JP6656052B2 - Immunoassay method, immunoassay kit and lateral flow type chromatographic test strip - Google Patents

Description

  The present invention relates to an immunoassay method capable of detecting and quantifying viruses, bacteria, and the like contained in a sample with high sensitivity, an immunoassay kit used for the method, and a lateral flow type chromatographic test strip.

  Since there are countless chemical substances in a living body, it is extremely important to qualitatively and quantitatively analyze a specific trace component in the living body. In the fields of medicine, pharmaceuticals, health foods, biotechnology, the environment, etc., medicines and foods that act only on specific parts (chemical substances) in the living body, analyzers and diagnostic agents that detect slight changes in the living body, It has evolved with the above technologies.

  One of the analysis techniques is an immunoassay. This is also called an immunological measurement method, and is a method of qualitatively and quantitatively analyzing a trace component using a specific reaction between an antigen and an antibody, which is one of the immune reactions. Antigen-antibody reactions are widely used in the above fields because of their high sensitivity and high selectivity. The immunoassay has various measurement methods depending on the measurement principle. For example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), latex agglutination (LIA, PA), immunochromatography ( ICA), the hemagglutination method (HA), the hemagglutination inhibition method (HI) and the like.

  The immunoassay qualitatively or quantitatively detects an antigen or an antibody from a change when the antigen and the antibody react to form a complex (a change in the concentration of the antigen, the antibody or the 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 that affects the detection ability in the immunoassay. In the immunoassays exemplified above, 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 substances (for CLIA) and the like are used.

  By the way, when colored fine particles are used as the labeling substance, the detection can be confirmed visually without using a special analyzer, so that it is expected that more simple measurement can be performed. As such colored fine particles, for example, Patent Literature 1 proposes a colored latex composed of gold nanoparticles bonded to the surface of polymer-based latex particles. By bonding the gold nanoparticles to the surface of the polymer-based latex particles, the gold nanoparticles themselves are useful as colorants to improve visibility and detection sensitivity, while the gold nanoparticles themselves are also excellent in binding to antigens or antibodies. From this, it is said that a sufficient amount of antigen or antibody can be bound even if the gold nanoparticles are bound to such an extent that a sufficiently dark color is obtained.

  The colored latex is obtained by irradiating a dispersion of styrene-acrylic acid copolymer latex and HAuCl, which is a precursor of gold nanoparticles, with gamma rays to bond gold nanoparticles to the surface of the latex. However, in the colored latex, since the gold nanoparticles are bonded only to the surface of the latex, the carrying amount of the gold nanoparticles exhibiting surface plasmon absorption is limited, and the gold nanoparticles are easily detached. As a result, the visibility and sensitivity of the immunological measurement reagent may not be sufficient. Further, irradiation with electromagnetic radiation such as gamma rays may damage latex. Furthermore, in the specification of Patent Document 1, the preferred ranges of the latex diameter and the gold nanoparticle diameter are disclosed. However, it is not clear whether or not the examples have been verified in these preferred ranges. There is no basis.

  Patent Literature 2 discloses polymer latex particles coated with metal gold, and suggests application to reagents that can be used for microscopy and immunoassay. However, the polymer latex particles coated with metal gold do not disclose the material or particle size of the polymer latex particles. Furthermore, there is no verification of the effect as a reagent usable for an immunoassay method. Therefore, its effect as a reagent on metal gold and polymer latex particles is unknown.

  Against this background, the present inventors have previously proposed a resin-metal complex having a specific structure as a labeling substance that enables highly sensitive immunological measurement (Patent Documents 3 and 4). And Japanese Patent Application No. 2015-139269).

  By the way, when detecting or quantifying an analyte contained in a biological sample such as blood, urine, saliva or an extracted sample of food, a lateral flow type chromatographic test strip (hereinafter, referred to as a “test strip” may be referred to as a “test strip”). ) Can quickly and easily detect an analyte. Therefore, test strips are widely used in various clinical tests and certification tests. So-called one-step and two-step immunochromatographs using such test strips are known.

  In the one-step immunochromatograph, first, a sample containing an analyte is added to the sample addition section. The added sample moves (develops) on the membrane from the sample addition section toward the determination section by capillary action. Since the intermediate reaction portion contains a labeled ligand that specifically binds to the analyte, the analyte binds to the labeled ligand to form a complex containing the analyte and the labeled ligand. . Next, the formed complex moves to the determination unit. In the determination section, a ligand (capture ligand) that specifically binds to the analyte in the complex is fixed. Therefore, when the complex moves to the determination unit, the complex is captured by the determination unit via a binding reaction between the analyte in the complex and the capture ligand. Next, in the determination unit, a signal due to the labeling substance in the captured complex is detected, and the analyte in the sample is qualitatively or quantitatively analyzed (for example, Patent Document 5).

  On the other hand, in the two-step immunochromatography, first, an analyte such as an antigen is specifically bound to the analyte, and a ligand such as a labeled antibody is brought into contact with the analyte to form a complex. Next, this complex was added as a mobile phase to a test strip, developed on a membrane by the principle of chromatography, and the complex was captured by a capture ligand (for example, a second antibody against the antigen) in the reaction part of the membrane. Thereafter, a signal emitted from the label is detected, and the analyte in the sample is qualitatively or quantitatively analyzed (for example, Patent Document 6).

JP 2009-168495 A JP-A-3-206959 International Publication WO2016 / 002742 International Publication WO2016 / 002743 JP 2011-27693 A JP 2005-522698 A

  In immunochromatography, sensitivity and visibility are affected by the color depth and the size of particles. In order to increase the sensitivity, it is important that a small number of particles be seen as a line even if caught by the test line. Therefore, a large particle is advantageous. In addition, in order for a person to visually recognize the small number of particles, it is desired to make the particles easily visible with dark colored particles. However, in the conventional immunochromatography using test strips, proteins containing enzymes, metal colloids, colored latex particles, and the like have been generally used as labeling substances. Immunochromatographs using these labeling substances are not always satisfactory in terms of color development, detection sensitivity, etc., and there is room for improvement.

For example, colloidal gold is a dark colored particle with high visibility, but small particles with a particle size of 70 nm or less are often used, so if the amount of antigen (such as virus) decreases, the visibility becomes extremely poor. , The sensitivity decreases. Further, when the size of the gold nanoparticles is set to 100 nm or more in order to increase the sensitivity, the original metallic color of gold appears, the dyeing degree of the colloid is remarkably reduced, and the visibility is reduced.
On the other hand, as the colored latex particles, relatively large particles of 200 nm to 300 nm are often used, so that even if the amount of antigen is small, the sensitivity can be expected to be increased by catching the large particles on the test line. However, the dyeing degree of the latex itself is limited, and a color development as deep as colloidal gold cannot be obtained. Accordingly, the colored latex particles are large particles, but have a low degree of dyeing, so that visibility is deteriorated and sensitivity cannot be increased. In addition, if the amount of the dye is increased in order to increase the dyeing degree of the colored latex particles, the dispersibility of the particles deteriorates, so that improvement in the dyeing degree cannot be expected.

  Therefore, an object of the present invention is to measure viruses and bacteria with high sensitivity by an immunoassay.

  The present inventors have conducted intensive studies, and as a result, have found that by using a resin-metal complex having a specific structure as a labeling substance, excellent color developability and detection sensitivity can be obtained and the above-mentioned problems can be solved. Heading, the present invention has been completed.

That is, the immunoassay method of the present invention is a method for detecting or quantifying a virus or a bacterium contained in a sample.
The immunoassay method of the present invention uses a lateral flow type chromatographic test strip including a membrane, and a determination section in which a capture ligand that specifically binds to the virus is immobilized on the membrane, using the following steps (I) to (III). );
Step (I): The virus or bacterium contained in the sample and an antibody that specifically binds to the virus or bacterium are labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. Contacting with a labeled antibody,
Step (II): a step of bringing the complex containing the virus or the bacterium and the labeled antibody, formed in step (I), into contact with the capture ligand in the determination unit;
Step (III): a step of measuring a coloring intensity derived from light energy absorption by localized surface plasmon resonance and electronic transition of the resin-metal composite,
Is performed.

  In the immunoassay method of the present invention, the metal particles may be gold particles or platinum particles.

  In the immunoassay method of the present invention, the metal particles in the resin-metal complex are a first particle having a portion exposed outside the resin particle, and a second particle entirely contained in the resin particle. And at least some of the first particles and the second particles may be three-dimensionally distributed in the surface layer of the resin particles.

  In the immunoassay method of the present invention, the resin particles may be polymer particles having a substituent capable of adsorbing a metal ion in the structure.

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

  In the immunoassay method of the present invention, the average particle size of the resin-metal complex may be in the range of 50 to 1000 nm.

  In the immunoassay method of the present invention, the antibody may be an anti-influenza A monoclonal antibody.

The immunoassay kit of the present invention is an immunoassay kit for detecting or quantifying a virus or a bacterium contained in a sample using a lateral flow-type chromatographic test strip.
The immunoassay kit of the present invention includes a lateral flow-type chromatographic test strip including a membrane, and a determination unit in which a capture ligand that specifically binds to the virus or bacterium is immobilized on the membrane. A detection reagent comprising a labeled antibody that specifically binds to an antibody that is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles.

  The lateral flow type chromatographic test strip of the present invention is for detecting or quantifying a virus or a bacterium contained in a sample. The lateral flow type chromatographic test strip of the present invention comprises a membrane, a determination unit in which a capture ligand that specifically binds to the virus or bacteria is fixed to the membrane in a direction in which the sample is developed, and the determination unit. On the upstream side, a reaction unit containing a labeled antibody labeled with an antibody that specifically binds to the virus or bacterium with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. ,including.

  According to the immunoassay method of the present invention, by using the resin-metal complex by labeling it with an antibody, an antigen, a protein, or the like, it is possible to achieve both excellent color development and high detection sensitivity, and to obtain blood, saliva, Antibodies, drugs, low molecular hormones (such as estrogen), high molecular hormones, cancer markers (such as hemoglobin in feces), and viruses (such as hepatitis B and C viruses) contained in biological fluids such as stool and urine Highly sensitive immunological measurements of influenza virus, etc., bacteria (Staphylococcus aureus, Legionella, periodontal disease, etc.), high molecular hormones (insulin, etc.), DNA and RNA, cytokines, etc. can be performed. Further, the immunoassay kit and the lateral flow type chromatographic test strip of the present invention are excellent in visibility, visual judgment, detection sensitivity, and are advantageous in immunological measurement of viruses and bacteria such as influenza virus and Legionella bacteria. Available.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram showing an outline of an immunoassay method using a lateral flow type chromatographic test strip according to one embodiment of the present invention. FIG. 1 is a schematic diagram illustrating a cross-sectional structure of a resin-metal composite used in an embodiment of the present invention. 9 is a scanning electron microscope (SEM) photograph of the resin-metal composite obtained in Production Example 2.

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

[Lateral flow type chromatographic test strip]
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. This test strip 200 can be preferably used for the immunoassay according to one embodiment of the present invention, as described later. Hereinafter, a case where the influenza virus is measured will be described as an example.

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

<Membrane>
As the membrane 110 used for the test strip 200, a membrane 110 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 influenza virus 160, various ligands, etc.) composed of a microporous substance that exhibits, for example, a capillary phenomenon and that develops at the same time as the sample is added. It is. Specific examples of the membrane 110 include polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, a fibrous or non-woven fibrous matrix composed of a cellulose derivative, a membrane, a filter paper, a glass fiber filter paper, a cloth, Cotton and the like. Among these, membranes composed of cellulose derivatives or nylon, filter paper, glass fiber filter paper, etc. are preferably used, and nitrocellulose membrane, mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membrane, nylon membrane are more preferred. , Filter paper is used.

  The test strip 200 preferably includes a support for supporting the membrane 110 for easier operation. As the support, for example, plastic or the like can be used.

<Sample addition section>
The test strip 200 may have a sample addition section 120 for adding a sample containing the influenza virus 160. The sample adding section 120 is a part for receiving a sample containing the influenza virus 160 in the test strip 200. The sample addition section 120 may be formed on the membrane 110 on the upstream side of the determination section 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 form the sample addition section 120.

<Judgment unit>
A capture ligand 131 that specifically binds to the influenza virus 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 influenza virus 160. For example, an antibody against the influenza virus 160 can be preferably used. The capture ligand 131 is immobilized so as not to move from the determination unit 130 even when the 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 influenza virus 160 is configured to contact the capture ligand 131 that specifically binds to the influenza virus 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 absorbing part>
The liquid absorbing portion 140 is formed of a pad of a water absorbing material such as cellulose filter paper, nonwoven fabric, cloth, cellulose acetate, and the like. The moving speed of the sample after the development front (front line) of the added sample reaches the liquid absorbing section 140 varies depending on the material and size of the liquid absorbing section 140. Therefore, by selecting the material, size, and the like of the liquid absorbing section 140, it is possible to set an optimal speed for detecting and quantifying the influenza virus 160. Note that the liquid absorbing section 140 has an optional configuration and may be omitted.

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

<Reaction part>
Although not shown, the test strip 200 may have a reaction portion including the labeled antibody 150 formed 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. 1 as a reaction part. When the test strip 200 has a reaction part, when the sample containing the influenza virus 160 is supplied to the reaction part or the sample addition part 120, the influenza virus 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, the complex 170 containing the influenza virus 160 and the labeled antibody 150 can be formed by simply supplying the sample to the reaction section or the sample addition section 120, so that a so-called one-step immunochromatography can be performed. Become.

  The reaction section is not particularly limited as long as it includes the labeled antibody 150 that specifically binds to the influenza virus 160, but may be one in which the labeled antibody 150 is directly applied to the membrane 110. Alternatively, the reaction section may be one in which a pad (conjugate pad) made of, for example, cellulose filter paper, glass fiber, non-woven fabric, etc. impregnated with the labeled antibody 150 is fixed to the membrane 110.

<Control part>
Although not shown, the test strip 200 may have a control unit formed by immobilizing a capture ligand that specifically binds to the labeled antibody 150 on the membrane 110 in the direction in which the sample is developed. The color intensity is measured by the control unit together with the judgment unit 130, so that the sample provided to the test strip 200 is developed, reaches the reaction unit and the judgment unit 130, and confirms that the test was performed normally. be able to. The control unit is created in the same manner as the above-described determination unit 130, except that another type of capture ligand that specifically binds to the labeled antibody 150 is used instead of the capture ligand 131. A configuration can be adopted. In addition, other than the above-described method, a substance showing a relationship between a key and a keyhole, for example, a substance such as avidin and biotin, an enzyme and a substrate, and an enzyme and a coenzyme can be used for the control section. Preferably, avidin and biotin may be used in place of capture ligand 131 and labeled antibody 150.

[Method for measuring influenza virus]
Next, a method for measuring the influenza virus 160 according to one embodiment of the present invention, which is performed using the test strip 200, will be described.

The method for measuring influenza virus 160 according to the present embodiment is a method for measuring influenza virus 160 for detecting or quantifying influenza virus 160 contained in a sample. Here, the influenza virus 160 includes not only humans but also those that infect animals other than humans, such as avian influenza viruses. The influenza virus 160 may have any antigenicity, and may be not only the whole virus but also a part thereof such as, for example, NP antigen, M1 antigen, HA antigen, and NA antigen. The method for measuring influenza virus 160 of the present embodiment uses a test strip 200 including a membrane 110 and a determination unit 130 in which a capture ligand 131 that specifically binds to the influenza virus 160 is fixed to the membrane 110. Steps (I) to (III);
Step (I): labeling the influenza virus 160 contained in the sample and an antibody that specifically binds to the influenza virus 160 with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. Contacting the labeled antibody 150,
Step (II): a step of bringing the complex containing the influenza virus 160 and the labeled antibody 150 formed in the step (I) into contact with the capture ligand 131 in the determination unit 130;
Step (III): a step of measuring the color intensity derived from light energy absorption due to localized surface plasmon resonance and electronic transition of the resin-metal complex,
Can be included.

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

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

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

  In addition, since the capture ligand 131 does not specifically bind to the labeled antibody 150, when the labeled antibody 150 not bound to the influenza virus 160 reaches the determination unit 130, the labeled antibody 150 not bound to the influenza virus 160 Pass through the determination unit 130. Here, if the test strip 200 has a control section (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 is unfolded. And bind to the other capture ligand in the control section. As a result, the labeled antibody 150 that does not form the complex 170 with the influenza virus 160 is captured by the control section.

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

  By performing the washing step, in the step (III), the determination unit 130 or the determination unit 130 and the determination unit 130 and the control unit measure the localized surface plasmon resonance of the resin-metal complex and the color development due to light energy absorption due to electron transition. In this case, the intensity of background coloring can be reduced, the signal / background ratio can be increased, and the detection sensitivity and quantification can be further improved.

Step (III):
The step (III) is a step of measuring the color intensity derived from light energy absorption due to localized surface plasmon resonance and electronic transition of the resin-metal composite. After the step (II) or the washing step as required, the color intensity derived from light energy absorption due to localized surface plasmon resonance and electronic transition of the resin-metal composite is measured in the test strip 200.

  When a control portion is formed on the test strip 200, the labeled antibody 150 is captured by another capture ligand in the control portion in step (II) to form a complex. Therefore, in the process (III), in the test strip 200, not only in the determination unit 130 but also in the control unit, it is possible to cause the localized surface plasmon resonance and the color development due to the light energy absorption by the electron transition. As described above, by measuring the coloring intensity in the control section together with the determination section 130, it is possible to confirm whether or not the sample provided on the test strip 200 has developed normally and has reached the reaction section and the determination section 130.

<Sample>
The sample in the method for measuring influenza virus 160 of the present embodiment is not particularly limited as long as it contains influenza virus 160. For example, a biological sample containing the influenza virus 160 of interest (ie, whole blood, serum, plasma, urine, saliva, sputum, nasal or pharyngeal swabs, cerebrospinal fluid, amniotic fluid, nipple secretions, tears, sweat, exudates from the skin) And extracts from tissues, cells, and stool). Prior to the above step (I), the influenza virus 160 contained in the sample is pretreated, if necessary, in order to facilitate a specific binding reaction between the labeled antibody 150 and the capture ligand 131 and the influenza virus 160. You may. Here, examples of the pretreatment include a chemical treatment using various chemicals such as an acid, a base, and a surfactant, and a physical treatment using heating, stirring, ultrasonic waves, and the like. In particular, when the substance is not normally exposed on the surface, such as 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 the specific binding reaction, for example, the binding reactivity between the ligand and the influenza virus 160 such as an antigen-antibody reaction. .

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

<Labeled antibody>
The labeled antibody 150 is used in step (I) to form a complex 170 containing the influenza virus 160 as an analyte and the labeled antibody 150 by contacting the influenza virus 160 contained in the sample. The labeled antibody 150 is obtained by labeling an antibody that specifically binds to the influenza virus 160 with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. Here, “labeling” means that the resin-metal complex is directly or indirectly attached to the antibody to the extent that the resin-metal complex is not detached from the labeled antibody 150 in the steps (I) to (III). Means that they are fixed by chemical or physical bonding or adsorption. For example, the labeled antibody 150 may be one in which a resin-metal complex is directly bonded to an antibody, or one in which an antibody and a resin-metal complex are bonded via an arbitrary linker molecule. Alternatively, each of them may be fixed to insoluble particles.

  Further, in the present embodiment, the resin-metal complex used as a label has a structure in which a plurality of metal particles are immobilized on resin particles. The details of the resin-metal composite will be described later.

In the present embodiment, the “antibody” is not particularly limited, and may be, for example, a polyclonal antibody, a monoclonal antibody, an antibody obtained by genetic recombination, or an antibody fragment capable of binding to an antigen [eg, H chain , L chain, Fab, Fab ′, F (ab ′) ₂ , SCFV, etc.]. Specific examples of preferred antibodies include, for example, anti-influenza virus monoclonal antibodies such as anti-influenza A monoclonal antibody, anti-influenza B monoclonal antibody, and anti-influenza C monoclonal antibody, and anti-Legionella polyclonal antibodies.

<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 includes at least the following step A;
Step A) including a step of obtaining the labeled antibody 150 by mixing and binding the resin-metal complex with the antibody under the first pH condition, and preferably further comprises a step B;
Step B) may include a step of treating the labeled antibody 150 under the second pH condition.

[Step A]
In step A, the labeled antibody 150 is obtained by mixing the resin-metal complex with the antibody under the first pH condition. In step A, the solid resin-metal complex is preferably brought into contact with the antibody in a state of being dispersed in the liquid phase. The first pH condition differs depending on the metal species of the metal particles in the resin-metal composite.

  When the metal particles of the resin-metal composite are gold particles (including gold alloy particles; the same applies hereinafter), the first pH condition can be applied under any of acidic, neutral and alkaline conditions. However, from the viewpoint of uniformly contacting the resin-metal complex and the antibody while maintaining the dispersion of the resin-metal complex and the activity of the antibody, conditions within the range of pH 2 to 7 are preferable, and further acidic conditions, for example, pH 2.0. More preferably, it is in the range of 5 to 5.5. When the metal particles are gold particles, when the conditions for binding the resin-metal complex and the antibody are less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and when the pH exceeds 7, the resin-metal complex may be inactivated. When the body and the antibody are mixed, they aggregate and become difficult to disperse. However, when the antibody is not deactivated due to strong acidity, the treatment can be performed even at a pH lower than 2.

  Further, when the metal particles of the resin-metal complex are particles other than gold, for example, platinum, palladium, or an alloy thereof, the first pH condition is to control the dispersion of the resin-metal complex and the activity of the antibody. From the viewpoint of uniformly contacting the antibody with the resin-metal complex while maintaining the same, conditions within the range of pH 2 to 10 are preferable, and more preferably within the range of pH 5 to 9, for example. When the metal particles are particles other than gold, when the conditions for binding the resin-metal complex and the antibody are less than pH 2, the antibody may be degraded and deactivated due to strong acidity, and if the pH exceeds 10, the resin- When the metal complex and the antibody are mixed, they aggregate and become difficult to disperse. However, when the antibody is not deactivated due to strong acidity, the treatment can be performed even at a pH lower 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 is mixed with the binding buffer 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, or the like.

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

[Step B]
In the step B, the labeled antibody 150 obtained in the step A is treated under the second pH condition to perform blocking for suppressing 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. The blocking conditions vary depending on the metal species of the metal particles in the resin-metal composite.

  When the metal particles of the resin-metal complex are gold particles, the second pH condition is preferably in the range of, for example, 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, 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 the pH exceeds 9, the labeled antibody 150 aggregates and dispersion becomes difficult.

  Further, when the metal particles of the resin-metal complex are particles other than gold, the second pH condition is, for example, a pH of 2 to 10 from the viewpoint of maintaining the activity of the antibody and suppressing the aggregation of the labeled antibody 150. It is preferably within the range, and more preferably within the range of pH 5 to 9, from the viewpoint of suppressing nonspecific adsorption of the labeled antibody 150. If the blocking condition is lower 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 dispersion becomes difficult.

  Step B is preferably performed using a blocking buffer (Blocking Buffer) 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, for example, it is preferable to use a solution of a protein that does not bind to the analyte. Examples of proteins that can be used in the blocking buffer include bovine serum albumin, ovalbumin, casein, gelatin and the like. In addition to proteins, artificial polymer such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and amino acid polymer may be used. 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, or the like. A known method can be used to disperse the labeled antibody 150, but it is preferable to use a dispersing means such as ultrasonic treatment or stirring with a rotator. Thus, a dispersion liquid in which the labeled antibody 150 is uniformly dispersed is obtained.

  As described above, a dispersion of the labeled antibody 150 is obtained. From the dispersion, only the labeled antibody 150 can be collected as a solid portion by a solid-liquid separation means such as centrifugation. Further, a cleaning process, a storage process, and the like can be performed as necessary. Hereinafter, the cleaning process and the storage process will be described.

(Washing treatment)
In the washing treatment, a washing 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 washing buffer. For dispersion, it is preferable to use dispersion means such as ultrasonic treatment. The washing buffer is not particularly limited. For example, a Tris buffer, a glycinamide buffer, an arginine buffer, or the like having a predetermined concentration adjusted to a range of pH 8 to 9 may be used. it can. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, or the like. The washing treatment of the labeled antibody 150 can be repeated a plurality of times as necessary.

(Save processing)
In the preservation treatment, 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, it is preferable to use 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 the anticoagulant include sugars represented by sucrose, maltose, lactose, and trehalose; artificial polymers such as glycerin, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG); and octylphenol ethoxylate. Surfactants, ionic surfactants such as polysorbates, and the like. Examples of the stabilizer include, but are not particularly limited to, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin, and saccharides represented by sucrose, maltose, lactose, and trehalose. Thus, the preservation treatment of the labeled antibody 150 can be performed.

  In each of the above steps, a surfactant and a preservative such as sodium azide and p-hydroxybenzoate can be used, if necessary.

<Resin-metal composite>
Next, the resin-metal complex used as a label in the influenza virus measurement method of the present embodiment will be described in detail. FIG. 2 is a schematic cross-sectional view of a resin-metal composite used as a marker in the present embodiment. The resin-metal composite 100 includes resin particles 10 and metal particles 20.

  In the resin-metal composite 100, the metal particles 20 are dispersed or fixed in the resin particles 10. Further, in the resin-metal composite 100, a part of the metal particles 20 is three-dimensionally distributed in the surface layer portion 60 of the resin particle 10, and a part of the three-dimensionally distributed metal particles 20 is partially It is 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 included in the resin particles 10 (hereinafter, also referred to as “encapsulated metal particles 30”), portions embedded in the resin particles 10, and portions outside the resin particles 10. Metal particles having an exposed portion (hereinafter, also referred to as "partially exposed metal particles 40") and metal particles adsorbed on the surface of the resin particles 10 (hereinafter, also referred to as "surface adsorbed metal particles 50"). Exists. The partially exposed metal particles 40 and the surface-adsorbed metal particles 50 correspond to “first particles” in the present invention, and the encapsulated metal particles 30 correspond to “second particles” in the present invention.

  For example, when the resin-metal complex 100 is used for immunological measurement, an antibody is immobilized on partially exposed metal particles 40 or surface-adsorbed metal particles 50 and used. At this time, the antibody is immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, but is not immobilized on the encapsulated metal particles 30. However, since all of the metal particles 20 including the encapsulated metal particles 30 exhibit absorption by localized surface plasmon resonance and light energy absorption by electron transition, not only the partially exposed metal particles 40 and the surface-adsorbed metal particles 50 but also In addition, the encapsulated metal particles 30 also contribute to the improvement of visibility as a label for immunological measurement. Further, the partially exposed metal particles 40 and the encapsulated metal particles 30 have a large contact area with the resin particles 10 as compared with the surface adsorbed metal particles 50, and have a physical adsorption force such as an anchor effect due to the embedded state. Strong and difficult to detach from the resin particles 10. Therefore, durability and stability as a label for immunological measurement using the resin-metal complex 100 can be improved.

  The encapsulating metal particles 30 are all covered with the resin constituting the resin particles 10. The partially exposed metal particles 40 have a surface area of 5% or more and less than 100% covered with the resin constituting the resin particles 10. In light of the durability of each of the above uses, the lower limit is preferably 20% or more of the surface area, and more preferably 30% or more. The surface-adsorbed metal particles 50 have a surface area of more than 0% and less than 5% covered by the resin constituting the resin particles 10.

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

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

  Further, 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 existing in the surface layer portion 60 are partially exposed metal particles 40 or surface adsorbed metal particles 50. This is preferable because the sufficient amount of the antibody immobilized on the metal particles 20 can be ensured, so that the sensitivity as a labeling substance increases. In other words, it is preferable that 10 wt% to 95 wt% of the metal particles 20 existing in the surface layer portion 60 are the encapsulated metal particles 30.

  Here, the “surface layer portion” means a range of 50% of the particle radius in the depth direction from the surface of the resin particle 10. 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.

The resin particles 10 are preferably polymer particles having a substituent capable of adsorbing metal ions in the structure. In particular, nitrogen-containing polymer particles are preferable. Nitrogen atoms in the nitrogen-containing polymer are preferable because they are excellent in visibility and easily chemisorb anionic metal ions, which are precursors of metal particles 20 such as gold, platinum, and palladium, which are easy to immobilize antibodies. 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 encapsulated 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, so that silver, nickel and copper can easily adsorb cationic metal ions which are precursors of metal particles 20 such as, Metal particles 20 such as silver, nickel, and copper can be formed, and alloys with metals such as gold, platinum, and palladium can also be formed.
On the other hand, in the case of resin particles other than the nitrogen-containing polymer having a substituent capable of adsorbing metal ions, such as polystyrene, it is difficult to adsorb the metal ions inside the resin. As a result, most of the generated metal particles 20 become the surface-adsorbed metal particles 50. As described above, since the contact area between the surface-adsorbed metal particles 50 and the resin particles 10 is small, the adhesive force between the resin and the metal is small, and the effect of the metal particles 20 detaching from the resin particles 10 tends to be large.
The nitrogen-containing polymer is a resin having a nitrogen atom in a main chain or a side chain, and examples thereof include polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, and polyaniline. Preferred are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. Further, when a nitrogen atom is present in the side chain, it can be widely used, for example, acrylic resin, phenol resin, epoxy resin and the like.

  As the material of the metal particles 20, for example, silver, nickel, copper, gold, platinum, palladium and the like can be applied. Preferably, they are gold, platinum and palladium which are excellent in visibility and easy to immobilize antibodies. These are preferable because they exhibit absorption derived from light energy due to localized surface plasmon resonance and electronic transition. More preferably, it is gold having good storage stability. These metals can be used alone or in a composite such as an alloy. Also, from the viewpoint that excellent color developability is obtained when the resin-metal composite 100 is combined with an antibody under conditions within a pH range of 2 to 7, gold, a gold alloy, platinum, Alternatively, platinum alloys are the most preferred metal species. Here, the gold alloy means an alloy made of, for example, gold and a metal species other than gold and containing 10% by weight or more of gold. Here, other metal species forming an alloy with gold are not particularly limited, but, for example, silver, nickel, copper, platinum, palladium and the like are preferable, and storage stability, platinum and palladium having excellent visibility are more preferable. preferable. Further, the platinum alloy means an alloy composed of platinum and a metal species other than platinum and containing 1% by weight or more of platinum. Here, other metal species that form an alloy with platinum are not particularly limited, but, for example, silver, nickel, copper, gold, palladium, and the like are preferable, and gold, palladium, and the like having excellent storage stability and visibility are more preferable. preferable.

  The average particle size of the metal particles 20 measured by scanning electron microscope (SEM) observation is preferably, for example, 1 to 80 nm from the viewpoint of obtaining high detection sensitivity in immunological measurement. When the average particle diameter of the metal particles 20 is less than 1 nm or more than 80 nm, the sensitivity tends to decrease because absorption due to localized surface plasmon resonance and light energy absorption due to electron transition are difficult to develop. For example, in the case of gold particles, the average particle diameter of the metal particles 20 is preferably 20 nm or more and less than 70 nm, and more preferably 22 nm or more and less than 50 nm, from the viewpoint of obtaining high detection sensitivity in immunological measurement. Further, for example, in the case of platinum particles, from the viewpoint of obtaining high detection sensitivity in immunological measurement, preferably from 1 nm to 50 nm, more preferably from 1 nm to 30 nm, and still more preferably from 1 nm to 20 nm, Most preferably, it is 1 nm or more and 15 nm or less. In particular, when the average particle diameter of the platinum particles is 15 nm or less, extremely excellent detection sensitivity is obtained when the resin-metal composite 100 is used as a labeling substance for an immunochromatography.

  The average particle size of the resin-metal composite 100 is preferably, for example, 50 to 1000 nm. If the average particle size is less than 50 nm, for example, the amount of metal carried tends to be reduced, so that coloring tends to be weaker than metal particles of the same size, and if it exceeds 1000 nm, when a labeling substance or a reagent is used, There is a tendency that the pores of a chromatographic medium such as a membrane filter are easily clogged, and the dispersibility tends to be reduced. The average particle size of the resin-metal composite 100 is preferably 100 nm or more and less than 700 nm, 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 size is 300 nm or more, excellent detection sensitivity can be obtained stably when used as a labeling substance for immunochromatography. Here, the particle diameter of the resin-metal composite 100 means a value obtained by adding the length of the projecting portion of the partially exposed metal particles 40 or the surface-adsorbed metal particles 50 to the particle diameter of the resin particles 10, / Scattering method, dynamic light scattering method, or centrifugal sedimentation method.

  The method for producing the resin-metal composite 100 is not particularly limited. For example, a solution containing metal ions is added to a dispersion of the resin particles 10 produced by the emulsion polymerization method, and the metal ions are adsorbed to the resin particles 10 (hereinafter, referred to as “metal ion-adsorbed resin particles”). Further, 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 resin-metal composite 100 is obtained.

Further, for example, when gold particles are used as the metal particles 20, examples of the solution containing metal ions include a chloroauric acid (HAuCl ₄ ) aqueous solution. Further, a metal complex may be used instead of the metal ion.
In addition, as a solvent for the solution containing the metal ion, instead of water, a water-containing alcohol or alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, and t-butanol, hydrochloric acid, sulfuric acid, and nitric acid are used. And the like.
In the solution, if necessary, for example, a water-soluble polymer compound such as polyvinyl alcohol, a surfactant, an alcohol; an ether such as tetrahydrofuran, diethyl ether, diisopropyl ether; an alkylene glycol, a polyalkylene glycol; And other water-miscible organic solvents such as polyols such as monoalkyl ethers or dialkyl ethers, and glycerin; and ketones such as acetone and methyl ethyl ketone. Such an additive is effective in accelerating the reduction reaction rate of the metal ions and controlling the size of the generated metal particles 20.

In addition, known reducing agents can be used. For example, sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, Rochelle salt and the like. No. Of these, sodium borohydride, dimethylamine borane, and citric acid are preferred. A surfactant can be added to the reducing agent solution as needed, 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.
Further, the particle diameter of the metal particles 20 to be formed can be controlled by adjusting the reduction rate of the metal ions according to the temperature of the reducing agent solution.

  When reducing metal ions in the metal ion-adsorbing resin particles to form 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 former is preferred from the viewpoint of the easiness of forming 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, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 (Big Chemie Japan) And the like. Further, 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 can be preferably applied to immunological measurement of viruses and bacteria by adsorbing an antibody on the surface of the metal particles 20. In particular, a labeling substance or a reagent for immunological measurement of an influenza virus which is excellent in visual judgment even in a low concentration region (high sensitivity region) and can achieve both excellent visibility and high detection sensitivity. It can be preferably applied as a material. The form of the labeling substance for immunological measurement or the reagent for immunological measurement is not particularly limited. For example, a dispersion in which the resin-metal complex 100 is dispersed in water or a buffer solution adjusted to pH is used. Can be used as

[Immunodetection / quantification kit]
The kit for immunoassay according to one embodiment of the present invention detects influenza virus 160 contained in a sample based on the method for measuring influenza virus according to the present embodiment using, for example, a lateral flow-type chromatographic test strip 200. Or a kit for quantification.

The kit for immunoassay according to the present embodiment comprises:
A membrane 110,
A lateral flow type chromatographic test strip 200 including a determination unit 130 in which a capture ligand 131 that specifically binds to the influenza virus 160 is immobilized on the membrane 110;
A detection reagent comprising a labeled antibody 150 in which an antibody that specifically binds to influenza virus 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;
Contains. The immunoassay kit of the present embodiment may further include other components as necessary.

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

  Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. In the following Examples and Comparative Examples, various measurements and evaluations are as follows unless otherwise specified.

<Absorbance measurement of resin-metal composite>
The absorbance of the resin-metal composite is measured by adding a resin-metal composite dispersion (dispersion medium: water) adjusted to 0.01 wt% to an optical white plate glass cell (optical path length 10 mm), and using an instantaneous multiphotometry system (Otsuka The absorbance at 570 nm for gold and 700 nm for platinum was measured using MCPD-3700 (manufactured by Denshi Co., Ltd.). In the case of gold, the absorbance at 570 nm was 0.9 or more (good), 0.5 to less than 0.9 (good), and less than 0.5 (bad). In the case of platinum, 吸 光 (good) when the absorbance at 700 nm was 0.6 or more, Δ (good) when 0.4 to less than 0.6, and × (improper) when less than 0.4.

<Solid concentration measurement and measurement of metal loading>
1 g of the dispersion before concentration adjustment was placed in a porcelain crucible and heat-treated 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 equation.

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

Further, the sample after the heat treatment was further subjected to a heat treatment at 500 ° C. for 5 hours, the weight before and after the heat treatment was measured, and the amount of metal carried was calculated from the following equation.
Metal loading (wt%) =
[Weight (g) after heat treatment at 500 ° C./weight (g) before heat treatment at 500 ° C.] × 100

<Measurement of average particle diameter of resin-metal composite>
It was measured using a disk centrifugal particle size distribution analyzer (CPS Instruments). The measurement was performed in a state where the resin-metal composite was dispersed in water.

<Measurement of average particle diameter of metal particles>
The average particle diameter of the metal particles was measured by dropping a resin-metal composite dispersion onto a metal mesh provided with a carbon support film and using a substrate prepared by a field emission scanning electron microscope (STEM; SU, manufactured by Hitachi High-Technologies Corporation). -9000), the area average diameter of the metal particles was measured.

[Production Example 1]
<Synthesis of resin particles>
Aliquat 336 [manufactured by Aldrich] (2.00 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 48.00 g) and divinyl were dissolved. Benzene (DVB, 2.00 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g) dissolved in 18.00 g of pure water was added dropwise, followed by stirring at 60 ° C. for 3.5 hours, Resin particles A-1 having an average particle diameter of 0.38 μm were obtained. After sedimentation by centrifugation and removal of the supernatant, an 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 a 10 wt% resin particle dispersion B-1.

<Synthesis of resin-metal composite>
After adding 1233 ml of pure water to the resin particle dispersion B-1 (50 ml), a 30 mM aqueous solution of chloroauric acid (100 ml) was added, and the mixture was allowed to stand at room temperature for 24 hours. Thereafter, the operation of precipitating the resin particles by centrifugation and removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare a 2.5 wt% gold ion-adsorbed resin particle dispersion C-1.
Next, the above-mentioned C-1 (42.4 ml) was added to 1580 ml of pure water, a 528 mM dimethylamine borane aqueous solution (10 ml) was added dropwise while stirring at 3 ° C., and the mixture was stirred at room temperature for 2 hours to give an average. A resin-gold composite D-1 having a particle diameter of 0.399 μm was obtained. After the above-mentioned D-1 was precipitated by centrifugation and the supernatant was removed, the operation of dispersing again in pure water was repeated three times, and then purified by dialysis to obtain a 1 wt% resin-gold composite dispersion E. -1 was obtained. The absorbance of the resin-gold complex F-1 in E-1 was 0.98 as measured by the method described above. The average particle diameter of the gold particles in F-1 was 25.1 nm, and the amount of gold carried was 53.2 wt%.

[Production Example 2]
<Synthesis of resin particles>
Aliquat 336 [manufactured by Aldrich] (1.00 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 48.00 g) and divinyl were dissolved. Benzene (DVB, 2.00 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g) dissolved in 18.00 g of pure water was added dropwise over 2 minutes, and the mixture was stirred at 60 ° C. for 3.5 hours. Thereby, resin particles A-2 having an average particle diameter of 0.42 μm were obtained. After sedimentation by centrifugation and removal of the supernatant, an 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 a resin particle dispersion B-2 of 10 wt%.

<Synthesis of resin-metal composite>
After adding 1233 ml of pure water to the resin particle dispersion B-2 (50 ml), a 30 mM aqueous solution of chloroauric acid (100 ml) was added, and the mixture was allowed to stand at room temperature for 24 hours. Thereafter, the operation of precipitating the resin particles by centrifugation and removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare a 2.5 wt% gold ion-adsorbed resin particle dispersion C-2.
Next, the above-mentioned C-2 (42.4 ml) was added to 1580 ml of pure water, a 528 mM dimethylamine borane aqueous solution (10 ml) was added dropwise while stirring at 3 ° C., and the mixture was stirred at room temperature for 2 hours to give an average. A resin-gold composite D-2 having a particle size of 0.438 μm was obtained. After the above-mentioned D-2 was precipitated by centrifugation, the supernatant was removed, and the operation of re-dispersion in pure water was repeated three times, and the resultant was purified by dialysis to obtain a 1 wt% resin-gold composite dispersion E. -2 was obtained. The absorbance of the resin-gold composite F-2 in E-2 was 0.98 as measured by the method described above. The average particle size of the gold particles in F-2 was 25.0 nm, and the amount of gold carried was 54.7% by weight. A scanning electron microscope (SEM) photograph of F-2 is shown in FIG.

[Production Example 3]
Aliquat 336 (manufactured by Aldrich) (3.00 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 49.50 g) and divinyl were dissolved. Benzene (DVB, 0.50 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.250 g) dissolved in 18.00 g of pure water was added dropwise, followed by stirring at 60 ° C. for 3.5 hours. Resin particles A-3 having an average particle diameter of 370 nm were obtained. After sedimentation by centrifugation and removal of the supernatant, an 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 a resin particle dispersion liquid B-3 of 10 wt%.

<Synthesis of resin-metal composite>
After 245 ml of pure water was added to the resin particle dispersion B-3 (90 ml), a 400 mM aqueous solution of chloroplatinic acid (90 ml) was added, and the mixture was stirred at 30 ° C. for 3 hours and left at room temperature for 24 hours. Thereafter, the operation of precipitating the resin particles by centrifugation and removing the supernatant was repeated three times to remove excess chloroplatinic acid. After that, the concentration was adjusted to prepare a 5 wt% platinum ion-adsorbed resin particle dispersion C-3.
Next, the above C-3 (55 ml) was added to 3825 ml of pure water, a 132 mM aqueous dimethylamine borane solution (110 ml) was added dropwise while stirring at 3 ° C., and the mixture was stirred at 3 ° C. for 1 hour. Thereafter, by stirring at 25 ° C. for 3 hours, a resin-platinum composite D-3 having an average particle diameter of 382 nm was obtained. After the above D-3 was precipitated by centrifugation and the supernatant was removed, the operation of re-dispersion in pure water was repeated three times, and then purified by dialysis to remove impurities. Thereafter, the concentration was adjusted to obtain a 1 wt% resin-platinum composite dispersion E-3. The absorbance of the resin-platinum composite F-3 in E-3 was 0.70 as a result of measurement according to the method described above. Further, the average particle diameter of the platinum particles of F-3 was 5 nm, and the supported amount of platinum was 38.5 wt%.

[Production Example 4]
<Synthesis of resin particles>
Aliquat 336 [manufactured by Aldrich] (1.50 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 49.50 g) and divinyl were dissolved. Benzene (DVB, 0.50 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.50 g) dissolved in 18.00 g of pure water was added dropwise, followed by stirring at 60 ° C. for 3.5 hours, Resin particles A-4 having an average particle diameter of 430 nm were obtained. After sedimentation by centrifugation and removal of the supernatant, an 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 a resin particle dispersion B-4 of 10 wt%.

<Synthesis of resin-metal composite>
After 245 ml of pure water was added to the resin particle dispersion B-4 (90 ml), a 400 mM aqueous solution of chloroplatinic acid (90 ml) was added, and the mixture was stirred at 30 ° C. for 3 hours and left at room temperature for 24 hours. Thereafter, the operation of precipitating the resin particles by centrifugation and removing the supernatant was repeated three times to remove excess chloroplatinic acid. Thereafter, the concentration was adjusted to prepare a 5 wt% platinum ion-adsorbed resin particle dispersion C-4.
Next, the above C-4 (55 ml) was added to 3825 ml of pure water, a 132 mM aqueous dimethylamine borane solution (110 ml) was added dropwise at 160 rpm and stirring at 3 ° C., and the mixture was stirred at 3 ° C. for 1 hour. Thereafter, by stirring at 25 ° C. for 3 hours, a resin-platinum composite D-4 having an average particle diameter of 454 nm was obtained. After the above D-4 was precipitated by centrifugation and the supernatant was removed, the operation of re-dispersion in pure water was repeated three times, and then purified by dialysis to remove impurities. Thereafter, the concentration was adjusted to obtain a 1 wt% resin-platinum composite dispersion E-4. As a result of measuring the absorbance of the resin-platinum composite F-4 in E-4 according to the method described above, it was 0.70. Further, the average particle diameter of the platinum particles of F-4 was 5 nm, and the carried amount of platinum was 37.7% by weight.

[Production Example 5]
<Synthesis of colloidal gold>
250 ml of a 1 mM aqueous solution of chloroauric acid was placed in a 500-ml three-necked round-bottomed flask, and the mixture was boiled with vigorous stirring using a heating reflux apparatus. After boiling, 25 ml of a 38.8 mM aqueous solution of sodium citrate was added, and the solution turned from pale yellow to concentrated. It was confirmed that the color changed to red. After heating was continued for 10 minutes with stirring, the mixture was allowed to cool with stirring at room temperature for about 30 minutes. The solution was filtered using a membrane filter having a pore size of 2 μm, transferred to an Erlenmeyer flask, and stored in a cool, dark place. The average particle size of the produced particles was 12.3 nm. The absorbance was 1.32 as measured according to the method described above.

[Reagents, etc.]
The following reagents and the like were used in Examples and Comparative Examples.
Anti-influenza A monoclonal antibody (7.15 mg / mL / PBS): Anti-Legionella polyclonal antibody manufactured by Adtec Co., Ltd. Binding buffer a: A 100 mM boric acid solution was adjusted to pH ≒ 3 with HCl.
Binding buffer b: 100 mM boric acid solution was adjusted to pH 8.5 with NaOH.
Blocking buffer a: A 1% by weight bovine serum albumin solution was adjusted to pH ≒ 5 with HCl.
Blocking buffer b: 1 wt% bovine serum albumin solution was adjusted to pH 8.5 with HCl.
Washing buffer: 5 mM Tris solution was adjusted to pHＨＣｌ8.5 with HCl.
Storage buffer: Sucrose was added to the washing buffer to a concentration of 10% by weight.
Influenza A positive control (APC): Influenza A virus inactivated antigen (manufactured by Adtech Co., Ltd.) was prepared by diluting 100 times using a sample processing solution (manufactured by Adtec Co., Ltd.). The antigen concentration of APC corresponds to 5000 FFU / ml.
Negative control: Sample processing solution (Adtech Co., Ltd.)
AuNCP beads (1): resin-gold composite obtained in Preparation Example 1 AuNCP beads (2): resin-gold composite obtained in Preparation Example 2 PtNCP beads (1): resin-platinum composite obtained in Preparation Example 3 PtNCP beads (2): resin-platinum composite obtained in Preparation Example 4

[Example 1]
(Coupling process)
0.1 mL of AuNCP beads (1) was added as a resin-metal complex to a microtube [2 mL of ibis (registered trademark; manufactured by AS ONE Corporation)], and 0.9 mL of a binding buffer a was added. After thorough mixing by inversion mixing, an anti-influenza A monoclonal antibody was added, and the mixture was inverted and stirred at room temperature for 3 hours to obtain a labeled antibody-containing solution containing an anti-influenza A monoclonal antibody labeled with a resin-metal complex. a-1 was obtained.

(Block process)
Next, the labeled antibody-containing solution a-1 was cooled on ice, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. Then, 1 mL of the blocking buffer a was added to the solid residue, and the mixture was applied for 10 to 20 seconds. Then, the mixture was subjected to ultrasonic dispersion treatment, and further overturned and stirred at room temperature for 2 hours to obtain a labeled antibody-containing solution b-1.

(Washing treatment)
Next, after cooling the labeled antibody-containing solution b-1 with ice, centrifugation is performed at 12,000 rpm for 5 minutes, and the supernatant is removed. Then, 1 mL of a washing buffer is added to the solid residue, and the solution is applied for 10 to 20 seconds. To perform an ultrasonic dispersion treatment. This operation was repeated three times to obtain a washing process.

(Save processing)
Next, after cooling on ice, centrifugation is performed at 12,000 rpm for 5 minutes, and the supernatant is removed. Then, 1 mL of a storage buffer is added to the solid residue, and ultrasonic dispersion treatment is performed for 10 to 20 seconds. As a result, a labeled antibody-containing liquid c-1 was obtained.

<Evaluation method>
The evaluation was carried out using a monochrome screen for evaluating influenza A (manufactured by Adtech), and the color development levels were compared after 5 minutes, 10 minutes, and 15 minutes. The color development level was determined using a gold colloid determination color sample (manufactured by Adtech). In the performance evaluation, a 2-fold dilution series (1- to 1024-fold) of an influenza A positive control (APC) was used as an antigen.

<Evaluation procedure>
In each well of the 96-well plate, 3 μL of the labeled antibody-containing solution c-1 was added, and a 2-fold dilution column of APC (1 to 1024-fold, represented as APC × 1 to APC × 1024, respectively) and a negative control, respectively, 100 μL was mixed. Next, 50 μL was added to a monochrome screen for evaluating influenza A, and the color development levels after 5 minutes, 10 minutes, and 15 minutes were evaluated. The results are shown in Table 1. It should be noted that the larger the numerical value in Table 1, the higher the coloring level (the stronger the coloring).

  From Table 1, it was confirmed that the labeled antibody-containing solution c-1 using the AuNCP beads (1) exhibited good color development even with a 256-fold diluted antigen, and had excellent labeling performance.

[Example 2]
A labeled antibody-containing solution c-2 was prepared in the same manner as in Example 1 except that the AuNCP beads (2) were used instead of the AuNCP beads (1), and the coloring level was evaluated. The results are shown in Table 2.

  From Table 2, it was confirmed that the labeled antibody-containing solution c-2 using the AuNCP beads (2) exhibited good color development even with a 256-fold diluted antigen, and had excellent labeling performance.

[Example 3]
A labeled antibody-containing solution c-3 was prepared in the same manner as in Example 1 except that the PtNCP beads (1) were used instead of the AuNCP beads (1), and the coloring level was evaluated. Table 3 shows the results.

  From Table 3, it was confirmed that the labeled antibody-containing solution c-3 using the PtNCP beads (1) exhibited good color development even with a 512-fold diluted antigen, and had excellent labeling performance.

[Example 4]
A labeled antibody-containing solution c-4 was prepared in the same manner as in Example 1 except that the PtNCP beads (2) were used instead of the AuNCP beads (1), and the color development level was evaluated. Table 4 shows the results.

  From Table 4, it was confirmed that the labeled antibody-containing solution c-4 using the PtNCP beads (2) exhibited good color development even with 1024-fold diluted antigen, and had excellent labeling performance.

[Example 5]
(Coupling process)
0.1 mL of PtNCP beads (1) was added as a resin-metal complex to a microtube [2 mL of ibis (registered trademark; manufactured by AS ONE Corporation)], and 0.9 mL of a binding buffer a was added. After sufficient mixing by inversion mixing, an anti-Legionella polyclonal antibody was added, and the mixture was stirred by inversion at room temperature for 3 hours to prepare a labeled antibody-containing solution a-5 containing an anti-Legionella polyclonal antibody labeled with a resin-metal complex. Obtained.

(Block process)
Next, the labeled antibody-containing solution a-5 was ice-cooled, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. Then, 1 mL of the blocking buffer a was added to the solid residue, and the mixture was applied for 10 to 20 seconds. Then, the mixture was subjected to ultrasonic dispersion treatment, and further overturned and stirred at room temperature for 2 hours to obtain a labeled antibody-containing solution b-5.

(Washing treatment)
Next, the labeled antibody-containing solution b-5 was ice-cooled, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. Then, 1 mL of a washing buffer was added to the solid residue, and the solution was applied for 10 to 20 seconds. To perform an ultrasonic dispersion treatment. This operation was repeated three times to obtain a washing process.

(Save processing)
Next, after cooling on ice, centrifugation is performed at 12,000 rpm for 5 minutes, and the supernatant is removed. Then, 1 mL of a storage buffer is added to the solid residue, and ultrasonic dispersion treatment is performed for 10 to 20 seconds. As a result, a labeled antibody-containing liquid c-5 was obtained.

(Pad)
The labeled antibody-containing liquid C-5 was diluted 4-fold, impregnated with a 30 cm × 1 cm glass fiber (Merck Millipore) pad, and dried at 20 ° C. for 60 minutes to obtain a labeled antibody-containing pad.

<Evaluation method>
The evaluation was performed using a reaction strip obtained by immobilizing an anti-Legionella polyclonal antibody on a nitrocellulose membrane (manufactured by Merck Millipore), and comparing the color development levels after 5 minutes, 10 minutes, and 15 minutes. The color development level was determined using a gold colloid determination color sample (manufactured by Adtech). In the performance evaluation, a two-fold dilution series of Legionella antigen (concentration: 2.0 × 10 ⁵ cfu / mL) was used.

<Evaluation procedure>
Each well of a 96-well plate was mixed with 100 μL of a 2-fold dilution column of APC (1- to 32-fold, respectively, represented as PC × 1 to PC × 32) and a negative control. Next, 100 μL of a buffer (100 mM Tris-HCl, 1% Triton X-100) was mixed, 100 μL of the mixture was added to the reaction strip, and the color development level after 15 minutes was evaluated. As a comparative example, a labeled antibody-containing pad labeled with colloidal gold prepared in Preparation Example 5 was used instead of the PtNCP beads (1). Table 5 shows the results.

A labeled antibody-containing solution containing an anti-Legionella polyclonal antibody labeled with colloidal gold was prepared by the following procedure.
100 μg of the anti-Legionella polyclonal antibody was mixed with 1 ml of gold colloid (OD = 10), and the mixture was stirred at room temperature for about 3 hours to bind the antibody to the gold colloid. A bovine serum albumin solution was added to a final concentration of 1%, and the mixture was stirred at room temperature for 2 hours to block the gold colloid surface. The cells were collected by centrifugation at 12,000 rpm at 4 ° C. for 5 minutes, and suspended in a buffer containing 0.2% bovine serum albumin to prepare a colloidal gold-labeled antibody.

  From Table 5, it can be seen that the labeled antibody-containing pad using PtNCP beads (1) showed good color development even with a 32-fold diluted antigen, and was superior in labeling to the labeled antibody-containing pad using colloidal gold. It was confirmed to have performance.

  The embodiments of the present invention have been described in detail for the purpose of illustration, but the present invention is not limited to the above embodiments.

Reference Signs List 10: resin particles, 20: metal particles, 30: encapsulated metal particles, 40: partially exposed metal particles, 50: surface-adsorbed metal particles, 60: surface layer, 100: resin-metal composite, 110: membrane, 120: Sample addition section, 130 determination section, 131 capture ligand, 140 liquid absorption section, 150 labeled antibody, 160 influenza virus, 170 complex, 200 test strip

Claims (9)

An immunoassay method for detecting or quantifying Legionella bacteria contained in a sample,
The following steps (I) to (III) are performed using a lateral flow-type chromatographic test strip including a membrane, and a determination section in which a capture ligand that specifically binds to the Legionella bacterium is immobilized on the membrane.
Step (I): a labeled antibody in which the Legionella bacterium contained in the sample and an antibody that specifically binds to the Legionella bacterium are labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. And the step of contacting
Step (II): a step of bringing the complex comprising the Legionella bacterium and the labeled antibody formed in step (I) into contact with a capture ligand in the determination unit;
Step (III): a step of measuring a coloring intensity derived from light energy absorption by localized surface plasmon resonance and electronic transition of the resin-metal composite,
An immunoassay method comprising performing a step comprising:   The immunoassay according to claim 1, wherein the metal particles are gold particles or platinum particles. The metal particles in the resin-metal composite,
First particles having a portion exposed outside the resin particles,
Second particles entirely contained in the resin particles,
And
The immunoassay according to claim 1, wherein at least some of the first particles and the second particles are three-dimensionally distributed in a surface layer of the resin particles. Method.   The immunoassay method according to any one of claims 1 to 3, wherein the resin particles are polymer particles having a substituent capable of adsorbing a metal ion in the structure.   The immunoassay according to any one of claims 1 to 4, wherein the metal particles have an average particle diameter in a range of 1 to 80 nm.   The immunoassay according to any one of claims 1 to 5, wherein the resin-metal complex has an average particle diameter in a range of 50 to 1000 nm. The immunoassay according to any one of claims 1 to 6, wherein the antibody is an anti-Legionella polyclonal antibody . Using a lateral flow chromatographic test strip, an immunoassay kit for detecting or quantifying Legionella bacteria contained in a sample,
A membrane, and a lateral flow-type chromatographic test strip including a determination unit in which a capture ligand that specifically binds to the Legionella bacterium is immobilized on the membrane,
An antibody that specifically binds to the Legionella bacterium , a detection reagent containing a labeled antibody labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles,
An immunoassay kit for detecting or quantifying Legionella bacteria comprising: A lateral flow type chromatographic test strip for detecting or quantifying Legionella bacteria contained in a sample,
With a membrane,
In the membrane, in the direction in which the sample is developed, a determination unit in which a capture ligand that specifically binds to the Legionella bacterium is fixed,
A reaction including a labeled antibody obtained by labeling an antibody that specifically binds to the Legionella bacterium with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles, upstream of the determination unit. Department and
A lateral flow type chromatographic test strip containing.