JP2016105066A - Magnetic silica particle, and method and reagent for measuring measurement object substance using the magnetic silica particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a magnetic silica particle that has excellent binding property when immobilizing a measurement object substance, an analog of the measurement object substance, or a substance that is specifically bound to the measurement object substance to a magnetic silica particle, shows excellent sensitivity in immunoassay, and can exhibit stable performance even when stored for the long term.SOLUTION: A magnetic silica particle (C) is a core-shell shaped particle composed of a core layer (P) being a silica particle comprising 60-95 wt.% of a superparamagnetic metal oxide particle (A) with an average particle diameter of 1-15 nm, and a shell layer (Q) being a silica layer formed on the surface of the core layer (P) and with an average thickness of 3-3000 nm.SELECTED DRAWING: None

Description

The present invention relates to magnetic silica particles, a method for measuring a substance to be measured using the magnetic silica particles, and a reagent used in the method. More specifically, the present invention relates to a measurement method and a reagent used for assisting diagnosis, assisting confirmation of therapeutic effect, confirmation of the degree of purification during protein purification and cell separation, and the like.

Conventionally, as a method of measuring or purifying a sample containing a biological substance, for example, a protein in a biological sample, the protein in the biological sample is bound to the particle surface to which the protein can bind, and other than the target protein in the biological sample. In order to remove impurities, there are known methods of washing particles, collecting particles to which proteins and the like are bound, measuring the amount of protein bound, and dissociating into a protein dissociation solution for purification.

In the above method, magnetic particles are used because they can be easily separated and collected by magnetic force. As such magnetic particles, for example, Patent Document 1 describes magnetic silica particles in which a silica film is formed on the surface of core particles made of iron oxide. When the magnetic silica particles described in Patent Document 1 are used, a magnetic field is applied when the magnetic silica particles are recovered. However, since the magnetic substance that forms the magnetic silica particles is ferromagnetic, even if the magnetic field at the time of recovery is removed, the magnetic substance itself exhibits a temporary magnetic field due to ferromagnetism, and the magnetic silica particles self-associate with each other, and the detergency Is bad and / or adversely affects subsequent operations (eg, immune response).

Furthermore, for the purpose of solving self-association between magnetic silica particles due to ferromagnetism, for example, Patent Document 2 discloses magnetic silica particles using a superparamagnetic magnetic material as a magnetic material. However, when the particle size of the magnetic silica particles is small, the content of the magnetic material is low, and it takes time to recover the magnetic silica particles by magnetic force. When the particle size is large, the specific surface area is small. Therefore, there is a problem that the amount of protein or the like to be bound is small.

Therefore, Patent Document 3 discloses a magnetic silica particle with an increased content of a superparamagnetic magnetic substance for the purpose of quickly recovering the magnetic silica particle with a magnetic force even when the particle diameter of the magnetic silica particle is small. Has been. However, the magnetic silica particles described in Patent Document 3 are not sufficiently satisfactory in sensitivity and storage stability when used as a reagent.

JP 2000-256388 A JP 2000-40608 A WO2012 / 173002 brochure

The present invention has excellent binding properties when immobilizing a substance to be measured, a substance similar to the substance to be measured, or a substance that specifically binds to the substance to be measured on the magnetic silica particles, and exhibits excellent sensitivity in immunoassay. Another object of the present invention is to provide magnetic silica particles that can exhibit stable performance even when stored for a long period of time, a method for measuring a substance to be measured using the magnetic silica particles, and a reagent for measuring the substance to be measured. .

The inventors of the present invention have reached the present invention as a result of intensive studies to achieve the above object. That is, the present invention relates to a core layer (P) which is a silica particle containing 60 to 95% by weight of superparamagnetic metal oxide particles (A) having an average particle diameter of 1 to 15 nm, and on the surface of the core layer (P). Magnetic silica particles (C) that are core-shell type particles composed of a shell layer (Q) that is a silica layer having an average thickness of 3 to 3000 nm formed on the surface of the magnetic silica particles (C) Measure the target substance in the sample using magnetic silica particles (D) to which the target substance, the similar substance to the target substance, or the target substance binding substance that specifically binds to the target substance are immobilized A measurement target substance measuring method, and a measurement target substance binding that specifically binds to the measurement target substance, a similar substance to the measurement target substance, or a measurement target substance on the surface of the magnetic silica particles (C) Magnetic material with immobilized substance Characterized in that it contains a silica particles (D), as the measurement substance measurement reagent.

In the measurement method and reagent of the present invention, a substance to be measured can be measured (detected) with high sensitivity, and can be stored as a reagent for a long time without impairing performance.

  The substance to be measured in the present invention is not particularly limited as long as it is usually measured in this field. For example, biological body fluids such as serum, blood, plasma and urine, lymph fluid, blood cells, various cells, etc. Typical examples include proteins, lipid proteins, nucleic acids, immunoglobulins, blood coagulation-related factors, antibodies, enzymes, hormones, cancer markers, heart disease markers, and various drugs contained in these samples. More specifically, for example, proteins such as albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), such as high density lipoprotein (HDL), low density lipoprotein (LDL), ultra low density lipo Lipid proteins such as proteins, such as nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as alkaline phosphatase, amylase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactic acid Enzymes such as dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase (PK), tyrosine kinase, such as IgG, IgM, IgA, I Blood coagulation-related factors such as immunoglobulins such as gD and IgE (or fragments thereof such as Fc part, Fab part and F (ab) 2 part) such as fibrinogen, fibrin degradation product (FDP), prothrombin and thrombin For example, anti-streptridine O antibody, anti-human H. Antibodies such as H. pylori antibody, anti-human hepatitis B virus surface antigen antibody (anti-HBs antigen antibody), anti-human hepatitis B core antigen antibody (anti-HBc antigen antibody), anti-human hepatitis C virus antibody, anti-rheumatic factor, etc. Hepatitis B virus surface antigen (HBsAg) such as thyroid stimulating hormone (TSH), thyroid hormone (FT3, FT4, T3, T4), parathyroid hormone (PTH), procalcitonin (PCT), adrenocorticotropic hormone (ACTH) , Hormones such as human chorionic gonadotropin (hCG), estradiol (E2), cortisol, aldosterone, for example, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, prostate specific antigen (PSA), etc. Markers such as troponin T (TnT), human brain natriuretic peptide Cardiac markers such precursor N-terminal fragment (NT-proBNP), for example, antiepileptic drugs, antibiotics, drugs such as theophylline and the like. Among those described above, antibodies, hormones, cancer markers, heart disease markers and the like are preferable.

A similar substance (analog) of a measurement target substance in the present invention is present when a measurement target substance and a substance that specifically binds to the measurement target substance (hereinafter also referred to as “measurement target substance binding substance”) are present. Any material that competes with the substance may be used.
For example, the similar substance may be any substance that binds to the binding site of the measurement target substance binding substance with the measurement target substance. Furthermore, the similar substance may have the same site as the binding site with the measurement target substance binding substance of the measurement target substance.

  Examples of the substance that specifically binds to the measurement target substance (measurement target substance binding substance) in the present invention include an “antigen”-“antibody” reaction, a “sugar chain”-“protein” reaction, and a “sugar chain” — Reaction between “lectin”, reaction between “enzyme” and “inhibitor”, reaction between “protein” and “peptide chain”, reaction between “protein” and “protein”, reaction between “chromosome or nucleotide chain” and “nucleotide chain” , A substance that binds to a substance to be measured or a similar substance by an interaction such as a reaction between “nucleotide chain” and “protein”. When one of the above combinations is a measurement target substance or a similar substance, the other is the measurement target substance binding substance. For example, when the measurement target substance or a similar substance is “antigen”, the measurement target substance binding substance is “antibody”, and when the measurement target substance or similar substance is “antibody”, the measurement target substance binding substance is It is an “antigen” (the same applies to the other combinations described above).

  Specifically, for example, nucleotide chain (for example, oligonucleotide chain, polynucleotide chain); chromosome; peptide chain (for example, C-peptide, angiotensin I, etc.), protein [for example, procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), β2-microglobulin, albumin, their degradation products, serum proteins such as ferritin]; enzymes [eg, amylases (eg, Pancreatic type, salivary gland type, X type, etc.), alkaline phosphatase (eg, hepatic, osseous, placental, small intestine, etc.), acid phosphatase (eg, PAP), γ-glutamyltransferase (eg, renal, pancreatic, hepatic) Etc.), lipase (eg pancreatic type, stomach type, etc.), creatine Kinase (for example, CK-1, CK-2, mCK, etc.), lactate dehydrogenase (for example, LDH1-LDH5, etc.), glutamate oxaloacetate transaminase (for example, ASTm, ASTs, etc.), glutamate pyruvate transaminase (for example, ALTm, ALTs, etc.) Cholinesterase (eg, ChE1-ChE5), leucine aminopeptidase (eg, C-LAP, AA, CAP, etc.), renin, protein kinase, tyrosine kinase, etc.) and inhibitors of these enzymes, hormones (eg, PTH, TSH, insulin, LH) , FSH, prolactin, etc.), receptor (eg, receptor for estrogen, TSH, etc.); ligand (eg, estrogen, TSH, etc.); bacteria (eg, tuberculosis, pneumococci, diphtheria, meningitis) , Neisseria gonorrhoeae, Staphylococcus, Streptococcus, Enterobacteriaceae, Escherichia coli, Helicobacter pylori, etc.), viruses (eg, rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus, enterovirus, poliovirus) , EB virus, HAV, HBV, HCV, HIV, HTLV, etc.), fungi (eg, Candida, cryptococcus, etc.), spirochetes (eg, leptospira, syphilis treponema, etc.), chlamydia, mycoplasma, etc .; a protein or peptide derived from the microorganism Or sugar chain antigens; various allergens that cause allergies such as bronchial asthma, allergic rhinitis, atopic dermatitis (eg house dust, mites such as white mite, mite, etc., eg It is derived from pollen of Japanese cedar, Japanese cypress, Japanese cypress, ragweed, blue-necked frog, hargaya, rye, etc., such as animals such as cats, dogs, crabs, etc., food such as rice, egg white, fungi, insects, wood, drugs, chemicals, etc. Lipids (eg, lipoproteins); proteases (eg, trypsin, plasmin, serine proteases, etc.); tumor marker protein antigens (eg, PSA, PGI, PGII, etc.); sugar chain antigens (eg, AFP (eg, L1 to L3, etc.) ), HCG (hCG family), transferrin, IgG, thyroglobulin, decay-accelerating-factor (DAF), carcinoembryonic antigen (eg CEA, NCA, NCA-2, NFA, etc.), CA19-9, PIVKA-II, CA125 Prostate specific antigen, Tumor marker sugar chain antigens, ABO sugar chain antigens, etc. having special sugar chains produced by cancer cells]; sugar chains (eg, hyaluronic acid, β-glucan, sugar chains possessed by the above sugar chain antigens, etc.); Proteins that bind (eg, hyaluronic acid binding protein, β-glucan binding protein, etc.); phospholipids (eg, cardiolipin, etc.); lipopolysaccharides (eg, endotoxin, etc.); chemicals (eg, T3, T4, eg, tributyltin, nonylphenol, 4-octylphenol, Environmental hormones such as di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate); various drugs administered and inoculated to the human body and their metabolites; aptamers; nucleic acid binding Sex substances; and antibodies against these substances. The antibodies used in the present invention also include proteolytic enzymes such as papain and pepsin, or Fab and F (ab ') 2 fragments that are antibody degradation products generated by chemical degradation.

  As the measurement target substance-binding substance as described above, a substance that binds to a measurement target substance or a similar substance by an “antigen”-“antibody” reaction or a “sugar chain”-“protein” reaction is preferable. Specifically, an antibody to the measurement target substance or a similar substance, an antigen to which the measurement target substance or the similar substance binds, or a protein that binds to the measurement target substance or the similar substance is preferable. More preferably, the antibody is a protein that binds to a target substance or a substance to be measured or a similar substance.

  The magnetic silica particles (C) in the present invention comprise a core layer (P) and a shell layer (Q) formed on the surface of the core layer (P), and the average thickness of the shell layer (Q) is 3 to 3. It is a core-shell type particle having a thickness of 3000 nm. The core layer (P) is a silica particle composed of spheres in which superparamagnetic metal oxide particles (A) having an average particle diameter of 1 to 15 nm and having superparamagnetism are dispersed in a silica matrix. The shell layer (Q) is made of silica and may contain other components.

  The average particle size of the superparamagnetic metal oxide particles (A) in the present invention is an average value of the particle sizes measured by observing any 200 superparamagnetic metal oxide particles (A) with a scanning electron microscope. is there. The average particle diameter of the superparamagnetic metal oxide particles (A) can be controlled by adjusting the metal ion concentration at the time of preparing the superparamagnetic metal oxide particles (A) described later. Moreover, the average particle diameter of the superparamagnetic metal oxide particles (A) can be set to a desired value also by a usual method such as classification.

  Superparamagnetism means that when a material is subjected to an external magnetic field, a temporary magnetic field from the material is induced by the external magnetic field, and the alignment of the individual atomic magnetic moments of the material occurs. Disappears and the alignment of partial atomic magnetic moments is impaired.

  Examples of superparamagnetic metal oxide particles (A) having superparamagnetism with an average particle diameter of 1 to 15 nm in the present invention include oxides such as iron, cobalt, nickel, and alloys thereof, but are sensitive to magnetic fields. Iron oxide is particularly preferred because of its superiority. The superparamagnetic metal oxide particles (A) may be used alone or in combination of two or more.

The average particle diameter of the superparamagnetic metal oxide particles (A) in the present invention is 1 to 15 nm, preferably 3 to 13 nm, and more preferably 5 to 13 nm. When the average particle size of the superparamagnetic metal oxide particles (A) is less than 1 nm, synthesis is difficult, and when the average particle size exceeds 15 nm, magnetic silica particles containing superparamagnetic metal oxide particles (A) ( The magnetic properties of C) become ferromagnetic, and superparamagnetic metal oxide particles (A) themselves exhibit a temporary magnetic field even when the magnetic field is removed in actual applications, and the magnetic silica particles (C) self-associate with each other to be washed. There is a problem that the sex is bad and / or the immune reaction is adversely affected.

  Various known iron oxides can be used as the iron oxide used for the superparamagnetic metal oxide particles (A). Among iron oxides, at least one selected from the group consisting of magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide and γ-hematite-α-hematite intermediate iron oxide because of its excellent chemical stability. Magnetite is more preferable because it has a large saturation magnetization and excellent sensitivity to an external magnetic field.

  The lower limit of the content of the superparamagnetic metal oxide particles (A) based on the weight of the core layer (P) in the present invention is 60% by weight, preferably 65% by weight, and the upper limit is 95% by weight, preferably 80% by weight. %. When the content of the superparamagnetic metal oxide particles (A) is less than 60% by weight, the obtained magnetic silica particles (C) are not sufficiently magnetized, so that it takes time for the separation operation in actual use. Moreover, when the content of the superparamagnetic metal oxide particles (A) exceeds 95% by weight, the synthesis is difficult.

  The production method of the superparamagnetic metal oxide particles (A) is not particularly limited, but based on the one reported by Massart, a coprecipitation method using a water-soluble iron salt and ammonia (R. Massart, IEEE Trans. Magn. 1981). , 17, 1247) and a method using an oxidation reaction in an aqueous solution of a water-soluble iron salt.

In the present invention, the shell layer (Q) is formed on the surface of the core layer (P).
As described above, the shell layer (Q) is a silica layer and has a silanol group on the surface thereof. Moreover, since the shell layer (Q) is located in the outermost layer of the magnetic silica particles (C), there are silanol groups on the surface of the magnetic silica particles (C). Therefore, many measurement target substances, similar substances to the measurement target substances, or measurement target substance binding substances can be immobilized on the surface.

  The average thickness of the shell layer (Q) in the present invention is measured from image analysis of an image obtained by observing with a transmission electron microscope a cross section obtained by embedding the magnetic silica particles (C) in a resin and cutting with a microtome. I can do it. The average thickness of the shell layer (Q) is the shell layer of any 100 magnetic silica particles (C) measured by observation with a transmission electron microscope (for example, “H-7100” manufactured by Hitachi, Ltd.). It is an average value of the thickness of (Q). The thickness of the shell layer (Q) is an average value of the thinnest part and the thickest part in one magnetic silica particle (C).

  The average thickness of the shell layer (Q) is 3 to 3000 nm, preferably 10 to 500 nm, and more preferably 50 to 200 nm. When the average thickness is less than 3 nm, the effect that the shell layer (Q) is formed cannot be obtained, the sensitivity and stability are lowered, and those having an average thickness exceeding 3000 nm are difficult to synthesize.

  The average particle diameter of the magnetic silica particles (C) is preferably 0.5 to 20 μm, more preferably 1 to 10 μm, and particularly preferably 1 to 5 μm. When the average particle size of the magnetic silica particles (C) is less than 0.5 μm, it tends to take time for separation and recovery. In addition, when the average particle diameter of the magnetic silica particles (C) exceeds 20 μm, the specific surface area becomes small, and the immobilization amount of the substance to be immobilized (target substance, similar substance to the measurement target substance or measurement target substance binding substance) is increased. The coupling efficiency tends to be low.

  The average particle diameter of the magnetic silica particles (C) in the present invention is measured by observing any 200 magnetic silica particles (C) with a scanning electron microscope (for example, “JSM-7000F” manufactured by JEOL Ltd.). It is the average value of the measured particle diameter.

  The average particle diameter of the magnetic silica particles (C) can be controlled by controlling the average particle diameter of the core layer (P) and the average thickness of the shell layer (Q). The average particle size of the core layer (P) can be controlled by adjusting the particle size of the oil-in-water emulsion by adjusting the mixing conditions (shearing force, etc.) when preparing the oil-in-water emulsion described later. The average thickness of the shell layer (Q) can be controlled by adjusting the amount of (alkyl) alkoxysilane, the amount of catalyst, and the reaction time when the shell layer (Q) described later is formed. In addition, the average particle diameter of the core layer (P) and the magnetic silica particles (C) can be set to a desired value by a method such as a change in conditions of a washing process at the time of production or a normal classification.

Next, the manufacturing method of the magnetic silica particle (C) in this invention is demonstrated.
The method for producing the magnetic silica particles (C) in the present invention can be produced by a production method that includes at least the following two steps.
(Step 1) An oil-in-water emulsion of (alkyl) alkoxysilane containing superparamagnetic metal oxide particles (A) was prepared and subjected to a condensation reaction, and the superparamagnetic metal oxide particles (A) were included in silica. The process of manufacturing a core layer (P).
(Step 2) A step of forming a shell layer (Q) by condensing (alkyl) alkoxysilane on the surface of the core layer (P).
Hereinafter, the above steps will be described.

(Process 1)
Examples of the method for producing the core layer (P) in the present invention include a superparamagnetic metal oxide particle (A) having an average particle diameter of 1 to 15 nm and a weight of 30 to 30 based on the weight of the superparamagnetic metal oxide particle (A). A dispersion (B1) containing 1000% by weight of (alkyl) alkoxysilane was mixed with a solution (B2) containing water, a nonionic surfactant and a catalyst for hydrolysis of (alkyl) alkoxysilane. An oil-in-water emulsion is prepared, and a hydrolysis reaction and a condensation reaction of (alkyl) alkoxysilane are performed to produce particles in which superparamagnetic metal oxide particles (A) are included in silica. After hydrolysis and condensation reaction of (alkyl) alkoxysilane, the core layer (P) is obtained by solid-liquid separation with a centrifugal separator and a magnet. Furthermore, in the synthesis of the core layer (P), a dispersant, a water-soluble organic solvent, and a water-insoluble organic solvent may be used, and these may be used in combination of two or more.
Above and below, (alkyl) alkoxysilane means alkylalkoxysilane or alkoxysilane.

  Examples of the dispersant include an organic compound having one or more carboxyl groups in the molecule, an organic compound having one or more sulfo groups, and an organic compound having one or more carboxyl groups and one or more sulfo groups. Can be mentioned. Specific examples include the organic compounds (a-1) to (a-5) exemplified below, and these may be used alone or in combination of two or more.

(A-1) Organic compound having two or more carboxyl groups:
C2-C30 aliphatic polycarboxylic acid (oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, propane-1,2,3-tricarboxylic acid, Saturated polycarboxylic acids such as citric acid and dodecanedioic acid and unsaturated polycarboxylic acids such as maleic acid, fumaric acid and itaconic acid), aromatic polycarboxylic acids having 8 to 30 carbon atoms (phthalic acid, isophthalic acid, terephthalic acid) Acid, trimellitic acid, pyromellitic acid and the like) and alicyclic polycarboxylic acid having 5 to 30 carbon atoms (cyclobutene-1,2-dicarboxylic acid, cyclopentene-1,2-dicarboxylic acid, furan-2,3-dicarboxylic acid) Acids, bicyclo [2,2,1] hept-2-ene-2,3-dicarboxylic acid and bicyclo [2,2,1] hepta-2,5-diene- , 3-dicarboxylic acid, etc.) and the like.

(A-2) Organic compound having two or more sulfo groups:
C1-C30 aliphatic polysulfonic acid (methionic acid, 1,1-ethanedisulfonic acid, 1,2-ethanedisulfonic acid, 1,1-propanedisulfonic acid, 1,3-propanedisulfonic acid, polyvinylsulfonic acid, etc. ), Aromatic polysulfonic acid having 6 to 30 carbon atoms (m-benzenedisulfonic acid, 1,4-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, 1,6-naphthalenedisulfonic acid, 2,6-naphthalenedisulfonic acid 2,7-naphthalenedisulfonic acid and sulfonated polystyrene, etc.), bis (fluorosulfonyl) imide and bis (perfluoroalkanesulfonyl) imide having 2 to 10 carbon atoms [bis (trifluoromethanesulfonyl) imide, bis (pentafluoroethane) Sulfonyl) imide, bis (nonafluorobutanesulfonate) ) Imide, pentafluoroethanesulfonyl trifluoromethanesulfonyl imide, trifluoromethanesulfonyl heptafluoropropanesulfonyl imide, nonafluorobutanesulfonyl trifluoromethanesulfonyl imide], and the like.

(A-3) Organic compound having at least one carboxyl group and one sulfo group:
C2-C30 sulfocarboxylic acid (such as sulfoacetic acid and sulfosuccinic acid) and C7-C30 sulfoaromatic mono- or polycarboxylic acid (o-, m- or p-sulfobenzoic acid, 2,4-disulfo) Benzoic acid, 3-sulfophthalic acid, 3,5-disulfophthalic acid, 4-sulfoisophthalic acid, 2-sulfoterephthalic acid, 2-methyl-4-sulfobenzoic acid, 2-methyl-3,5-disulfobenzoic acid, 4 -Propyl-3-sulfobenzoic acid, 4-isopropyl-3-sulfobenzoic acid, 2,4,6-trimethyl-3-sulfobenzoic acid, 2-methyl-5-sulfoterephthalic acid, 5-methyl-4-sulfo Isophthalic acid, 5-sulfosalicylic acid, 3-oxy-4-sulfobenzoic acid and the like).

(A-4) Organic compound having one carboxyl group:
C1-C30 aliphatic saturated monocarboxylic acid (formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, verargonic acid, lauric acid, myristic acid, stearic acid and behenine Acid etc.), C3-C30 aliphatic unsaturated monocarboxylic acid (acrylic acid, methacrylic acid, oleic acid, stearic acid, etc.), C3-C30 hydroxy aliphatic monocarboxylic acid (glycolic acid, lactic acid and the like) Tartaric acid, etc.), C4-30 alicyclic monocarboxylic acids (cyclopropanecarboxylic acid, cyclopentanecarboxylic acid, cyclohexanecarboxylic acid, etc.), C7-30 aromatic monocarboxylic acids (benzoic acid, cinnamon) Acid, naphthoic acid, etc.), C7-20 hydroxy aromatic monocarboxylic acids (salicylic acid, mandelic acid, etc.) and carbon number To 20 perfluorocarboxylic acids (trifluoroacetic acid, undecafluorohexanoic acid, tridecafluoroheptanoic acid, perfluorooctanoic acid, pentadecafluorooctanoic acid, peptadecafluorononanoic acid, nonadecafluorodecanoic acid, etc.) .

(A-5) Organic compound having one sulfo group:
C1-C30 aliphatic monosulfonic acid (methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, hexanesulfonic acid, decanesulfonic acid, undecanesulfonic acid, dodecanesulfonic acid, etc.), C6-C30 Aromatic monosulfonic acid (benzenesulfonic acid, p-toluenesulfonic acid, o-toluenesulfonic acid, m-toluenesulfonic acid, 4-dodecylbenzenesulfonic acid, 4-octylbenzenesulfonic acid, naphthalenesulfonic acid, etc.) and carbon number 1-20 perfluoroalkanesulfonic acid (such as trifluoromethanesulfonic acid) and the like.

  Among these, an organic compound having 10 to 30 carbon atoms in (a-4) is preferable from the viewpoint of compatibility with (alkyl) alkoxysilane.

  The amount of the dispersant used is preferably 100 to 2,000% by weight, particularly 250 to 1,000% by weight, based on the weight of the superparamagnetic metal oxide particles (A). When the amount of the dispersant used is less than 100% by weight based on the weight of the superparamagnetic metal oxide particles (A), the superparamagnetic metal oxide particles (A) tend not to be dispersed in the (alkyl) alkoxysilane solution. is there. Further, when the amount of the dispersant used exceeds 2,000% by weight based on the weight of the superparamagnetic metal oxide particles (A), an emulsion tends to hardly form during dispersion in an aqueous solution in a later step. .

Examples of the (alkyl) alkoxysilane used include compounds represented by the following general formula (1).
R ¹ _(4-n) Si (OR ² ) _n (1)
In General Formula (1), R ¹ and R ² represent a monovalent hydrocarbon group having 1 to 10 carbon atoms that may be substituted with an amino group, a carboxyl group, a hydroxyl group, a mercapto group, or a glycidyloxy group.

  Examples of the monovalent hydrocarbon group having 1 to 10 carbon atoms include an aliphatic hydrocarbon group having 1 to 10 carbon atoms (methyl group, ethyl group, n- or iso-propyl group, n- or iso-butyl group, n -Or iso-pentyl group and vinyl group), aromatic hydrocarbon groups having 6 to 10 carbon atoms (phenyl group and the like), and aromatic aliphatic groups having 7 to 10 carbon atoms (benzyl group and the like).

  N in General formula (1) represents the integer of 1-4. However, when using an alkylalkoxysilane with n = 1, it is necessary to use it together with an (alkyl) alkoxysilane with n = 2-4. From the viewpoint of the strength of the particles after the reaction and the amount of silanol groups on the particle surface, n is preferably 4.

Specific examples of the compound represented by the general formula (1) include alkoxysilanes such as tetramethoxysilane, tetraethoxysilane, tetraisopropylsilane and tetrabutoxysilane; alkylalkoxysilanes such as methyltrimethoxysilane and methyltriethoxysilane. An amino group such as 3-aminopropyltrimethoxysilane, 3-aminopropylethoxysilane, N-2 (aminoethyl) 3-aminopropyltrimethoxysilane and N-2 (aminoethyl) 3-aminopropyltriethoxysilane; Alkylalkoxysilanes having substituted alkyl groups; alkylalkoxysilanes having alkyl groups substituted with carboxyl groups such as 7-carboxy-heptyltriethoxysilane and 5-carboxy-pentyltriethoxysilane; 3 Alkyl alkoxysilanes having alkyl groups substituted with hydroxyl groups such as hydroxypropyltrimethoxysilane and 3-hydroxypropylethoxysilane; substituted with mercapto groups such as 3-mercaptopropyltrimethoxysilane and 3-mercaptopropyltriethoxysilane Examples include alkylalkoxysilanes having an alkyl group; alkylalkoxysilanes having an alkyl group substituted with a glycidyloxy group such as 3-glycidyloxypropyltrimethoxysilane and 3-glycidyloxypropyltriethoxysilane.
(Alkyl) alkoxysilane may be used alone or in combination of two or more.

  The amount of (alkyl) alkoxysilane used is usually 30 to 1,000% by weight, preferably 40 to 500% by weight, based on the weight of the superparamagnetic metal oxide particles (A). When the amount of (alkyl) alkoxysilane used is less than 30% by weight based on the weight of the superparamagnetic metal oxide particles (A), the surface of the superparamagnetic metal oxide particles (A) is difficult to be uniformly coated. Further, when the amount of (alkyl) alkoxysilane used exceeds 1000% by weight with respect to the weight of the superparamagnetic metal oxide particles (A), the content of the superparamagnetic metal oxide particles (A) becomes small, and the magnetic force The collection time by becomes longer.

  The amount of water used is preferably 500 to 50,000% by weight, particularly 1,000 to 10,000% by weight, based on the weight of the superparamagnetic metal oxide particles (A).

  Examples of the water-insoluble organic solvent include an aromatic hydrocarbon solvent having 6 to 16 carbon atoms (toluene, xylene and the like) having a solubility in water at 25 ° C. of 0.1 g / 100 g or less, and 5 to 16 carbon atoms. Aliphatic hydrocarbon solvents (n-heptane, n-hexane, cyclohexane, n-octane, decane, decahydronaphthalene, etc.) and the like. These may be used alone or in combination of two or more.

  The amount of the water-insoluble organic solvent used is preferably 200 to 1,000% by weight, particularly 250 to 500% by weight, based on the weight of the superparamagnetic metal oxide particles (A). When the amount of the organic solvent used is less than 200% by weight based on the weight of the superparamagnetic metal oxide particles (A), the dispersibility of the superparamagnetic metal oxide particles (A) tends to deteriorate. Further, when the amount of the organic solvent used exceeds 1,000% by weight with respect to the weight of the superparamagnetic metal oxide particles (A), the particle diameter of the magnetic silica particles (C) tends to be non-uniform.

  As a water-soluble organic solvent, solubility in water at 25 ° C. is 100 g / 100 g or more of water, alcohol having 1 to 4 carbon atoms (such as methanol, ethanol and n- or iso-propanol), or having 2 to 9 carbon atoms. Glycol (ethylene glycol and diethylene glycol etc.), amide (N-methylpyrrolidone etc.), ketone (acetone etc.), cyclic ether (tetrahydrofuran and tetrahydropyran etc.), lactone (γ-butyrolactone etc.), sulfoxide (dimethyl sulfoxide etc.) and nitrile (Acetonitrile and the like). Among these, C1-C4 alcohol is preferable from a viewpoint of the uniformity of the particle diameter of a magnetic silica particle (C). A water-soluble organic solvent may be used individually by 1 type, or may use 2 or more types together.

  It is preferable that the usage-amount of a water-soluble organic solvent is 100 to 500 weight% with respect to the weight of water.

Nonionic surfactants include higher alcohol alkylene oxide (hereinafter, alkylene oxide is abbreviated as AO) adduct; higher alcohols having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol and oleyl). 1-20 mol of ethylene oxide (hereinafter abbreviated as EO) and / or 1-20 mol of propylene oxide (hereinafter abbreviated as PO) (including block adducts and / or random adducts). ), AO adducts of alkylphenols having 6 to 24 carbon alkyls; EO 1-20 mol and / or PO 1-20 mol adducts of octyl or nonylphenol, polypropylene glycol EO adducts and polyethylene glycol PO adducts; Pluronic Fatty acid AO adducts such as surfactants; EO 1 to 20 mol of fatty acids having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.) and / or PO1 -20 mol adducts, etc. and polyhydric alcohol type nonionic surfactants; EO of 2 to 8 polyhydric alcohols having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan) and / Or PO adduct; fatty acid ester of polyhydric alcohol and EO adduct thereof [TWEEN® 20 and TWEEN® 80 etc.]; alkyl glucoside (N-octyl-β-D-maltoside, n- Dodecanoyl sucrose and n-octyl-β-D-glucopyranoside); fatty acid ester of sucrose Fatty acid alkanolamides and their AO adducts (polyoxyethylene fatty acid alkanolamide, etc.); non-ionic surfactants, and the like. These may be used alone or in combination of two or more.
Of these, an EO 1-20 mol adduct of a higher alcohol having 8 to 24 carbon atoms is preferred, and polyoxyethylene alkyl ether is more preferred.

  The amount of the nonionic surfactant used is preferably 10 to 1,000% by weight, particularly preferably 100 to 500% by weight, based on the weight of the superparamagnetic metal oxide particles (A). When the amount of the nonionic surfactant used is less than 10% by weight or more than 1,000% by weight based on the weight of the superparamagnetic metal oxide particles (A), the emulsion is not stable, and the particle size of the generated particles The distribution tends to be wide.

  The amount of the aqueous solution containing the nonionic surfactant is 1,000 to 10,000% by weight, particularly 1,500 to 4,000% by weight, based on the weight of the superparamagnetic metal oxide particles (A). preferable. When the amount of the aqueous solution containing the nonionic surfactant is less than 1,000% by weight or more than 10,000% by weight based on the weight of the superparamagnetic metal oxide particles (A), the emulsion is not stable, There is a tendency for the particle size distribution of the generated particles to be broadened.

As a catalyst for hydrolysis of (alkyl) alkoxysilane, Lewis acid or hydrochloric acid can be used. Specifically, inorganic acid such as hydrochloric acid, organic acid such as acetic acid, inorganic basic compound such as ammonia, ethanolamine, etc. An amine compound such as can be used.
The amount of the catalyst for hydrolysis is preferably 1 to 1000% by weight, particularly preferably 2 to 500% by weight, based on the weight of the (alkyl) alkoxysilane.

  The mixing method of the dispersion liquid (B1) and the solution (B2) is not particularly limited, and can be mixed at once using the equipment described below, but from the viewpoint of the uniformity of the particle diameter of the magnetic silica particles (C). A method of dropping the dispersion (B1) while stirring the solution (B2) is preferable.

  The equipment for mixing the dispersion (B1) and the solution (B2) is not particularly limited as long as it is generally commercially available as an emulsifier or a disperser. For example, a homogenizer (manufactured by IKA), Polytron Batch type emulsifiers such as Kinematica (manufactured by Kinematica Co., Ltd.) and TK auto homomixer (manufactured by Special Machine Industries Co., Ltd.), Ebara Milder (manufactured by Aihara Seisakusho Co., Ltd.), TK Philmix, TK Pipeline Homo Mixer (Special Machine Industries Co. ), Colloid mill (manufactured by Shinko Pantech Co., Ltd.), clear mix (manufactured by M Technique Co., Ltd.), slasher, trigonal wet pulverizer (manufactured by Mitsui Miike Chemical Co., Ltd.), Captron (manufactured by Eurotech) and fine flow mill ( Continuous emulsifiers such as Taiheiyo Kiko Co., Ltd., Microfluidizer (Mizuho Kogyo Co., Ltd.), Nanomizer (Nanomizer Co. High-pressure emulsifiers such as APV Gaurin (manufactured by Gaurin), membrane emulsifiers such as membrane emulsifiers (manufactured by Chilling Industries), vibratory emulsifiers such as Vibro mixers (manufactured by Chilling Industries), ultrasonic homogenizers (Branson) From the viewpoint of homogenization of the particle size, APV Gaurin, Homogenizer, TK Auto Homo Mixer, Ebara Milder, TK Fill Mix, TK Pipeline Homo Mixer and Clear Mix ( Mtechnic Co., Ltd.) is preferable.

  The temperature for the hydrolysis and condensation reaction of the (alkyl) alkoxysilane is preferably 10 to 100 ° C, more preferably 25 to 60 ° C. Moreover, reaction time becomes like this. Preferably it is 0.5 to 5 hours, More preferably, it is 1-2 hours.

(Process 2)
Examples of the method for forming the shell layer (Q) in the present invention include a core layer (P) obtained in (Step 1), a (alkyl) alkoxysilane, a catalyst for hydrolysis of (alkyl) alkoxysilane, and water. For example, a method of forming a shell layer (Q) containing silica on the surface of the core layer (P) by further mixing with a water-soluble organic solvent, carrying out hydrolysis reaction and condensation reaction of (alkyl) alkoxysilane. It is done.

Examples of the (alkyl) alkoxysilane used include compounds represented by the following general formula (1).
R ¹ _(4-n) Si (OR ² ) _n (1)
In General Formula (1), R ¹ and R ² represent a monovalent hydrocarbon group having 1 to 10 carbon atoms that may be substituted with an amino group, a carboxyl group, a hydroxyl group, a mercapto group, or a glycidyloxy group.

  Examples of the monovalent hydrocarbon group having 1 to 10 carbon atoms include monovalent aliphatic hydrocarbon groups having 1 to 10 carbon atoms (methyl group, ethyl group, n- or iso-propyl group, n- or iso-butyl group). Group, n- or iso-pentyl group and vinyl group), aromatic hydrocarbon group having 6 to 10 carbon atoms (phenyl group and the like), and aromatic aliphatic group having 7 to 10 carbon atoms (benzyl group and the like). It is done.

  N in General formula (1) represents the integer of 1-4. However, when using an alkylalkoxysilane with n = 1, it is necessary to use it together with an (alkyl) alkoxysilane with n = 2-4. From the viewpoint of the strength of the particles after the reaction and the amount of silanol groups on the particle surface, n is preferably 4.

Specific examples of the compound represented by the general formula (1) include alkoxysilanes such as tetramethoxysilane, tetraethoxysilane, tetraisopropylsilane and tetrabutoxysilane; alkylalkoxysilanes such as methyltrimethoxysilane and methyltriethoxysilane. An amino group such as 3-aminopropyltrimethoxysilane, 3-aminopropylethoxysilane, N-2 (aminoethyl) 3-aminopropyltrimethoxysilane and N-2 (aminoethyl) 3-aminopropyltriethoxysilane; Alkylalkoxysilanes having substituted alkyl groups; alkylalkoxysilanes having alkyl groups substituted with carboxyl groups such as 7-carboxy-heptyltriethoxysilane and 5-carboxy-pentyltriethoxysilane; 3 Alkyl alkoxysilanes having alkyl groups substituted with hydroxyl groups such as hydroxypropyltrimethoxysilane and 3-hydroxypropylethoxysilane; substituted with mercapto groups such as 3-mercaptopropyltrimethoxysilane and 3-mercaptopropyltriethoxysilane Examples include alkylalkoxysilanes having an alkyl group; alkylalkoxysilanes having an alkyl group substituted with a glycidyloxy group such as 3-glycidyloxypropyltrimethoxysilane and 3-glycidyloxypropyltriethoxysilane.
(Alkyl) alkoxysilane may be used alone or in combination of two or more.

The concentration of the core layer (P) is preferably less than 50% by weight in the solution, and more preferably less than 20% by weight. When the concentration of the core layer (P) is 50% by weight or more in the solution, the core layer (P) is not uniformly dispersed in the solution, and the shell layer (Q) is not uniformly formed. Particles in which the core layers (P) are aggregated tend to be generated.

  The concentration of the (alkyl) alkoxysilane is preferably less than 50% by weight in the solution, and more preferably less than 20% by weight. When the concentration of the (alkyl) alkoxysilane is 50% by weight or more in the solution, particles in which the core layers (P) are aggregated through silica are obtained, and particles composed only of silica, aggregates thereof, and the like And a large amount of aggregates composed of the core layer (P) are formed.

As a catalyst for hydrolysis of (alkyl) alkoxysilane, Lewis acid or hydrochloric acid can be used. Specifically, inorganic acid such as hydrochloric acid, organic acid such as acetic acid, inorganic basic compound such as ammonia, ethanolamine, etc. An amine compound such as can be used.
The amount of the hydrolysis catalyst used is preferably 1 to 2000% by weight, particularly 2 to 1000% by weight, based on the weight of the (alkyl) alkoxysilane.

The amount of water used is preferably 0.01% by weight or more, more preferably 0.1% by weight or more based on the weight of the (alkyl) alkoxysilane. When the amount of water used is less than 0.01% by weight based on the weight of the (alkyl) alkoxysilane, the reaction rate of hydrolysis of the (alkyl) alkoxysilane is slowed down, and the shell layer (Q ) Requires a long reaction time.

    A water-soluble organic solvent may or may not be used. When used, one type may be used alone or two or more types may be used in combination. As a water-soluble organic solvent, solubility in water at 25 ° C. is 100 g / 100 g or more of water, alcohol having 1 to 4 carbon atoms (such as methanol, ethanol and n- or iso-propanol), or having 2 to 9 carbon atoms. Glycol (ethylene glycol and diethylene glycol etc.), amide (N-methylpyrrolidone etc.), ketone (acetone etc.), cyclic ether (tetrahydrofuran and tetrahydropyran etc.), lactone (γ-butyrolactone etc.), sulfoxide (dimethyl sulfoxide etc.) and nitrile (Acetonitrile and the like). Among these, C1-C4 alcohol is preferable from a viewpoint of the uniformity of the particle diameter of a magnetic silica particle (C).

  In addition to the above, a surfactant or a dispersant can be used to improve the dispersibility of the core layer (P) during the reaction.

  The temperature of the hydrolysis reaction and condensation reaction of (alkyl) alkoxysilane is preferably 0 to 90 ° C., more preferably 15 to 50 ° C., and the reaction time is 1 to 5 hours, preferably 1 to 3 hours.

  Since the magnetic silica particles (C) in the present invention have silanol groups on the surface, many measurement target substances, similar substances to the measurement target substances, or measurement target substance binding substances can be immobilized on the surface.

Next, the measurement target substance measurement method of the present invention, which is a method of using the magnetic silica particles (C) in the present invention, will be described.
In the measurement target substance measurement method of the present invention, a measurement target substance, a similar substance of the measurement target substance, or a measurement target substance binding substance that specifically binds to the measurement target substance is formed on the surface of the magnetic silica particle (C) of the present invention. The measurement target substance in a sample is measured using the immobilized magnetic silica particles (D).

  The measurement target substance, a similar substance to the measurement target substance, or a measurement target substance binding substance (hereinafter, these may be collectively referred to as an immobilization substance) is fixed to the magnetic silica particles (C) in the present invention. Examples of the method for producing the magnetic silica particles (D) include a method of physically adsorbing the immobilizing substance on the magnetic silica particles (C) described above. From the viewpoint of more efficiently immobilizing the immobilizing substance, magnetic silica particles (at least one organic compound selected from the group consisting of glutaraldehyde, albumin, carbodiimide, streptavidin, biotin, and alkylalkoxysilane having a functional group are used. It is preferable that the substance for immobilization is immobilized on the magnetic silica particles (C) through binding to the surface of C). Of these organic compounds, an alkylalkoxysilane having a functional group is more preferable from the viewpoint of binding a specific immobilizing substance. Examples of the alkylalkoxysilane having such a functional group include vinyltrimethoxysilane, vinyltriethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3- Glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyl Trimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2- (aminoethyl) -3-aminopro Rumethyldimethoxysilane, N-2- (aminoethyl) -3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N- (1,3 -Dimethyl-butylidene) propylamine, N-phenyl-3-aminopropyltrimethoxysilane, tris- (trimethoxysilylpropyl) isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercapto Examples thereof include propyltrimethoxysilane and 3-isocyanatopropyltriethoxysilane.

  Examples of the functional group of the alkyl alkoxysilane include at least one functional group selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, a mercapto group, a glycidyloxy group, and a hydrocarbon group having 1 to 18 carbon atoms. In addition, one molecule of alkylalkoxysilane may have different types of functional groups.

Furthermore, as the functional group possessed by the alkylalkoxysilane, the above functional group may be further modified, for example, a functional group in which an amino group and succinic acid are bonded by an amide bond.

  As a method of bonding the alkylalkoxysilane having a functional group to the surface of the magnetic silica particle (C), the above-mentioned amino group, carboxyl group, hydroxyl group can be used as the (alkyl) alkoxysilane when the magnetic silica particle (C) is produced. , A method using an alkylalkoxysilane having an alkyl group substituted with a mercapto group glycidyloxy group or a hydrocarbon group having 1 to 18 carbon atoms, or using an (alkyl) alkoxysilane which does not have these substituents After producing the magnetic silica particles (C), the magnetic silica particles (C) have an alkyl group substituted with an amino group, carboxyl group, hydroxyl group, mercapto group glycidyloxy group or hydrocarbon group having 1 to 18 carbon atoms. Examples include a method of treating with alkylalkoxysilane.

  As a specific example of the latter method, magnetic silica particles (C) are dispersed in a solvent so that the concentration thereof is 0.1 to 50% by weight, and an amino group, a carboxyl group, a hydroxyl group, a mercapto group, Examples thereof include a method of adding a solution of an alkylalkoxysilane having a glycidyloxy group or an alkyl group substituted with a hydrocarbon group having 1 to 18 carbon atoms, and performing a hydrolysis reaction and a condensation reaction at room temperature.

  The solvent in this method is appropriately selected according to the solubility of the alkylalkoxysilane used, and when an alkylalkoxysilane having an alkyl group substituted with an amino group, carboxyl group, hydroxyl group or mercapto group soluble in water is used. It is preferable to use water, a mixed solvent of water-alcohol, or the like. When using an alkylalkoxysilane having an alkyl group substituted with a glycidyloxy group that is difficult to dissolve in water, it is preferable to use butyl acetate or the like.

  The amount of alkylalkoxysilane having an amino group, carboxyl group, hydroxyl group, mercapto group, glycidyloxy group or alkyl group substituted with a hydrocarbon group having 1 to 18 carbon atoms depends on the weight of the magnetic silica particles (C). On the other hand, it is preferably 0.01% by weight or more. If the proportion of this amount used is less than 0.01% by weight, the number of functional groups introduced onto the surface of the magnetic silica particles (C) may not be sufficient.

A method for binding glutaraldehyde, albumin, carbodiimide, streptavidin, or biotin to the surface of the magnetic silica particles (C) is not particularly limited, and for example, the binding can be performed as follows.
Glutaraldehyde having an aldehyde group and biotin having a carboxyl group are bonded to the surface of the magnetic silica particle (C) by reacting with an alkylalkoxysilane having an amino group bonded to the surface of the magnetic silica particle (C). Can do. In addition, albumin and streptavidin having an amino group and carbodiimide having a carbodiimide group are reacted with an alkylalkoxysilane having a carboxyl group bonded to the surface of the magnetic silica particle (C), whereby the surface of the magnetic silica particle (C). Can be combined.

  The method for measuring a substance to be measured of the present invention is usually performed in this field except that the magnetic silica particles (D) in the present invention are used. Document [for example, enzyme immunoassay second edition (edited by Eiji Ishikawa et al. , Medical School), 1982], and may be performed according to the sandwich method, the competition method, and the measurement method described in JP-A-6-130063.

First, the sandwich method will be described.
In the sandwich method, for example, the measurement target substance binding substance is immobilized on the surface of the magnetic silica particle (D), and the sample, the magnetic silica particle (D), and the labeling substance are labeled. A labeled measurement target substance binding substance that is a measurement target substance binding substance is mixed, and the immobilized measurement target substance binding substance, the measurement target substance in the sample, and the labeled measurement target substance binding substance are brought into contact with each other. And forming a labeled complex which is a complex of the immobilized measurement target substance binding substance, the measurement target substance in the sample, and the labeled measurement target substance binding substance, and carrying the label complex The magnetic silica particles (D) are subjected to B / F separation, the amount of the labeling substance in the labeling complex is measured, and the measurement target in the sample is based on the amount of the labeling substance in the labeling complex Measure the amount of a substance It is done by.

Specifically, for example, first, the measurement target substance in the sample and the measurement target substance binding substance immobilized on the surface of the magnetic silica particle (D) are brought into contact with each other to be immobilized on the surface of the magnetic silica particle (D). A complex of the measurement target substance binding substance and the measurement target substance in the sample is formed.
Next, the measurement target substance binding substance immobilized on the magnetic silica particles (D), the measurement target substance in the sample, and the label measurement target substance binding substance are further contacted with the complex and the target substance binding substance. To form a complex (labeled complex).
The labeled complex is subjected to B / F separation, the amount of the labeling substance in the labeling complex is measured, and the amount of the substance to be measured in the sample is measured based on the amount of the labeling substance in the labeling complex. .
In this method, the measurement target substance in the sample is reacted with the immobilized measurement target binding substance, and then the labeled measurement target substance binding substance is reacted. However, the labeled measurement target substance binding substance and the sample are reacted. The measurement target substance binding substance immobilized after reacting with the measurement target substance therein may be reacted, or these three may be reacted simultaneously.

B / F separation (Bond / Free separation) in the sandwich method means separation of a substance supported on the magnetic silica particles (D) and other substances.
That is, it means separation of the labeled complex from the labeled measurement substance-binding substance that was not involved in the formation of the labeled complex. Specifically, the measurement target binding substance immobilized on the magnetic silica particles (D), the complex of the measurement target binding substance immobilized on the magnetic silica particles (D) and the measurement target substance in the sample, and the above-mentioned This means separation of the labeled complex from other components (components other than the measurement target substance in the sample, labeled measurement target substance-binding substances that were not involved in the formation of the labeled complex, etc.).
In addition, the B / F separation step is an essential step after the formation of the labeled complex, and as the implementation timing, the measurement target substance binding substance and the measurement target substance immobilized on the surface of the magnetic silica particles (D) are used. It is also possible to carry out after forming the complex.

Next, the competition method will be described.
The competition method is, for example, a method in which a measurement target substance binding substance that specifically binds to a measurement target substance competes with the measurement target substance and the same substance as the measurement target substance, or is similar to the measurement target substance and the same substance as the measurement target substance. A method for measuring the amount of a substance to be measured in a sample by using a reaction for competing substances, and a reaction for competing two kinds of substance to be measured for binding to a measurement target substance, This refers to a method for measuring the amount of a substance to be measured.

  As an example of the competition method, a measurement target substance or a similar substance is immobilized on the surface of the magnetic silica particle (D), and the sample is labeled with the sample, the magnetic silica particle (D), and a labeling substance. The labeled measurement target substance binding substance, which is a measured measurement target substance binding substance, is mixed, and the measurement target substance in the sample competes with the immobilized measurement target substance or a similar substance to thereby measure the labeled measurement target. A label complex which is a complex containing the immobilized measurement target substance or a similar substance and the labeled measurement target substance binding substance is formed in contact with the substance binding substance, and the label complex is supported. The magnetic silica particles (D) are subjected to B / F separation, the amount of the labeling substance in the labeling complex is measured, and the measurement target in the sample is based on the amount of the labeling substance in the labeling complex Measure the amount of a substance It is made by Rukoto.

Specifically, for example, first, the surface of the magnetic silica particles (D) is prepared by competing the measurement target substance in the sample with the immobilized measurement target substance or a similar substance and bringing it into contact with the labeled measurement target substance binding substance. A labeled complex containing the measurement target substance or its similar substance immobilized on the label and the labeled measurement target substance binding substance is formed.
Next, the magnetic silica particles (D) carrying the labeling complex are subjected to B / F separation, the amount of the labeling substance in the labeling complex is measured, and the sample is determined based on the amount of the labeling substance in the labeling complex. What is necessary is just to measure the quantity of a to-be-measured substance.
In this method, a measurement target substance in a sample, a labeled measurement target substance binding substance, and an immobilized measurement target substance or a similar substance are simultaneously competitively reacted. Alternatively, after mixing the immobilized measurement target substance or a similar substance, a labeled measurement target substance binding substance may be added to cause a competitive reaction. Further, after bringing the measurement target substance in the sample into contact with the labeled measurement target substance-binding substance, the immobilized measurement target substance or a similar substance is added to the magnetic silica particles (D) to cause a competitive reaction. Also good.

  The B / F separation in the competitive method means separation of a substance supported on the magnetic silica particles (D) and other substances. That is, it means separation of the labeled complex, the labeled measurement substance binding substance that was not involved in the formation of the labeled complex, and the complex of the labeled measurement substance binding substance and the measurement target substance. Specifically, magnetic silica particles (D) on which a measurement target substance or a similar substance is immobilized, and magnetic silica particles (D) on which a measurement target substance or a similar substance is immobilized and a labeled measurement target substance binding substance This means separation of the complex from other components (components other than the measurement target substance in the sample, labeled measurement target substance binding substance, complex of the measurement target substance, etc.).

  As another aspect of the competitive method, the substance to be measured-binding substance is immobilized on the surface of the magnetic silica particles (D), and the sample, the magnetic silica particles (D), the label A measurement target substance labeled with a substance or a similar substance is mixed with a labeled measurement target substance or a similar substance, and the measurement target substance in the sample competes with the labeled measurement target substance or a similar substance. Contacting the immobilized measurement target substance binding substance to form a labeled complex that is a complex containing the immobilized measurement target substance binding substance and the labeled measurement target substance or a similar substance, The magnetic silica particles (D) carrying the labeling complex are subjected to B / F separation, the amount of the labeling substance in the labeling complex is measured, and based on the amount of the labeling substance in the labeling complex Measurement in the sample Done by measuring the target substance.

Specifically, for example, first, a measurement target substance binding substance immobilized by contacting a measurement target substance in a sample, a labeled measurement target substance or a similar substance, and an immobilized measurement target substance binding substance. In addition, the measurement target substance in the sample and the labeled measurement target substance or a similar substance are subjected to a competitive reaction, and the measurement target substance binding substance and the labeled measurement target substance or the similar substance immobilized on the surface of the magnetic silica particles (D) And a labeled complex.
Next, the magnetic silica particles (D) carrying the labeling complex are subjected to B / F separation, the amount of the labeling substance in the labeling complex is measured, and the sample is determined based on the amount of the labeling substance in the labeling complex. What is necessary is just to measure the substance to be measured.
In the competition method, a measurement target substance in a sample, a labeled measurement target substance or a similar substance, and an immobilized measurement target substance binding substance are simultaneously subjected to a competitive reaction. After contacting the immobilized measurement target substance binding substance, a labeled measurement target substance or a similar substance may be added to cause a competitive reaction. Further, after mixing the measurement target substance in the sample with the labeled measurement target substance or a similar substance, the magnetic silica particles (D) on which the measurement target substance binding substance is immobilized may be added to cause a competitive reaction.

The B / F separation in the competitive method means separation of a substance supported on the magnetic silica particles (D) and other substances. That is, it means separation of the labeled complex from the label measurement target substance that was not involved in the formation of the labeled complex or a similar substance. Specifically, the magnetic silica particles (D) on which the measurement target substance binding substance is immobilized, the composite of the magnetic silica particles (D) on which the measurement target substance binding substance is immobilized and the measurement target substance in the sample, and measurement Complex of magnetic silica particle (D) with immobilized target substance binding substance and labeled measurement target substance or similar substance, and other components (participating in the formation of other components (components other than the measurement target substance in the sample, labeled complex) It means the separation from the labeling measurement target substance or its similar substance that did not exist.
The B / F separation step is an indispensable step after the formation of the labeled complex, but the timing of its implementation is that after the measurement target substance in the sample is brought into contact with the immobilized measurement target substance binding substance. May also be implemented.

When the measurement target substance is an enzyme, the amount of the enzyme as the measurement target substance may be measured by a method using an enzyme activity method other than the sandwich method or the competition method.
For example, a sample containing a measurement target substance is brought into contact with a measurement target substance binding substance (for example, a substance capable of binding to an enzyme such as an antibody) immobilized on the surface of the magnetic silica particle (D), thereby bringing the magnetic silica particle into contact. (D) A complex of an enzyme and an immobilized substance to be measured is formed on the surface, B / F separation of the magnetic silica particles (D) supporting the complex is performed, and the type of enzyme is determined. Substrate or color-developing (light) agent according to the substrate or the type of enzyme, and if necessary, a conjugated enzyme is added, and the enzyme in the sample based on the change of the substrate or the color-developing (light) result of the color developing (light) agent It can measure by the method of measuring the quantity of. For the substrate, the coloring (light) agent and the conjugated enzyme, known ones may be used. For example, when the enzyme to be measured is peroxidase, hydrogen peroxide as the substrate, luminol luminescence reagent as the luminescent agent, etc. May be used. These amounts may be used within the range usually used in this field. B / F separation in the above method refers to a complex of a measurement target substance in a sample and a magnetic silica particle (D) on which the measurement target substance binding substance is immobilized, and other components (other than the measurement target substance in the sample). Means separation from components, etc.).

  In the measurement target substance measurement method of the present invention, the measurement target substance in the sample, the immobilization substance immobilized on the surface of the magnetic silica particles (D), the labeled measurement target substance binding substance, the labeled measurement target substance, or the same As a method of bringing a similar substance or the like into contact, it may be brought into contact by a usual process such as stirring and mixing. The reaction time may be appropriately set according to the difference between the measurement target substance, the measurement target substance binding substance used, the sandwich method, the competition method, etc., but is usually 1 to 10 minutes, preferably 1 to 5 minutes.

  The B / F separation in the method for measuring a substance to be measured of the present invention uses, for example, the magnetic properties of the magnetic silica particles (D), collects the magnetic silica particles (D) with a magnet or the like from the outside of the reaction tank, and the like. The magnet is removed, the magnetic silica particles (D) are mixed and dispersed, and washed. The above operation may be repeated 1 to 3 times. The cleaning liquid is not particularly limited as long as it is usually used in this field.

Examples of labeling substances used for labeling a substance to be measured binding substance, a substance to be measured, or a similar substance include alkaline phosphatase, β-galactosidase, peroxidase, and microperoxidase used in enzyme immunoassay (EIA). , Glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, acid phosphatase and other enzymes such as ^99m Tc, ¹³¹ I used in radioimmunoassay (RIA), Radioisotopes such as ¹²⁵ I, ¹⁴ C, ³ H, ³² P, such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or derivatives thereof used in fluorescent immunoassay (FIA), green fluorescent protein ( GFP ) Such as luciferin, isoluminol, luminol, luminescent materials such as bis (2,4,6-trifluorophenyl) oxalate, such as phenol, naphthol, anthracene, or derivatives thereof. For example, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di Spin labels typified by compounds having an oxyl group such as -t-butyl-α- (3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene) -p-trioxyl Examples include substances having properties as agents.
Among these, from the viewpoint of sensitivity and the like, enzymes and fluorescent substances are preferable, alkaline phosphatase, peroxidase and glucose oxidase are more preferable, and peroxidase is particularly preferable.

  In order to bind a labeling substance as described above to a substance to be measured, a substance to be measured, a substance to be measured, or a similar substance, it is generally performed by a method used in this field, for example, a known EIA, RIA or FIA. Known labeling methods [for example, Medical Chemistry Laboratory, Vol. 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawaio, 1st edition, Soft Science Co., Ltd. 1983; Enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, medical school, 1982, etc.] may be used.

The amount of labeling substance used varies depending on the type of labeling substance to be used, so it cannot be said unconditionally. For example, when peroxidase is used as the labeling substance, the substance to be measured and the labeling substance are usually combined with, for example, 1: 1. It is preferable that the molar ratio is ˜20, preferably 1: 1 to 10, more preferably 1: 1 to 2. In addition, the analyte binding substance labeled with peroxidase is contained in the buffers usually used in this field such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, etc. It can be used. Examples of the buffer solution include Tris buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution, Good buffer solution and the like which are usually used in this field.
Further, when an antigen or antibody is used as the substance to be measured binding substance, the pH of the buffer solution may be in a range that does not suppress the antigen-antibody reaction, and is usually 5 to 9. Further, in such a buffer solution, if it does not inhibit the target antigen-antibody reaction, for example, a stabilizer such as albumin, globulin, water-soluble gelatin, polyethylene glycol, surfactant, saccharide, etc. You may keep it. In addition, in this specification, pH means the numerical value (measuring temperature 25 degreeC) measured based on JISK0400-12-10: 2000.

  Examples of methods for measuring the labeling substance or its activity include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), and chemiluminescence immunoassay (CLIA and CLEIA). From the viewpoint of sensitivity in immunoassay over time, EIA, CLIA and CLEIA are preferable, and CLEIA is more preferable.

  The measurement method of the present invention may be performed as follows, for example, when measuring PSA by the sandwich method.

That is, for example, 10-25 μL of a sample containing PSA, and a phosphate buffer containing 0.1-10 mg / mL of magnetic silica particles (D) in which an anti-PSA polyclonal antibody is immobilized on the magnetic silica particles (C) of the present invention, etc. 40 to 50 μL of a buffer solution (reagent containing magnetic silica particles (D)) is added to the reaction vessel, the reaction solution is stirred, and the magnetic silica particles (D) are dispersed to obtain magnetic silica particles (D). Of anti-PSA polyclonal antibody immobilized on the surface of PSA and PSA in the sample are brought into contact with and reacted to form a composite of anti-PSA polyclonal antibody immobilized on the surface of magnetic silica particles (D) and PSA in the sample Form the body. Next, for example, the magnetic silica particles (D) are collected from the outside of the reaction tank with a magnet or the like, the reaction solution is discharged, and a cleaning solution such as physiological saline is added. Thereafter, the magnet is removed, and the magnetic silica particles (D) are dispersed and washed. This operation may be repeated 1 to 10 times. In this washing operation, an anti-PSA polyclonal antibody (hereinafter abbreviated as labeling reagent) labeled with horseradish-derived peroxidase (hereinafter abbreviated as POD) or the like was left with the sample or the reagent containing magnetic silica particles (D) remaining. When adding and reacting, this washing operation may be omitted. Thereafter, for example, a labeling reagent is added, the reaction solution is stirred, the magnetic silica particles (D) are dispersed, and the POD-labeled anti-PSA polyclonal antibody and the complex are reacted and bonded. Next, washing is performed by adding and dispersing a washing solution such as physiological saline in the same manner as the washing described above. Finally, for example, luminol and hydrogen peroxide are added, and the amount of accumulated chemiluminescence for 1 second is measured with a chemiluminescence meter, and the amount of PSA in the sample is calculated based on the measured value.
In this case, the amount of PSA in the sample can be easily calculated by using a calibration curve indicating the relationship between the amount of PSA and the accumulated amount of chemiluminescence for 1 second. The calibration curve can be prepared in advance by using the prescribed PSA-containing solution as a sample and performing the same operation as described above.

The reagent of the present invention comprises the magnetic silica particles (D) in the present invention, and is used in the measurement method. Specifically, for example, a buffer solution containing the magnetic silica particles (D) in the present invention can be mentioned. As the buffer solution, a buffer solution usually used for immunoassay or the like is preferably used. Examples include piperazine diethanesulfonic acid / sodium hydroxide buffer, MOPS [3- (N-morpholino) propanesulfonic acid] / sodium hydroxide buffer, triethanolamine / hydrochloric acid buffer, and PBS (phosphate buffer). It is done.
The concentration of the buffering agent in these buffers is preferably 1 to 500 mM, more preferably 5 to 300 mM, and particularly preferably 10 to 200 mM.
Above and below, mM represents the concentration (mmol / liter) at 25 ° C.
The amount of the magnetic silica particles (D) is not particularly limited, and can be appropriately selected depending on the type of the measurement target substance binding substance or the similar substance to the measurement target substance used, the type of the measurement target substance, and the like.
The reagent of the present invention is preferably in a form in which the magnetic silica particles (D) of the present invention are dispersed in a buffer solution.

  The reagent of the present invention is a reagent containing a measurement target substance binding substance labeled with a labeling substance, or a measurement target substance or a similar substance labeled with a labeling substance in addition to the magnetic silica particles (D) of the present invention. (Hereinafter may be abbreviated as a labeling reagent). Examples of the labeling substance are the same as those described in the section of the measurement method of the present invention, and preferable ones are also the same.

  The labeling reagent can contain a buffer solution in addition to the measurement target substance binding substance labeled with the labeling substance, the measurement target substance labeled with the labeling substance, or a similar substance. As the buffer solution contained in the labeling reagent, a buffer solution usually used for immunoassay is preferable, and for example, the same buffer solution used for the reagent containing the above-mentioned magnetic silica particles (D) can be mentioned. The concentration of the buffer in the liquid is preferably the same as that used in the reagent containing the magnetic silica particles (D).

  The reagent of the present invention may contain a chemiluminescent reagent in addition to the reagent containing the magnetic silica particles (D) and the labeling reagent of the present invention. The chemiluminescent reagent is selected based on the above-described labeling substance. For example, when the labeling substance is POD, a 2,3-dihydro-1,4-phthalazinedione compound and a chemiluminescence enhancer are essential components. It is preferable that the chemiluminescent reagent 1st liquid and the chemiluminescent reagent 2nd liquid which have an oxidizing agent and water as an essential component are included.

Examples of the 2,3-dihydro-1,4-phthalazinedione compound include those disclosed in JP-A-2-291299, JP-A-10-319015, JP-A-2000-279196, and the like. 3-dihydro-1,4-phthalazinedione compounds and mixtures thereof can be used.
Of these, luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof (alkali metal salts, etc.) are preferable. Further preferred is luminol and its metal salt, particularly preferred is the sodium salt of luminol.

  The content of the 2,3-dihydro-1,4-phthalazinedione compound in the chemiluminescent reagent is appropriately set depending on the type and the applied measuring method, measuring conditions, etc., but the chemiluminescent enhancing effect, storage stability, etc. In view of the above, 0.5 to 80 mM is preferable, more preferably 1.8 to 40 mM, and particularly preferably 3.5 to 21 mM.

Examples of the chemiluminescence enhancer include known chemiluminescence enhancers described in JP-A-59-500262, JP-A-59-171839 and JP-A-2-291299, and mixtures thereof. Can be used.
Among these, from the viewpoint of the chemiluminescence enhancing effect, etc., phenol is preferable, P-iodophenol, 4- (cyanomethylthio) phenol and 4-cyanomethylthio-2-chlorophenol are particularly preferable, and 4- (Cyanomethylthio) phenol.

  The content of the chemiluminescent enhancer in the chemiluminescent reagent is appropriately set depending on the type and applied measurement method, measurement conditions, etc., but from the viewpoint of the chemiluminescence enhancing effect and storage stability, 0.1 to 15 mM is preferable. More preferably, it is 0.3-7.0 mM, Most preferably, it is 0.6-3.4 mM.

  In addition to the 2,3-dihydro-1,4-phthalazinedione compound and the chemiluminescence enhancer, the first chemiluminescent reagent solution may contain a buffer solution and / or a chelating agent.

As the buffer solution, for example, known buffer solutions described in JP-A-10-31915 and JP-A-2003-279489 can be used.
Among these, from the viewpoint of chemiluminescence enhancing effect and storage stability, 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer, 2-hydroxy-3- [ 4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid monohydrate / sodium hydroxide buffer and piperazinyl-1,4-bis (2-hydroxy-3-propanesulfonic acid) dihydrate Product / sodium hydroxide buffer, more preferably 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer and 2-hydroxy-3- [4- ( 2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid monohydrate / sodium hydroxide buffer, particularly preferred is 3- [4- (2- Dorokishiechiru) -1-piperazinyl] propane sulfonic acid / sodium hydroxide buffer.
The content of the buffering agent in these buffers is preferably 1 to 500 mM, more preferably 5 to 300 mM, and particularly preferably 10 to 200 mM, from the viewpoints of chemiluminescence enhancing effect and storage stability.

As the chelating agent, for example, known chelating agents described in JP-A-9-75099 and JP-A-2003-279489 can be used.
Of these, 4-coordinate chelating agents are preferred from the viewpoint of chemiluminescence enhancing effect and storage stability, and more preferred are ethylenediaminetetraacetic acid (EDTA) and salts thereof (ethylenediaminetetraacetic acid disodium, ethylenediaminetetraacetic acid trisodium. , Ethylenediaminetetraacetic acid tetrasodium, ethylenediaminetetraacetic acid dipotassium and ethylenediaminetetraacetic acid tripotassium, etc.) and trans-1,2 diaminocyclohexane-N, N, N ′, N′-tetraacetic acid (CyDTA), particularly preferably ethylenediamine Tetraacetic acid (EDTA) and its salts.

  When the chelating agent is contained, the content is 0.001 to 4% by weight based on the weight of the 2,3-dihydro-1,4-phthalazinedione compound from the viewpoint of chemiluminescence enhancing effect and storage stability. Is preferable, more preferably 0.01 to 2% by weight, and particularly preferably 0.05 to 1% by weight.

  The first chemiluminescent reagent liquid is preferably a liquid, and is preferably alkaline from the viewpoint of the fluorescence intensity of the enzyme. The pH of the first liquid is preferably 7 to 11, more preferably 8 to 10.

  The first chemiluminescent reagent liquid can be easily obtained by uniformly mixing a 2,3-dihydro-1,4-phthalazinedione compound, a chemiluminescence enhancer and, if necessary, a buffer and / or a chelating agent.

Examples of the oxidizing agent contained in the second liquid of the chemiluminescent reagent include known oxidizing agents described in JP-A Nos. 8-261194 and 2000-279196, etc. [Inorganic peroxide (hydrogen peroxide) , Sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxide, acyl peroxide, etc.), peroxo acid compounds (peroxosulfuric acid, peroxophosphoric acid, etc.), etc.].
Among these, from the viewpoint of storage stability and the like, a hydrogen peroxide aqueous solution, a sodium perborate aqueous solution and a potassium perborate aqueous solution are preferable, and a hydrogen peroxide aqueous solution is more preferable.

  The concentration of the oxidizing agent in the second liquid of the chemiluminescent reagent is appropriately set depending on the type and applied measurement method, measurement conditions, etc., but from the viewpoint of the chemiluminescence enhancing effect, etc., 0.5 to 40 mM is preferable, and more preferable. Is 1-20 mM, particularly preferably 2.5-10 mM.

  Examples of the water contained in the second liquid of the chemiluminescent reagent include distilled water, reverse osmosis water, and deionized water. Of these, distilled water and deionized water are preferable from the viewpoint of chemiluminescence enhancing effect and storage stability, and more preferable is deionized water.

The chemiluminescent reagent second liquid can contain a chelating agent in addition to the oxidizing agent and water.
As a chelating agent, the thing similar to what was illustrated as a chelating agent which can be contained in the above-mentioned 1st liquid is mentioned, A preferable thing is also the same.
In the case of containing a chelating agent, the content is preferably 0.2 to 100% by weight, more preferably 0.5 to 20%, based on the weight of the oxidizing agent, from the viewpoint of chemiluminescence enhancing effect and storage stability. % By weight, particularly preferably 1 to 10% by weight.
The second chemiluminescent reagent liquid can be easily obtained by uniformly mixing an oxidizing agent, water and, if necessary, a chelating agent.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these. Hereinafter, unless otherwise specified, “%” means “% by weight” and “part” means “part by weight”.

Example 1-1
Preparation of superparamagnetic metal oxide particles (A):
In a reaction vessel, 186 parts of iron (III) chloride hexahydrate, 68 parts of iron (II) chloride tetrahydrate and 1288 parts of water are charged and dissolved, and the temperature is raised to 50 ° C. While maintaining the temperature, 280 parts of 25% ammonia water was added dropwise over 1 hour to obtain magnetite particles in water. 64 parts of oleic acid as a dispersant was added to the obtained magnetite particles, and stirring was continued for 2 hours. After cooling to room temperature, magnetite particles adsorbed with oleic acid obtained by solid-liquid separation by decantation were washed three times with 1000 parts of water, and further washed twice with 1000 parts of acetone. Superparamagnetic metal oxide particles (A-1) were obtained by drying at 0 ° C. for 2 days.

Preparation of core layer (P):
80 parts of superparamagnetic metal oxide particles (A-1) were added to 240 parts of tetraethoxysilane and dispersed to prepare a dispersion (B1). Next, 5050 parts of water, 3500 parts of 25% ammonia aqueous solution, 400 parts of polyoxyethylene (added mole number 20 moles) alkyl ether (product name “Emalmine 200”, manufactured by Sanyo Chemical Industries, Ltd.) are added to the reaction vessel to clear it. The mixture (M technique company make) was mixed and the solution (B2) was obtained. After raising the temperature to 50 ° C., the dispersion (B1) was added dropwise to the solution (B2) over 1 hour while stirring the clear mix at a rotation speed of 6,000 rpm, and then reacted at 50 ° C. for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant containing fine particles, thereby obtaining a core layer (P-1).

Production of magnetic silica particles (C):
(1) Formation of shell layer (Q) Clear mix by adding 80 parts of core layer (P-1), 2500 parts of deionized water, 260 parts of 25% aqueous ammonia solution, 2500 parts of ethanol, and 1200 parts of tetraethoxysilane to the reaction vessel. (Mtechnic Co., Ltd.) was mixed, and the mixture was reacted for 2 hours while stirring at 6,000 rpm of the clear mix.
(2) Classification (i) After the reaction, centrifugation was performed at 2,000 rpm for 20 minutes to remove the supernatant containing fine particles, and the operation of collecting the particles using a magnet and removing the supernatant was performed 10 times.
(Ii) Next, 5000 parts of water was added to the obtained solid phase to disperse the particles, and after centrifugation at 600 rpm for 10 minutes, the operation of removing the supernatant containing fine particles was performed 20 times.
(Iii) Subsequently, 5000 parts of water was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 300 rpm for 10 minutes to settle and remove particles having a large particle size, thereby performing classification.
(3) Purification Further, the operation of collecting the particles using a magnet and removing the supernatant was performed 10 times to obtain the magnetic silica particles (C-1) according to Example 1-1 having the target volume average particle diameter. It was.

Production of magnetic silica particles (D):
5 mg of magnetic silica particles (C-1) after classification are added to a polyethylene bottle with a lid containing 40 mL of an aqueous solution containing 1% by weight of 3-aminopropyltriethoxysilane, reacted at 25 ° C. for 1 hour, and the particles are collected by a magnet. Thereafter, the liquid was removed by suction with an aspirator. Next, 40 mL of deionized water was added to disperse the magnetic silica particles (C-1). After collecting the particles with a magnet, the liquid was attracted and removed with an aspirator to wash the magnetic silica particles (C-1). This washing operation was performed 4 times. Next, the washed magnetic silica particles (C-1) were added to a polyethylene bottle with a lid containing 10 mL of an ethanol solution containing 0.5 wt% succinic anhydride, and reacted at 25 ° C. for 2 hours. Then, after collecting the particles with a magnet, the liquid was sucked and removed with an aspirator to wash the magnetic silica particles (C-1). This washing operation was performed three times. Subsequently, the washed magnetic silica particles (C-1) were mixed with 0.5 wt% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.5 wt% N-hydroxysuccinimide. The solution was added to a polyethylene bottle with a lid containing 40 mL of an aqueous solution containing (NHS) and reacted at 25 ° C. for 1 hour. Then, after collecting the particles with a magnet, the liquid was sucked and removed with an aspirator to wash the magnetic silica particles (C-1). This washing operation was performed three times. Furthermore, the magnetic silica particles (C-1) after the washing were added to 40 mL of 100 mM morpholinoethanesulfonic acid buffer (pH 5.0) containing an anti-PSA polyclonal antibody (manufactured by Dako Cytomation) at a concentration of 20 μg / mL. Was added to a polyethylene bottle with a lid and reacted at 25 ° C. for 3 hours. By this operation, the anti-PSA polyclonal antibody can be immobilized on the surface of the magnetic silica particles (C-1). After the reaction, the particles were collected with a magnet, and the liquid was sucked and removed with an aspirator to wash the magnetic silica particles (C-1). This washing operation was performed three times to obtain antibody-immobilized magnetic silica particles (D-1). This was immersed in 40 mL of 0.02 M phosphate buffer (pH 7.2) containing 0.1% bovine serum albumin and stored at 4 ° C.

Example 1-2
In (2) classification (ii) and (iii) of the production of magnetic silica particles (C), 5000 parts of water are added to disperse the particles, and after centrifugation at 2500 rpm for 10 minutes, the supernatant containing fine particles is removed. Then, 5000 parts of water is added to the obtained solid phase to disperse the particles, and the mixture is centrifuged at 900 rpm for 10 minutes to settle and remove particles having a large particle size. Magnetic silica particles (C-2) according to Example 1-2 were obtained. Other operations than using magnetic silica particles (C-2) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-2) Got.

Example 1-3
In (2) classification (ii) and (iii) of the production of magnetic silica particles (C), 5000 parts of water is added to disperse the particles, and after centrifugation at 200 rpm for 10 minutes, the supernatant containing fine particles is removed. Then, 5000 parts of water is added to the obtained solid phase to disperse the particles, and the mixture is centrifuged at 100 rpm for 10 minutes to settle and remove particles having a large particle size. Magnetic silica particles (C-3) according to Example 1-3 were obtained. Other operations than using magnetic silica particles (C-3) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-3) Got.

Example 1-4
In (2) classification (ii) and (iii) of the production of magnetic silica particles (C), 5000 parts of water is added to disperse the particles, and after centrifugation at 300 rpm for 1 minute, the supernatant containing fine particles is removed. Then, 5000 parts of water is added to the obtained solid phase to disperse the particles and centrifuged at 200 rpm for 1 minute to classify particles by settling and removing large particles. And the magnetic silica particle (C-4) which concerns on Example 1-4 was obtained. Other operations than using magnetic silica particles (C-4) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-4) Got.

Example 1-5
In (2) classification (ii) and (iii) of production of magnetic silica particles (C), 5000 parts of water is added to disperse the particles, and after centrifugation at 300 rpm for 30 seconds, the supernatant containing fine particles is removed. Then, 5000 parts of water is added to the obtained solid phase to disperse the particles and centrifuged at 200 rpm for 30 seconds to settle and remove particles having a large particle size. Magnetic silica particles (C-5) according to Example 1-5 were obtained. Other operations than using magnetic silica particles (C-5) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-5) Got.

Example 1-6
Superparamagnetic metal oxide particles (A) were prepared in the same manner as in Example 1-1 except that 5000 parts of 1% ammonia water was used instead of 280 parts of 25% ammonia water. Oxide particles (A-2) were obtained. Production of the core layer (P) was performed in the same manner as in Example 1-1 except that the superparamagnetic metal oxide particles (A-2) were used to obtain the core layer (P-2). The production of the magnetic silica particles (C) was performed in the same manner as in Example 1-1 except that the core layer (P-2) was used to obtain the magnetic silica particles (C-6) according to Example 1-6. . Other operations than using magnetic silica particles (C-6) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-6) Got.

Example 1-7
In preparation of a core layer (P), except having used 360 parts of tetraethoxysilane, operation similar to Example 1-1 was performed and the core layer (P-3) was obtained. The production of the magnetic silica particles (C) was performed in the same manner as in Example 1-1 except that the core layer (P-3) was used, and magnetic silica particles (C-7) according to Example 1-7 were obtained. . Other operations than using magnetic silica particles (C-7) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-7) Got.

Example 1-8
In preparation of a core layer (P), except having used 160 parts of tetraethoxysilane, operation similar to Example 1-1 was performed and the core layer (P-4) was obtained. The production of the magnetic silica particles (C) was performed in the same manner as in Example 1-1 except that the core layer (P-4) was used, and magnetic silica particles (C-8) according to Example 1-8 were obtained. . Other operations than using magnetic silica particles (C-8) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-8). Got.

Example 1-9
In the production of the magnetic silica particles (C), the same operation as in Example 1-1 was performed except that 30 parts of a 25% aqueous ammonia solution was used, and the magnetic silica particles (C-9) according to Example 1-9 were obtained. Obtained. Other operations than using magnetic silica particles (C-9) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-9) Got.

Example 1-10
In the production of the magnetic silica particles (C), the same operation as in Example 1-1 was performed except that 15 parts of tetraethoxysilane was used to obtain the magnetic silica particles (C-10) according to Example 1-10. It was. Other operations than using magnetic silica particles (C-10) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-10) Got.

Example 1-11
In the production of the magnetic silica particles (C), the same operation as in Example 1-1 was performed except that 5800 parts of deionized water, 5800 parts of ethanol, and 2500 parts of tetraethoxysilane were used. Magnetic silica particles (C-11) were obtained. Other operations than using magnetic silica particles (C-11) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-11) Got.

Example 1-12
In the production of the magnetic silica particles (C), the same operation as in Example 1-1 was performed except that 58000 parts of deionized water, 58000 parts of ethanol, and 25,000 parts of tetraethoxysilane were used, and Example 1-12 was applied. Magnetic silica particles (C-12) were obtained. Other operations than using magnetic silica particles (C-12) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-12) Got.

Example 1-13
(1) In the formation of the magnetic silica particles (C), in the formation of the shell layer (Q), 58000 parts of deionized water, 58000 parts of ethanol, and 25,000 parts of tetraethoxysilane were used. The solution after the reaction was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant in which fine particles were present, and the operation of collecting the particles using a magnet and removing the supernatant was performed 10 times, and then 5000 parts of water was added. After dispersing the particles and centrifuging at 300 rpm for 1 minute, the operation of removing the supernatant containing the fine particles was performed 20 times. Subsequently, 5000 parts of water was added to the obtained solid phase to disperse the particles, and 1 at 200 rpm. The magnetic silica particles (C-13) according to Example 1-13 were obtained in the same manner as in Example 1-1 except that the particles having a large particle size were settled and removed by centrifuging for 1 minute. Other operations than using magnetic silica particles (C-13) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-13) Got.

Example 1-14
(1) In the formation of the magnetic silica particles (C), in the formation of the shell layer (Q), 30 parts of 25% ammonia aqueous solution was used, and (2) in the classification, the solution after the reaction was 2,000 rpm. Centrifuge for 20 minutes to remove the supernatant containing fine particles, collect the particles using a magnet and remove the supernatant 10 times, and then add 5000 parts of water to disperse the particles. After centrifugation for 20 minutes, the operation of removing the supernatant containing fine particles was performed 20 times. Subsequently, 5000 parts of water was added to the obtained solid phase to disperse the particles, followed by centrifugation at 900 rpm for 10 minutes to obtain large particles. Magnetic silica particles (C-14) according to Example 1-14 were obtained in the same manner as in Example 1-1 except that the particles having a diameter were settled and removed. Other operations than using magnetic silica particles (C-14) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-14). Got.

Example 1-15
(1) In the formation of the magnetic silica particles (C), in the formation of the shell layer (Q), 30 parts of 25% ammonia aqueous solution was used, and (2) in the classification, the solution after the reaction was 2,000 rpm. Centrifuge for 20 minutes to remove the supernatant containing fine particles, collect the particles using a magnet and remove the supernatant 10 times, and then add 5000 parts of water to disperse the particles. After centrifugation for 20 minutes, the operation of removing the supernatant containing fine particles was performed 20 times. Subsequently, 5000 parts of water was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 200 rpm for 1 minute. Magnetic silica particles (C-15) according to Example 1-15 were obtained in the same manner as in Example 1-1 except that the particles having a diameter were settled and removed. Other operations than using magnetic silica particles (C-15) instead of magnetic silica particles (C-1) were performed in the same manner as in Example 1-1, and antibody-immobilized magnetic silica particles (D-15) Got.

Example 1-16
The production of magnetic silica particles (D) was carried out except that 20 μg / mL of anti-AFP monoclonal antibody (manufactured by Dako Cytomation) was used instead of anti-PSA polyclonal antibody (manufactured by Dako Cymation) In the same manner as in Example 1-1, antibody-immobilized magnetic silica particles (D-16) were obtained.

Comparative Example 1-1
Instead of producing the magnetic silica particles (C) in Example 1-1, 5000 parts of water was added to the core layer (P-1) to disperse the particles, and after centrifugation at 600 rpm for 10 minutes, fine particles were present. The operation of removing the liquid was carried out 20 times, and then 5000 parts of water was added to the obtained solid phase to disperse the particles, followed by centrifugation at 300 rpm for 10 minutes to precipitate and remove particles having a large particle size. Furthermore, the operation of collecting particles using a magnet and removing the supernatant was performed 10 times to obtain magnetic silica particles (I-1) according to Comparative Example 1-1 after classification. In preparing the antibody-supporting magnetic silica particles (D), the same operation as in Example 1-1 was performed except that 5 mg of the magnetic silica particles (I-1) were used instead of the magnetic silica particles (C-1). Antibody-immobilized magnetic silica particles (H-1) were obtained.

Comparative Example 1-2
In the classification of the core layer (P-1) in Comparative Example 1-1, 5000 parts of water was added to disperse the particles, and after centrifugation at 300 rpm for 1 minute, the operation of removing the supernatant containing fine particles was performed 20 times, Subsequently, 5000 parts of water was added to the obtained solid phase to disperse the particles, followed by centrifugation at 200 rpm for 1 minute to precipitate and remove particles having a large particle size. Furthermore, the operation of collecting the particles using a magnet and removing the supernatant was performed 10 times to obtain magnetic silica particles (I-2) after classification according to Comparative Example 1-2. In the production of the magnetic silica particles (D), the same procedure as in Comparative Example 1-1 was performed, except that 5 mg of the magnetic silica particles (I-2) was used instead of the magnetic silica particles (I-1). Magnetic silica particles (H-2) were obtained.

Comparative Example 1-3
Compared with the production of magnetic silica particles (D), 20 μg / mL of anti-AFP monoclonal antibody (manufactured by DACO CITATION Co., Ltd.) was used in place of anti-PSA polyclonal antibody (manufactured by DACO Cymation Co., Ltd.). In the same manner as in Example 1-1, antibody-immobilized magnetic silica particles (H-3) were obtained.

<Method for measuring volume average particle diameter of superparamagnetic metal oxide particles (A)>
Using a scanning electron microscope (model number JSM-7000F, manufacturer name JEOL Ltd.), any 200 superparamagnetic metal oxide particles [in the examples, solid-liquid separation of underwater magnetite particles by decantation, Obtained after washing with water and drying. ] Was measured and the particle diameter was measured to determine the volume average particle diameter.

<Method for measuring volume average particle diameter of magnetic silica particles (C)>
Using a scanning electron microscope (model number JSM-7000F, manufacturer name JEOL Ltd.), arbitrary 200 magnetic silica particles (C-1) to (C-15) and magnetic silica particles (I-1 ) And (I-2) were observed to measure the particle diameter, and the volume average particle diameter was determined. The results are shown in Table 1.

<Method for Measuring Content of Superparamagnetic Metal Oxide Particles (A) in Core Layer (P)>
With respect to the magnetic silica particles (C-1) to (C-15) and any 20 core layers (P) of the magnetic silica particles (I-1) and (I-2), the above scanning electron microscope is used. The content of superparamagnetic metal oxide particles (A) was measured by an energy dispersive X-ray spectrometer (model number INCA Wave / Energy, manufacturer: Oxford), and the average value was defined as content S. In addition, the content of silica was measured by the same measurement, and the average value was defined as content T. The content of the superparamagnetic metal oxide particles (A) was determined by the following calculation formula (1). The results are shown in Table 1.
Content (%) of superparamagnetic metal oxide particles (A) = (S) / (S + T) (1)

<Measurement method of average film thickness in shell layer (Q)>
The cross section obtained by embedding the magnetic silica particles (C-1) to (C-15) and the magnetic silica particles (I-1) and (I-2) in an epoxy resin and cutting with a microtome is shown in FIG. [Model number “H-7100”, manufactured by Hitachi, Ltd.]), and the film thickness was determined from the average value of the thickest part and the thinnest part of each magnetic silica particle. The film thickness was determined in the same manner as described above for each of 100 arbitrary magnetic silica particles, and the average value was taken as the average film thickness. The results are shown in Table 1.

  Volume average particle diameter of magnetic silica particles (C), volume average particle diameter of superparamagnetic metal oxide particles (A), content of superparamagnetic metal oxide particles (A) in core layer (P), shell layer (Q Table 1 shows the results of measuring the average film thickness. From Table 1, the silica layer having a sufficient thickness is formed on the surface of the magnetic silica particles (C-1) to (C-15) by producing the shell layer (Q). On the other hand, in the magnetic silica particles (I-1 and I-2) in which the shell layer (Q) is not prepared, a film derived from the silica component at the time of preparing the core layer (P) is formed. The thickness is less than 3 nm.

Preparation of labeling reagent:
Anti-PSA polyclonal antibody (manufactured by Dako Cytomation), horseradish-derived peroxidase (manufactured by Toyobo, hereinafter abbreviated as POD), and literature (S Yoshitake, M Imagawa, Yi Ishikawa, Etol; POD-labeled antibody PSA polyclonal antibody was prepared by the method described in Biochem, Vol. In the same manner as described above, a POD-labeled anti-AFP monoclonal antibody was prepared using an anti-AFP polyclonal antibody (manufactured by DAKO CITATION Co., Ltd.). These were diluted with a 0.02M phosphate buffer solution (pH 7.0) containing 1% bovine serum albumin to a concentration of 200 nM as a POD-labeled antibody concentration, a labeling reagent was prepared, and refrigerated (2-10 ° C.). saved.

Preparation of chemiluminescent reagent first solution:
A 1,000 mL volumetric flask was charged with 0.7 g of a sodium salt of luminol [manufactured by Sigma Aldrich Japan Co., Ltd.] and 0.1 g of 4- (cyanomethylthio) phenol. 3- [4- (2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer (10 mM, pH 8.6) was charged so that the volume of the solution was 1,000 mL, and even at 25 ° C. A first chemiluminescent reagent solution was prepared by mixing. It was stored refrigerated (2-10 ° C.) until used for measurement.

Preparation of chemiluminescent reagent second solution:
1,000 mL and 6.6 g of hydrogen peroxide [manufactured by Wako Pure Chemical Industries, Ltd., reagent grade, concentration 30 wt%] were charged into a 1,000 mL volumetric flask. Deionized water was charged so that the volume of the solution was 1,000 mL, and the mixture was uniformly mixed at 25 ° C. to prepare a second solution of the chemiluminescent reagent. It was stored refrigerated (2-10 ° C.) until used for measurement.

Example 2-1
Immunoassay:
After collecting the particles in the above-mentioned bovine serum albumin-containing phosphate buffer containing 25 μg of antibody-immobilized magnetic silica particles (D-1) with a magnet, the bovine serum albumin-containing phosphate buffer is removed with an aspirator, After washing the particles by adding 0.5 mL of water and collecting the particles with an aspirator, the antibody-immobilized magnetic silica particles (D-1) and 1% by weight of bovine serum were removed. 0.025 mL of standard PSA antigen solution with 0.1 ng / mL antigen concentration prepared with phosphate buffer containing albumin and buffer for immune reaction [0.1% bovine serum albumin, 0.85% sodium chloride And 0.02 mL of 0.02 M phosphate buffer (pH 7.2) containing 0.5% polyoxyethylene sorbitan monolaurate (surfactant) in a test tube. In reacted for 3 minutes to form an anti-PSA polyclonal antibody immobilized magnetic silica particles (D-1) / PSA complex. After the reaction, collect the particles from the outside of the test tube with a magnet for 10 seconds, remove the liquid in the test tube with an aspirator, add 0.5 mL of physiological saline to disperse the particles, collect the magnetic flux, and then remove the liquid with an aspirator The operation was performed 3 times.

Subsequently, 0.050 mL of an immune reaction buffer containing 4 μg / mL of POD-labeled anti-PSA polyclonal antibody was injected into the test tube, reacted at 37 ° C. for 3 minutes in the test tube, and anti-PSA polyclonal antibody-immobilized silica particles ( D-1) A / PSA / POD-labeled anti-PSA polyclonal antibody complex was formed. After the reaction, the complex containing antibody-immobilized magnetic silica particles (D-1) is collected with a magnet from the outside of the test tube for 10 seconds, the solution in the test tube is removed with an aspirator, and 0.5 mL of physiological saline is added to the particles. After the magnetic flux was dispersed and the magnetic flux was collected, the washing operation for removing the liquid with an aspirator was performed twice.
Finally, 0.07 mL of a luminescent reagent [0.1 M Tris / HCl buffer (pH 9.0) containing 0.18 g / L luminol and 0.1 g / L 4- (cyanomethylthio) phenol] and 0 0.07 mL of 0.01% hydrogen peroxide solution was added at the same time, and a luminescence reaction was carried out at 37 ° C. for 43 seconds. After adding a luminescent reagent, an average luminescence amount of 43 to 45 seconds was measured with a luminescence detector [BLR-201 (Aloka) : Using a photomultiplier tube]. In addition, instead of the standard PSA solution having a PSA concentration of 0.1 ng / mL, a standard PSA solution having a PSA concentration of 0 ng / mL was used to perform the same operation as above and used as a background.

Examples 2-2 to 2-15
This was performed in the same manner as in Example 2-1, except that the antibody-immobilized magnetic silica particles (D-1) were replaced with antibody-immobilized magnetic silica particles (D-2) to (D-15).

Example 2-16
Standard AFP using magnetic silica particles (D-16) instead of magnetic silica particles (D-1) and having an antigen concentration of 2.0 ng / mL instead of a standard PSA antigen solution having an antigen concentration of 0.1 ng / mL Anti-AFP monoclonal antibody-immobilized magnetic silica particles (D-16) / AFP complex was formed using the antigen solution. Anti-AFP monoclonal antibody-immobilized magnetic silica particles (D-16) / AFP / POD-labeled anti-AFP monoclonal antibody complex was formed using POD-labeled anti-AFP monoclonal antibody instead of POD-labeled anti-PSA polyclonal antibody. Furthermore, a standard AFP solution having an AFP concentration of 0 ng / mL was used as a background instead of a standard PSA solution having a PSA concentration of 0 ng / mL. Others were the same as in Example 2-1.

Comparative Examples 2-1 to 2-2
The same procedure as in Example 2-1 was performed except that the antibody-immobilized magnetic silica particles (D-1) were replaced with antibody-immobilized magnetic silica particles (H-1) and (H-2).

Comparative Example 2-3
The same procedure as in Example 2-16 was carried out except that the antibody-immobilized magnetic silica particles (D-16) were replaced with antibody-immobilized magnetic silica particles (H-3).

<Calculation method of luminescence ratio>
For Examples 2-1 to 2-15 and Comparative Examples 2-1 to 2-2, the average luminescence amount when using a standard PSA solution with a PSA concentration of 0.1 ng / mL is M, and the PSA concentration is 0 ng / mL. The average light emission amount when using the standard PSA solution was N, and the light emission amount ratio was determined by the following calculation formula (2). For Example 2-16 and Comparative Example 2-3, the average amount of luminescence when a standard AFP solution having an AFP concentration of 2.0 ng / mL was used was M, and a standard AFP solution having an AFP concentration of 0 ng / mL was used. In this case, the average light emission amount was N, and the light emission amount ratio was determined by the following calculation formula (2). The results are shown in Table 2.
Light emission ratio = M / N (2)

<Calculation method of storage stability>
About Example 2-1 to 2-15 and Comparative Examples 2-1 to 2-2, it was immersed in 40 mL of 0.02M phosphate buffer (pH 7.2) containing 0.1% bovine serum albumin at 4 ° C. Using the antibody-immobilized magnetic silica particles (D-1) to (D-15) and the antibody-immobilized magnetic silica particles (H-1) and (H-2) stored for 2 weeks, the PSA concentration was 0.1 ng / Antibody-immobilized magnetic silica particles (D-1) to (D-15) and antibody-immobilized magnetic silica particles (H-), which were stored for 2 weeks at X, 31 ° C. When 1) and (H-2) are used, the average luminescence amount when using a standard PSA solution with a PSA concentration of 0.1 ng / mL is Y, and storage stability is obtained by the following calculation formula (3). Asked.
For Example 2-16 and Comparative Example 2-3, antibody immobilization was immersed in 40 mL of 0.02 M phosphate buffer (pH 7.2) containing 0.1% bovine serum albumin and stored at 4 ° C. for 2 weeks. Using the magnetic silica particles (D-16) and the antibody-immobilized magnetic silica particles (H-3), the average amount of luminescence when using a standard AFP solution having an AFP concentration of 2.0 ng / mL at V and 31 ° C. When using a standard AFP solution having an AFP concentration of 2.0 ng / mL when using antibody-immobilized magnetic silica particles (D-16) and antibody-immobilized magnetic silica particles (H-3) stored for 2 weeks Storage stability was calculated | required by the following formula (4) by making average light-emission quantity into W. The results are shown in Table 2.
Storage stability (%) = Y / X × 100 (3)
Storage stability (%) = W / V × 100 (4)

Table 2 shows the results of evaluating the sensitivity and storage stability in the immunoassay using the obtained reagents. From the results of Table 2, antibody-immobilized magnetic silica particles (D-1) to (D-15) obtained by immobilizing anti-PSA polyclonal antibodies on each magnetic silica particle (C) having a shell layer (Q) are sufficient. Compared to antibody-immobilized magnetic silica particles (H-1) and (H-2) in which an anti-PSA polyclonal antibody is immobilized on a core layer (P) having no thickness shell layer (Q), PSA It can be seen that the sensitivity and stability in measurement are excellent (Examples 2-1 to 2-15 and Comparative Examples 2-1 to 2-2). Further, the antibody-immobilized magnetic silica particles (D-16) obtained by immobilizing the anti-AFP monoclonal antibody on the magnetic silica particles (C-1) having the shell layer (Q) have a sufficiently thick shell layer (Q). Compared to the antibody-immobilized magnetic silica particles (H-3) in which the anti-AFP monoclonal antibody is immobilized on the core layer (P) that is not included, it is understood that the sensitivity and stability in the measurement of AFP are superior ( Example 2-16 and Comparative Example 2-3).

  Since the measurement target substance measurement method using the magnetic silica particles of the present invention is excellent in sensitivity and stability, the measurement target substance can be measured with high sensitivity and good reproducibility. Therefore, radioimmunoassay, enzyme immunoassay It can be widely applied to clinical tests such as immunoassay, fluorescence immunoassay and chemiluminescence immunoassay. In addition, the reagent of the present invention is suitable for use in the above-described measurement method, and similarly, as a clinical test agent such as radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, chemiluminescence immunoassay and the like. Can be used.

Claims (13)

A core layer (P) which is a silica particle containing 60 to 95% by weight of superparamagnetic metal oxide particles (A) having an average particle diameter of 1 to 15 nm;
Magnetic silica particles (C) which are core-shell type particles composed of a shell layer (Q) which is a silica layer having an average thickness of 3 to 3000 nm formed on the surface of the core layer (P). The magnetic silica particles (C) according to claim 1, wherein the magnetic silica particles (C) have an average particle size of 0.5 to 20 µm. The magnetic silica particle (C) according to claim 1 or 2, wherein the superparamagnetic metal oxide contained in the superparamagnetic metal oxide particle (A) is iron oxide. 4. The iron oxide according to claim 3, wherein the iron oxide is at least one iron oxide selected from the group consisting of magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide and γ-hematite-α-hematite intermediate iron oxide. Magnetic silica particles (C). A measurement target substance, a similar substance of the measurement target substance, or a measurement target substance binding substance that specifically binds to the measurement target substance on the surface of the magnetic silica particle (C) according to any one of claims 1 to 4. A method for measuring a substance to be measured, comprising measuring the substance to be measured in a sample using the immobilized magnetic silica particles (D). The measurement object binding substance is immobilized on the surface of the magnetic silica particles (D),
Mixing the sample, the magnetic silica particles (D), and a labeled measurement target substance binding substance that is a measurement target substance binding substance labeled with a labeling substance;
The immobilized measurement target substance binding substance, the measurement target substance in the sample, and the labeled measurement target substance binding substance are brought into contact with each other, and the immobilized measurement target substance binding substance and the sample Forming a labeled complex that is a complex of the measurement target substance and the labeled measurement target substance binding substance;
B / F separation of the magnetic silica particles (D) supporting the labeling complex, and measuring the amount of the labeling substance in the labeling complex,
The method for measuring a substance to be measured according to claim 5, wherein the amount of the substance to be measured in the sample is measured based on the amount of the labeling substance in the label complex. On the surface of the magnetic silica particles (D), a substance to be measured or a similar substance is immobilized,
Mixing the sample, the magnetic silica particles (D), and a labeled measurement target substance binding substance that is a measurement target substance binding substance labeled with a labeling substance;
The measurement target substance in the sample and the immobilized measurement target substance or a similar substance are caused to compete with each other and contacted with the labeled measurement target substance binding substance, so that the immobilized measurement target substance or the similar substance is obtained. And a labeled complex that is a complex containing the label measurement target substance binding substance,
B / F separation of the magnetic silica particles (D) supporting the labeling complex, and measuring the amount of the labeling substance in the labeling complex,
The method for measuring a substance to be measured according to claim 5, wherein the amount of the substance to be measured in the sample is measured based on the amount of the labeling substance in the label complex. The measurement target substance binding substance is immobilized on the surface of the magnetic silica particles (D),
Mixing the sample, the magnetic silica particles (D), and a measurement target substance or a similar substance labeled with a labeling substance;
The measurement target substance in the sample and the labeled measurement target substance or a similar substance are caused to compete with each other to contact the immobilized measurement target substance binding substance, and the immobilized measurement target substance binding substance and the Forming a labeled complex, which is a complex containing the labeling target substance or a similar substance,
B / F separation of the magnetic silica particles (D) supporting the labeling complex, and measuring the amount of the labeling substance in the labeling complex,
The measurement target substance measurement method according to claim 5, wherein the measurement target substance in the sample is measured based on the amount of the labeling substance in the label complex. The method for measuring a substance to be measured according to any one of claims 5 to 8, wherein the B / F separation is performed using magnetism of the magnetic silica particles (D). Glutaraldehyde, albumin, carbodiimide, streptavidin, biotin and the like on the surface of the magnetic silica particle (C) before the measurement target substance, the similar substance of the measurement target substance, or the measurement target substance binding substance is immobilized The method for measuring a substance to be measured according to any one of claims 5 to 9, wherein at least one organic compound selected from the group consisting of an alkylalkoxysilane having a functional group is bonded. The functional group possessed by the alkylalkoxysilane is at least one functional group selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, a mercapto group, a glycidyloxy group, and a hydrocarbon group having 1 to 18 carbon atoms. The method for measuring a substance to be measured according to claim 10. The measurement target substance binding substance is an antibody against the measurement target substance or a similar substance, an antigen to which the measurement target substance or a similar substance binds, or a protein that binds to the measurement target substance or a similar substance. Item 12. The method for measuring a substance to be measured according to any one of Items 5 to 11. A measurement target substance, a similar substance of the measurement target substance, or a measurement target substance binding substance that specifically binds to the measurement target substance on the surface of the magnetic silica particle (C) according to any one of claims 1 to 4. A reagent for measuring a substance to be measured, comprising immobilized magnetic silica particles (D).