JP2018141779A - Immunity measuring method and kit for immunity measurement used therein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunity measuring method and a kit for immunity measurement having high sensitivity and excellent reproducibility achieved by effectively reducing the nonspecific reaction of a specimen-derived component or a labeling substance to a sold-phase carrier surface, a substance immobilized to the sold-phase carrier, and the inner wall of a reaction vessel, and further improving the dispersibility of magnetic particles.SOLUTION: Provided is an immunity measuring method for performing an immune reaction using magnetic particles (B) as solid-phase carriers in the presence of alkyl glycoside (A) whose hydrophilic part is monosaccharide (a). It is preferable that the alkyl glycoside (A) be an alkyl glycoside having a 6-36C alkyl group as a hydrophilic part.SELECTED DRAWING: None

Description

  The present invention relates to an immunoassay method and an immunoassay kit used therefor.

  In immunoassay, when measuring a serum sample, a nonspecific reaction of a component derived from the sample to a reaction container, a solid phase carrier surface and a substance immobilized on the solid phase carrier surface is observed in a specific sample, These non-specific reactions hinder accurate measurement and are problematic (for example, Non-Patent Document 1). In order to prevent non-specific reactions, immunoassay reagents containing various proteins, surfactants, salts, and the like have been proposed (for example, Patent Documents 1 to 3).

  In immunoassay using magnetic particles, when particles are collected by a magnetic force with an antibody or antigen bound to the surface of the magnetic particles, the particles may aggregate and become difficult to disperse again. As a result, the measurement values are likely to vary due to the influence of the presence of aggregated particles and the like, and the reproducibility may be deteriorated, and there is a problem that the methods described in Patent Documents 1 to 3 cannot be sufficiently handled. In the development of an automated immunoassay device for the purpose of simultaneous rapid testing of a large number of samples, the development of an immunoassay reagent with good reproducibility has become an increasingly important issue.

Japan Journal of Clinical Laboratory Automation, Vol. 36, no. 2, P208-213, 2011

JP 2004-117341 A JP 2010-1227827 A JP 2010-216970 A

  The object of the present invention is to effectively reduce the non-specific reaction of the component immobilized on the surface of the solid-phase carrier, the substance immobilized on the solid-phase carrier, and the analyte or the labeled substance on the inner wall of the reaction vessel, and further the dispersibility of the magnetic particles. It is an object to provide an immunoassay method and an immunoassay kit that are highly sensitive and have excellent reproducibility.

  The inventors of the present invention have arrived at the present invention as a result of intensive studies to achieve the above object. That is, the present invention relates to an immunoassay method in which an immunoreaction is performed using magnetic particles (B) as a solid phase carrier in the presence of an alkyl glycoside (A) whose hydrophilic part is a monosaccharide (a); a solid phase carrier reagent A kit for immunoassay comprising (E) and a labeling reagent (F), wherein the solid phase carrier reagent (E) contains magnetic particles (B) having the antigen (C) or antibody (D) immobilized on the surface. The solid phase carrier reagent (E) contains a substance (F1) that specifically binds to a measurement target substance labeled with a labeling substance (F1) or a measurement target substance (F2) labeled with a labeling substance. Or at least any one of a labeling reagent (F) is an immunoassay kit used for the said immunoassay method containing the alkyl glycoside (A) whose hydrophilic part is monosaccharide (a).

  By using the immunoassay method of the present invention, the measurement target substance can be measured with high sensitivity and high reproducibility.

<Immunoassay method>
The immunoassay method of the present invention is characterized in that an immune reaction is carried out using magnetic particles (B) as a solid phase carrier in the presence of an alkyl glycoside (A) whose hydrophilic part is a monosaccharide (a). This is an immunoassay method.
By performing an immune reaction in the presence of an alkyl glycoside (A) whose hydrophilic part is a monosaccharide (a), measurement with high sensitivity and excellent reproducibility becomes possible.

The alkyl glycoside (A) in the present invention is a glycoside bond of a monosaccharide (a) and an alkyl alcohol or alkyl thiol.
Examples of the monosaccharide (a) include triose, tetrose, pentose, hexose and heptose, and hexose is preferable from the viewpoint of reproducibility.
Examples of hexose include ketohexose (such as fructose), aldohexose (such as glucose, mannose, and galactose), and deoxy sugar (such as fucose, fucose, and rhamnose).
Among these, from the viewpoint of reproducibility, hexose is preferable, aldohexose is more preferable, and glucose is particularly preferable.

As the alkyl alcohol, an alcohol having an alkyl group having 6 to 36 carbon atoms [alcohol having a linear alkyl group (hexanol, heptanol, octanol, nonanol, decanol, undecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol, penta Those having a saturated linear alkyl group such as decyl alcohol, tetradecyl alcohol, hexadecyl alcohol, octadecyl alcohol, nonadecyl alcohol and behenyl alcohol, octenyl alcohol, decenyl alcohol, dodecenyl alcohol, tridecenyl alcohol, Pentadecenyl alcohol, oleyl alcohol, gadrelyl alcohol, linoleyl alcohol and other unsaturated linear alkyl groups) and branched alkyls Alcohols (isostearyl alcohol) or the like having a group.
Among these, from the viewpoints of solubility and detergency, alcohols having a linear alkyl group having 6 to 36 carbon atoms are preferable, and alcohols having a linear alkyl group having 6 to 18 carbon atoms are more preferable.

As alkyl thiol, thiol having an alkyl group having 6 to 36 carbon atoms [thiol having linear alkyl group {hexyl thiol, octyl thiol, nonyl thiol, decyl thiol (decane thiol), undecyl thiol, dodecyl thiol (dodecane thiol) ), Those having saturated linear alkyl groups such as tetradecylthiol (tetradecanethiol), hexadecanethiol and octadecylthiol, and unsaturated linear alkyl groups such as 7-octene-1-thiol and 9-decen-1-thiol Thiol having a branched alkyl group (t-dodecanethiol etc.)] and the like.
Among these, from the viewpoints of solubility and detergency, a thiol having a linear alkyl group having 6 to 36 carbon atoms is preferable, and a thiol having a linear alkyl group having 6 to 18 carbon atoms is more preferable.

  Specific examples of the alkyl glycoside (A) include those having an O-glycoside bond (having an alkyl group having 6 to 36 carbon atoms, specifically, n-hexyl β-D-glucopyranoside, n-heptyl β- D-glucopyranoside, n-octyl β-D-glucopyranoside, n-nonyl β-D-glucopyranoside, n-decyl β-D-glucopyranoside, n-dodecyl β-D-glucopyranoside, n-tetradecyl β-D-glucopyranoside, n -Hexadecyl β-D-glucopyranoside and n-octadecyl β-D-glucopyranoside and the like) and those having an S-glycoside bond (having an alkyl group having 6 to 36 carbon atoms, specifically, n-hexyl β-D -Thioglycosides, n-heptyl β-D-thioglycosides, n-octyl β-D-thioglycosides n-nonyl β-D-thioglycoside, n-decyl β-D-thioglycoside, n-dodecyl β-D-thioglycoside, n-tetradecyl β-D-thioglycoside, n-hexadecyl β-D-thioglycoside and n-octadecyl β-D-thioglycoside) and the like.

  (A) is preferably an alkylglycoside having a C6-C36 alkyl group as a hydrophobic part from the viewpoint of sensitivity and reproducibility, and more preferably an alkylglycoside having a C6-C18 linear alkyl group. Particularly preferred is an alkyl glycoside having a linear alkyl group having 6 to 18 carbon atoms and having an O-glycoside bond. Alkyl glycoside (A) may be used individually by 1 type, or may use 2 or more types together.

In the immunoassay method of the present invention, a solid phase carrier reagent (E) and a labeling reagent (F) are used.
The magnetic particle (B) as a solid phase carrier in the present invention is used for the solid phase carrier reagent (E) as a magnetic particle (B1) having an antigen (C) or antibody (D) immobilized on its surface.

  The magnetic particles (B) are not particularly limited as long as they are generally used in the field of immunoassay, but from the viewpoint of shortening the measurement time in immunoassay, JP-A-2014-210680 and JP-A Magnetic silica particles described in JP2013-019889A are preferred.

  When the magnetic particles (B) are used as the solid phase carrier, the magnetic collection and dispersion by the magnet are repeated. However, when the dispersibility is poor, the measured values are likely to vary due to the presence of aggregated particles and the reproducibility. Decreases. On the other hand, by using the alkyl glycoside (A) at the time of immunoassay, the dispersibility of the magnetic particles becomes good and the reproducibility can be improved.

As the magnetic silica particles, those in which a metal oxide having a volume average particle diameter of 1 to 20 nm and superparamagnetism is dispersed in a silica matrix from the viewpoint of shortening measurement time in immunoassay. Superparamagnetism refers to a temporary magnetic field that is induced by the individual atomic magnetic moments of a material being aligned in the presence of an external magnetic field, and that when the external magnetic field is removed, partial alignment is impaired and no magnetic field is exhibited. Say.
In addition, the volume average particle diameter of the metal oxide and magnetic silica particles in the present invention was measured by observing an arbitrary 200 particles with a scanning electron microscope (“JSM-7000F” manufactured by JEOL Ltd.). The average value of diameters.

  Examples of superparamagnetic metal oxides having a volume average particle diameter of 1 to 20 nm and exhibiting superparamagnetism include oxides such as iron, cobalt, nickel, and alloys thereof. Iron oxide is particularly preferred. A superparamagnetic metal oxide may be used individually by 1 type, or may use 2 or more types together.

The lower limit of the content of superparamagnetic metal oxide in the magnetic silica particles is preferably 60% by weight, more preferably 65% by weight, and the upper limit is preferably 95% by weight, more preferably 80% by weight.
When the content of the superparamagnetic metal oxide is 60% by weight or more, the obtained magnetic silica particles are sufficiently magnetized, so that it does not take time for the separation operation in actual use. Those having a content of 95% by weight or less are easy to synthesize.
In addition, when the content of the superparamagnetic metal oxide in the magnetic silica particles is in the above range, the magnetism is strong, and thus the measurement value variation due to repeated magnetism collection and dispersion by the magnet is more likely to occur. Even in such a case, by using the alkyl glycoside (A) at the time of immunoassay, the dispersibility of the magnetic silica particles becomes good and the reproducibility can be improved.

  The antigen (C) in the present invention is not particularly limited as long as it is generally used in the field of immunoassay. Specifically, the nucleotide chain (oligonucleotide chain, polynucleotide chain); chromosome; peptide Chain (eg C-peptide, angiotensin I etc.), protein [eg procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D ( IgD), β2-microglobulin, albumin, degradation products thereof, serum proteins such as ferritin]; enzymes [eg amylase (eg pancreatic type, salivary gland type, X type etc.), alkaline phosphatase (eg hepatic, osseous, placental) Small intestine, etc.), acid phosphatase (eg PAP), γ-glutamyl Lancephalase (eg, renal, pancreatic, hepatic, etc.), lipase (eg, pancreatic type, stomach type, etc.), creatine kinase (eg, CK-1, CK-2, mCK, etc.), lactate dehydrogenase (eg, LDH1 to LDH5) Etc.), glutamate oxaloacetate transaminases (eg ASTm, ASTs etc.), glutamate pyruvate transaminases (eg ALTm, ALTs etc.), cholinesterases (eg ChEl to ChE5 etc.), leucine aminopeptidases (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.), receptors (eg, receptors for estrogen, TSH, etc.); (For example, estrogen, TSH, etc.); for example, bacteria (for example, tuberculosis, pneumococci, diphtheria, meningococcus, bacilli, staphylococci, streptococci, enterobacteria, Escherichia coli, Helicobacter pylori, etc.), viruses (for example, le Vera 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.), Microorganisms such as spirochetes (eg leptospira, syphilis treponema), chlamydia, mycoplasma, etc .; proteins or peptides derived from the microorganisms or sugar chain antigens; causes of allergies such as bronchial asthma, allergic rhinitis, atopic dermatitis Various allergens (e.g. house dust, mites such as cypress mite, mushroom mite, pollen such as cedar, cypress, sparrow mite, ragweed, prickly frog, hurghaya, rye, e.g. animals such as cats, dogs, crabs, e.g. rice, egg white, etc. Allergens derived from food, fungi, insects, wood, drugs, chemicals, etc.); lipids (eg, lipoproteins, etc.); proteases (eg, trypsin, plasmin, serine proteases, etc.); tumor marker protein antigens (eg, PSA, PGI, etc.) , PGII, etc.); sugar chain antigens (eg, AFP (eg, L1 to L3, etc.), hCG (hCG family), transferrin, IgG, thyroglobulin, decay-accelerating-factor (DAF), carcinoembryonic antigens (eg, CEA, N CA, NCA-2, NFA, etc.), CA19-9, PIVKA-II, CA125, prostate specific antigen, tumor marker sugar chain antigen having a special sugar chain produced by cancer cells, ABO sugar chain antigen, etc.]; (For example, hyaluronic acid, β-glucan, sugar chain possessed by the above-mentioned sugar chain antigen, etc.); protein that binds to sugar chain (for example, hyaluronic acid binding protein, β-glucan binding protein, etc.); phospholipid (for example, cardiolipin, etc.); Polysaccharides (for example, endotoxin); Chemical substances (for example, T3, T4, for example, tributyltin, nonylphenol, 4-octylphenol, di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate, etc. Environmental hormones); various drugs administered and inoculated to the human body and their Metabolites; aptamers; nucleic acid binding substances and the like.

  The antibody (D) in the present invention is not particularly limited as long as it is used in the field of general immunoassay, and specifically includes antibodies to those exemplified as the antigen (A). The antibodies used in the present invention include proteolytic enzymes such as papain and pepsin, and degradation products such as Fab and F (ab ') 2 fragments generated by chemical degradation.

In the present invention, as a method of immobilizing the antigen (C) or antibody (D) on the magnetic particle (B) to obtain the magnetic particle (B1), the antigen (C) or antibody (D) is added to the magnetic particle (B). From the viewpoint of immobilizing the antigen (C) or antibody (D) more efficiently, but consisting of glutaraldehyde, albumin, carbodiimide, streptavidin, biotin and an alkylalkoxysilane having a functional group. It is preferable that at least one organic compound selected from the group is bound to the surface of the magnetic particle (B), and the antigen (C) or the antibody (D) is immobilized on the magnetic particle (B) through them, More preferable is immobilization via an alkylalkoxysilane having a functional group.
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.

  In addition to the magnetic particles (B) as the solid phase carrier, the solid phase carrier reagent (E) is an aqueous solvent [water, physiological saline, various buffer solutions (phosphate buffer, borate buffer, glycine buffer, and Tris buffer). Liquid, etc.), albumin solution, dextran solution, normal human or animal serum solution and synthetic polymer solution, and a mixed solution of two or more thereof.

  The content of the magnetic particles (B) in the solid phase carrier reagent (E) is preferably 0.0001 to 10% by weight based on the weight of the solid phase carrier reagent (E), from the viewpoint of detergency of the particles. More preferably, it is 0.001-1 weight%.

  The labeling reagent (F) contains a substance (F1) that specifically binds to a measurement target substance labeled with a labeling substance or a measurement target substance (F2) labeled with a labeling substance.

The substance that specifically binds to the measurement target substance labeled with the labeling substance (F1) and the labeling substance in the measurement target substance labeled with the labeling substance (F2) is a substance or measurement target that specifically binds to the measurement target substance. Used for labeling substances such as alkaline phosphatase, β-galactosidase, peroxidase (hereinafter sometimes abbreviated as POD), microperoxidase, glucose oxidase, glucose used in enzyme immunoassay (EIA) -6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, acid phosphatase and other enzymes such as 99mTc, 131I, 125I, 14C, 3H, and 32P used in radioimmunoassay (RIA) Radioisotopes, such as fluorescence Fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or derivatives thereof used in the measurement method (FIA), fluorescent substances such as green fluorescent protein (GFP), such as luciferin, isoluminol, luminol, bis (2, 4, Luminescent substances such as 6-trifluorophenyl) oxalate, such as substances having absorption in the ultraviolet region such as phenol, naphthol, anthracene or derivatives thereof, such as 4-amino-2,2,6,6-tetramethylpiperidine- 1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α- (3,5-di-t-butyl-4-oxo Compound having an oxyl group such as -2,5-cyclohexadiene-1-ylidene) -p-trioxyl Substances having properties as spin labeling agents represented the like.
Among these, from the viewpoint of sensitivity and the like, enzymes and fluorescent substances are preferable, alkaline phosphatase, POD and glucose oxidase are more preferable, and POD is particularly preferable.

  In order to bind a labeling substance to a substance that specifically binds to a substance to be measured or a substance to be measured, it is generally performed by a method generally used in the field of immunoassay, for example, 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 Muroi, Second Edition, Medical School, 1982, etc.] may be used.

Although the amount of labeling substance used varies depending on the type of labeling substance used, it cannot be said unconditionally. For example, when POD is used as the labeling substance, the substance that specifically binds to the substance to be measured or the substance to be measured and the label For example, the substance may be contained in a buffer solution so that the molar ratio is preferably 1: 1 to 20 (more preferably 1: 1 to 10, particularly preferably 1: 1 to 2).
Examples of the buffer solution generally used in the field of immunoassay include, for example, Tris buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution, Good buffer solution, and the like. It may be in a range that does not suppress the antibody reaction, and 5 to 10 is preferable.
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, a surfactant, a saccharide and the like are contained. You may keep it.

  Examples of the measurement target substance in the substance (F1) that specifically binds to the measurement target substance labeled with the labeling substance or the measurement target substance (F2) labeled with the labeling substance include the measurement target substance described later, (F1) The substance that specifically binds to the substance to be measured is the “antibody” when the substance to be measured is “antigen”, and the substance to be measured when the substance to be measured is “antibody”. The binding substance is an “antigen”.

  The content of the substance (F1) that specifically binds to the measurement target substance labeled with the labeling substance in the labeling reagent (F) and the measurement target substance (F2) labeled with the labeling substance is 0 from the viewpoint of sensitivity. 0.01 to 40 μg / mL is preferable, and more preferably 0.1 to 20 μg / mL.

In the solid phase carrier reagent (E) and the labeling reagent (F), other components {sugars other than (A) (glucose, sucrose, etc.), inorganic salts (sodium chloride, etc.), as long as they do not interfere with the immune reaction , Selected from the group consisting of surfactants other than (A) (polyoxyethylene sorbitan monostearate, etc.), preservatives (sodium azide, etc.) and nonspecific reaction inhibitors (eg, IgG antibodies derived from normal animals). At least one kind or the like} may be included.
When the saccharide, inorganic salt, surfactant, preservative and nonspecific reaction inhibitor in the solid phase carrier reagent (E) and / or labeling reagent (F) are contained, the respective contents are (E) or (F) Based on the respective weights, the saccharide content is 0.1 to 10% by weight, the inorganic salt content is 0.01 to 5% by weight, the surfactant content is 0.02 to 5% by weight, antiseptic The content of the agent is preferably 0.001 to 0.1% by weight, and the content of the nonspecific reaction inhibitor is preferably 0.001 to 5% by weight.

The immunoassay method of the present invention is a known immunoassay method [for example, chemiluminescent enzyme immunoassay (CLEIA), except that alkylglycoside (A) whose hydrophilic moiety is a monosaccharide (a) is used during an immune reaction. Enzyme immunoassay (EIA), radioimmunoassay (RIA), electrochemiluminescence immunoassay (ECLIA), fluorescent enzyme immunoassay (FEIA), chemiluminescence immunoassay (CLIA), latex optical immunoassay ( LPIA) and latex particle counting immunoagglutination assay (CIA)].
In addition, as a method of immune reaction, a sandwich method, a competitive method and a literature generally described in the field of immunoassay [for example, the enzyme immunoassay second edition (edited by Eiji Ishikawa et al., Medical School) 1982] The method described in JP-A-6-130063, specifically, the following three methods are included.

<First method: sandwich method>
Magnetic particle (B) (preferably magnetic silica particle) has immobilized on its surface an antigen (C) or antibody (D) as a substance that specifically binds to the substance to be measured (measuring substance-binding substance). A substance [magnetic particle (B1)] that specifically binds to a measurement target substance labeled with a sample containing a measurement target substance (for example, a biological sample), magnetic particles (B1), and a labeling substance (label) A measurement target substance binding substance) (F1) or a measurement target substance labeled with a labeling substance (labeled measurement target substance) (F2) is brought into contact with the antigen as a measurement target substance binding substance on the magnetic particle (B) ( C) Magnetic particle carrying a complex (labeled complex) of antibody (D), measurement target substance and labeled measurement target substance binding substance (F1) or labeled measurement target substance (F2) and carrying the labeled complex (B) B / F separation Te, the amount of the labeling substance labeled complex was measured to measure the analyte in the sample based on the result.

<Second Method: Competition Method 1>
Magnetic particles (B) (preferably magnetic silica particles) in which antigen (C) or antibody (D) as a measurement target substance or a similar substance of the measurement target substance is immobilized on the surface thereof [magnetic particle (B1) ] A sample containing a measurement target substance (for example, a biological sample), a substance that specifically binds to a measurement target substance labeled with a labeling substance (labeled measurement target substance binding substance) (F1), and magnetic particles ( B1) is contacted, and the labeled measurement target substance binding substance (F1) is allowed to undergo a competitive reaction between the antigen (C) or antibody (D) immobilized on the magnetic particles (B) and the measurement target substance in the sample, After forming a complex (label complex) of the antigen (C) or antibody (D) and the labeled measurement substance-binding substance (F1) on the magnetic particle (B), the magnetic particle carrying the label complex ( B) B / F separated and labeled substance in the labeled complex It was measured, to measure the analyte in the sample based on the result.

<Third method: competitive method 2>
Magnetic particle (B) (preferably magnetic silica particle) has immobilized on its surface an antigen (C) or antibody (D) as a substance that specifically binds to the substance to be measured (measuring substance-binding substance). [Magnetic particles (B1)], a sample containing a substance to be measured (for example, a biological sample) and a substance to be measured (F2) labeled with a labeling substance or a substance similar to a substance to be measured labeled with a labeling substance (F3) Antigen (C) or antibody (D) immobilized on magnetic particle (B) by contacting [labeled substance to be measured (F2) or its similar substance (F3)] with magnetic particle (B1) In addition, the measurement target substance and the labeled measurement target substance (F2) or a similar substance (F3) are subjected to a competitive reaction, and the measurement target substance binding substance and the labeled measurement target substance (F2) or a similar substance are formed on the magnetic particles (B). Complex with (F3) After forming the composite), the magnetic particles (B) carrying the labeled complex are subjected to B / F separation, the amount of the labeled substance in the labeled complex is measured, and the measurement target in the sample is based on the result. Measure the substance.

The substance to be measured in the immunoassay method of the present invention is not particularly limited as long as it is generally measured in the field of immunoassay. For example, biological body fluids such as serum, blood, plasma, urine, lymph fluid, blood cells Nucleotide chains, peptide chains, proteins, lipid proteins, nucleic acids, immunoglobulins, blood coagulation-related factors, antibodies, enzymes, hormones, cancer markers, heart disease markers and various drugs contained in biological samples such as various cells Etc. are typical examples. More specifically, for example, peptide chains such as C-peptide and angiotensin I, proteins such as albumin, hemoglobin, myoglobin, transferrin, protein A, and C-reactive protein (CRP) such as C-peptide and angiotensin I. Peptide chains such as lipid proteins such as high density lipoprotein (HDL), low density lipoprotein (LDL), ultra low density lipoprotein, nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), such as alkaline phosphatase, Amylase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminator Enzymes such as ze (GPT), renin, protein kinase (PK), tyrosine kinase, eg immunoglobulins such as IgG, IgM, IgA, IgD, IgE (or their Fc part, Fab part, F (ab) Fragments of 2 parts), for example, blood coagulation-related factors such as fibrinogen, fibrin degradation product (FDP), prothrombin, thrombin, such as anti-streptolysin O antibody, anti-human hepatitis B virus surface antigen antibody (HBs antigen), anti-human Antibodies such as hepatitis C virus antibody and anti-rheumatic factor such as thyroid stimulating hormone (TSH), thyroid hormone (FT3, FT4, T3, T4), insulin, parathyroid hormone (PTH), human chorionic gonadotropin (hCG) estradiol (E2) hormones such as α-fetoprotein (A P), carcinoembryonic antigen (CEA), CA19-9, prostate specific antigen (PSA) and other cancer markers such as troponin T (TnT), human brain natriuretic peptide precursor N-terminal fragment (NT-proBNP), etc. Heart disease markers such as drugs such as antiepileptic drugs, antibiotics, theophylline and the like can be mentioned.
Among the above, at least one selected from the group consisting of an antibody, a peptide chain, a hormone, a cancer marker, and a heart disease marker is preferable and more preferable from the viewpoint of the stability of the antigen (C) or antibody (D). It is at least one selected from the group consisting of peptide chains, hormones, cancer markers and heart disease markers.

In the immunoassay method of the present invention, the concentration of the alkyl glycoside (A) in the solution at the time of performing the immune reaction is preferably 0.001 to 5% by weight based on the weight of the reaction solution from the viewpoint of sensitivity and reproducibility. More preferably, it is 0.01 to 1% by weight.
The alkyl glycoside (A) may be mixed in advance with either or both of the solid phase carrier reagent (E) and the labeling reagent (F), or when performing an immune reaction, the solid phase carrier reagent (E). In addition, it may be added together with the labeling reagent (F), but is preferably mixed with the labeling reagent (F) in advance from the viewpoint of reproducibility. Moreover, when using (A) without premixing with (E) or (F), you may melt | dissolve and use in the said aqueous solvent.
In addition, the pH of the solution during the immune reaction is preferably 5 to 10 from the viewpoint of the reactivity of the immune reaction.

  The method for measuring the amount of the labeling substance in the labeling complex is selected based on the labeling substance used. For example, the known chemiluminescent enzyme immunoassay (CLEIA), enzyme immunoassay (EIA), radioimmunoassay (RIA) , Electrochemiluminescence immunoassay (ECLIA), fluorescent enzyme immunoassay (FEIA), chemiluminescence immunoassay (CLIA), latex optical immunoassay (LPIA), latex particle count immunoagglutination assay (CIA), etc. This can be done based on the method used.

For example, when the amount of the labeling substance is measured by the chemiluminescence method, the chemiluminescence reagent (G) is used.
The chemiluminescent reagent (G) is selected based on the labeling substance used. For example, when the labeling substance is peroxidase, the 2,3-dihydro-1,4-phthalazinedione compound and the chemiluminescence enhancer are essential components. A first chemiluminescent reagent solution and a second chemiluminescent reagent solution containing oxidant and water as essential components.

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.
Among these, from the viewpoint of sensitivity, luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof (alkali metal salts) Etc.) are preferred, more preferred are luminol and its metal salts, and particularly preferred is the sodium salt of luminol.

  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 first chemiluminescent reagent solution can contain water and various buffers (phosphate buffer, borate buffer, glycine buffer, Tris buffer, and the like).

The first chemiluminescent reagent solution is preferably alkaline from the viewpoint of the fluorescence intensity of the enzyme, and the pH of the first solution is preferably 7 to 11, more preferably 8 to 10.
The pH is measured at a measurement temperature of 25 ° C. according to JIS K0400-12-10: 2000.

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.) and peroxo acid compounds (peroxosulfuric acid, peroxophosphoric acid, etc.).
Of these, hydrogen peroxide, sodium perborate, and potassium perborate are preferable from the viewpoint of storage stability and the like, and hydrogen peroxide is more preferable.

<Immunoassay kit>
The immunoassay kit of the present invention is a reagent kit composed of the above-mentioned various reagents and alkylglycoside (A) used in the immunoassay method of the present invention, and comprises a solid phase carrier reagent (E) and a labeling reagent (F). Although it is preferable that at least one of the solid phase carrier reagent (E) and the labeling reagent (F) contains the alkyl glycoside (A) as an essential constituent reagent, (A) is converted into (E) and (F). It is also possible to make a kit as a separate component.
The amount of the alkyl glycoside (A) in the immunoassay kit is preferably in the range where the concentration in the solution when the immunoreaction is performed using the immunoassay kit of the present invention is 0.001 to 5% by weight.
Moreover, the composition of each component in the alkyl glycoside (A) and the solid phase carrier reagent (E) and the labeling reagent (F) in the immunoassay kit, the content thereof, and preferred ranges thereof were described in the above immunoassay method. It is the same as that.

  In the immunoassay kit of the present invention, in addition to the solid phase carrier reagent (E) and the labeling reagent (F), a chemiluminescent reagent (G), a standard reagent (measuring solution with a known concentration), a dilution reagent ( A reagent for diluting a high-concentration sample), a cleaning reagent (for example, a reagent for cleaning a reaction container), and the like, and further an instruction manual, a jig necessary for measurement (for example, a sample container).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these. Hereinafter, unless otherwise specified, parts are parts by weight.

<Example 1>
The present invention comprises a solid phase carrier reagent (anti-CEA antibody-bound magnetic silica particle-containing reagent), a labeling reagent (POD-labeled anti-CEA antibody-containing reagent), a chemiluminescent reagent first solution, and a chemiluminescent reagent second solution as follows. The immunoassay kit (S1) was obtained.

Production of magnetic silica particles:
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 wt% aqueous ammonia 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 having a volume average particle diameter of 15 nm were obtained by drying at 2 ° C. for 2 days.

  80 parts of superparamagnetic metal oxide particles were added to 240 parts of tetraethoxysilane and dispersed to prepare dispersion (1). Next, 5050 parts of water, 3500 parts of a 25 wt% aqueous ammonia solution, and 400 parts of a nonionic surfactant [“NSA-17” manufactured by Sanyo Chemical Industries, Ltd.] were added to the reaction vessel, and Clearmix [M Technique Co., Ltd. The solution (2) was obtained by mixing the mixture (1) with the solution (2) for 1 hour while stirring the clear mix at 6,000 rpm. After dropping, the mixture was reacted for 1 hour at 50 ° C. 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.

  Add 80 parts of the core layer, 2500 parts of deionized water, 260 parts of 25% by weight ammonia aqueous solution, 2500 parts of ethanol, 1200 parts of tetraethoxysilane to the reaction vessel, and mix using Clear Mix [M Technique Co., Ltd.] The reaction was carried out for 2 hours while stirring at a clear mix rotation speed of 6,000 rpm. 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. 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. Classification was performed by adding 5000 parts of water to disperse the particles and centrifuging at 300 rpm for 10 minutes to settle 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 the desired magnetic silica particles having a volume average particle diameter of 2.0 μm. As a result of measuring the content of superparamagnetic metal oxide particles in the obtained magnetic silica particles, the content was 81% by weight.

<Method for measuring content of superparamagnetic metal oxide particles>
Any 20 of the magnetic silica particles were observed with a scanning electron microscope [model number JSM-7000F, manufacturer name JEOL Ltd.], and an energy dispersive X-ray spectrometer (model number INCA Wave / Energy, manufacturer name Oxford) ) To measure the content of the superparamagnetic metal oxide particles, and the average value was defined as the content S. Further, the content of silica was measured by the same measurement, and the average value was defined as the content T. The content of superparamagnetic metal oxide particles was determined by the following calculation formula (1).
Content of superparamagnetic metal oxide particles (% by weight) = {(S) / (S + T)} × 100 (1)

Preparation of solid support reagent:
40 mg of magnetic silica particles produced in a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1% by weight γ-aminopropyltriethoxysilane was added, reacted at 25 ° C. for 1 hour, and after collecting the magnetic silica particles with a neodymium magnet, The liquid was removed by suction with an aspirator. Next, 40 mL of deionized water was added, the cap was capped, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic silica particles with a neodymium magnet, the liquid was sucked and removed with an aspirator to wash the magnetic silica particles. This washing operation was performed 5 times. Next, the washed magnetic silica particles were added to a polyethylene bottle with a lid containing 40 mL of an aqueous solution containing 2% by weight glutaraldehyde, and reacted at 25 ° C. for 1 hour. Then, 40 mL of deionized water was added, the cap was closed, the polystyrene bottle was slowly inverted and stirred twice, and after collecting the magnetic silica particles with a neodymium magnet, the liquid was sucked and removed with an aspirator to wash the magnetic silica particles. . This washing operation was performed 10 times. Further, the magnetic silica particles after washing were placed in a polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-CEA monoclonal antibody [Dako Japan Co., Ltd.] at a concentration of 10 μg / mL. In addition, the mixture was reacted at 25 ° C. for 1 hour. After the reaction, the magnetic silica particles were collected with a neodymium magnet, and then the anti-CEA monoclonal antibody-containing phosphate buffer was removed. Next, the magnetic silica particles were added to a polyethylene bottle with a lid containing 40 mL of 0.02 M phosphate buffer (pH 7.0) containing 1% by weight of bovine serum albumin, and immersed for 12 hours at 25 ° C., and then a neodymium magnet. After collecting the silica particles, the phosphate buffer containing 1% by weight of bovine serum albumin was removed. 0.02M phosphate buffer containing 0.85 wt% NaCl and 0.01 wt% n-octyl β-D-glucopyranoside so that the concentration of anti-CEA antibody-bound magnetic silica particles is 1.0 mg / mL The solution was diluted with (pH 7.0) to prepare a solid phase carrier reagent (anti-CEA antibody-bound magnetic silica particle-containing reagent) (E-1) and stored in a refrigerator (2 to 10 ° C.).

Preparation of labeling reagent:
Using anti-CEA monoclonal antibody [manufactured by Dako Japan Co., Ltd.] and horseradish-derived POD [manufactured by Toyobo Co., Ltd.], literature (S Yoshitake, M Imagawa, E Ishikawa, Etol; J. Biochem, Vol. 92) , 1982, 1413-1424), a POD-labeled anti-CEA antibody was prepared. This was 0.02M phosphate buffer (pH 7.0) containing 0.5% by weight bovine serum albumin and 1% by weight NAROACTY CL-100 as a surfactant, and the concentration of POD-labeled anti-CEA antibody was 100 nM. It diluted to the density | concentration, the labeling reagent (F-1) was prepared, and it preserve | saved by refrigeration (2-10 degreeC).

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. The mixture was mixed to prepare a chemiluminescent reagent first liquid (G-1). 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 chemiluminescent reagent second liquid (G-2). It was stored refrigerated (2-10 ° C.) until used for measurement.

<Examples 2 to 13 and Comparative Examples 1 to 5>
The solid phase carrier reagents (E-2) to (E-13) and the solid phase carrier for comparison were the same as in Example 1 except that the type and / or amount of raw materials used were changed to those shown in Table 1. Reagents (HE-1) to (HE-5), labeling reagents (F-2) to (F-13) and comparative labeling reagents (HF-1) to (HF-5) were prepared and chemically The immunoassay kits (S2) to (S13) and the comparative immunoassay kits (H1) to (S13) are combined with the luminescent reagent first liquid (G-1) and the chemiluminescent reagent second liquid (G-2). H5) was obtained.

In Table 1, the following components were used.
-Alkyl glycosides (A) whose hydrophilic part is a monosaccharide
n-octyl β-D-glucopyranoside: n-dodecyl β-D-glucopyranoside manufactured by Wako Pure Chemical Industries, Ltd .: Sigma-Aldrich Co., Ltd. ○ alkyl glycoside n-dodecyl β-D-maltopyranoside whose hydrophilic part is a disaccharide N-octyl β-D-maltopyranoside manufactured by Kojun Pharmaceutical Co., Ltd .: Surfactant NAROACTY CL-100 other than ○ (A) manufactured by Wako Pure Chemical Industries, Ltd. “NAROACTY CL” manufactured by Sanyo Chemical Industries, Ltd. -100 "
○ Antibody (D)
CEA: anti-CEA monoclonal antibody [Dako Japan Co., Ltd.]
PSA: anti-PSA polyclonal antibody [manufactured by Dako Cytomation Co., Ltd.]
CA19-9: anti-CA19-9 antibody [manufactured by Dako Japan Co., Ltd.]

<Examples 14 to 24>
Using the obtained immunoassay kits (S1) to (S11), sensitivity and simultaneous reproducibility in immunoassay were evaluated by the following methods. The results are shown in Table 1.

<Evaluation method of sensitivity and simultaneous reproducibility in immunoassay>
Using the solid phase carrier reagents (E-1) to (E-11), the CEA concentration prepared with 0.025 mL of the solid phase carrier reagent and 0.02 M phosphate buffer (pH 7.0) was 1.0 ng / 0.025 mL of a standard CEA solution of mL was placed in a test tube, mixed, and reacted in the test tube at 37 ° C. for 3 minutes to form an anti-CEA antibody-bound magnetic silica particle / CEA complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times.

Subsequently, 0.025 mL of the standard reagent was injected into each test tube using the labeling reagents (F-1) to (F-11) containing 0.5% by weight of bovine serum albumin corresponding to the solid phase carrier reagent. The mixture was reacted in a test tube at 37 ° C. for 3 minutes to form an anti-CEA antibody-bound magnetic silica particle / CEA / POD-labeled anti-CEA antibody complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica 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 the chemiluminescent reagent first liquid (G-1) and 0.07 mL of the chemiluminescent reagent second liquid (G-2) are added simultaneously, and the luminescent reagent is allowed to react for 43 seconds at 37 ° C. The average amount of luminescence for 43 to 45 seconds after the addition was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.]. In addition, instead of the standard CEA solution having a CEA concentration of 1.0 ng / mL, a standard CEA solution having a CEA concentration of 0 ng / mL was used and the same operation as described above was performed and used as a background.

  As for sensitivity, the ratio of luminescence when immunoassay was performed using standard solutions with CEA concentrations of 1.0 ng / mL and 0 ng / mL {(luminescence level when using 1.0 ng / mL standard solution) ) / (Amount of luminescence when using a standard solution of 0 ng / mL)} is shown in Table 1 as sensitivity.

For simultaneous reproducibility, immunoassay was performed using a standard solution with a CEA concentration of 1 ng / mL, and this was repeated 10 times. From the average luminescence (X1) and standard deviation (X2), the coefficient of variation ( CV) was calculated.
CV (%) = (X2 / X1) × 100

<Example 25>
Using the obtained immunoassay kit (S12), sensitivity and simultaneous reproducibility in immunoassay were evaluated by the following methods. The results are shown in Table 1.

<Evaluation method of sensitivity and simultaneous reproducibility in immunoassay>
Put 0.025 mL of the solid phase carrier reagent (E-12) and 0.025 mL of a standard PSA solution with a PSA concentration of 50 mAU / mL prepared with 0.02 M phosphate buffer (pH 7.0) in a test tube, and mix. The mixture was reacted at 37 ° C. for 3 minutes in a test tube to form an anti-PSA antibody-bound magnetic silica particle / PSA complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times.

Subsequently, 0.025 mL of a labeling reagent (F-12) containing 0.5% by weight of bovine serum albumin was injected into the test tube, reacted in the test tube at 37 ° C. for 3 minutes, and anti-PSA antibody-conjugated magnetic silica particles / A PSA / POD-labeled anti-PSA antibody complex was formed. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica 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 the chemiluminescent reagent first liquid (G-1) and 0.07 mL of the chemiluminescent reagent second liquid (G-2) are added simultaneously, and the luminescent reagent is allowed to react for 43 seconds at 37 ° C. The average amount of luminescence for 43 to 45 seconds after the addition was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.]. In addition, instead of the standard PSA solution having a PSA concentration of 0.01 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.

  As for the sensitivity, the ratio of the amount of luminescence when the immunoassay was performed using standard solutions with PSA concentrations of 0.01 ng / mL and 0 ng / mL {(the amount of luminescence when using the standard solution of 0.01 ng / mL ) / (Amount of luminescence when using a standard solution of 0 ng / mL)} is shown in Table 1 as sensitivity.

For simultaneous reproducibility, immunoassay was performed using a standard solution with a PSA concentration of 0.01 ng / mL, and the measurement was repeated 10 times, varying from the average luminescence (X3) and standard deviation (X4) according to the following formula: The coefficient (CV) was calculated.
CV (%) = (X4 / X3) × 100

<Example 26>
Using the obtained immunoassay kit (S13), the sensitivity and simultaneous reproducibility in the immunoassay were evaluated by the following methods. The results are shown in Table 1.

<Evaluation method of sensitivity and simultaneous reproducibility in immunoassay>
Prepare 0.025 mL of CA19-9 solution prepared with 0.025 mL of solid phase carrier reagent (E-13) and 0.02 M phosphate buffer (pH 7.0), and 0.025 mL of standard CA19-9 solution with 1.0 U / mL. And the mixture was reacted in a test tube at 37 ° C. for 3 minutes to form an anti-CA19-9 antibody-bound magnetic silica particle / CA19-9 complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times.

Subsequently, 0.025 mL of a labeling reagent (F-13) containing 0.5% by weight of bovine serum albumin was injected into the test tube, reacted in the test tube at 37 ° C. for 3 minutes, and anti-CA19-9 antibody-bound magnetic silica. Particle / CA19-9 / POD labeled anti-CA19-9 antibody complex was formed. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica 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 the chemiluminescent reagent first liquid (G-1) and 0.07 mL of the chemiluminescent reagent second liquid (G-2) are added simultaneously, and the luminescent reagent is allowed to react for 43 seconds at 37 ° C. The average amount of luminescence for 43 to 45 seconds after the addition was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.]. Use the standard CA19-9 solution with a CA19-9 concentration of 0 U / mL instead of the standard CA19-9 solution with a CA19-9 concentration of 1.0 U / mL as the background. It was.

  Regarding the sensitivity, the ratio of luminescence when the immunoassay was performed using standard solutions with CA19-9 concentrations of 1.0 U / mL and 0 U / mL {(in the case of using a 1.0 U / mL standard solution) Luminous amount) / (Luminous amount when 0 U / mL standard solution is used)} is listed in Table 1 as sensitivity.

For simultaneous reproducibility, immunoassay was performed using a standard solution with a CA19-9 concentration of 1 U / mL, and this was repeated 10 times, varying from the average luminescence (X5) and standard deviation (X6) according to the following formula: The coefficient (CV) was calculated.
CV (%) = (X5 / X6) × 100

<Comparative Examples 6 to 10>
In Example 14, in place of “Immunoassay kit (S1)”, except for using “Comparative immunoassay kits (H1) to (H5)”, sensitivity and simultaneous immunoassay were performed in the same manner as in Example 14. Reproducibility was evaluated. The results are shown in Table 1.

  In Table 1, Examples 14 to 26 using the immunoassay kits of Examples 1 to 13 of the present invention containing the alkyl glycoside (A) in the solid phase carrier reagent and / or the labeling reagent as the immunoassay reagent. In the immunoassay method, the coefficient of variation is 2.4% or less, and the coefficient of variation of the immunoassay method of Comparative Examples 6 to 10 using the immunoassay kit of Comparative Examples 1 to 5 that does not contain alkylglycoside (A) It can be seen that the coefficient of variation is small and the reproducibility is excellent as compared with 4.4 to 6.2. Moreover, the sensitivity in the immunoassay methods of Examples 14 to 26 is 2.5 or more, which is equivalent to or higher than that of the comparative example, and it can be seen that the sensitivity is high and the reproducibility is excellent. Furthermore, in Examples 8 to 11 containing the alkylglycoside (A) in the labeling reagent, it can be seen that the coefficient of variation is as extremely small as 1.5 to 1.7, and the reproducibility is extremely excellent.

  Since the immunoassay method and immunoassay kit of the present invention are highly sensitive and excellent in reproducibility, they are suitable for clinical tests such as radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, and chemiluminescence immunoassay. Widely applicable.

Claims (6)

  An immunoassay method in which an immune reaction is carried out using magnetic particles (B) as a solid phase carrier in the presence of an alkyl glycoside (A) whose hydrophilic part is a monosaccharide (a).   The immunoassay method according to claim 1, wherein the alkyl glycoside (A) is an alkyl glycoside having an alkyl group having 6 to 36 carbon atoms as a hydrophobic portion.   The immunoassay method according to claim 1 or 2, wherein the immune reaction is carried out in a solution having an alkylglycoside (A) concentration of 0.001 to 5% by weight.   The immunoassay method according to any one of claims 1 to 3, wherein the magnetic particle (B) is a magnetic particle (B1) having an antigen (C) or antibody (D) immobilized on the surface thereof.   The immunoassay method according to any one of claims 1 to 4, wherein the magnetic particles (B) are magnetic silica particles containing 60 to 95% by weight of a superparamagnetic metal oxide having a volume average particle diameter of 1 to 20 nm.   A kit for immunoassay comprising a solid phase carrier reagent (E) and a labeling reagent (F), wherein the solid phase carrier reagent (E) has an antigen (C) or antibody (D) immobilized on its surface (B ), A labeling reagent (F) containing a substance (F1) that specifically binds to a measurement target substance labeled with a labeling substance or a measurement target substance (F2) labeled with a labeling substance, and a solid phase carrier The kit for immunoassay used for the immunoassay method of Claim 1 in which at least any one of a reagent (E) or a labeling reagent (F) contains the alkylglycoside (A) whose hydrophilic part is a monosaccharide (a).