JP2009288069A - Target substance detection method - Google Patents

Abstract

An object of the present invention is to provide a detection method that is simple and has a high S / N ratio in a heterogeneous detection method by minimizing nonspecific adsorption of impurities and ligand molecules to a solid phase. It is to provide.
The present invention performs B / F separation after forming a complex including a first ligand molecule, a target substance, and a second ligand molecule. Then, the light irradiation binding site in the second ligand molecule is activated by light irradiation, and the complex is immobilized on the non-specific adsorption preventing film formed on the substrate.
[Selection] Figure 1

Description

  The present invention relates to a method for detecting a target substance such as a physiologically active substance, and a detection kit.

  In the field of clinical examination, a method for detecting a target substance related to a disease from a specimen such as blood is widely used as a diagnostic method for cancer, infectious diseases, lifestyle-related diseases and the like. In recent clinical examinations, in order to detect these diseases at an early stage, there is a demand for a method for accurately quantifying a very small amount of a target substance in a specimen in a short time. In order to detect a very small amount of a target substance in a specimen, a detection method with a high S / N (signal / noise) ratio is required, and so far a heterogeneous detection method based on an immunoassay method has been used. I came. In this detection method, first, a first ligand molecule that can be specifically captured on a target substance on a solid phase, a target substance, a target substance that can be specifically captured on the target substance, and has a labeling substance. Sandwich reaction with two ligand molecules. Next, after the B / F separation, the target substance is detected by measuring the presence and amount of the labeling substance immobilized on the solid phase.

On the other hand, in order to detect a very small amount of target substance in a specimen, it is also important to reduce noise during measurement. In order to reduce noise, it is necessary to remove impurities and second ligand molecules that adsorb nonspecifically to the solid phase, and generally the solid phase surface is blocked with a nonspecific adsorption inhibitor. The method is used. In Japanese Patent Application Laid-Open No. 01-209370 (Patent Document 1), a target substance is sandwiched with a target substance in a liquid phase and then blocked on a solid phase previously blocked with a hydrophilic protein via a biotin-avidin bond. A method for detecting a target substance by immobilization and competitive reaction is disclosed. In this detection method, nonspecific adsorption of impurities and ligand molecules to the solid phase can be suppressed by blocking the solid surface.
Japanese Patent Laid-Open No. 01-209370

  In the solid-phase surface blocking method as shown in Patent Document 1, it is possible to suppress nonspecific adsorption of contaminants and second ligand molecules on the solid-phase surface. However, it is not possible to suppress the non-specific adsorption of contaminants to the first ligand molecule existing on the solid phase and the second ligand molecule. In particular, it is difficult to prevent nonspecific adsorption due to the cross-reaction between the first ligand molecule and the second ligand molecule, which causes a large noise. For example, in Patent Document 1, it is known that a cross-reaction occurs between avidin (first ligand molecule) immobilized on a solid phase and an antibody (second ligand molecule), causing noise. Yes.

  On the other hand, methods for preventing cross-reactions between ligand molecules have been reported so far, but many problems remain in terms of prevention ability, simplicity, and cost performance. For example, the group of Lu et al. (Non-Patent Document 1) prepared an antibody previously treated with papain and pepsin in order to suppress the cross-reaction between ligand molecules, and detected the target substance on the solid phase on which this antibody was immobilized. How to do it has been reported. In this method, the cross-reaction between the first antibody and the second antibody is suppressed by removing the Fc portion of the antibody that is the first ligand molecule. However, the above-described antibody preparation method requires complicated operations. In addition, it is impossible to completely remove non-specifically adsorbed substances contained in the sample.

Therefore, the present specification provides a simple and high S / N ratio detection method that suppresses non-specific adsorption of contaminants and second ligand molecules on a solid phase in a heterogeneous detection method. It is for the purpose.
Analytical Chemistry, 67, 83, 1995.

The method for detecting the presence or amount of a target substance according to the present invention comprises:
A method for detecting the presence or amount of a target substance using particles,
(A) a substrate having a non-specific adsorption preventing film, a particle having a first ligand molecule that specifically captures the target substance, a site that specifically captures the target substance, and the non-specific adsorption by light irradiation Providing a second ligand molecule having a light irradiation binding site that binds to the protective film;
(B) The first ligand molecule and the second ligand molecule are bound to the target substance in a reaction medium, and the particles having the first ligand molecule, the target substance, and the second ligand molecule are combined. Forming a complex comprising: and
(C) separating the complex by B / F separation;
(D) a step of binding a light irradiation binding site in the second ligand molecule contained in the complex to the non-specific adsorption preventing film by light irradiation;
(E) detecting the presence or absence or amount of particles fixed to the non-specific adsorption preventing film;
It is characterized by having.

  According to the present invention, nonspecific adsorption of a ligand molecule or a foreign substance to a solid phase can be suppressed, and noise can be further reduced as compared with the prior art. In addition, the present invention can efficiently and selectively immobilize a complex containing a target substance on a solid phase via a photoreaction, and can achieve detection with a high S / N ratio.

  Hereinafter, the present invention will be described in detail.

The present invention is a method for detecting the presence or amount of a target substance using particles,
(A) a substrate having a non-specific adsorption preventing film, a particle having a first ligand molecule that specifically captures the target substance, a site that specifically captures the target substance, and the non-specific adsorption by light irradiation Providing a second ligand molecule having a light irradiation binding site that binds to the protective film;
(B) The first ligand molecule and the second ligand molecule are bound to the target substance in a reaction medium, and the particles having the first ligand molecule, the target substance, and the second ligand molecule are combined. Forming a complex comprising: and
(C) separating the complex by B / F separation;
(D) a step of binding a light irradiation binding site in the second ligand molecule contained in the complex to the non-specific adsorption preventing film by light irradiation;
(E) detecting the presence or absence or amount of particles fixed to the non-specific adsorption preventing film;
It is characterized by having.

  In the present invention, a B / F separation is performed after forming a complex including particles having a first ligand molecule, a target substance, and a second ligand molecule having a light irradiation binding site in a liquid phase. Remove ligand molecules and contaminants that did not form a complex. Then, after performing the B / F separation, the complex is irradiated with light, the light irradiation binding site in the second ligand molecule is activated, and a bond is formed on the non-specific adsorption prevention film, whereby the complex To fix. According to the present invention, B / F separation after formation of a complex and use of a nonspecific adsorption-preventing membrane can significantly reduce nonspecific adsorption of a ligand molecule or a foreign substance to a solid phase. Moreover, since the complex is formed by a liquid-liquid reaction, there is an advantage that the reaction time can be shortened.

  Furthermore, in the present invention, as a preferred embodiment, complex formation, B / F separation, complex immobilization, and particle detection can be performed on the same solid phase. That is, in the present invention, since the substrate is covered with the non-specific adsorption preventing film, non-specific adsorption of ligand molecules, impurities, etc. is suppressed even when the complex is formed on the solid phase. Then, by performing B / F separation by an appropriate method, after removing ligand molecules, contaminants, and the like, the complex can be selectively fixed to the nonspecific adsorption-preventing membrane by light irradiation.

  Although details will be described later, in the present invention, by using magnetic particles, B / F separation, complex immobilization, and particle detection can be easily and accurately performed on the same solid phase. it can.

  The outline of the method for detecting a target substance according to the present invention will be described with reference to FIG. In the present invention, first, the particle 101 having the first ligand molecule 102, the second ligand molecule 103 having the light irradiation binding site, and the target substance 109 are mixed to form the complex 107. Thereafter, B / F separation is performed to remove ligand molecules, contaminants, and the like that have not formed a complex. Then, the complex 107 is immobilized by irradiating the complex with light, activating the light irradiation binding site in the second ligand molecule, and forming a bond with the non-specific adsorption preventing film 105. In order to remove the particles 101 having the first ligand molecules 102, washing is preferably performed. In the present invention, the step of forming a complex, the step of performing B / F separation, and the step of immobilizing the complex on a solid phase are not particularly limited to those performed on the same solid phase. For example, after the formation of the complex and the B / F separation are performed in another container, the complex can be placed on the substrate 106 having the non-specific adsorption preventing film 105 and immobilized by light irradiation.

  Hereinafter, each configuration will be described in detail.

<Target substance>
The target substance in the present invention refers to a substance whose presence or amount is to be measured, and is not particularly limited, and examples thereof include biological substances. Examples of biological substances include substances selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof. Specific examples of the biological material include DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide or sphingolipid. In addition, bacteria and cells themselves that produce these substances are also included in biological substances.

  For example, when the target substance is a specific nucleic acid (target nucleic acid), examples of the source of the target nucleic acid include artificial synthetic products, animals such as humans and mice, plants, bacteria, fungi, archaea, viruses, etc. Examples include microorganisms.

  Further, the target substance is not limited to the biological substance as described above, and includes non-biological substances. An antigen is a substance that induces the antibody, and can be divided into, for example, non-biological substances and biological substances. Examples of non-biological substances that are of great industrial utility include PCBs with different chlorine substitution numbers / positions as environmental pollutants, dioxins with different chlorine substitution numbers / positions, and endocrine disruptors called environmental hormones Substances and the like. In addition, the biological material antigen includes at least a biological material selected from the nucleic acids, proteins, sugar chains, lipids, and complexes thereof. More specifically, a substance selected from any one of DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide and glycosphingolipid, etc. included.

  The target substance is contained in a sample, for example. Examples of the sample include a sample solution containing a target substance desired to be analyzed in a field including medical diagnosis, environmental measurement, or food management. Examples of sample solutions for medical diagnosis include body fluids including blood, lymph, DNA extract or tissue fluid. Examples of sample solutions for environmental measurement include solutions containing materials collected from soil, rivers, air, and the like. Examples of the sample solution for food management include an extract from minced meat and an extract from the cutting board surface. Moreover, the solvent used for sample preparation can be suitably determined according to the kind of target substance, for example, water, a phosphate buffer, an acetate buffer, a Tris buffer etc. can be used.

<Composite>
The complex in the present invention includes a particle having a first ligand molecule, a target substance, and a second ligand molecule having a light irradiation binding site. Although each component will be described later, in the present invention, after this complex is first formed, B / F separation is performed so as to leave this complex. Then, this complex is immobilized by light irradiation on a non-specific adsorption preventing film disposed on the substrate, and the immobilized complex is detected by observation of particles. The second ligand molecule in this complex has a light irradiation binding site capable of binding to a nonspecific adsorption film disposed on the substrate by light irradiation, and this site prevents nonspecific adsorption by light irradiation. Bind to the membrane.

  For example, when the target substance is an antigen and the first and second ligand molecules are antibodies, it is preferable to form a sandwich structure so that the first ligand molecule and the second ligand molecule sandwich the target substance.

<Particle>
The particle | grains used by this invention should just have the structure which can detect the presence and quantity of particle | grains using a detection apparatus. As such particles, for example, particles that can detect the presence and amount of particles using a microscope or the like are desirable. It is also desirable to have a structure having a labeling substance on the surface and / or inside of the particles. The particles are preferably insoluble in a liquid medium such as a sample solution or dispersion (for example, a buffer solution). The particles used in the present invention preferably have a configuration capable of supporting the first ligand molecule on the surface thereof.

  Here, the particles that can detect the presence and amount of particles using a microscope or the like are particles that can be observed using an optical microscope, an electron microscope, an atomic force microscope, or the like, and are particles having a diameter of at least 5 nm or more. It is preferable.

  For example, examples of the particles include particles composed of polystyrene, nylon, glass, metal, porous material, sugar derivatives such as dextran, and the like. Other examples include silica, polystyrene latex, latex, polylactic acid, agarose, chitosan, albumin or starch. Among these particles, polystyrene particles such as latex particles, polystyrene particles having a functional group on the particle surface, carboxylic acid-modified styrene particles, etc., from the viewpoint of easy fixation of the first ligand molecule to the particle surface. Particles and the like are preferred. As such polystyrene particles, commercially available products can also be used. For example, Dynaspheres (registered trademark) XP-5002 (Dyno Industries, Inc.) can be used as having an amino group on the particle surface. IMMUTX G series (JSR Corporation) can be used as having a carboxyl group. Products having an carbamoyl group such as estapor (registered trademark) PSI 181 (Prolaboratory) and those having an amidino group are known.

  Moreover, in the structure which has a labeling substance on the surface and / or inside of the particle, for example, a magnetic substance can be used as the labeling substance. For example, particles in which a magnetic substance is contained inside the material can be used as the particles. The particles containing the magnetic substance inside may have a configuration in which the magnetic substance is covered with the material such as polystyrene, silica, latex, etc., or a material in which the magnetic substance is mixed may be formed into particles. . Although it does not specifically limit as a magnetic substance, For example, the material etc. which mixed at least 2 sort (s) among these materials, such as iron oxide, chromium oxide, cobalt, a ferrite, etc. are mentioned.

  Note that in this specification, the structure having a labeling substance on the surface and / or inside of a particle includes a case where the particle is composed only of the labeling substance. The “particles containing a labeling substance” are used synonymously with “particles having a labeling substance on the surface and / or inside”. In the present specification, “particles having a magnetic substance as a labeling substance on the surface and / or inside” are referred to as magnetic particles. The magnetic particles include particles containing the above-described magnetic substance and particles composed only of the magnetic substance.

  In addition to the magnetic substance, the labeling substance may be a light emitting substance, an electrochemically active substance, a heavy metal substance, a metal semiconductor substance, or the like. In addition, an enzyme can be bound to the particle surface using an enzyme as a labeling substance.

  Here, in the present specification, light emission includes, for example, chemical light emission, physical light emission, biological light emission, and the like, and the light emitting materials include fluorescent materials, phosphorescent materials, delayed fluorescent materials, and the like. The light emission is a concept including fluorescence and phosphorescence. Note that fluorescence refers to light emitted from a fluorescent material excited by excitation light. Phosphorescence refers to light emitted when electrons that have transitioned to the triplet excited state return to the ground state. Further, the delayed fluorescent substance refers to a substance that emits fluorescence longer than the fluorescent substance. Examples of the fluorescent substance among the luminescent substances include fluorescein derivatives, cyanine dyes, rhodamine dyes, coumarin dyes, dansyl dyes, anthracene derivatives, pyrene derivatives, rare earth metal complexes, and the like. Examples of phosphorescent substances among luminescent substances include iridium complexes. Examples of the delayed fluorescent substance among the luminescent substances include rare earth fluorescent complexes such as europium complexes. Examples of other luminescent substances include luminol, lophine, lucigenin, oxalate luminol, and the like. These luminescent substances can promote a chemical reaction using an enzyme or the like to increase luminous efficiency.

  Examples of the electrochemically active substance include heavy metal complex substances such as an iron complex, a cobalt complex, a ruthenium complex, or a nickel complex. Examples of the heavy metal substance include nano metal particles such as gold, silver, or platinum. Examples of the metal semiconductor material include quantum dot materials such as CdSe-based particles, ZnSe-based particles, and In (Ga) As-based particles.

  The particle size is preferably a particle diameter of 5 nm to 100 μm.

  As described above, the first ligand molecule is immobilized on the surface of the particle. As a method for immobilizing the first ligand molecule on the particle surface, many methods have already been proposed, and the method is not particularly limited and may be appropriately selected depending on the surface condition of the first ligand molecule or the particle. Can do. For example, the method of immobilizing the first ligand molecule to the particle can be performed using chemical binding or affinity binding. Here, the chemical bond is a bond formed with transfer of electrons between atoms or ions, and examples thereof include a covalent bond, a coordinate bond, and a metal bond. Examples of the method using a chemical bond include a method of fixing using a condensing agent, a method of fixing using a silane agent, a method of fixing via an aldehyde, and fixing via an amide bond with N-hydroxysuccinimide ester. Examples thereof include a method, a method of fixing by maleimide with a thiol bond, and a method of fixing by forming a complex between a nickel complex and a histidine site in a ligand molecule. In addition, as a method of using affinity binding, a method of fixing by biotin-avidin binding, a method of fixing via Protein A or Protein G, a method of fixing by antigen-antibody reaction, or a sandwich binding that combines these methods is used. And the like, a method of fixing by DNA-DNA, DNA-PNA hybridization, a method of fixing a ligand molecule via an aptamer, and the like can be used. Also known is a method of binding spacer molecules between them. When the first ligand molecule is immobilized, the temperature and chemical conditions are so mild that the first ligand molecule does not denature so as not to impair the activity required for the detection and separation of the target substance. It is desirable to be performed at.

  In order to immobilize the first ligand molecule on the surface of the particle, for example, the particle may have a configuration in which a portion capable of immobilizing the first ligand molecule is provided on the surface. Hereinafter, a site where the first ligand molecule existing on the particle surface can be fixed is abbreviated as a particle surface fixing site. The particle surface immobilization site is preferably a site where the first ligand molecule can be chemically immobilized, or a site with high affinity for the first ligand molecule. The site that can be chemically fixed refers to a site having a functional group or an active group capable of binding to the first ligand molecule. Examples of chemically fixable sites include carboxyl groups, primary to quaternary amine groups, hydroxyl groups, sulfonyl groups, phosphate groups, phosphonyl groups, aldehyde groups, thiol groups, carbodiimide sites, and maleimide groups. , Isocyanate site, isothiocyanate site, N-hydroxysuccinimide ester site, nickel complex or biotin. Moreover, a site | part with high affinity shows a site | part which has high affinity with respect to a 1st ligand molecule, and can fix a 1st ligand molecule to particle | grains. The site having high affinity includes avidin, streptavidin, neutravidin, protein G, protein A, DNA, PNA, RNA, aptamer and the like, depending on the type of the first ligand molecule. Here, as a preferable combination of the site having high affinity and the first ligand molecule, avidin and a biotinylated antibody, streptavidin and a biotinylated antibody, protein G and an antibody, and protein A and an antibody are preferable.

  For example, as described above, Dynaspheres (registered trademark) XP-5002 (Dyno Industries Co., Ltd.) may be mentioned as a polystyrene particle having an amino group-containing site as a particle surface fixing site. Similarly, IMMUTEX G series (JSR) can be used as polystyrene particles having a carboxyl group-containing site as a particle surface fixing site. Similarly, products such as estapor (registered trademark) PSI 181 (Prolaboratory) having a carbamoyl group-containing site as a particle surface fixing site, and estapor (registered trademark) PSI 629 (Prolaboratory) having an amidino group It has been known.

<Ligand molecule>
The first ligand molecule or the second ligand molecule used in the present invention is a substance that can specifically recognize and capture a target substance. Examples of the combination of the target substance and the first or second ligand molecule include, for example, antigen-antibody, peptide-antibody, virus-antibody, low molecular compound-antibody, cell-antibody, antigen-aptamer, antigen-enzyme, peptide- Examples include protein, peptide-peptide, DNA-DNA, DNA-RNA, DNA-PNA, DNA-protein, RNA-protein, sugar-protein, protein-protein, sugar-sugar and the like. In the case where “the combination of the target substance and the first or second ligand molecule is AB” is described, the case where the target substance is A and the first or second ligand molecule is B; Both the case where the target substance is B and the first or second ligand molecule is A are included. For example, when an antigen is used as the target substance and an antibody is used as the first or second ligand molecule, it was obtained by immunization with a monoclonal antibody or antigen obtained from a clone derived from a single antibody-producing cell. Polyclonal antibodies that are purified by serum can be used. The first and second ligand molecules are not particularly limited, but are preferably the same type of ligand molecules.
First ligand molecule The first ligand molecule used in the present invention specifically captures a target substance and is immobilized on the surface of the particle. For example, when the target substance is an antigen, the first ligand molecule can be an antibody against the antigen. When the target substance is a nucleic acid, the first ligand molecule can be a probe having a sequence complementary to the nucleic acid. When the target substance is an antibody, it can be used as an antigen for the antibody.

  The method for immobilizing the first ligand molecule on the surface of the particle is not particularly limited. For example, using linker chemistry, hybridization, affinity capture by biotin / avidin affinity, combinatorial chemistry, and other well-known techniques in the prior art, the surface state of the first ligand molecule or particle can be It can be prepared accordingly. For example, as described above, a particle surface fixing site can be provided on the surface of the particle, and in this case, a site capable of binding to the particle surface fixing site can be provided in the first ligand molecule. The site capable of binding to the particle surface fixing site depends on the type of the particle surface fixing site, for example, a site having a functional group such as a carboxyl group, amino group, thiol group, disulfide group or hydroxyl group, or a biotin derivative. Is mentioned.

Here, as a preferable combination of the functional group possessed by the particle surface fixing site and the site capable of binding to the particle surface fixing site, carboxyl group and amino group, aldehyde group and amino group, N-hydroxysuccinimide ester site and amino group, maleimide A group and a thiol group, avidin and biotin, streptavidin and biotin, protein A and antibody, and protein G and antibody are preferred.
Second Ligand Molecule The second ligand molecule used in the present invention has a site that specifically captures the target substance and binds to the nonspecific adsorption preventing film by light irradiation. In addition, in this specification, the site | part couple | bonded with a nonspecific adsorption | suction prevention film | membrane by light irradiation is called a light irradiation binding site.

  The second ligand molecule desirably has a photoaffinity compound as a light irradiation binding site. Here, in this specification, the photoaffinity compound is activated by light irradiation and can form a bond with a compound (for example, a compound constituting a nonspecific adsorption preventing film). The photoaffinity compound is particularly preferably one that is activated by light irradiation and can form a covalent bond in the nonspecific adsorption-preventing film. As a photoaffinity compound, the compound which generate | occur | produces a nitrene, a carbene, or a radical is mentioned, for example. These compounds are described in JP 2001-178472 A (Patent Document 2) and the like. Specific examples of the photoaffinity compound include compounds having an azide group such as aromatic azide, alkyl azide, and heterocyclic azide, compounds having a diazo group or a diazirine ring (diazirine derivatives), benzophenones and enones. Conjugated ketones, aromatic halides, olefins, aromatic diazonium salts, nitrobenzenes, sulfonium salts, phosphonium salts, ammonium salts, and the like. Moreover, the compound which has a diazonium group, an azide group, a diazirine ring, or a diazo group as a partial structure is also mentioned. Among them, the diazirine derivative is used as a highly useful compound because it generates a very active carbene derivative by irradiating light with a wavelength near 360 nm that does not damage the biomolecule (Non-patent Document 2). .

  Moreover, the photoaffinity compound activated by light irradiation can be covalently bonded at random to the non-specific adsorption preventing film. Therefore, it can be combined with any non-specific adsorption preventing film. Particularly preferred combinations of photoaffinity compounds and non-specific adsorption-preventing films include diazirine derivatives and albumin, diazirine derivatives and casein, diazirine derivatives and skim milk, diazirine derivatives and heparin, diazirine derivatives and phospholipids, diazirine derivatives and oligoethylene glycol. , A diazirine derivative and polyethylene glycol, a diazirine derivative and polymethacrylate, a diazirine derivative and polyacrylate, a diazirine derivative and dextran.

Further, the photoaffinity compound can be bound to the second ligand molecule by using a photoaffinity compound having a functional group or an active group. For example, the functional group includes a carboxyl group, an amino group, a thiol group, an aldehyde group, a hydroxyl group, a sulfonyl group, and the active group includes an N-succinimide ester moiety, a maleimide group, a carbodiimide moiety, an isocyanate moiety, Examples include isothiocyanate moieties. Here, preferred combinations of the functional group or active group and the second ligand molecule include carboxyl group and antibody, aldehyde group and antibody, N-succinimide ester site and antibody, maleimide group and antibody, carbodiimide site and antibody, isocyanate Sites and antibodies, isothiocyanate sites and antibodies are preferred.
JP 2001-178472 A Yakugaku Zashi, 127, 1693, 2007.

<Non-specific adsorption prevention membrane>
The nonspecific adsorption preventing film used in the present invention is provided on a substrate. The non-specific adsorption preventing film suppresses adsorption of particles having at least the first ligand molecule. It is also preferable to suppress the adsorption of the second ligand molecule that does not form a contaminant or complex. Further, the non-specific adsorption preventing film is preferably a film that can also suppress the adsorption of the target substance when there is a possibility that an unreacted target substance exists. The non-specific adsorption preventing film may be any material that can form a bond with the light irradiation binding site by light irradiation to the second ligand molecule in the complex. In addition, a contaminant shows substances other than the target substance which exists in a sample. Examples of contaminants include immunoglobulin G, immunoglobulin M, immunoglobulin A, lipoprotein, cholesterol, albumin, hemoglobin, metabolites, and the like.

  As the non-specific adsorption preventing film, a hydrophilic and highly polar compound is used. The main cause of non-specific adsorption is adsorption by hydrophobic interaction or electrostatic interaction. In particular, non-specific adsorption due to hydrophobic interaction is the most likely adsorption. Therefore, in order to prevent hydrophobic interaction, a hydrophilic and highly polar compound is used as the prevention film. In particular, examples of hydrophilic and highly polar compounds include albumin, casein, skim milk, heparin, phospholipid, oligoethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, dextran and the like. The nonspecific adsorption preventing film is preferably immobilized on the substrate. As the immobilization method, physical adsorption or chemical bonding is used. For example, when physical adsorption is used, preferred non-specific adsorption preventing film and substrate combinations include albumin and polystyrene resin, or metal substrate, casein and polystyrene resin, or metal substrate, skim milk and polystyrene resin, or metal substrate. Phospholipid and polystyrene resin, or metal substrate. When chemical bonding is used, the nonspecific adsorption preventing film is immobilized using a substrate having a functional group or an active group. Particularly preferred combinations of non-specific anti-adsorption membranes with functional groups or active groups include albumin, casein, skim milk, phospholipid, oligoethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, dextran and carboxyl group, albumin Casein, skim milk, phospholipid, oligoethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, dextran and amino groups, albumin, casein, skim milk, phospholipid, oligoethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, dextran Aldehyde group, albumin, casein, skim milk, phospholipid, oligoethylene glycol, polyethylene glycol, polymetac Rate, polyacrylate, dextran and N-succinimide ester moiety, albumin, casein, skim milk, phospholipid, oligoethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, dextran and maleimide moiety, albumin, casein, skim milk, phospholipid, oligo Ethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, dextran and carbodiimide moiety, albumin, casein, skim milk, phospholipid, oligoethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, dextran and isocyanate moiety, albumin, casein, skim milk, Phospholipid, oligoethylene glycol, polyethylene glycol , Polymethacrylates, polyacrylates, dextran and isothiocyanate site.

  The non-specific adsorption preventing film is preferably disposed on the entire surface of the substrate, but is not particularly limited. As long as it has the effect of the present invention, the case where it is partially disposed on the substrate is included.

  The structure and the like of the substrate are not particularly limited as long as the nonspecific adsorption preventing film is a material that can be immobilized by physical adsorption or chemical bonding. For example, examples of the substrate include polystyrene resin, gold, silver, metal oxide, and glass. Moreover, according to various measuring methods, the thing which has a light transmittance, a thing which has light reflectivity, an electrode substrate, the base | substrate with which the gold thin film or the gold dot was formed in the surface, etc. are used.

<Detection method>
The detection method used in the present invention is not particularly limited as long as it is a method capable of detecting the presence or absence or amount of particles immobilized on a non-specific adsorption preventing film (solid phase).

  For example, when the particles are polystyrene particles, nylon particles, glass particles, metal particles, various particles coated with metal, or porous particles, the change in the refractive index or dielectric constant of the substrate accompanying the immobilization of these particles Can be detected by an optical technique. In addition, it can be detected by directly observing particles with an optical microscope, an electron microscope, an atomic force microscope, or the like.

  In the case where the particles are metal particles or particles coated with metal, the change in absorption spectrum due to these particles can be detected by an optical technique.

  In the case where the particles contain a magnetic substance, the magnetic field effect can be detected using a magnetoresistive element, Hall effect element, magnetic impedance element, or superconducting quantum interferometer element as a base.

  In addition, when the particle contains a luminescent material such as a fluorescent material, a phosphorescent material, or a delayed fluorescent material (when a luminescent material is used as a labeling material), a photodetector such as a plate reader, a spectrophotometer, or a fluorescence measuring device is used. Can be detected.

  In addition, when the particle contains an electrochemically active substance, it can be detected by using an electrochemical measurement method such as cyclic voltammetry, pulse voltammetry, amperometry, coulometry using an electrode as a substrate. In addition, if a molecular weight measuring instrument such as MALDI TOF MS measurement or TOF SIMS measurement is used, the particles 101 shown here can be detected.

  When the particles have an enzyme (when an enzyme is used as a labeling substance), the particles can be detected by measuring the enzyme activity. For example, a method of measuring fluorescence generated by dephosphorylation of 4-methylumbelliferyl phosphate using alkaline phosphatase as an enzyme, a method of measuring chemiluminescence generated by oxidation of luminol using peroxidase as an enzyme, etc. It is done.

<B / F separation>
In the present invention, after the complex is formed, the second ligand molecule and the contaminants not forming the complex are removed by B / F separation. Here, B / F separation means separation of bound (complex) and free (free), and in this specification, a substance bound or fixed to particles, The separation of free material not bound or immobilized on the particles is shown.

  Specific B / F separation methods include a separation method using particle sedimentation by centrifugation, a separation method using natural sedimentation of particles, a particle separation method using a filter capable of size exclusion, and the like. . When using these separation methods, it is preferable to provide a sufficient difference in physical properties that affect separation such as density and size suitable for effective separation between the second ligand and the particles. . When the particles contain a magnetic substance, a magnetic separation method using a magnetic field generator such as a magnet or an electromagnet can be used.

(Embodiment 1)
Hereinafter, an embodiment of the present invention will be described with reference to FIG. 2 or FIG. However, the present invention is not limited to the following embodiments.

  In the present invention, the method of immobilizing the complex to the solid phase is preferably a method capable of selectively and efficiently immobilizing the complex on the non-specific adsorption preventing film by light irradiation. In the present embodiment, a method capable of selectively and efficiently immobilizing a complex on the same solid phase will be described with reference to FIG. FIG. 2 shows an example of a process until the complex 207 is fixed on the non-specific adsorption preventing film 205 when the particle 201 is a magnetic particle.

  As shown in FIG. 2A, the target substance 209, the particle 201 having the first ligand molecule 202, and the second ligand molecule 203 are bound in a solution on the non-specific adsorption preventing film 205. To form a complex 207.

  Next, as shown in FIG. 2B, a magnetic field generator 210 such as a magnet or an electromagnet is installed in the vicinity of the base 206 to generate a magnetic field, and the magnetic particles are attracted to the nonspecific adsorption preventing film 205. Then, the solution is removed and B / F separation is performed. By this step, the second ligand molecule 203 and the contaminant 208 that do not form the complex 207 are removed.

  Next, as shown in FIG. 2C, light irradiation is performed on the complex 207 concentrated on the non-specific adsorption preventing film 205 by the magnetic field generator 210 using the light irradiation device 211 having a light source. The complex 207 is fixed to the nonspecific adsorption preventing film 205. The light irradiation used here is to irradiate light including a wavelength appropriately determined according to the type of light irradiation binding site (for example, photoaffinity compound). As the light, sunlight, electric lamp light, laser light such as a semiconductor laser or a gas laser, light emission of a light emitting diode or light emission of an electroluminescent element can be used. In the light irradiation method, the composite 207 can be irradiated with light from a light source through an appropriate filter as necessary. Further, pattern exposure of a desired shape can be performed using a mask or the like. It is also possible to collect light using a lens or a mirror and irradiate it in a fine shape, or to perform scanning exposure of the collected light beam. The irradiation time is not particularly limited. Further, light irradiation can be performed while concentrating the complex 207 on the solid phase by the magnetic field generator 210. Thereby, improvement of the fixation efficiency of the complex 207 to the solid phase can be expected. It is also possible to fix the complex 207 on the non-specific adsorption preventing film 205 by light irradiation after the complex 207 is concentrated and dried on the non-specific adsorption preventing film 205. As a result, the non-specific adsorption preventing film 205 and the complex 207 closely approach each other, and the complex 207 can be bound at various sites on the non-specific adsorption preventing film 205, so that improvement in fixation efficiency can be expected. The method of making it dry is not specifically limited.

  Next, as shown in FIG. 2D, the non-specific adsorption preventing film 205 is washed with an appropriate solvent or buffer to remove the particles 201 that have not been immobilized. It is also possible to remove particles that are not fixed by the magnetic field generator 210. After washing, the target substance 209 in the sample can be detected by detecting the presence or amount of the complex 207 immobilized by light irradiation, for example, by the detection method described above.

  In the example shown in FIG. 2 of the present embodiment, it is possible to perform from complex formation to particle detection on the same solid phase. Thereby, since the container etc. which perform B / F separation separately are not required, the simplification and speeding-up of a detection process are attained.

(Embodiment 2)
FIG. 3 is a diagram showing an example of a target substance detection method according to the present invention. The particle 301 may be a magnetic particle or a particle containing a substance other than a magnetic substance as a labeling substance. For example, particles such as polystyrene, nylon, glass, metal, or porous body exemplified above are listed. In addition, for example, particles composed of a sugar derivative such as dextran or a fluorescein derivative are also included. In addition, for example, fluorescent particles containing a cyanine dye, a rhodamine dye, a coumarin dye, a dansyl dye, an anthracene derivative, a pyrene derivative, a rare earth metal complex, or the like can be given. Further, for example, electrochemically active particles containing a heavy metal complex substance such as an iron complex, a cobalt complex, a ruthenium complex, or a nickel complex are also included. Moreover, nano metal particles, such as gold, silver, or platinum, are also mentioned, for example. Further, for example, quantum dot particles such as CdSe-based particles, ZnSe-based particles, or In (Ga) As-based particles may be used.

  First, as shown in FIG. 3A, a target substance 308, a particle 301 having a first ligand molecule 302, and a second ligand molecule 303 are bound in a container 305 in a solution to form a complex. 306 is formed.

  Next, as shown in FIG. 3B, B / F separation is performed by a centrifugal separation method or a natural sedimentation method. By this step, the second ligand molecule 303 and the contaminant 307 that do not form the complex 306 are removed.

  Next, as shown in FIG. 3C, the complex 306 purified in the step shown in FIG. 3B is dropped on the non-specific adsorption preventing film 310, and a light irradiation device 309 having a light source is used. Then, the complex 306 is irradiated with light to fix the complex 306 on the non-specific adsorption preventing film 310. The light irradiation used here is to irradiate light including a wavelength determined according to the type of the photoaffinity compound. As light, for example, sunlight, electric lamp light, laser light such as a semiconductor laser or a gas laser, light emission of a light emitting diode or light emission of an electroluminescent element can be used. In the light irradiation method, the composite 306 can be irradiated with light from a light source through an appropriate filter as necessary. Further, pattern exposure of a desired shape can be performed using a mask or the like. It is also possible to collect light using a lens or a mirror and irradiate it in a fine shape, or to perform scanning exposure of the collected light beam. The irradiation time is not particularly limited. It is also possible to fix the complex 306 on the non-specific adsorption prevention film 310 by light irradiation after the complex 306 is dropped on the non-specific adsorption prevention film 310 and dried. As a result, the non-specific adsorption preventing film 310 and the complex 306 closely approach each other, and the complex 306 can be bound at various sites on the non-specific adsorption preventing film 310, so that it can be expected that the immobilization efficiency is improved. The method of making it dry is not specifically limited.

  Next, as shown in FIG. 3D, the non-specific adsorption preventing film 310 is washed with an appropriate solvent or buffer to remove the particles 301 that have not been immobilized. After washing, the target substance 308 in the sample can be detected by detecting the presence / absence and amount of the complex 306 fixed by light irradiation by the detection method described above.

<Kit>
The present invention can also be used as a target substance detection kit using the above-mentioned materials, substrates and the like. For example,
1-1) Substrate having a nonspecific adsorption-preventing film 1-2) Particles having a first ligand molecule 1-3) Second ligand molecules having a light irradiation binding site or in the case where the particles are magnetic particles 2-1) Substrate having non-specific adsorption preventing film 2-2) Particle having first ligand molecule 2-3) Second ligand molecule having light irradiation binding site 2-4) Provided with magnetic field generator It is the kit characterized by having.

  In the detection kit, a sample containing a target substance, a particle having a first ligand molecule, and a second ligand molecule having a light irradiation binding site are mixed on a substrate having a non-specific adsorption preventing film to form a composite. After the body is formed, B / F separation is performed. After the B / F separation, the target substance in the sample can be detected by fixing the complex formed by light irradiation to the non-specific adsorption preventing film and detecting the presence or amount of particles. In the detection kit, when the particles are magnetic particles, a complex is formed by the above method, and then a magnetic field generator is installed in the vicinity of the substrate to prevent nonspecific adsorption of the complex using magnetic force. It can be concentrated on the membrane. As a result, the complex can be efficiently immobilized on the nonspecific adsorption-preventing membrane, and detection with a high S / N ratio becomes possible.

  The detection kit according to the present invention further includes at least one of a reaction container for forming a complex, a container for performing B / F separation, and a detection container for detecting a target substance. Also good. Further, the reaction container and the detection container may be separate from each other or the same. The shape of the container is not particularly limited as long as the reaction for forming a complex, B / F separation, and detection of a target substance can be performed. Further, the reaction vessel for forming the complex may be configured so that the first ligand molecule-fixed particles and the second ligand molecule having the light irradiation binding site are included in advance. The container used in the present invention is not particularly limited as long as it plays the role of forming a complex, and the size thereof is not particularly limited.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

Example 1
In this example, the process was performed as shown in FIG. Magnetic particles are used as the particles 201. As the first ligand molecule 202, an α subunit recognition antibody (manufactured by Medix Biochemica) is used. Further, as the second ligand molecule 203, an anti-human chorionic gonatotropin antibody (manufactured by Medix Biochemica) having a diazirine site (light irradiation binding site 204) is used. Then, a solution containing these is mixed to form a sandwich complex 207 composed of magnetic particles 201 to which the antibody 202 is immobilized—human chorionic gonatotropin 209 (manufactured by Rohto Pharmaceutical Co., Ltd.) — The antibody 203 having a diazirine site 204. Let Then, it is selectively and efficiently fixed on a sensor element having a function as the substrate 206 (specifically, a non-specific adsorption preventing film formed on the sensor element), and the fixed complex is detected by a magnetic sensor.

1-1. Preparation of antibody-immobilized magnetic particles First, a biotinylated α subunit recognition antibody as the first ligand molecule 202 is prepared. 500 μg of α subunit-recognizing antibody is dissolved in 100 μL of a borate buffer solution at pH 9.0, and 1.0 mg of NHS-dPEG12Biotin (manufactured by QuantaBioDesign) is added to this solution, followed by stirring at room temperature for 3 hours. After stirring, unreacted NHS-dPEG12Biotin is removed from the reaction solution using Microcon YM100 (Millipore). The supernatant is recovered to obtain a biotinylated α subunit recognition antibody. The concentration of the obtained antibody is determined with a spectrophotometer.

  Next, the produced biotinylated α subunit recognition antibody is immobilized on a magnetic particle, and a particle 201 having a first ligand molecule 202 is produced. Magnetic particle having a particle diameter of about 1 μm and coated with avidin: “Product name” Dynabeads MyOne Streptavidin C1 (manufactured by Invitrogen) 1 mg / mL of the prepared biotinylated α-subunit-recognizing antibody 1 nmol is added at room temperature. Stir for more than an hour. After stirring, B / F separation is performed using a magnet to remove unreacted biotinylated α subunit recognition antibody. Finally, the solution is replaced with 1 × PBST solution to obtain magnetic particles on which the α subunit recognition antibody is immobilized.

  From the obtained particles, the amount of biotinylated α subunit recognition antibody immobilized on the surface of the magnetic particles and the average particle size are determined by UV measurement and dynamic light scattering measurement.

1-2. Preparation of antibody having photoaffinity compound p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid (manufactured by BACHEM) is used as the photoaffinity compound. 5 mg of p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid was dissolved in a DMF solution, and then 25 mg of N-hydroxysuccinimide (manufactured by Aldrich) and 1-ethyl-3- (3 -An aqueous solution in which 25 mg of (dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Aldrich) is dissolved is gradually added and stirred for 6 hours or more. After stirring, the target product is extracted with an organic solvent, and the solvent is distilled off under reduced pressure to obtain a succinimide ester of p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid.

  Next, a photoaffinity compound is introduced into the anti-human chorionic gonatotropin antibody using the prepared p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid succinimide ester according to the scheme shown in Formula 1. To do. 500 μg of anti-human chorionic gonatotropin antibody was dissolved in 100 μL of borate buffer at pH 9.0, and 1.0 mg of p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid succinimide ester was dissolved in this solution. Add DMSO solution or DMF solution and stir at room temperature for 3 hours. After stirring, unreacted p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid succinimide ester is removed from the reaction solution using Microcon YM100 (manufactured by Millipore). The supernatant is collected, and an antibody 203 having a photoaffinity compound as a light irradiation binding site is obtained. The concentration of the obtained antibody is determined with a spectrophotometer.

1-3. Fabrication of sensor element 213 having a portion for preventing adsorption of foreign substances Gold is coated on the sensor element used as substrate 206 by gold vapor deposition. The non-specific adsorption preventing film 205 is formed on the gold surface on the sensor element using BSA, skim milk, N101 (“trade name”, manufactured by Nippon Oil & Fats Co., Ltd.) or Thiol-dPEG-acid (manufactured by QuantaBioDesign). As a forming method, for example, a solution containing a protein or a synthetic compound is dropped on a sensor element and fixed under high humidity for 3 hours or more. After the formation, the nonspecific adsorption preventing film is sufficiently washed with a solvent or a buffer solution to remove proteins or synthetic compounds not immobilized on the element to obtain a sensor element having the nonspecific adsorption preventing film.

1-4. Detection of Human Chorionic Gonatotropin 210 Human chorionic gonatotropin, which is the target substance 209, is detected through the steps shown in FIG. First, on the sensor element prepared in 1-3, a solution containing human chorionic gonatotropin having a predetermined concentration, antibody-immobilized magnetic particles having a predetermined concentration prepared in 1-1, and a predetermined concentration prepared in 1-2 An antibody having a photoaffinity compound is mixed to form a complex (FIG. 2A). Next, after reacting for a predetermined time, the magnetic field generator 210 is brought close to the lower part of the sensor element, and the complex and the unreacted antibody-immobilized magnetic particles are concentrated on the sensor element. In this state, the surface of the sensor element is washed with a solvent or a buffer solution, and the antibody or contaminant 208 having a photoaffinity compound not forming a complex is removed from the sensor element (2 (B)). Next, with the magnetic field generator 210 being brought close to the lower part of the sensor element, the upper part of the sensor element is irradiated with laser light having a wavelength of 360 nm for 1 minute or more (FIG. 2C). After the light irradiation, the magnetic field generator 210 is separated from the lower part of the sensor element, and the sensor element is washed with a solvent or a buffer solution to remove antibody-immobilized magnetic particles not immobilized on the non-specific adsorption preventing film (FIG. 2 ( D)). Finally, the complex immobilized on the nonspecific adsorption-preventing film is detected by magnetoresistance measurement using a sensor element, and human chorionic gonatotropin is quantified.

  The detection process shown above is performed using the following measurement operations. FIG. 4 is a block diagram of the measurement system of the present invention. The sample solution 401 containing the target substance is dispensed by the sampling probe 407 into a container 410 having a sensor element 411 having a non-specific adsorption preventing film. The diluent 404 is also dispensed into the container 410 in the same manner. Further, a reagent 402 containing antibody-immobilized magnetic particles and a reagent 403 containing an antibody having a photoaffinity compound are similarly dispensed into the container 410. After forming a sandwich complex composed of an antibody having an antibody-immobilized magnetic particle-target substance-photoaffinity compound, a magnetic field is applied by a magnetic field generator 412 installed outside the container 410 to immobilize the complex and the antibody. Magnetic particles are collected on the sensor element 411. Samples and reagents other than the complex and unreacted antibody-immobilized magnetic particles are transferred from the container 410 to the waste liquid container 406 by the sampling probe 407. Further, the cleaning liquid 405 is filled into the container 410 by the sampling probe 407 and cleaning is performed. The used cleaning solution is transferred from the container 410 to the waste liquid container 406 by the sampling probe 407. The diluent 403 is dispensed into the container 410 by the sampling probe 407. In a state where a magnetic field is applied by the magnetic field generator 412, the sensor element 411 is irradiated with light using the light irradiation probe 414, the photoaffinity compound is activated, and a non-specific adsorption preventing film formed on the sensor element A bond is formed. Next, specimens and reagents other than the complex immobilized on the sensor element 411 are washed and transferred from the container 410 to the waste liquid container 406 by the sampling probe 407. Further, the cleaning liquid 405 is filled into the container 410 by the sampling probe 407 and cleaning is performed. The used cleaning solution is transferred from the container 410 to the waste liquid container 406 by the sampling probe 407. The diluent 403 is dispensed into the container 410 by the sampling probe 407. A magnetic field generator 412 generates a magnetic field perpendicular to the sensor element 411. After the initial value is measured by the magnetic signal detector 413 without a sample in advance, the magnetic signal generated from the magnetic particles of the complex immobilized on the sensor element 411 (non-specific adsorption preventing film) Measured by the magnetic signal detector 413. Based on a calibration curve prepared in advance using a target substance having a known concentration, the target substance concentration in the specimen is calculated from the amount of change in the magnetic signal compared to the initial value.

(Example 2)
In this example, the process was performed as shown in FIG. As the particles 301, particles containing a fluorescent substance as a labeling substance are used. As the first ligand molecule 302, an α subunit recognition antibody (manufactured by Medix Biochemica) is used. An anti-human chorionic gonatotropin antibody (Medix Biochemica) having a diazirine site (light irradiation binding site 304) is used as the second ligand molecule 303. Non-specific adsorption-preventing film formed on a plate as a substrate with a sandwich complex 306 composed of fluorescent particles 301 having immobilized antibodies-human chorionic gonatotropin 308 (manufactured by Rohto Pharmaceutical Co., Ltd.)-Antibody 303 having a diazirine moiety The immobilized complex 306 is selectively and efficiently fixed to 310, and the fixed complex 306 is detected by a fluorescence microscope or a fluorescence plate reader.

2-1. Preparation of antibody-immobilized fluorescent particles First, a biotinylated α subunit recognition antibody as the first ligand molecule 303 is prepared. 500 μg of α subunit-recognizing antibody is dissolved in 100 μL of a borate buffer solution at pH 9.0, and 1.0 mg of NHS-dPEG12Biotin (manufactured by QuantaBioDesign) is added to this solution, followed by stirring at room temperature for 3 hours. After stirring, unreacted NHS-dPEG12Biotin is removed from the reaction solution using Microcon YM100 (Millipore). The supernatant is recovered to obtain a biotinylated α subunit recognition antibody. The concentration of the obtained antibody is determined with a spectrophotometer.

  Next, the prepared biotinylated α subunit recognition antibody is immobilized on a fluorescent particle, and a particle 301 having a first ligand molecule 302 is prepared. Fluorescein-containing fluorescent particle having a particle diameter of about 1 μm and coated with avidin: “trade name” NeutrAvidinTM-Labeled Microspheres (manufactured by Invitrogen) 1 mg / mL solution, 1 nmol of the prepared biotinylated α subunit recognition antibody was added, Stir at room temperature for over 1 hour. After the stirring is completed, B / F separation is performed using a centrifuge to remove unreacted biotinylated α subunit recognition antibody. Finally, the solution is replaced with 1 × PBST solution to obtain α subunit-recognizing antibody-immobilized fluorescent particles.

  From the obtained particles, the amount of biotinylated α subunit recognition antibody immobilized on the surface of the fluorescent particles and the average particle size are determined by UV measurement and dynamic light scattering measurement.

2-2. Production of Antibody 306 Having Photoaffinity Compound As a photoaffinity compound, p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid (manufactured by BACHEM) is used. 5 mg of p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid was dissolved in a DMF solution, and then 25 mg of N-hydroxysuccinimide (manufactured by Aldrich) and 1-ethyl-3- An aqueous solution in which 25 mg of (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Aldrich) is dissolved is gradually added and stirred for 6 hours or more. After stirring, the target product is extracted with an organic solvent, and the solvent is distilled off under reduced pressure to obtain a succinimide ester of p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid.

  Next, a photoaffinity compound is introduced into the anti-human chorionic gonatotropin antibody using the prepared p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid succinimide ester according to the scheme shown in Formula 1. To do. 500 μg of anti-human chorionic gonatotropin antibody was dissolved in 100 μL of borate buffer at pH 9.0, and 1.0 mg of p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid succinimide ester was dissolved in this solution. Add DMSO solution or DMF solution and stir at room temperature for 3 hours. After stirring, unreacted p- (3- (Trifluoromethyl) -3H-diazirin-3-yl) benzoic acid succinimide ester is removed from the reaction solution using Microcon YM100 (manufactured by Millipore). The supernatant is collected, and an antibody 303 having a photoaffinity compound is obtained. The concentration of the obtained antibody is determined with a spectrophotometer.

2-3. Production of Plate 311 Having Sites to Prevent Adsorption of Contaminants On a polystyrene microwell plate (Perkin Elmer) used as a substrate, non-specific adsorption preventing film 310 is made of B101, skim milk, or synthetic compound N101 (Nippon Oils and Fats). For example). In the formation method, a solution containing a protein or a synthetic compound is dropped on a plate and fixed under high humidity for 3 hours or more. After the formation, the plate is thoroughly washed with a solvent or a buffer solution, and the non-immobilized protein or synthetic compound is removed from the plate to obtain a plate having a nonspecific adsorption-preventing membrane.

2-4. Detection of Human Chorionic Gonatotropin 310 Human chorionic gonatotropin, which is the target substance 308, is detected through the steps shown in FIG. First, a solution containing a human chorionic gonatotropin at a predetermined concentration in a B / F separable container 305, a predetermined concentration of antibody-immobilized fluorescent particles prepared at 2-1 and a photoaffinity of a predetermined concentration prepared at 2-2 An antibody having a compound is added and mixed to form a complex (FIG. 3 (a)). After reacting for a predetermined time, the container 305 is placed in a centrifugal separator, and the antibody having a photoaffinity compound and the contaminant 307 not forming the complex 306 are removed from the container 305 (FIG. 3B). . Next, a solution purified by B / F separation is dropped on the plate prepared in 2-3, and a laser beam 312 having a wavelength of 360 nm is irradiated to the upper part of the plate for 1 minute or longer, and the complex is prevented from nonspecific adsorption. (FIG. 3C). After light irradiation, the surface of the plate is washed with a solvent or a buffer solution to remove antibody-immobilized fluorescent particles that are not immobilized on the nonspecific adsorption-preventing membrane (FIG. 3D). Finally, the complex 306 immobilized on the plate is detected by a fluorescence microscope or a fluorescence plate reader, and human chorionic gonatotropin is quantified.

  The detection process shown above is performed using the following measurement operations. FIG. 5 is a block diagram of the measurement system of the present invention. The sample solution 501 containing the target substance is dispensed into the reaction vessel 510 by the sampling probe 507. The diluent 504 is dispensed into the container 510 in the same manner. Further, the reagent 502 containing antibody-immobilized fluorescent particles and the reagent 503 containing an antibody having a photoaffinity compound are also dispensed into the container 510 in the same manner. After forming a sandwich complex composed of an antibody having an antibody-immobilized fluorescent particle-target substance-photoaffinity compound, the container 510 is centrifuged by a centrifuge 511 installed outside, and the complex is precipitated at the bottom of the container, Samples and reagents other than the unreacted antibody-immobilized fluorescent particles are transferred from the container 510 to the waste liquid container 506 by the sampling probe 507. Further, the cleaning liquid 505 is filled into the container 510 by the sampling probe 507, and cleaning is performed. The used cleaning solution is transferred from the container 510 to the waste liquid container 506 by the sampling probe 507. The diluent 503 is dispensed into the container 510 by the sampling probe 507. The complex and the antibody-immobilized fluorescent particles in the container 510 are redispersed in the solution. The solution containing the complex in the container 510 and the antibody-immobilized fluorescent particles is transferred from the reaction container 510 to the detection container 514 by the sampling probe 507. The container 514 is provided with a plate on which a non-specific adsorption prevention film is formed (not shown). The plate is irradiated with light using a light irradiation probe 513 to immobilize the complex on the non-specific adsorption prevention film. To do. Samples and reagents other than the complex immobilized on the non-specific adsorption preventing film are transferred from the container 514 to the waste liquid container 506 by the sampling probe 507. Further, the cleaning liquid 505 is filled into the container 514 by the sampling probe 507, and cleaning is performed. The used cleaning solution is transferred from the container 514 to the waste liquid container 506 by the sampling probe 507. The diluent 503 is dispensed into the container 514 by the sampling probe 507. For example, the light source 516 irradiates light having a wavelength of 485 nm to the non-specific adsorption preventing film on which the complex is immobilized, and the fluorescence (for example, wavelength 515 nm) emitted from the immobilized fluorescent particles is measured using the detection probe 515 To do. Based on a calibration curve prepared in advance using a target substance having a known concentration, the target substance concentration in the specimen is calculated from the amount of change in fluorescence compared with the initial value.

It is a schematic diagram showing the outline | summary of the detection method of the target substance based on this invention. It is a schematic diagram explaining an example of embodiment of the detection method of the target substance which concerns on this invention. It is a schematic diagram explaining an example of embodiment of the detection method of the target substance which concerns on this invention. 1 is a block diagram illustrating an example of a measurement system in Embodiment 1. FIG. 10 is a block diagram illustrating an example of a measurement system in Embodiment 2. FIG.

Explanation of symbols

101. Particle 102. First ligand molecule 103. Second ligand molecule 104. Light irradiation binding site 105. Non-specific adsorption preventing film 106. Substrate 107. Complex 108. Foreign matter 109. Target substance 201. Particle (magnetic particle)
202. First ligand molecule 203. Second ligand molecule 204. Light irradiation binding site (eg photoaffinity compound)
205. Non-specific adsorption preventing film 206. Substrate 207. Complex 208. Foreign matter 209. Target substance 210. Magnetic field generator (eg magnet)
211. Light irradiation device 301. Particle 302. First ligand molecule 303. Second ligand molecule 304. Light irradiation binding site (eg photoaffinity compound)
305. Container 306. Complex 307. Foreign matter 308. Target substance 309. Light irradiation device 310. Non-specific adsorption preventing film 401. Sample liquid 402. Reagent including first ligand molecule-fixed magnetic particles 403. A reagent comprising a second ligand molecule having a photoaffinity compound 404. Diluent 405. Cleaning solution 406. Waste liquid container 407. Sampling probe 408. Tube 409. Pump 410. Detection container 411. Sensor element 412. Magnetic field generator 413. Magnetic signal detector 414. Light irradiation probe 415. Light source 501. Sample liquid 502. Reagent including first ligand molecule-immobilized fluorescent particle 503. A reagent comprising a second ligand molecule having a photoaffinity compound; Diluent 505. Cleaning solution 506. Waste liquid container 507. Sampling probe 508. Tube 509. Pump 510. Reaction vessel 511. Centrifuge 512. Light source 513. Light irradiation probe 514. Container for detection (well plate)
515. Detection probe 516. light source

Claims (10)

A method for detecting the presence or amount of a target substance using particles,
(A) a substrate having a non-specific adsorption preventing film, a particle having a first ligand molecule that specifically captures the target substance, a site that specifically captures the target substance, and the non-specific adsorption by light irradiation Providing a second ligand molecule having a light irradiation binding site that binds to the protective film;
(B) The first ligand molecule and the second ligand molecule are bound to the target substance in a reaction medium, and the particles having the first ligand molecule, the target substance, and the second ligand molecule are combined. Forming a complex comprising: and
(C) separating the complex by B / F separation;
(D) a step of binding a light irradiation binding site in the second ligand molecule contained in the complex to the non-specific adsorption preventing film by light irradiation;
(E) detecting the presence or absence or amount of particles fixed to the non-specific adsorption preventing film;
A method for detecting the presence or absence or amount of a target substance, characterized by comprising:   The method for detecting the presence or absence or amount of a target substance according to claim 1, wherein the non-specific adsorption preventing film is made of at least a material that suppresses non-specific adsorption of the particles.   The method for detecting the presence or absence or amount of a target substance according to claim 2, wherein the non-specific adsorption preventing film is made of a compound having high hydrophilicity and polarity.   The non-specific adsorption-preventing membrane comprises albumin, casein, skim milk, heparin, phospholipid, oligoethylene glycol, polyethylene glycol, polymethacrylate, polyacrylate, or dextran. Of detecting the presence or amount of the target substance.   The method for detecting the presence or absence or amount of a target substance according to any one of claims 1 to 4, wherein the light irradiation binding site comprises a photoaffinity compound.   6. The method for detecting the presence or absence or amount of a target substance according to claim 5, wherein the photoaffinity compound is a diazirine derivative.   The method for detecting the presence or absence or amount of a target substance according to any one of claims 1 to 6, wherein the particles are magnetic particles.   The method for detecting the presence or absence or amount of a target substance according to claim 7, wherein the step (c) is performed using magnetic force.   The method for detecting the presence or absence or amount of a target substance according to claim 8, wherein the steps (b) to (e) are performed in the same order in the same container. A detection kit for detecting the presence or amount of a target substance using particles,
A substrate having the non-specific adsorption-preventing film;
Particles having a first ligand molecule that specifically captures the target substance;
A second ligand molecule having a site that specifically captures the target substance and a light irradiation binding site that binds to the non-specific adsorption-preventing film by light irradiation;
A detection kit comprising: