JP2006162467A - Immunity measurement method using light transmissive magnetic particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for sensitively detecting signals from a label and a reagent used for this method, in a method of measuring substances by using magnetic carrier particles and a label, based on a specific reaction between a substance specifically bonding to a substance to be measured and the substance to be measured. <P>SOLUTION: This method for measuring substances to be measured includes a process of generating a label which is a complex formed out of the magnetic carrier particles (i) by making a substance specifically bonded to a measured substance carried by the carrier particles or the measured substance react with a substance specifically bonded to a labeled substance to be measured with a substance to be measured in a reaction liquid, or (ii) by making a substance specifically bonding to a measured substance carried by the carrier particles react with a labeled substance to be measured with a substance to be measured in a reaction liquid; and a process of measuring the label which is the complex formed out of the magnetic carrier particles. A reagent is also disclosed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a substance measurement method and a reagent using a substance that specifically binds to a substance to be measured.

  An immunoassay method using an antigen-antibody reaction is used for detection of diagnostic markers and trace substances showing various diseases, and its principle is widely known. Among these, representative immunoassay methods include radioimmunoassay (RIA), enzyme immunoassay (EIA), and fluorescent immunoassay (FIA) using labeling substances such as radioisotopes (RI), enzymes, fluorescent substances, and luminescent substances, respectively. Luminescence immunoassay (LIA) and the like are known. These methods have been used as extremely sensitive measuring methods by taking advantage of the characteristics of the labeling substance. On the other hand, in order to expand the reaction area of the carrier particles, the magnetic particles are used as the carrier particles, and their dispersion and magnetic collection. A method characterized by this has also been developed. While this method has greatly improved performance such as reactivity and sensitivity, it can be supplied as a reagent in a state where the particles are dispersed in a liquid, so it can be used like an ELISA (Enzyme-Linked Immunosorbent Assay) plate. Compared with the case where the carrier particles and the reaction vessel are integrated, the versatility is very high. In particular, the combination of labels with magnetic particles and luminescent substances is still widely used as a highly sensitive and practical method.

  However, this method has various problems. For example, magnetic particles that have been used for immunoassays have been based on iron oxide, which does not transmit light, as a main component. Therefore, labels of fluorescent substances or luminescent substances in immune complexes formed on magnetic particles are detected. In some cases, it is always accompanied by a shielding phenomenon in which the light signal is weakened. Such a shielding phenomenon not only shields a weak light signal, but also reduces the slope of the calibration curve when using a high concentration of magnetic particles, thereby greatly degrading accuracy. Therefore, the amount of magnetic particles used is generally limited to a range where the shielding phenomenon is small.

  However, in order to avoid such a shielding effect, reducing the amount used is not an effective means. That is, a decrease in the amount of magnetic particles used has a great influence on measurement performance. First, since the reaction area of the solid phase is proportional to the amount of carrier particles, reducing the amount of particles affects performance such as reactivity and measurement sensitivity. Secondly, in a measurement system including a cleaning step, after collecting magnetic particles, the loss of particles due to physical impact is involved in the step of removing the reaction liquid, which deteriorates the measurement accuracy. The loss ratio of particles due to this physical impact is large when the amount of magnetic particles is small, and the influence on the performance becomes remarkable. Therefore, the shielding effect of the magnetic particles greatly limits the adjustment range for maintaining the reactivity and accuracy.

As a countermeasure against this problem, Patent Document 1 describes a method of dissociating specific binding of an immune complex during detection. In this method, in order to detect the label in the immune complex formed on the magnetic particle by the antigen-antibody reaction, a dissociation solution is added to dissociate the specific binding due to the antigen-antibody reaction at the time of detection to dissociate the label. Measure the intensity of the dissociated label. However, these methods are not preferable because steps such as addition of a dissociation solution and re-separation are necessary and are complicated, and the separation solution may affect the measurement of the label.
JP-A-8-43391

  An object of the present invention is to use a magnetic carrier particle and a label, in a method for measuring a substance based on a specific reaction between a substance that specifically binds to the substance to be measured and the substance to be measured, and a signal from the label with high sensitivity. It is to provide a method of detection and a reagent used in the method.

The present invention relates to the following [1] to [10].
(1) (i) A substance that specifically binds to a substance to be measured supported on magnetic carrier particles or a substance to be measured and a substance that specifically binds to a labeled substance to be measured in a reaction solution. React with the substance to be measured, or (ii) react the substance that specifically binds to the substance to be measured supported on the magnetic carrier particles and the labeled substance to be measured with the substance to be measured in the reaction solution And generating a label that forms a complex with the magnetic carrier particle, and measuring a label that forms a complex with the magnetic carrier particle, and transmits a signal that the magnetic carrier particle uses to detect the label. A method for measuring a substance to be measured, characterized by being possible magnetic carrier particles.

(2) The measuring method according to (1), wherein the step of measuring the label is performed in a state where a complex with the magnetic carrier particles is formed.
(3) The magnetic carrier particles are magnetic particles having a transmittance of 20% or more at 1 cm of a signal used to detect a label in a state suspended in distilled water at a concentration of 0.1 mg / mL. The method according to (1) or (2).

(4) The method according to any one of (1) to (3), wherein the label is a label selected from an enzyme, a luminescent substance, a fluorescent substance, and a chromogen.
(5) The method according to any one of (1) to (4), wherein the substance that specifically binds to the substance to be measured is an antibody.
(6) A substance that specifically binds to a substance to be measured supported on a magnetic carrier particle or a substance to be measured, and a substance that specifically binds to a labeled substance to be measured. A reagent for measuring a substance to be measured, which is a magnetic carrier particle capable of transmitting a signal used to detect a label.

(7) Contains a substance that specifically binds to the substance to be measured carried on the magnetic carrier particles and a labeled substance to be measured, and transmits the signal used by the magnetic carrier particles to detect the label A reagent for measuring a substance to be measured, which is a magnetic carrier particle.
(8) The magnetic carrier particles are magnetic particles having a transmittance of 20% or more at 1 cm of a signal used for detecting a label in a state suspended in distilled water at a concentration of 0.1 mg / mL. (6) or the reagent according to (7).

(9) The reagent according to any one of (6) to (8), wherein the label is an enzyme, a luminescent substance, a fluorescent substance, or a chromogen.
(10) The reagent according to any one of (6) to (9), wherein the substance that specifically binds to the substance to be measured is an antibody.

  The present invention provides an accurate immunoassay method and reagent with high measurement accuracy.

The magnetic particle used in the present invention is not particularly limited as long as it can transmit a signal used for detecting a label. For example, Tb ₂ O ₃ is 25 to 50%, SiO ₂ is 5 to 40%, B ₂ O ₃ is 5 to 45%, and Ga ₂ O ₃ is 5 to 35 in mol% described in JP-A-10-297933. %, more P ₂ O ₅ is 0 to 7%, GeO ₂ 0 to 20%, also MgO, CaO, at Less than six% SrO and BaO, respectively, MgO, CaO, the total sum of SrO and BaO Less than six %, La ₂ O ₃ is 0 to 6%, Gd ₂ O ₃ is 0 to 6%, Yb ₂ O ₃ is 0 to 6%, Dy ₂ O ₃ is 0 to 15%, Tb ₂ O ₃ , La ₂ O ₃ , a glass for a Faraday rotator having a composition in which the sum of Gd ₂ O ₃ , Yb ₂ O ₃ and Dy ₂ O ₃ is 25 to 60% and ZrO ₂ is 0 to 8%, disclosed in JP-A-2002-145622 Chemical formula: Ti _1-x Co _x O ₂ ; represented by 0 <x ≦ 0.3, Co is substituted at the Ti lattice position, and single crystal Titanium dioxide / cobalt magnetic film epitaxially grown on a substrate, compound composed of yttrium / iron / garnet (Proc. 12th Int. Symp. Plasma Chem., / Minneapolis, USA, 1995), magnetic glass (Japanese Patent Laid-Open No. 04-170338) ), Magneto-optical recording material (Japanese Patent Laid-Open No. 01-261235), gallium nitride, and the like.

  Further, as the magnetic carrier particles, the above-mentioned permeable magnetic fine particles are made of a hydrophobic polymer such as polystyrene, polyacrylonitrile, polymethacrylonitrile, polymethyl methacrylate, polycapramide, polyethylene terephthalate, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, Polyvinyl alcohol, poly (2-oxyethyl acrylate), poly (2-oxyethyl methacrylate), poly (2,3-dioxypropyl acrylate), poly (2,3-dioxypropyl methacrylate), polyethylene glycol methacrylate, etc. Latex, gelatin, liposomes such as crosslinked hydrophilic polymers, or about 2 to 4 types of copolymers of the respective monomers, or latex, gelatin, liposomes, etc. , Such as packaging particles and the like.

  The magnetic carrier particle is not particularly limited as long as it is a magnetic carrier particle that can transmit a signal used to detect a label, but the suspension is suspended in distilled water at a concentration of 0.1 mg / mL, for example. In the state, the magnetic particles having a transmittance of 20% or more at 1 cm of the signal used for detecting the label are preferably 35%, more preferably 50%, and particularly preferably 50% or more.

The particle size of the magnetic particles is not particularly limited as long as it is a particle size that can be suspended in the reaction solution, but is preferably 0.05 μm to 300 μm, more preferably 0.1 μm to 30 μm, and particularly preferably 0.5 to 10 μm. preferable.
The label used in the present invention is not particularly limited as long as it can be detected directly using a signal that permeates magnetic particles or indirectly using a supplementary reagent. For example, an enzyme, a luminescent substance, a fluorescent substance, a color Examples of the active ingredient and enzyme include those that can be measured by reaction with an auxiliary reagent.

The signal is not particularly limited as long as the label can be detected. For example, an optical signal is preferable, and examples thereof include light and fluorescence.
Examples of the luminescent substance include acridium derivatives such as 4- (2-succinimidyloxycarbonylethyl) phenyl-10-methylacridium-9-carboxylic acid fluorosulfuric acid (hereinafter abbreviated as acridinium-I), dioxetanes, and luminol. , Isoluminol, lucigenin, coumarin derivatives, pyrazolopyridopyridazine derivatives and the like.

Examples of the fluorescent substance include 3- (4-hydroxyphenyl) propionic acid.
The chromogen may be a compound that forms a dye alone, or a compound that forms a dye by combining two compounds.
As a compound that forms a dye alone, for example, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (hereinafter abbreviated as CCAP), 10-N-methylcarbamoyl- 3,7-bis (dimethylamino) -10H-phenothiazine (hereinafter abbreviated as MCDP), N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt (hereinafter DA- 64, abbreviated as 64), 4,4′-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (hereinafter abbreviated as BCMA), and the like. It is done.

Examples of compounds that combine two compounds to form a dye include compounds that combine to form a dye in the presence of aqueous hydrogen peroxide and peroxidase.
Specifically, couplers such as 4-aminoantipyrine (hereinafter abbreviated as 4-AA) and 3-methyl-2-benzothiazolinone hydrazine and aniline compounds such as N-ethyl-N- (3- Methylphenyl) -N′-succinylethylenediamine (hereinafter abbreviated as EMSE), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N-ethyl-N-sulfopropylaniline, N— Ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl -M-Toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine, N-ethyl-N- (2-hydro Xyl-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt dihydrate (hereinafter abbreviated as TOOS), N-ethyl- N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy -3-Sulfopropyl) -3,5-dimethylaniline, N-sulfopropylaniline, N-ethyl-N-sulfopropylanilinepropyl-m-anidin and the like. In addition, combinations of 4-AA and phenol or 3-hydroxy-2,4,6-triiodoacetic acid can be mentioned.

Examples of the enzyme include peroxidase and alkaline phosphatase (hereinafter abbreviated as ALP).
The auxiliary reagent is a reagent used for measuring the aforementioned label, and can be appropriately selected by those skilled in the art depending on the type of the aforementioned label. For example, the aforementioned luminescent substance, fluorescent substance, chromogen, enzyme Etc. Moreover, an alkali, hydrogen peroxide, etc. are also mentioned.

In the invention, examples of the substance that specifically binds to the substance to be measured include antibodies, nucleic acids, glycoproteins, aptamers and the like that bind to the substance to be measured.
Examples of the antibody include IgM, IgG, IgA, IgE, and the like, and fragments such as Fab, Fab ′, or F (ab ′) ₂ obtained by treating these antibodies with an enzyme treatment or the like. As the antibody, IgG is preferable. As the antibody, either a monoclonal antibody or a polyclonal antibody can be used. When using a monoclonal antibody, it is preferable to use a combination of two or more types of monoclonal antibodies with different epitopes to be recognized. However, a single type of monoclonal antibody can be used for an antigen having a plurality of epitopes on the same antigen.

The substance to be measured is not particularly limited as long as it is a substance that specifically binds to a specific substance, and includes components measured using an antigen-antibody reaction, components measured by other specific reactions, and the like. .
Components measured by antigen-antibody reaction include, for example, IgG, IgM, IgA, IgE, apoprotein AI, apoprotein AII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, oxidized LDL, glycoalbumin, chicken Drugs such as iodothyronine (T3), total thyroxine (T4), anti-tencan agents, C-reactive protein (hereinafter abbreviated as CRP), cytokines, α-fetoprotein (AFP), carcinoembryonic antigen (CEA) ), CA19-9 (carbohydrate antigen 19-9), CA15-3 (carbohydrate antigen 15-3), CA-125 (carbohydrate antigen 125), PIVKA-II (Protein induced by vitamin K absence-II), parathyroid hormone (PTH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), insulin, C-peptide, est Rogen, anti-antiglutamate decarboxylase antibody (GAD) antibody, pepsinogen, HBV antigen, anti-hepatitis B virus (HBV) antibody, hepatitis C virus (HCV) antigen, anti-HCV antibody, adult T-cell leukemia virus (HTLV) -I) Antigen, anti-HTLV-I antibody, human immunodeficiency virus (HIV) antibody, tuberculosis antibody, Mycobacterium tuberculosis antigen (TBGL) mycoplasma antibody, hemoglobin A1c, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) ), Troponin T, troponin I, creatinine kinase-MB (CK-MB), myoglobin, H-FABP (human heart-derived fatty acid binding protein), deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2) Mold toxins such as bisphenol A, nonylphenol, Foods such as dibutyl lurate, polychlorinated biphenyls (PCBs), dioxins, endocrine disruptors such as p, p'-dichlorodiphenyltrichloroethane, tributyltin, fungi such as E. coli, eggs, milk, wheat, buckwheat, and peanuts Examples include allergic substances, allergic substances such as mites, such as mite and mite, and anti-allergic antibodies.

Examples of other components to be measured by specific binding include nucleic acids, lectins and the like. For example, DNA or RNA encoding cancer genes such as ras, cancer suppressor genes such as p53, peptide nucleic acids, aptamers, glycoproteins, etc. Can be given.
In the present invention, the target sample is not particularly limited as long as it is suspected of containing the aforementioned substance to be measured. For example, whole blood, plasma, serum, spinal fluid, saliva, amniotic fluid, urine, sweat And biological samples such as pancreatic juice, food, soil and the like.

The method of binding the substance to be measured or the substance that specifically binds to the substance to be measured to the magnetic carrier particles or the label can be performed, for example, by physical adsorption, chemical binding, or the like.
Examples of the physical binding method include a method in which an antigen or an antibody is directly immobilized on a magnetic carrier particle or label by a hydrophobic bond or the like. Further, there is a method of chemically binding to other proteins such as albumin and physically binding.

As a method of chemically binding, the amino group, carboxyl group, thiol group, etc. present on the magnetic carrier particle or label are chemically modified to directly immobilize the same functional group on the antigen or antibody molecule. A method is mentioned. A method of chemically binding an antigen or antibody via other proteins such as spacer molecules and albumin is also included.
The amount of the substance to be bound or the substance specifically bound to the substance to be measured varies depending on the surface area, functional group amount, etc. of the magnetic carrier particles to be used, but usually 1 μg to 500 μg, preferably 10 μg per 1 mg of magnetic carrier particles. ~ 200 μg. In the case of a label, the amount of the enzyme or luminescent substance supported varies depending on the type of the reactive functional group or the label, but 1 to 50 per molecule of the substance to be measured or the substance that specifically binds to the substance to be measured. , Preferably 5-20.

When immobilizing the label on the magnetic carrier particle, there are a method of chemically bonding using a functional group on the surface of the magnetic carrier particle or a method of physically bonding to the surface.
In the present invention, the binding between the magnetic carrier particle or the label and the substance to be measured or the substance that specifically binds to the substance to be measured is a bioactive substance such as biotin or streptavidin, a sugar chain and a lectin, or a chemical substance. It is also possible to carry out via a substance having such an interaction.

Further, there is a method of binding to a magnetic carrier particle or a label using a substance that can bind to a substance to be immobilized or a substance that specifically binds to the substance to be measured, such as an antibody or protein A. .
Regarding the separation of the magnetic carrier particles, the strength of the magnetic field and the shape of the container are preferred so that the magnetic carrier particles can be separated in about 1 to 3 minutes. A permanent magnet, an electromagnet, etc. can be used for a magnet. Separation is preferably carried out promptly. For this reason, the reaction vessel is preferably relatively small. The material of the reaction vessel is not particularly limited, and examples thereof include plastics such as polystyrene and acrylic, tubes made of glass, and cuvettes.

The reaction solution is not particularly limited as long as it is a solvent that can react with the substance to be measured and the substance that specifically binds to the substance to be measured. However, if the label is measured using an auxiliary reagent, further labeling is required. A solvent capable of reacting with the auxiliary reagent is preferable, and examples thereof include an aqueous medium.
Examples of the aqueous medium include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable. The buffer used in the buffer is not particularly limited as long as it has a buffer capacity, but has a pH of 1 to 11, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphorus Acid buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer Borate buffer, carbonate buffer, glycine buffer, Good buffer, and the like.

  Examples of the good buffer include 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane Phosphonic acid (HEPES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), N- [tris (hydroxymethyl) methyl] -2-hydroxy-3- Aminopropanesulfonic acid (TAPSO), piperazine-N, N′-bis (2-hydroxypropanesulfonic acid) (POPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] -2-hydroxypropanesulfone Acid (HEPPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid [(H) EPPS], N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N- Bis (2-hydroxyethyl) glycine (Bicine), N-tris (hydroxymethyl) methyl -3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), N-cyclohexyl-3-amino Examples thereof include propanesulfonic acid (CAPS).

The concentration of the buffer solution is not particularly limited as long as it is a concentration suitable for measurement. For example, 0.001 to 2.0 mol / L is preferable, 0.005 to 1.0 mol / L is more preferable, and 0.01 to 0 is preferable. .1 mol / L is particularly preferred.
The measurement method of the present invention includes the following steps, for example.
(I) The substance that specifically binds to the substance to be measured supported on the magnetic carrier particles or the substance to be measured and the substance that specifically binds to the labeled substance to be measured should be measured in the reaction solution. React with a substance, or (ii) react a substance that specifically binds to a substance to be measured supported on magnetic carrier particles and a substance to be measured supported on a label with the substance to be measured in a reaction solution A step of generating a label that forms a complex with magnetic carrier particles, and a step that measures the label that forms a complex with magnetic carrier particles.

The magnetic carrier particle is a magnetic carrier particle capable of transmitting a signal used for detecting the aforementioned label.
For the measurement of the label formed in a complex with the magnetic carrier particles, the label may be directly measured optically, but a method of indirectly measuring by generating an optical signal using an auxiliary reagent is preferable. The measurement of the label is preferably performed optically using an optical signal that can pass through the magnetic carrier particles to be used.

The concentration of the measurement object can be determined by comparing the product amount with a calibration curve indicating the relationship between the concentration of the measurement object and the product amount, which has been created in advance using a measurement object having a known concentration.
The reaction conditions are not particularly limited as long as specific binding occurs, but are preferably 2 ° C to 50 ° C, more preferably 20 ° C to 40 ° C, and particularly preferably 25 ° C to 37 ° C. The reaction time is not particularly limited, and is preferably one day after the reagent is mixed, more preferably 2 to 60 minutes, and particularly preferably 3 to 30 minutes. The carrier particles are used in an amount of 0.001 to 1.0 mg / mL, preferably 0.05 to 0.2 mg / mL with respect to the reaction solution. Although mixing at the time of reagent addition needs to be performed quickly, after mixing uniformly, it may be left still. There is no restriction | limiting in particular in pH of a reaction liquid, 1-11 are preferable, 2-10 are more preferable, 3-9 are especially preferable, The above-mentioned buffering agent can be used.

In order to avoid non-specific reaction, the reaction solution may contain salts such as sodium chloride and proteins such as bovine serum albumin.
When the magnetic carrier particle and the labeled antibody are added, the antigen in the sample binds to the antibody on the magnetic carrier particle and the labeled antibody to form an immune complex. Next, the magnetic carrier particles containing the immune complex are moved to the side of the container in the reaction solution by magnetic force, and after removing the reaction solution, washing can be repeated several times as necessary. The cleaning method is not particularly limited, and examples thereof include a method of removing by suction after adding a cleaning liquid as necessary. Examples of the cleaning liquid include the reaction liquid described above, and a surfactant such as Triton X-100 can be added as necessary.

  Next, the magnetic carrier particles remaining in the reaction vessel are redispersed in an appropriate dispersion, for example, the aforementioned reaction solution, and the amount of the label on the magnetic carrier particle is measured by a method specific to the label. When the label is a fluorescent substance, it can be irradiated with excitation light unique to the fluorescent dye, and the fluorescence intensity emitted at the same time can be measured. For example, when a fluorescene compound is used as a label, it can be excited at 490 nm and measure fluorescence at 520 nm.

  Optically performing the labeling strength in a dispersed suspension state of the magnetic carrier particles is subject to optical interference by the dispersed magnetic carrier particles. That is, if the shielding effect by the magnetic carrier particles is too high, the optical detection of the label becomes extremely difficult, so that the concentration of the magnetic carrier particles used is limited. In the present invention, in order to improve this point, light-transmitting magnetic carrier particles are used in place of the conventional magnetic carrier particles, thereby enabling more accurate measurement of the label intensity with a measurement procedure that is completely different from the conventional one. It is something to be made.

Examples of the measuring reagent of the present invention include the following reagents.
A substance that specifically binds to a substance to be measured or a substance to be measured supported on a magnetic carrier particle that can transmit a signal used to detect the label, and specifically binds to a substance to be measured or a labeled substance to be measured. A reagent for measuring a substance to be measured containing a substance.
A reagent for measuring a substance to be measured, comprising a substance that specifically binds to the substance to be measured supported on a magnetic carrier particle that can transmit a signal used for detecting the label, and a labeled substance to be measured .

The reagent of the present invention may be in the form of a kit as described below. A substance that specifically binds to a substance to be measured supported on a magnetic carrier particle capable of transmitting a signal used to detect a label or should be measured. A kit comprising a reagent containing a substance and a reagent containing a substance that specifically binds to the substance to be measured carried on the label.
A kit comprising a reagent containing a substance to be measured supported on a magnetic carrier particle capable of transmitting a signal used to detect a label and a substance that specifically binds to the substance to be measured carried on the label.

A kit including a reagent containing a substance that specifically binds to a substance to be measured supported on a magnetic carrier particle capable of transmitting a signal used to detect the label, and a reagent containing a substance to be measured supported on a label .
These reagents and kits may contain the aforementioned buffer, salts, proteins, and the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to the following.

Comparison of shielding effect between magnetic carrier particles made of iron oxide and light-transmitting magnetic carrier particles In order to prepare light-transmitting magnetic carrier particles, a commercially available thickness of 10.1 mm, transmittance of 80% in the visible region, and specific gravity of 6. The powdered magnetic glass (made by Sumita Optical Glass Co., Ltd.) having a refractive index of 04 and a refractive index of 1.83 was used to make a fine powder in a menor mortar. At this time, as a guide for sizing, a fraction that did not settle even after 5 minutes in a state of being dispersed in an aqueous solution was collected to obtain magnetic carrier particles having a fixed particle size or less that can be used for immunoassay. Further, these magnetic carrier particles were collected with a magnet, and magnetic carrier particles that were not collected even after 3 minutes and were dispersed in the aqueous solution were removed, thereby obtaining light-transmitting magnetic carrier particles for immunoassay. As for the particle diameter of these magnetic carrier particles, it was confirmed that the average particle diameter was 1 to 3 μm from observation under a microscope.

  200 μL of 0.02N NaOH was added to 1 g of these light transmissive magnetic carrier particles, and 200 μL of N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane (manufactured by Shin-Etsu Silicone) as a silane coupling agent was added, The reaction was carried out at 150 ° C. for 2 hours. The obtained amino group-containing light-transmitting magnetic carrier particles were washed 5 times with 50 mL of 1% Triton X-100 aqueous solution to remove unnecessary silane coupling agents, and then stored in the aqueous solution.

  The prepared light-transmitting magnetic carrier particles and commercially available amino group-added magnetic carrier particles (MagnaBind Beads; manufactured by PIERCE; hereinafter referred to as magnetic carrier particles) containing iron oxide having an average particle size of 0.5 to 1.0 μm as a main component are adjusted to pH 7. .4 of 10 mmol / L phosphate buffer (PB) at 0.3 mg / mL, and 4-mL of luminescent substance 4- (2-succinimidyloxycarbonylethyl) phenyl-10-methyl was dispersed in 5 mL of this solution. 10 μL of 0.001 μg / μL dimethylformamide solution of acridium-9-carboxylic acid fluorosulfuric acid (hereinafter abbreviated as acridinium-I: manufactured by Dojin Chemical Co., Ltd.) was added and reacted at 37 ° C. for 1 hour. After the reaction, the magnetic carrier particles were washed 5 times with 50 mL of 1% Triton X-100 aqueous solution to remove unnecessary acridinium-I. All the washings were recovered and the total amount of acridinium-I not bound to the magnetic carrier particles was measured in the same procedure as described below.

  The obtained acridinium-I bonded light transmitting magnetic carrier particles and acridinium-I bonded magnetic carrier particles were again dispersed in an aqueous solution at a concentration of 0.3 mg / mL. 200 μL of an aqueous solution containing 3% hydrogen peroxide was added to cause the bound acridinium-I to emit light. This detection was performed using a luminescence photometer (Biormatt LB9500T; manufactured by Bertolud), and the integrated value of all luminescence counts captured by the luminescence photometer was calculated as the label intensity.

  Table 1 shows the luminescence counts of the acridinium-I-coupled light-transmitting magnetic carrier particles and the acridinium-I-coupled magnetic carrier particles, and the luminescence counts extracted into the cleaning solution without being bound when acridinium-I was immobilized. The light emission intensity indicates a relative value (RLU).

  As a result, the emission counts observed on each of the fixed amount of magnetic carrier particles were higher for the acridinium-I bonded light transmissive magnetic carrier particles. Here, since the amount of unbound acridinium-I extracted into the washing solution was very small, the amount of acridinium-I immobilized on both magnetic carrier particles can be regarded as constant. Therefore, the difference in the obtained luminescence count can be regarded as a difference depending on the shielding effect of each magnetic carrier particle, suggesting that an accurate luminescence count can be obtained by using the light-transmitting magnetic carrier particles.

  When the light-transmitting magnetic particles used in this test were suspended in distilled water at a concentration of 0.1 mg / ml, the transmittance of 1 cm of 600 nm light was 51.1%. On the other hand, when the magnetic carrier particles were suspended in distilled water at a concentration of 0.1 mg / ml, the transmittance of 1 cm of 600 nm light was 5.1%.

Immunoassay system using light transmissive magnetic carrier particles An example in which a CRP measurement system is constructed using light transmissive magnetic carrier particles and an antibody labeled with a fluorescent substance, fluorescein, is shown.
Preparation of reagent containing carrier particles 100 mg of the light transmissive magnetic carrier particles prepared in Example 1 were dispersed in 5 mL of an acetic acid buffer solution having a pH of 5.5 and 10 mmol / mL, and 1 mL of glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) was added. And activated by shaking at 37 ° C. for 1 hour. The light-transmitting magnetic carrier particles were washed 5 times with 100 mL of acetate buffer and adjusted to a concentration of 10 mg / mL in acetate buffer. The anti-CRP antibody to be immobilized was prepared to 1 mg / mL in an acetate buffer, and 20 mL of this was added to the light-transmitting magnetic carrier particle dispersion solution. The dispersion solution was put into a sealed solution and allowed to react overnight in a constant temperature bath at 37 ° C. After the reaction, the anti-CRP antibody-immobilized light-transmitting magnetic carrier particles are washed 5 times with 100 mL of acetate buffer solution, and 10 mmol / L phosphate buffer solution of pH 7.4 containing 0.1% BSA and 0.1% NaN _3. It added to (BSA / PBS) so that it might become 0.1 mg / mL, and it was set as the carrier particle containing reagent.

Preparation of Labeling Reagent Anti-CRP monoclonal antibody (hereinafter referred to as anti-CRP antibody) was dissolved in 100 mmol / L carbonate buffer (pH 8.5) to a concentration of 1.0 mg / mL, and Texas Red (manufactured by PIERCE) DMSO. 100 μL of the solution (1 mg / mL) was added and reacted at room temperature for 1 hour. After the reaction, in order to remove the unreacted label, the product was purified by Sephadex G-25 column chromatography (Pharmacia) to obtain a Texas Red labeled anti-CRP antibody. This was added to BSA / PBS at 0.01 mg / mL to obtain a labeling reagent.

Measurement of Standard Antigen Solution As a standard CRP antigen solution, CRP recombinant (rCRP; manufactured by Oriental Yeast Co., Ltd.) was diluted with a phosphate buffer solution of pH 7.2 containing 2 mmol / L calcium chloride and 0.1% BSA. The solution was used.
For measurement of this standard antigen, 50 μL of a standard antigen solution was mixed with 300 μL of a carrier particle-containing reagent and 100 μL of a labeling reagent in a reaction vessel, and reacted at 37 ° C. for 5 minutes. After the reaction, magnets were arranged on both sides of the reaction vessel to collect the light-transmitting magnetic carrier particles dispersed in the reaction solution, and the reaction solution and unreacted labeled antibody were removed. Furthermore, addition and removal of 1 mL of distilled water were repeated twice to sufficiently remove the reaction solution and unreacted labeled antibody. Next, in order to measure the amount of the label in the immune complex formed on the magnetic carrier particles, 100 μL of distilled water is added to disperse the light-transmitting magnetic carrier particles, and a spectrofluorophotometer (F-4000; (Manufactured by Hitachi, Ltd.), and measured with excitation light (Ex: 596 nm) and fluorescence (Em: 615 nm) specific to the label. As a result, the measurable CRP concentration range was 0.001 to 5.0 mg / dL based on at least the relationship between fluorescence intensity and antigen concentration. The results are shown in FIG.

  In addition, three types of healthy human serum to which CRP was added (CRP concentrations: 0.50 mg / dL for serum 1, 1.0 mg / dL for serum 2, 2.0 mg / dL for serum 3) were measured, and the fluorescence intensity and antigen were measured. When each concentration was calculated from the relationship of the concentration, each concentration value showed an equivalent numerical value. The results are shown in Table 2.

It is a figure which shows the CRP density | concentration and fluorescence intensity which were obtained in Example 2.

Claims (10)

(I) The substance that specifically binds to the substance to be measured supported on the magnetic carrier particles or the substance to be measured and the substance that specifically binds to the labeled substance to be measured should be measured in the reaction solution. React with a substance, or (ii) react a substance that specifically binds to the substance to be measured supported on the magnetic carrier particles and a labeled substance to be measured with the substance to be measured in the reaction solution, A magnetic material capable of transmitting a signal used by the magnetic carrier particles to detect the label, including a step of generating a label that forms a complex with the carrier particle, and a step of measuring the label that forms a complex with the magnetic carrier particle. A method for measuring a substance to be measured, which is a carrier particle. The measurement method according to claim 1, wherein the step of measuring the label is performed in a state where a complex is formed with the magnetic carrier particles. The magnetic carrier particle is a magnetic particle having a transmittance of 20% or more at 1 cm of a signal used for detecting a label in a state suspended in distilled water at a concentration of 0.1 mg / mL. 2. The method according to 2. The method according to any one of claims 1 to 3, wherein the label is a label selected from an enzyme, a luminescent substance, a fluorescent substance, and a chromogen. The method according to claim 1, wherein the substance that specifically binds to the substance to be measured is an antibody. Contains a substance that specifically binds to a substance to be measured, or a substance to be measured, and a substance that specifically binds to a labeled substance to be measured, and the magnetic carrier particles detect the label. A reagent for measuring a substance to be measured, which is a magnetic carrier particle capable of transmitting a signal to be used. A magnetic carrier that contains a substance that specifically binds to the substance to be measured carried on the magnetic carrier particles, and a labeled substance to be measured and that is permeable to the signal used by the magnetic carrier particles to detect the label A reagent for measuring a substance to be measured which is a particle. The magnetic carrier particles are magnetic particles having a transmittance of 20% or more at 1 cm of a signal used for detecting a label in a state suspended in distilled water at a concentration of 0.1 mg / mL. 7. The reagent according to 7. The reagent according to any one of claims 6 to 8, wherein the label is an enzyme, a luminescent substance, a fluorescent substance, or a chromogen. The reagent according to any one of claims 6 to 9, wherein the substance that specifically binds to the substance to be measured is an antibody.

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