20 are surface-adsorbed metal particles 50 .

Known resin particles such as polystyrene, polyvinyl alcohol, poly(p-sodium p-styrenesulfonate), melamine resin, urea resin, acrylic resin, and cellulose can be applied to the resin particles 10, but they are capable of adsorbing metal ions. It is preferably a polymer particle having a substituent in its structure. Nitrogen-containing polymer particles are particularly preferred. Nitrogen atoms in nitrogen-containing polymers chemisorb anionic metal complex ions, which are precursors of metal particles such as silver, nickel, copper, gold, platinum, and palladium, which have excellent visibility and are easy to immobilize antibodies. preferred because it is easy. In the present embodiment, since the metal ions adsorbed in the nitrogen-containing polymer are reduced to form metal nanoparticles, a part of the generated metal particles 20 becomes the included metal particles 30 or the partially exposed metal particles 40. . In addition, like acrylic acid polymers, carboxylic acids and the like can adsorb cationic metal ions. and can form metal particles 20 of silver, nickel, copper, gold, platinum, palladium, etc., and can be alloyed with any of the above metals.
On the other hand, in the case of resin particles other than nitrogen-containing polymers having substituents capable of adsorbing metal ions in their structure, such as polystyrene, the metal ions are less likely to be adsorbed inside the resin. As a result, most of the generated metal particles 20 become surface-adsorbed metal particles 50 . As described above, since the surface-adsorbed metal particles 50 have a small contact area with the resin particles 10, the adhesion between the resin and the metal is small, and the detachment of the metal particles 20 from the resin particles 10 tends to have a large effect.
The nitrogen-containing polymer is a resin having nitrogen atoms in its main chain or side chain, and includes, for example, polyamines, polyamides, polypeptides, polyurethanes, polyureas, polyimides, polyimidazoles, polyoxazoles, polypyrroles, and polyanilines. Polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine and poly-4-vinylpyridine are preferred. Moreover, when the side chain has a nitrogen atom, it can be widely used, for example, an acrylic resin, a phenolic resin, an epoxy resin, and the like.

As the material of the metal particles 20, for example, silver, nickel, copper, gold, platinum, and palladium can be applied. These metals can be used singly or in composites such as alloys. Preferred are gold, platinum, and palladium, which have excellent visibility and facilitate antibody immobilization. These are preferable because they exhibit absorption derived from localized surface plasmon resonance. More preferred are gold and platinum, which have good storage stability. In addition, from the viewpoint that excellent color development is obtained when the metal-resin composite 100 is bound to an antibody under conditions within the pH range of 2 to 9, the material of the metal particles 20 is gold, platinum, a gold alloy, or Platinum alloys are the most preferred metal species. Here, the gold alloy means, for example, an alloy composed of gold and a metal other than gold and containing 10% by weight or more of gold. The platinum alloy means an alloy composed of platinum and a metal species other than platinum and containing 10% by weight or more of platinum.

Further, the average value (average particle diameter) of the particle diameter D3 of the metal particles 20 measured by scanning electron microscope (SEM) observation is preferably 1 to 80 nm, for example. When the average particle size of the metal particles 20 is less than 1 nm or more than 80 nm, localized surface plasmon is less likely to occur, and sensitivity tends to decrease. The average particle diameter of the metal particles 20 is preferably 1 nm or more and less than 70 nm, more preferably 1 nm or more and less than 50 nm.

Further, the average value (average particle diameter) of the particle diameter D1 of the metal-resin composite 100 is, for example, 100 to 1000 nm. If the average particle diameter of the metal-resin composite 100 is less than 100 nm, for example, when gold particles or platinum particles are used as the metal particles 20, the amount of metal particles supported tends to be small. Coloration tends to be weaker, and if it exceeds 1000 nm, when used as a labeling substance or reagent, the pores of a chromatographic medium such as a membrane filter tend to be easily clogged, and the dispersibility tends to decrease. The average particle size of the metal-resin composite 100 is preferably 100 nm or more and less than 700 nm, more preferably 100 nm or more and less than 650 nm. Here, the particle diameter D1 of the metal-resin composite 100 means a value obtained by adding the length of the protruding portion of the partially exposed metal particles 40 or the surface-adsorbed metal particles 50 to the particle diameter D2 of the resin particles 10, It can be measured by laser diffraction/scattering, dynamic light scattering, or centrifugal sedimentation.

A method for manufacturing the metal-resin composite 100 is not particularly limited. For example, a solution containing metal ions or metal complex ions is added to a dispersion liquid of resin particles 10 produced by an emulsion polymerization method to cause the resin particles 10 to adsorb the metal ions or metal complex ions (hereinafter referred to as “metal ion adsorption "resin particles"). Furthermore, by adding the metal ion-adsorbing resin particles to a reducing agent solution, the metal ions are reduced to generate metal particles 20, and the metal-resin composite 100 is obtained.

Further, for example, when gold particles are used as the metal particles 20, examples of the solution containing metal complex ions include a chloroauric acid (HAuCl ₄ ) aqueous solution. Moreover, when platinum particles are used, a chloroplatinic acid (H ₂ PtCl ₆ ) aqueous solution and the like can be used. Also, a metal complex may be used instead of the metal ion.
In addition, water-containing alcohols such as methanol, ethanol, n-propanol, 2-propanol, n-butanol, sec-butanol and t-butanol or alcohols can be used instead of water as the solvent for the solution containing metal ions or metal complex ions. , hydrochloric acid, sulfuric acid, nitric acid and the like may be used.

In addition, if necessary, the solution may be added, 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 monoalkyl ethers or dialkyl ethers, polyols such as glycerin, and various water-miscible organic solvents such as ketones such as acetone and methyl ethyl ketone may be added. Such additives are effective in promoting the reduction reaction rate of metal ions and controlling the size of the metal particles 20 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, sodium phosphinate, hydroquinone, hydrazine sulfate, formaldehyde , Rochelle salt, and the like. Among these, sodium borohydride, dimethylamine borane, and citric acid are preferred. If necessary, a surfactant can be added to the reducing agent solution, or the pH of the solution can be adjusted. The pH can be adjusted 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.
Furthermore, the particle size of the metal particles to be formed can be controlled by adjusting the reduction rate of the metal ions by adjusting the temperature of the reducing agent solution.

When the metal ions in the metal ion-adsorbing resin particles are reduced to generate the metal particles 20, the metal ion-adsorbing resin particles may be added to a reducing agent solution, or the reducing agent may be added to the metal ion-adsorbing resin. Although it may be added to the particles, the latter is preferred from the viewpoint of ease of generation of the encapsulated metal particles 30 and the partially exposed metal particles 40 .

In order to maintain dispersibility in water, the metal-resin composite 100 obtained as described above is treated with, for example, citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, A dispersing agent such as DISPERBYK184 (manufactured by Big Chemie Japan) may be added to the sample treatment liquid. 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 metal-resin composite 100 having the above configuration can be produced by, for example, EIA: enzyme immunoassay, RIA: radioimmunoassay, CLIA: chemiluminescence measurement method, FIA: fluorescence immunoassay method, LIA: latex agglutination immunoassay method, PA: particle agglutination method, ICA: immunochromatography method, HA: hemagglutination method, HI: hemagglutination inhibition method, etc. It can be preferably applied. In addition, it can be preferably applied as a material for an immunological measurement labeling substance or an immunological measurement reagent, which is excellent in visual determination, particularly in a low concentration range (high sensitivity range). The form of the labeling substance for immunological measurement or the reagent for immunological measurement is not particularly limited. can be used as

In addition, since the labeling substance provided with the metal-resin complex 100 has remarkably strong chromogenicity, the background of the membrane, which could not be detected with a conventional immunochromatographic kit using a labeling substance provided with colloidal gold or colored latex, was reduced. Coloring and non-specific adsorption tend to cause color development in the test line, making it impossible to use conventional sample treatment solutions. In the sample treatment solution of the present invention, background coloring of the membrane and coloring of the test line due to non-specific adsorption are suppressed.

<Specimen treatment liquid>
The sample treatment solution of the present invention comprises buffer, salt, serum-derived protein, 0.3-5 wt % polysorbate surfactant and water as main components.
Also, known buffers such as Tris buffers, glycinamide buffers, arginine buffers, and phosphate buffers can be used as buffers. For pH stability, the concentration is preferably 0.3 to 1.0 wt%.
Also, the salt is preferably a component close to that of physiological saline, for example 0.5 to 2.0 wt % sodium chloride.
Moreover, the serum-derived protein is preferably a highly hydrophilic, water-soluble protein that does not react with antibodies or antigens. For example, 0.1-3.0 wt % bovine serum albumin is preferred.
In addition, 0.3 to 5 wt% polysorbate surfactant is more preferably at a concentration of 0.5 wt% or more in order to obtain a sufficient effect of preventing background coloring, and in order to prevent a decrease in sensitivity of the immunochromatographic kit, the concentration is 2 wt%. It is more preferably less than. Polysorbate surfactants are more preferably highly water soluble, for example Tween 20 and Tween 80.
In addition, it is preferable to further add 0.01 to 5 wt % of a milk-derived blocking agent to the sample treatment solution of the present invention. Milk-derived blocking agents include, for example, casein, sodium caseinate, skimmed milk, and blockace. The concentration is more preferably 0.05 wt% or more in order to obtain a sufficient effect of suppressing non-specific adsorption, and the concentration is less than 2 wt% in order to prevent a decrease in the sensitivity of the immunochromatographic kit and an increase in the viscosity of the sample treatment solution. is more preferable. Further, the pH of the sample treatment liquid of the present invention is preferably 7-9. Within this range, an immune reaction occurs normally and detection by immunochromatography is possible.

Further, the sample treatment liquid of the present invention preferably contains 0.20 wt % or less of a polyoxyethylene (alkylphenyl) ether surfactant represented by Triton X100. With this concentration range, it is possible to prevent the background from being colored.
A plurality of buffers, salts, serum-derived proteins, surfactants, blocking agents and additives may be added to the sample treatment solution of the present invention, if necessary. In addition, the sample treatment solution of the present invention may optionally contain a known preservative such as sodium azide.

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

[Test strips for lateral flow chromatography]
First, with reference to FIG. 2 , a test strip for lateral flow chromatography (hereinafter sometimes simply referred to as "test strip") according to the form of use of the sample treatment liquid of the present invention will be described. As will be described later, this test strip 200 can be preferably used in a method for measuring an analyte, which is a utilization form of the sample treatment liquid of the present invention.

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

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

The test strip 200 preferably includes a support 190 that supports the membrane 110 for easier handling. As the support, for example, plastic or the like can be used.

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

<Determination part>
A capture ligand 131 that specifically binds to the analyte 160 is immobilized on the determination section 130 . The capture ligand 131 can be used without any 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 that it does not move from the determination section 130 even when the test strip 200 is provided with a sample. The capture ligand 131 may be directly or indirectly immobilized on the membrane 110 by physical or chemical bonding, adsorption, or the like.

Moreover, determination unit 130 is not particularly limited as long as it is configured such that complex 170 containing labeled antibody 150 and analyte 160 contacts capture ligand 131 that specifically binds to analyte 160 . For example, the capture ligand 131 may be directly immobilized on the membrane 110, or the capture ligand 131 may be immobilized on a pad made of cellulose filter paper, glass fiber, non-woven fabric, etc., immobilized on the membrane 110. .

<Liquid absorbing part>
The liquid absorbing portion 140 is formed of, for example, a pad of a water absorbing material such as cellulose filter paper, nonwoven fabric, cloth, cellulose acetate, or the like. The movement speed of the sample after the development front (front line) of the added sample reaches the liquid absorbing portion 140 varies depending on the material, size, and the like of the liquid absorbing portion 140 . Therefore, by selecting the material, size, etc. of the liquid absorbing portion 140, the optimum speed for detecting and quantifying the analyte 160 can be set. Note that the liquid absorbing portion 140 has 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, if necessary.

<Reaction part>
The test strip 200 may have a membrane 110 formed with a reaction portion 180 containing the labeled antibody 150 . The reaction section 180 can be provided upstream of the determination section 130 in the direction in which the sample flows. Note that the sample addition section 120 in FIG. 2 may be used as the reaction section. When the test strip 200 has the reaction portion 180, when the sample containing the analyte 160 is provided to the reaction portion 180 or the sample application portion 120, the analyte 160 contained in the sample and the labeled antibody 150 are brought into contact with each other in the reaction portion 180. can be made In this case, by simply providing the sample to the reaction section 180 or the sample addition section 120, the complex 170 containing the analyte 160 and the labeled antibody 150 can be formed, so that so-called one-step immunochromatography is possible. become.

The reaction section 180 is not particularly limited as long as it contains the labeled antibody 150 that specifically binds to the analyte 160 , but may be formed by directly applying the labeled antibody 150 to the membrane 110 . Alternatively, the reaction section 180 may be formed by fixing a pad (conjugate pad) made of, for example, cellulose filter paper, glass fiber, non-woven fabric, etc., impregnated with the labeled antibody 150 to the membrane 110 .

<Control section>
Although illustration is omitted, the test strip 200 has a control section 210 in which a capture ligand that specifically binds to the labeled antibody 150 is immobilized on the membrane 110 downstream of the determination section 130 in the direction in which the sample develops. may be formed. By measuring the color intensity in the control unit 210 together with the determination unit 130, the sample applied to the test strip 200 develops and reaches the reaction unit 180 and the determination unit 130, thereby confirming that the test was performed normally. can be confirmed. Note that the control section 210 is fabricated in the same manner as the determination section 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. configuration can be adopted.

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

The method for measuring analyte 160 of the present embodiment is a method for measuring analyte 160 for detecting or quantifying analyte 160 contained in a sample. The method for measuring the analyte 160 of the present embodiment uses a test strip 200 that includes a membrane 110 and a determination section 130 in which a capture ligand 131 that specifically binds to the analyte 160 is immobilized on the membrane 110 . Then, the method for measuring the analyte 160 of the present embodiment includes the following steps (I) to (III);
Step (I): A metal-resin composite 100 having a structure in which a plurality of metal particles 20 are immobilized on a resin particle 10 with the analyte 160 contained in the sample and an antibody that specifically binds to the analyte 160. contacting with a labeled antibody 150 labeled with
Step (II): A step of contacting the complex 170 formed in the step (I) containing the analyte 160 and the labeled antibody 150 with the capture ligand 131 in the determination unit 130;
Step (III): A step of measuring the color intensity derived from light energy absorption by localized surface plasmon resonance and/or electronic transition of the metal-resin composite 100;
can include

Process (I):
Step (I) is a step of contacting the analyte 160 contained in the sample 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, a sample may be supplied to the sample application portion 120 or the reaction portion 180 (not shown) of the test strip 200, and the analyte 160 may be brought into contact with the labeled antibody 150 in the sample application portion 120 or the reaction portion 180. Analyte 160 in the sample may be contacted with labeled antibody 150 prior to subjecting the sample to 200 .

The complex 170 formed in step (I) develops and moves on the test strip 200 to reach the determination section 130 .

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

Since the capturing 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 passes through the determination unit 130 . Here, if the test strip 200 is formed with a control section 210 (not shown) to which another capture ligand that specifically binds to the labeled antibody 150 is immobilized, the labeled antibody 150 that has passed through the determination section 130 It continues to develop and binds with the other capture ligand at control section 210 . As a result, labeled antibody 150 not forming complex 170 with analyte 160 is captured by control section 210 .

After step (II) and before step (III) if necessary, the test strip 200 is washed with a buffer commonly used in biochemical tests, such as water, physiological saline, and phosphate buffer. A washing step may be performed. The washing step removes the labeled antibody 150 not captured by the determination unit 130 or the determination unit 130 and the control unit 210 (the labeled antibody 150 that is not bound to the analyte 160 and does not form the complex 170). can do.

By performing the washing step, in the step (III), the metal-resin composite 100 in the determination unit 130 or the determination unit 130 and the control unit localized surface plasmon resonance and / or light energy absorption by electronic transition When measuring color development, the intensity of background color development can be reduced, the signal/background ratio can be increased, and detection sensitivity and quantification can be further improved.

Step (III):
Step (III) is a step of measuring the intensity of color development resulting from optical energy absorption by localized surface plasmon resonance and/or electronic transition of the metal-resin composite 100 . After performing the step (II) or the washing step as necessary, the test strip 200 measures the intensity of color development derived from light energy absorption by localized surface plasmon resonance and/or electronic transition of the metal-resin composite 100. Measure.

If the test strip 200 is formed with the control portion 210, the labeled antibody 150 is captured by another capturing ligand in the control portion 210 to form a complex in step (II). Therefore, in the step (III), in the test strip 200, not only the determination section 130 but also the control section 210 can cause color development due to optical energy absorption by localized surface plasmon resonance and/or electronic transition. In this way, by measuring the color development intensity in the control unit 210 as well as in the determination unit 130, it is possible to confirm whether or not the sample applied to the test strip 200 is normally developed and reaches the reaction unit and the determination unit 130. .

<Sample and analyte>
The sample in the method for measuring an analyte of the present embodiment is not particularly limited as long as it contains a substance that can serve as an antigen, such as protein, as the analyte 160 . For example, a biological sample containing the analyte of interest 160 (i.e., whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swabs, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, exudates from the skin) , extracts from tissues, cells, and stool) and food extracts. If necessary, the analyte 160 contained in the sample is pretreated prior to the above step (I) in order to facilitate the specific binding reaction between the labeled antibody 150 and the capture ligand 131 and the analyte 160. may Examples of the pretreatment include chemical treatment using various chemicals such as acids, bases, and surfactants, 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 should be used in consideration of the specific binding reaction, for example, the binding reactivity between the capture ligand 131 and the analyte 160 such as an antigen-antibody reaction. can be done.

In addition, the sample may be appropriately diluted with a solvent (water, physiological saline, buffer solution, etc.) or a water-miscible organic solvent used in ordinary immunological analysis methods.

As the analyte 160, known ones can be used without any particular limitation. Examples of the analyte 160 include proteins such as tumor markers, signaling substances, and hormones (including polypeptides, oligopeptides, etc.), nucleic acids (single-stranded or double-stranded DNA: deoxyribonucleic acid, RNA: ribonucleic acid , polynucleotides, oligonucleotides, PNAs (peptide nucleic acids), etc.) or substances having nucleic acids, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or substances having sugar chains, and other molecules such as lipids. It is not particularly limited as long as it specifically binds to the labeled antibody 150 and the capture ligand 131. For example, carcinoembryonic antigen (CEA), HER2 protein (human epidermal growth factor receptor 2), prostate specific antigen ( PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive acidic protein (IAP), CA15-3, CA125, estrogen receptor, progesterone receptor, 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-hemolytic streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, saccharified albumin and the like. Among these, antigens that are solubilized by nonionic surfactants are preferred, and antigens that form self-aggregates like viral nucleoproteins are more preferred.

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

In addition, in the present embodiment, the "antibody" is not particularly limited and known ones can be used, and those with strong anionic properties, those with strong cationic properties, and others can also be used. For example, in addition to polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, antibody fragments capable of binding to antigens [eg, H chain, L chain, Fab, F(ab') ₂ , etc.] can be used. can. Moreover, any of IgG, IgM, IgA, IgE, and IgD may be used as the immunoglobulin. Antibody-producing animal species include humans and non-human animals (eg, mice, rats, rabbits, goats, horses, etc.). Specific examples of antibodies include anti-PSA antibodies, anti-AFP antibodies, anti-CEA antibodies, anti-adenovirus antibodies, anti-influenza virus antibodies, anti-HCV antibodies, anti-IgG antibodies, anti-human IgE antibodies and the like.

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

[Step A]
In step A, a labeled antibody 150 is obtained by mixing the metal-resin complex 100 with an antibody under a first pH condition. In step A, the solid metal-resin composite 100 is preferably dispersed in a liquid phase and brought into contact with the antibody.

The first pH condition is preferably in the range of pH 2 to 10 from the viewpoint of uniform contact between the metal-resin composite 100 and the antibody while maintaining the dispersion of the metal-resin composite 100 and the activity of the antibody. Furthermore, for example, the range of pH 5 to 9 is more preferable. If the conditions for binding the metal-resin complex 100 and the antibody are less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if the pH exceeds 10, when the metal-resin complex 100 and the antibody are mixed. It aggregates to a large extent and becomes difficult to disperse. However, if the antibody is not deactivated by strong acidity, the treatment can be performed even at pH less than 2.

Step A is preferably carried out in a binding buffer adjusted to the first pH condition. For example, a predetermined amount of the metal-resin composite 100 is mixed with the binding buffer solution adjusted to the above pH and thoroughly mixed. 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, sodium hydroxide, and the like.

Next, a predetermined amount of antibody is added to the resulting mixed solution, and the mixture is sufficiently stirred and mixed to obtain a labeled antibody-containing solution. From the labeled antibody-containing liquid thus obtained, only the labeled antibody 150 can be fractionated as a solid portion by solid-liquid separation means such as centrifugation.

[Step B]
In step B, blocking for suppressing non-specific adsorption to the labeled antibody 150 is performed by treating the labeled antibody 150 obtained in step A under a second pH condition. 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.

The second pH condition is, for example, preferably in the range of pH 2 to 10 from the viewpoint of maintaining the activity of the antibody and suppressing aggregation of the labeled antibody 150, and pH 5 from the viewpoint of suppressing non-specific adsorption of the labeled antibody 150. It is more preferably in the range of ~9. If the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if the pH exceeds 10, the labeled antibody 150 aggregates and becomes difficult to disperse.

Step B is preferably carried out using a blocking buffer in which the blocking agent is adjusted to the second pH condition. For example, a blocking buffer adjusted to the above pH is added to a predetermined amount of the labeled antibody 150, and the labeled antibody 150 is uniformly dispersed in the blocking buffer. As the blocking buffer, it is preferable to use, for example, a protein solution that does not bind to the substance to be detected. The blocking agent that can be used in the blocking buffer solution is not particularly limited and known ones can be used. Examples of proteins include bovine serum albumin, ovalbumin, casein, gelatin, and whey whey. More specifically, it is preferable to use a bovine serum albumin solution or the like adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, and the like. Dispersion means such as sonication is preferably used for dispersing the labeled antibody 150 . Thus, a dispersion liquid in which the labeled antibody 150 is uniformly dispersed is obtained.

In the above steps A and B, the metal-resin composite 100 is unlikely to aggregate due to pH, and can be treated in a wide range of pH from acidic to alkaline. Therefore, the metal-resin composite 100 used in the present invention also has the advantage of being less subject to restrictions on the conditions for producing labeled antibodies.

As described above, a dispersion liquid of the labeled antibody 150 is obtained. From this dispersion, only the labeled antibody 150 can be fractionated as a solid portion by solid-liquid separation means such as centrifugation. In addition, washing treatment, storage treatment, and the like can be carried out as necessary. The cleaning treatment and storage treatment will be described below.

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

(preservation processing)
In the storage treatment, a storage buffer is added to the labeled antibody 150 collected by solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the storage buffer. For dispersion, it is preferable to use a dispersion means such as ultrasonic treatment. As the storage buffer, for example, a solution obtained by adding a predetermined concentration of an anti-aggregation agent and/or a stabilizer to a washing buffer can be used. Examples of anti-aggregating agents that can be used include sugars typified by sucrose, maltose, lactose and trehalose, and polyhydric alcohols typified by glycerin and polyvinyl alcohol. Examples of the stabilizer include, but are not limited to, proteins such as bovine serum albumin, ovalbumin, casein and gelatin. In this manner, storage treatment of the labeled antibody 150 can be performed.

In each of the above steps, if necessary, a surfactant and a preservative such as sodium azide and paraoxybenzoic acid ester can be used.

[Analyte measurement kit]
The analyte measurement kit according to one embodiment of the present invention detects or quantifies the analyte 160 contained in the sample based on the analyte measurement method of the present embodiment using, for example, the test strip 200. It is a kit for

The analyte measurement kit of the present embodiment includes
a membrane 110;
a test strip 200 including a determination section 130 in which a capture ligand 131 that specifically binds to the analyte 160 is immobilized on a membrane 110;
a detection reagent containing a labeled antibody 150 in which an antibody that specifically binds to an analyte 160 is labeled with a metal-resin complex 100 having a structure in which a plurality of metal particles 20 are immobilized on a resin particle 10;
contains. The analyte measurement kit of the present embodiment may further contain other components as necessary.

In using the analyte measurement kit according to the present embodiment, the analyte 160 in the sample and the labeled antibody 150 in the detection reagent are brought into contact with each other to carry out the step (I), and then the test strip 200 reacts. A sample may be supplied to the section 180 or the sample addition section 120 to sequentially perform steps (II) and (III). Alternatively, after the detection reagent is applied to the upstream side of the determination portion 130 of the test strip 200 and dried as appropriate to form the reaction portion 180, the reaction portion 180 formed or the reaction portion 180 upstream of the reaction portion 180 is formed. The steps (I) to (III) may be sequentially performed by adding a sample to a position (for example, the sample adding section 120).

In addition, the metal-resin composite 100 can be preferably applied as solid catalysts, pigments, paints, conductive materials, electrodes, and sensor elements other than labeling substances for immunological measurements or reagents for immunological measurements.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these.

[Production example 1]
<Synthesis of resin particles>
After dissolving trioctylammonium chloride (0.39 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) in 300 g of pure water, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added. , under a stream of nitrogen, at 30° C. for 50 minutes and then at 60° C. for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.50 g) dissolved in 18.00 g of pure water was added dropwise and stirred at 60°C for 3.5 hours to obtain an average particle size. Resin particles A-1 having a diameter of 439 nm were obtained. It was precipitated by centrifugation (9000 rpm, 40 minutes), and after removing the supernatant, it was dispersed again in pure water, and impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt % resin particle dispersion B-1.

[Example 1]
<Synthesis of gold-resin composite particles>
After adding 255 g of pure water to B-1 (91.5 g), 400 mM chloroauric acid aqueous solution (147 g) was added and stirred at room temperature for 3 hours. This mixture was centrifuged (3000 rpm, 30 minutes) to precipitate A-1, and the supernatant was removed to remove excess chloroauric acid. After that, the concentration was adjusted to obtain a 2.5 wt % gold ion-adsorbing resin particle dispersion liquid C-1.

Next, C-1 (43.3 g) was added to 1580 g of pure water, and while stirring at 3°C, a 528 mM dimethylamine borane aqueous solution (10.0 g) was added dropwise over 2 minutes, followed by 1 hour at 3°C. , and stirred at room temperature for 3 hours to obtain gold-resin composite particles D-1 having an average particle size of 455 nm. After concentrating D-1 by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain 1 wt % gold-resin composite particle dispersion E-1. The absorbance of the gold-resin composite particles F-1 in E-1 was 1.36. The average particle size of gold particles in F-1 was 20 nm, and the amount of gold supported was 48.3 wt %.

(Binding process)
After mixing 100 μg of anti-influenza A monoclonal antibody and 0.9 mL of 100 mM boric acid aqueous solution (pH 8.5), 0.1 mL of 1 wt % gold-resin composite particle dispersion E-1 was added, and the mixture was allowed to stand at room temperature for 3 hours. The mixture was overturned and stirred to obtain a labeled antibody dispersion liquid J-1 containing the anti-influenza A monoclonal antibody labeled with the gold-resin composite particles F-1.

(block process)
Next, after cooling the labeled antibody dispersion J-1 with ice, it was centrifuged at 3000 rpm for 5 minutes. 1 mL of an aqueous Tris solution (pH 8.5) was added, subjected to ultrasonic dispersion treatment for 10 to 20 seconds, and further stirred over 2 hours at room temperature to obtain labeled antibody dispersion K-1.

(washing treatment)
Next, after ice-cooling the labeled antibody dispersion K-1, centrifugation was performed at 3000 rpm for 5 minutes, and after removing the supernatant, 5 mM Tris containing less than 0.1 wt% surfactant was added to the sediment. 1 mL of an aqueous solution (pH 8.5) was added, and ultrasonic dispersion treatment was performed for 10 to 20 seconds. This operation was repeated three times, and was used as a washing treatment.

(preservation processing)
Next, after ice-cooling, centrifugation was performed at 3000 rpm for 5 minutes, and after removing the supernatant, the sediment was treated with a 5 mM Tris aqueous solution containing less than 0.1 wt% surfactant and 10 wt% sucrose ( pH 8.5) (1 mL) was added and subjected to ultrasonic dispersion treatment for 10 to 20 seconds to obtain labeled antibody dispersion L-1.

(Preparation of reaction part)
After centrifuging 0.3 ml of the labeled antibody dispersion L-1 at 3000 rpm for 5 minutes and removing the supernatant, the precipitate was added to an aqueous solution containing 5 wt% sucrose and 2.5 wt% bovine serum albumin ( pH 8.0) (0.833 mL) was added, and ultrasonic dispersion treatment was performed for 10 to 20 seconds to obtain a labeled antibody dispersion M-1. A glass fiber nonwoven fabric was uniformly impregnated with the labeled antibody dispersion M-1, and then freeze-dried at -10°C for 24 hours to prepare a reaction portion 180; N-1. At this time, the liquid volume of the labeled antibody dispersion liquid M-1 was adjusted so that the content of the gold-resin composite particles in the reaction section 180; N-1 was 9 μg per evaluation test by the immunochromatographic method described later.

(Preparation of immunochromatographic strip)
An anti-influenza type A monoclonal antibody was applied to the center of a nitrocellulose membrane 110 having a width of 25 mm, and a determination portion 130 was drawn. After drying at room temperature, as shown in the cross-sectional view of the immunochromatographic strip 200 shown in FIG. 2, the support 190, the reaction section 180; was laminated. Finally, an immunochromatographic strip P-1 was prepared by cutting into a width of 5 mm.

(Preparation of sample treatment solution)
0.60 wt% trishydroxymethylaminomethane (hereinafter Tris), 0.88 wt% sodium chloride (hereinafter NaCl), 1.0 wt% bovine serum albumin (hereinafter BSA), 1.0 wt% Tween 20 and 0.01 wt % of sodium caseinate (pH 7.1) was prepared as sample treatment liquid Q-1. Table 1 shows the liquid composition of the sample treatment liquid.

(Evaluation by immunochromatography)
Influenza A positive control (equivalent to antigen concentration of 2500 FFU/ml) and negative control (antigen-free) sample solutions were prepared by diluting the antigen using the sample treatment solution Q-1, and 100 μl of the sample solution was applied to an immunochromatographic strip. It was dropped into the sample addition portion 120 of P-1 to develop the sample. After 15 minutes from the dropping of the specimen solution, the color development level of the test line and the presence or absence of background were evaluated using a gold colloid determination color sample (manufactured by ADTEC Co., Ltd.). As for the presence or absence of background, if the part other than the judgment part 130 of the nitrocellulose membrane 110 after 15 minutes has a coloring of 0.5 or more of the color sample, it is judged to be present, and if it is less than 0.5, it is absent. and In addition, when the coloring level of the positive control was 3 or more, the coloring level of the negative control was less than 0.5, and there was no background, the judgment was judged as good, and the other cases were judged as unsatisfactory. Table 2 shows the evaluation results by immunochromatography.

[Examples 2 to 10]
(Preparation of sample treatment solution)
Specimen treatment liquids Q-2 to Q-10 were prepared so as to have liquid compositions shown in Table 1. In addition, 0.60 wt% trishydroxymethylaminomethane (hereinafter, Tris), 0.88 wt% sodium chloride (hereinafter, NaCl), and 1.0 wt% bovine serum albumin (hereinafter, BSA) are all Q- 1 and common composition.
(Evaluation by immunochromatography)
Evaluation by immunochromatography was carried out in the same manner as in Example 1, except that the sample-treated liquids Q-2 to Q-10 were used instead of the sample-treated liquid Q-1. Table 2 shows the evaluation results. All judgments of the evaluation results were good.

[Example 11]
<Synthesis of platinum-resin composite particles>
After adding 54 g of pure water to B-1 (91.5 g), 400 mM chloroplatinic acid aqueous solution (100 g) was added and stirred at 30° C. for 3 hours. After this mixture was allowed to stand for 24 hours, A-1 was precipitated by centrifugation (3000 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. After that, the concentration was adjusted to obtain 5 wt % platinum ion-adsorbing resin particle dispersion liquid C-11.

Next, C-11 (20.6 g) was added to 1392 g of pure water, and while stirring at 3° C., a 132 mM dimethylamine borane aqueous solution (40 g) was added dropwise over 20 minutes. By stirring for 3 hours, platinum-resin composite particles D-11 having an average particle size of 461 nm were obtained. After concentrating D-11 by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain 1 wt % platinum-resin composite particle dispersion E-11. The absorbance of platinum-resin composite particles F-11 in E-11 was 1.77. The average particle size of the platinum particles in F-11 was 5.0 nm, and the amount of platinum supported was 37.6 wt%.

(Binding step, blocking step, washing treatment, storage treatment, preparation of reaction part, preparation of immunochromatographic strip)
Immunochromato strip P-11 was prepared in the same manner as in Example 1, except that 1 wt% platinum-resin composite particle dispersion E-11 was used instead of 1 wt% gold-resin composite particle dispersion E-1. made.

(Evaluation by immunochromatography)
Evaluation by the immunochromatographic method was performed in the same manner as in Example 1, except that the immunochromatographic strip P-11 was used instead of the immunochromatographic strip P-1. Table 2 shows the evaluation results. The judgment of the evaluation result was good.

[Comparative Examples 1 to 13]
(Preparation of sample treatment solution)
Specimen treatment liquids R-1 to R-13 were prepared so as to have liquid compositions shown in Table 1. In addition, 0.60 wt% trishydroxymethylaminomethane (hereinafter, Tris), 0.88 wt% sodium chloride (hereinafter, NaCl), and 1.0 wt% bovine serum albumin (hereinafter, BSA) are all Q- 1 and common composition.
(Evaluation by immunochromatography)
Evaluation by immunochromatography was performed in the same manner as in Example 1, except that the sample treatment solutions R-1 to R-13 were used instead of the sample treatment solution Q-1. Table 2 shows the evaluation results. All judgments of the evaluation results were unsatisfactory.

(Comparative Example 2) PVA: polyvinyl alcohol 500
(Comparative Example 3) PEG: polyethylene glycol 20000
(Comparative Example 4) PVP: polyvinylpyrrolidone K90
(Comparative Example 7) SDS: sodium dodecyl sulfate (Comparative Example 8) TBAB: tetrabutylammonium bromide (Comparative Example 9) TEAB: tetraethylammonium bromide

Claims (7)

A sample treatment solution for an immunochromatographic kit using a labeling substance comprising metal-resin composite particles, comprising 0.3 wt% or more and 5 wt% or less of a polysorbate surfactant and 0.01 wt% or more and less than 2 wt% as main components contains a milk-derived blocking agent, provided that the content of polyoxyethylene (alkylphenyl) ether surfactant is 0.20 wt% or less (when it does not contain polyoxyethylene (alkylphenyl) ether surfactant ) sample treatment liquid, which is an aqueous solution. A sample treatment solution for an immunochromatographic kit using a labeling substance provided with metal-resin composite particles, comprising 0.3 to 1.0 wt% buffer, salt, serum-derived protein, and 0.3 wt% as main components Polysorbate surfactant at least 2 wt%, milk-derived blocking agent at least 0.01 wt% and less than 2 wt%, and water, provided that the polyoxyethylene (alkylphenyl) ether surfactant content is 0.20 wt% A specimen treatment liquid characterized by the following (including cases where it does not contain a polyoxyethylene (alkylphenyl) ether surfactant) . The sample treatment liquid according to claim 1, further comprising 0.3 to 1.0 wt% of a buffering agent. The sample treatment liquid according to any one of claims 1 to 3, containing 0.5 to 2.0 wt% of salt. The sample treatment liquid according to any one of claims 1 to 4, which contains 0.1 to 3.0 wt% serum-derived protein. 6. The sample treatment liquid according to any one of claims 1 to 5, which has a pH of 7 to 9. An immunochromatography kit comprising the sample treatment liquid according to any one of claims 1 to 6.

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