JP2022056909A - Immunoassay measurement method and immunoassay measurement kit - Google Patents

Abstract

To provide an immunoassay measurement method and immunoassay measurement kit that are high in precision, and high in sensitivity.SOLUTION: An immunoassay measurement method, which measures density of a measurement object substance (G) in a specimen, includes a step (1) of causing a solid-phase carrier (a1) having a substance (D) specifically bonding to the measurement object substance immobilized, and the measurement object substance (G) to react with each other in a solution in which a content of a compound (C) having a specific structure is 3 to 10 wt.%, and causing a composite (J1) of the substance (D) and the measurement object substance (G) to form, or a step (2) of causing a substance (F3) labeled by a labeled substance (b) and specifically bonding to the measurement object substance and the measurement object substance (G) to react with each other in the solution in which the content of the compound (C) having the specific structure is 3 to 10 wt.%, and causing a composite (J2) of the substance (F3) and the measurement object substance (G) to form.SELECTED DRAWING: None

Description

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

Conventionally, as a method for measuring a sample containing a biological substance, for example, a protein in a biological sample, a protein or the like in the biological sample is bound to the surface of a magnetic particle to which the protein can be bound, and the protein or the like in the biological sample is not used. In order to remove the impurities of the above, an immunoassay method including a step of washing magnetic particles, recovering particles to which proteins and the like are bound, and measuring the amount of protein bound is known. (Patent Documents 1 and 2 etc.)
In the above immunoassay, there is a problem that the signal (luminous amount, etc.) when a positive sample is measured is not sufficient due to the insufficient reaction rate with the substance to be measured.
Therefore, the difference between the signal when measuring a positive sample and the signal when measuring a negative sample is not sufficient, so accurate measurement cannot be performed (the sample that should be judged as a normal value is erroneously abnormal. It is judged as a value, etc.) is a big problem.
In addition, there is a problem that the measured value fluctuates greatly and the measurement cannot be performed with good reproducibility.

Japanese Unexamined Patent Publication No. 2000-40608 International Publication No. 2012/173002

An object of the present invention is to provide an immunoassay method and an immunoassay kit with high accuracy and high sensitivity.

The present inventors have reached the present invention as a result of diligent studies to achieve the above object. That is, the present invention is an immunoassay method for measuring the concentration of the substance (G) to be measured in a sample.
The content of the solid phase carrier (a1) on which the substance (D) specifically bound to the substance to be measured is immobilized and the substance (G) to be measured are the compound (C) represented by the general formula (1). The step (1) of forming a complex (J1) of the substance (D) and the substance to be measured (G) by reacting in a solution containing 3 to 10% by weight, or
A substance (F3) labeled with a labeling substance (b) that specifically binds to the substance to be measured and a substance (G) to be measured are represented by the compound (C) represented by the general formula (1). An immunoassay method comprising a step (2) of reacting in a solution having a content of 3 to 10% by weight to form a complex (J2) of a substance (F3) and a substance to be measured (G); immunity. An immunoassay kit comprising a solid phase carrier reagent (A), a labeling reagent (B), and an immunoassay buffer (W) as measurement reagents.
The solid phase carrier reagent (A) immobilizes the solid phase carrier (a1) or the substance to be measured (G) or a similar substance (G') on which the substance (D) specifically bound to the substance to be measured is immobilized. Containing the solid phase carrier (a2)
The labeling reagent (B) is a substance labeled with the labeling substance (b), a substance to be measured (F1), a similar substance (F2) or a substance labeled with the labeling substance (b), and is specific to the substance to be measured. Contains the binding substance (F3) and
The immunoassay buffer solution (W) is an immunoassay kit used in the above-mentioned immunoassay method containing the compound (C) represented by the general formula (1).
HO-[(A ¹ O) _x / (A ² O) _y ] -H (1)
[In the general formula (1), A ¹ is an ethylene group; A ² is a propylene group; x is an integer of 250 to 800; y is an integer of 55 to 200; [(A ¹ ). O) _x / (A ² O) _y ] has an arbitrary binding order of (A ¹ O) units having x divalent groups and (A ² O) units having y divalent groups. Indicates that. ]

The immunoassay method and immunoassay kit of the present invention enable highly accurate and highly sensitive clinical tests.

The present invention is an immunoassay method for measuring the concentration of a substance (G) to be measured in a sample.
The solid phase carrier (a1) on which the substance (D) specifically bound to the substance to be measured is immobilized and the substance (G) to be measured are contained in the compound (C) represented by the above general formula (1). The step (1) of reacting in a solution having an amount of 3 to 10% by weight to form a complex (J1) of the substance (D) and the substance to be measured (G), or
The substance (F3) labeled by the labeling substance (b) and specifically bound to the substance to be measured and the substance (G) to be measured are represented by the compound (C) represented by the general formula (1). This is an immunoassay method comprising a step (2) of reacting in a solution having a content of 3 to 10% by weight to form a complex (J2) of the substance (F3) and the substance to be measured (G). ..

The reaction between the substance to be measured (G) and the substance (D) that specifically binds to the substance to be measured, or the substance (D1) that specifically binds to the substance to be measured labeled by the labeling substance, and the measurement target. By performing the reaction with the substance (G) [reaction in the presence of the substance (G) to be measured] in the presence of the compound (C) represented by the general formula (1), the substance to be measured and each of them in the immunoassay. It is presumed that by improving the reaction rate and reaction efficiency with a substance, the signal (emission amount, etc.) when a positive sample is measured increases, and as a result, highly accurate immunomeasurement becomes possible.

The immunoassay method of the present invention
The solid phase carrier (a1) on which the substance (D) specifically bound to the substance to be measured is immobilized and the substance (G) to be measured are subjected to the presence of the compound (C) represented by the general formula (1). A method including a step (1) of reacting to form a complex (J1) of the substance (D) and the substance to be measured (G), or
The substance (F3) labeled by the labeling substance (b) and specifically bound to the substance to be measured and the substance (G) to be measured are represented by the compound (C) represented by the general formula (1). As long as the method includes the step (2) of reacting in a solution having a content of 3 to 10% by weight to form a complex (J2) of the substance (F3) and the substance to be measured (G). It can be used as a method generally used in the field of immunoassay as an immunoassay method for quantifying a substance (G) to be measured in a sample. It can be used for the sandwich method described in Eiji Ishikawa et al., Medical Shoin) 1982], the competitive method, and the immunoassay method described in JP-A-6-130063.

Among the immunoassay methods in the present invention, the solid phase carrier reagent (A) containing the solid phase carrier (a1) is used, and as the solid phase carrier (a1), the antigen (X) or antibody (Y) described later is used. As a method using the magnetic particles (H) having the above-mentioned [the antigen (X) or the antibody (Y) described later is used as the substance (D) or the substance to be measured (G) or a similar substance (G')]. Specifically, the following sandwich method and competitive methods (1-1), (1-2), (2-1) and (2-2) are included.

<Sandwich method: A method including step (1) in the present invention>
Specific examples of applying the immunoassay method of the present invention [method including step (1) in the present invention] to the sandwich method include the following methods.
That is, the sample containing the substance to be measured (G) and the substance (D) that specifically binds to the substance to be measured (using the antigen (X) or antibody (Y) described later as the substance (D)] are immobilized. The magnetic particles (H) {solid phase carrier (a1) in the solid phase carrier reagent (A)} are brought into contact with each other in the presence of the compound (C), and the surface of the magnetic particles (H) is specifically matched with the substance to be measured. A complex (J1) is formed between the substance (D) to be bound and the substance (G) to be measured.
After that, the complex (J1) is contacted with a substance (F3) {(F3) in the labeling reagent (B)} which is a substance labeled by the labeling substance (b) and specifically binds to the substance to be measured. A substance (D) that specifically binds to the substance to be measured immobilized on the magnetic particles (H) and a substance labeled with the substance to be measured (G) that specifically binds to the substance to be measured. A complex {(D) / (G) / (F3)} [labeled complex (L)] with the substance (F3) is formed, and the labeled complex (L) is separated by B / F to form a labeled complex. The amount of the labeled substance (b) in (L) is measured, and the amount of the substance to be measured in the sample is measured based on the result.
The B / F separation in the sandwich method means the separation of the labeled complex (L) and the substance (F3) that was not involved in the formation of the labeled complex (L), specifically. , Labeled complex (L), complex (J1), and magnetic particles (H) on which the substance (D) is immobilized, and other components [components other than the substance to be measured (G) in the sample, labeled complex. It means separation from substances (F3) etc. that were not involved in the formation of the body (L)].
The B / F separation step is an essential step after the formation of the labeled complex (L), but the complex (J1) of the substance (D) and the substance to be measured (G) on the surface of the magnetic particles (H). It can be carried out even after the formation of the above. Specifically, the B / F separation in this case is the separation of the complex (J1) and the magnetic particles (H) from other components [components other than the substance to be measured (G) in the sample, etc.]. Means.

<Competitive method (1-1): Method including step (1) in the present invention>
Specific examples of applying the immunoassay method of the present invention [method including step (1) in the present invention] to a competitive method include the following methods.
That is, the sample containing the substance to be measured (G) and the substance (D) that specifically binds to the substance to be measured (using the antigen (X) or antibody (Y) described later as the substance (D)] are immobilized. The magnetic particles (H) {solid phase carrier (a1) in the solid phase carrier reagent (A)} are brought into contact with each other in the presence of the compound (C), and the surface of the magnetic particles (H) is specifically matched with the substance to be measured. A complex (J1) is formed between the substance (D) to be bound and the substance (G) to be measured.
After that, the substance (D) that has not reacted with (G) immobilized on the magnetic particles (H) and the substance to be measured (F1) labeled with the labeling substance (b) or a similar substance (F2) {knowledge. By contacting (F1) or (F2)} in the reagent (B) with the substance (D) and the labeling substance (b) that specifically bind to the substance to be measured immobilized on the magnetic particles (H). A complex {(D) / (F1) or (F2)} [labeled complex (M)] with the labeled substance to be measured (F1) or a similar substance (F2) is formed to form a labeled complex (M). ) Is immobilized by B / F separation, the amount of the labeled substance (b) in the complex (M) is measured, and the amount of the substance to be measured in the sample is measured based on the result. Will be done.
The B / F separation in the competitive method (1-1) refers to the labeled complex (M) and other components [substance (F1) or a substance (F1) thereof that were not involved in the formation of the labeled complex (M). It means separation from a similar substance (F2)], specifically, a magnetic particle (H), a complex (J1), and a labeled complex (M) on which the substance (D) is immobilized, and other substances. It means separation from the component [components other than the substance to be measured (G) in the sample, the substance (F1) or a similar substance (F2), etc. that did not participate in the formation of the labeled complex (M)].
The B / F separation step is an essential step after the formation of the labeled complex (M), but the complex (J1) of the substance (D) and the substance to be measured (G) on the surface of the magnetic particles (H). It can be carried out even after the formation of the above. Specifically, the B / F separation in this case is the separation of the complex (J1) and the magnetic particles (H) from other components [components other than the substance to be measured (G) in the sample, etc.]. Means.

<Competitive method (1-2): Method including step (1) in the present invention>
Another specific example of the method including the step (1) in the present invention is the following method.
That is, the sample containing the measurement target substance (G) and the measurement target substance (F1) labeled with the labeling substance (b) or a similar substance (F2) {(F1) or (F2) in the knowledge reagent (B). } And a substance (D) that specifically binds to the substance to be measured [using the antigen (X) or antibody (Y) described later as the substance (D)] immobilized magnetic particles (H) {solid phase carrier reagent The solid phase carrier (a1)} in (A) is brought into contact with the substance (D) on the magnetic particles (H) in the presence of the compound (C), and the substance (G) to be measured and the substance (G) in the sample are brought into contact with each other. Competitive reaction with (F1) or a similar substance (F2).
Due to this competitive reaction, a complex (J1) of a substance (D) and a substance (G) and a substance (D) and a substance (F1) or a similar substance (F2) are formed on the magnetic particles (H). The labeled complex (M) is formed.
Then, the magnetic particles (H) carrying the labeled complex (M) are separated by B / F, the amount of the labeled substance (b) in the labeled complex (M) is measured, and the amount in the sample is based on the result. The substance to be measured may be measured.
The B / F separation in the competitive method (1-2) refers to the labeled complex (M) and other components [substance (F1) or a substance (F1) thereof that were not involved in the formation of the labeled complex (M). It means separation from a similar substance (F2)], specifically, a magnetic particle (H), a complex (J1), and a labeled complex (M) on which the substance (D) is immobilized, and other substances. It means separation from the component [components other than the substance to be measured (G) in the sample, the substance (F1) or a similar substance (F2), etc. that did not participate in the formation of the labeled complex (M)].

<Competitive method (2-1): Method including step (2) in the present invention>
Specific examples of applying the immunoassay method of the present invention [method including step (2) in the present invention] to a competitive method include the following methods.
That is, the sample containing the substance to be measured (G) and the substance labeled with the labeling substance (b) that specifically binds to the substance to be measured (F3) {(F3) in the labeling reagent (B). , The antigen (X) or antibody (Y) labeled with the labeling substance (b) is used as (F3)} is brought into contact with the substance (F3) and the substance to be measured (G) in the presence of the compound (C). ) To form a complex (J2).
After that, the substance (F3) that has not reacted with (G) and the substance to be measured (G) and / or a similar substance (G') [the substance (G) and the substance (G') described later are the antigens (X). Alternatively, the antibody (Y) is used] is immobilized on the magnetic particles (H) {solid phase carrier (a2) in the solid phase carrier reagent (A)}, and the measurement is immobilized on the magnetic particles (H). A complex of the target substance (G) or a similar substance (G') and a substance (F3) labeled with the labeling substance (b) and specifically binding to the target substance (F3) G') / (F3)} [labeled complex (N)] is formed, and the magnetic particles (H) on which the labeled complex (N) is immobilized are B / F separated into the complex (N). The amount of the labeled substance (b) is measured, and the amount of the substance to be measured in the sample is measured based on the result.
The B / F separation in the competitive method (2-1) includes the labeled complex (N) and other components [substance (F3)] that were not involved in the formation of the labeled complex (N). Means the separation of, specifically, the substance (G) or the magnetic particles (H) on which the substance (G') is immobilized, the labeled complex (N), and other components [measurement target in the sample]. It means separation from a component other than the substance (G), a substance (F3) that was not involved in the formation of the labeled complex (N), a complex (J2), etc.].
The B / F separation step is an essential step after the formation of the labeled complex (N), but it is also after the complex (J2) of the substance (F3) and the substance to be measured (G) is formed. Can be carried out. Specifically, the B / F separation in this case is performed by the magnetic particles (H), other components [unreacted substance to be measured (G), components other than the substance to be measured (G) in the sample, and a complex. (J2) etc.] means separation.

<Competitive method (2-2): Method including step (2) in the present invention>
Another specific example of the method including the step (2) in the present invention is the following method.
That is, a sample containing the substance to be measured (G) and a substance labeled with the labeling substance (b) that specifically binds to the substance to be measured (F3) {(F3) in the labeling reagent (B). , The antigen (X) or antibody (Y) labeled with the labeling substance (b) is used as (F3)}, and the substance to be measured (G) and / or a similar substance (G') [substance (G) and Use the antigen (X) or antibody (Y) described below as the substance (G')] in the immobilized magnetic particles (H) {solid phase carrier (a2) in the solid phase carrier reagent (A)} and compound ( In the presence of C), the substance (F3) is brought into a competitive reaction between the substance (G) to be measured in the sample and the substance (G) or the substance (G') on the magnetic particles (H).
Due to this competitive reaction, a complex (J2) of a substance (F3) and a substance (G), and a labeled complex of a substance (G) or a substance (G') and a substance (F3) on a magnetic particle (H). Form the body (N).
Then, the magnetic particles (H) carrying the labeled complex (N) are separated by B / F, the amount of the labeled substance (b) in the labeled complex (N) is measured, and the amount in the sample is based on the result. The substance to be measured may be measured.
The B / F separation in the competitive method (2-2) refers to the above-mentioned labeled complex (N) and other components [substance (F3)] that were not involved in the formation of the labeled complex (N). Means the separation of, specifically, the substance (G) or the magnetic particles (H) on which the substance (G') is immobilized, the labeled complex (N), and other components [measurement target in the sample]. It means separation from a component other than the substance (G), a substance (F3) that was not involved in the formation of the labeled complex (N), a complex (J2), etc.].

The substance (G) to be measured in the sample to be measured by 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, serum, blood, and the like. Substance chains (oligonucleotide chains, polynucleotide chains) contained in biological samples such as plasma, urine and other biological fluids, lymph fluids, blood cells, and various cells; chromosomes; nucleic acids (deoxyribonucleic acid (DNA), ribonucleic acid (ribonucleic acid) RNA) etc.); Peptide chains (eg C-peptide, angiotensin I etc.); Proteins [eg procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM) ), Immunoglobulin D (IgD), β2-microglobulin, albumin, hemoglobulin, myoglobin, transferase, protein A, C reactive protein (CRP), ferritin, troponin T (TnT), human brain sodium diuretic peptide precursor N-end Fragments (NT-proBNP), degradation products thereof]; Blood coagulation-related factors (eg, fibrinogen, fibrin degradation products, prothrombin, thrombin, etc.); Enzymes [eg, amylases (eg, pancreatic, salivary gland, X, etc.), alkaline phosphatase (eg, pancreatic, salivary gland, X, etc.) For example, hepatic, osseous, placenta, small intestinal, etc.), acidic phosphatase (eg, PAP, etc.), γ-glutamyl transferase (eg, renal, pancreatic, hepatic, etc.), lipase (eg, pancreatic, gastric, etc.), Cleatin kinase (eg, CK-1, CK-2, mCK, etc.), lactic acid dehydrogenase (eg, LDH1 to LDH5, etc.), glutamate oxaloacetate transaminase (eg, ASTm, ASTs, etc.), glutamate pyruvate transaminase (eg, ALTm, ALTs, etc.) ), Colin esterase (eg ChE1 to ChE5 etc.), leucine aminopeptidase (eg C-LAP, AA, CAP etc.), renin, protein kinase, tyrosine kinase etc.] and inhibitors of these enzymes; hormones (eg PTH, TSH, insulin, etc.) LH, FSH, estradiol, prolactin, etc.); Receptors (eg, receptors for estrogen, TSH, etc.); Ligands (eg, estrogen, TSH, etc.); Bacteria (eg, tuberculosis, pneumonia, diphtheria, meningitis, gonococcus, grapes) Globulin, Lenza globulin, intestinal bacterium, Escherichia coli, Helicobacter pylori, etc.); Virus (eg, rubella virus, herpe) Svirus, hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus, enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV, HTLV, etc.; Leptspira, syphilis treponema, etc.); Microorganisms such as chlamydia, mycoplasma; proteins or peptides or sugar chain antigens derived from the microorganisms; various allergens causing allergies such as bronchial asthma, allergic rhinitis, and atopic dermatitis (eg, house) Dust, such as ticks such as Kona leopard mite and Yake leopard mite, such as cedar, hinoki, suzumenohie, pigfish, pollen of Oawagaeri, Harugaya, limegi, etc., animals such as cats, dogs, crabs, etc. , Wood, drugs, allergens derived from chemicals, etc.); Lipids (eg, lipoproteins, etc.); Proteas (eg, trypsin, plasmin, serine protease, etc.); Tumor marker protein antigens (eg, PSA, PGI, PGII, etc.); Sugars Chain antigens [eg, AFP (eg, L1 to L3, etc.), hCG (hCG family), transferase, IgG, thyroglobulin, Decay-accelerating-factor (DAF), cancer fetal antigens (eg, CEA, NCA, NCA-2, NFA, etc.), 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.]; Sugar chain (for example, hyaluronic acid, β-glucan) , Sugar chains of the above sugar chain antigens, etc.); Proteins that bind to sugar chains (eg, hyaluronic acid-binding protein, β-glucan-binding protein, etc.); Substances (eg, environmental hormones such as T3, T4, FT3, FT4, tributyltin, nonylphenol, 4-octylphenol, di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate); Examples thereof include various drugs administered / inoculated to the human body and their metabolites; antigeners; nucleic acid-binding substances; antibodies thereof and the like.
For the sensitivity improvement by the compound represented by the general formula (1) of the present invention, the substance to be measured (G) is the substance to be measured (preferably antibody, hormone, cancer marker, heart disease marker, etc.) contained in serum. This is especially effective.

The substance (G') (analog) similar to the substance to be measured (G) in the above can be bound to the binding site with the substance to be measured (G) possessed by the substance (D) that specifically binds to the substance to be measured. In other words, a substance having a binding site with a substance (D) that specifically binds to the substance to be measured (D) possessed by the substance to be measured (G), in other words, a substance (G) to be measured and a substance to be measured specifically. Any substance can be used as long as it can compete with the reaction between (G) and (D) when coexisting with the substance (D) to which the substance binds.

Examples of the substance (D) that specifically binds to the substance to be measured, which will be described in detail later, include a reaction between “antigen (X)” and “antibody (Y)”, a reaction between “sugar chain” and “protein”, and “sugar”. Reaction between "chain"-"lectin", reaction between "enzyme"-"inhibitor", reaction between "protein"-"peptide chain", reaction between "chromosome or nucleotide chain"-"nucleotide chain", or "nucleotide chain" -A substance that binds to a substance to be measured (G) or a similar substance (G') thereof in a sample by a mutual reaction such as a reaction between "proteins" can be mentioned, and in each of the above combinations, one of them is a substance to be measured (a substance to be measured (G'). When it is G) or a similar substance (G'), the other one is a substance (D) that specifically binds to this substance to be measured.
The substance (D) that specifically binds to the substance to be measured (G) is the substance to be measured (G) or a similar substance (G') by the reaction between "antigen (X)" and "antibody (Y)". Those that combine with are preferable. [Preferably, when (G) is the antigen (X), (D) is the antibody, and when (G) is the antibody (Y), (G) is the antigen. ]

In the immunoassay method of the present invention, a solid phase carrier (a1) on which a substance (D) specifically bound to the substance to be measured is immobilized, or a substance to be measured (G) or a similar substance (G') is immobilized. It is preferable to use the solid phase carrier reagent (A) containing the solid phase carrier (a2).

The solid phase carriers (a1) and (a2) are not particularly limited as long as they are generally measured in the field of immunoassay, and examples thereof include glass beads, polystyrene beads, magnetic particles, microplates, latex and the like. It can be mentioned as a typical one. Of these, silica particles containing metal oxides described in JP-A-2014-210680 and JP-A-2013-019889 can be used from the viewpoint of shortening the measurement time and accuracy in immunoassay. preferable.
Further, the magnetic particles are magnetic particles (H) having an antigen (X) or antibody (Y), which will be described later, from the viewpoint of measurement sensitivity and shortening of measurement time [a substance (D) that specifically binds to a substance to be measured or As the substance to be measured (G) or a substance similar thereto (G'), magnetic particles or the like on which an antigen (X) or an antibody (Y) is immobilized] is preferable.

As the silica particles containing the metal oxide, those in which the metal oxide having a volume average particle diameter of 1 to 20 nm and having superparamagnetism is dispersed in the silica matrix are preferable. Supernormal magnetism refers to a temporary magnetic field in which individual atomic magnetic moments of a substance are aligned and induced in the presence of an external magnetic field, and when the external magnetic field is removed, partial alignment is impaired and the magnetic field is no longer shown. say.
The volume average particle diameter of the metal oxide and the silica particles containing the metal oxide in the present invention is observed with a scanning electron microscope (“JSM-7000F” manufactured by JEOL Ltd.) for any 200 particles. It is the average value of the particle diameters measured in the above.

Examples of the superparamagnetic metal oxide exhibiting superparamagnetism with a volume average particle diameter of 1 to 20 nm include oxides such as iron, cobalt, nickel and alloys thereof, but since they are excellent in sensitivity to a magnetic field, they are excellent. Iron oxide is particularly preferred. As the superparamagnetic metal oxide, one type may be used alone or two or more types may be used in combination.

As the iron oxide, various known iron oxides can be used.
Of 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. Is preferable, and magnetite is more preferable because it has a large saturation magnetization and is excellent in sensitivity to an external magnetic field.

The lower limit of the content of the ultranormal magnetic metal oxide in the silica particles containing the metal oxide is preferably 60% by weight, more preferably 65% by weight, based on the weight of the silica particles containing the metal oxide. 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 magnetism of the obtained silica particles containing the metal oxide is sufficient, and the separation operation in the actual application can be performed in a short time, which is preferable. Further, when it is 95% by weight or less, synthesis is easy.

The method for producing the ultranormal magnetic metal oxide is not particularly limited, but is a co-precipitation method using a water-soluble iron salt and ammonia based on the method reported by Massart (R. Massart, IEEE Trans. Magn. 1981, 17, 1247). ) Or a method using an oxidation reaction in an aqueous solution of a water-soluble iron salt.

The volume average particle diameter of the silica particles containing the metal oxide is preferably 1 to 5 μm, more preferably 1 to 3 μm.
When the volume average particle size is 1 μm or more, separation and recovery tend to be performed in a short time, and when it is 5 μm or less, the surface area is appropriate and the substance to be immobilized [a substance that specifically binds to the substance to be measured (substance to be specifically bound to the substance to be measured (substance to be measured). D), the substance to be measured (G) and its similar substance (G'), etc.] can be appropriately bonded, and the binding efficiency is good.

The volume average particle size of the silica particles containing the metal oxide is controlled by adjusting the particle size of the oil-in-water emulsion by adjusting the mixing conditions (shearing force, etc.) when producing the oil-in-water emulsion described later. can do. Further, the volume average particle diameter can be set to a desired value by changing the conditions of the washing step at the time of producing silica particles containing a metal oxide or by a method such as ordinary classification.

The silica particles containing the metal oxide in the present invention are, for example, supernormal magnetic metal oxide particles having a volume average particle diameter of 1 to 20 nm, and 30 to 500% by weight (alkyl) based on the weight of the supernormal magnetic metal oxide particles. ) A dispersion containing alkoxysilane and, if necessary, a dispersant, and a solution containing water, a water-soluble organic solvent, a nonionic surfactant and a catalyst for hydrolysis of (alkyl) alkoxysilane are mixed. After forming an oil-in-water emulsion, a hydrolysis reaction and a condensation reaction of (alkyl) alkoxysilane are carried out to obtain an aqueous dispersion of magnetic particles in which a supernormal magnetic metal oxide is contained in silica, and then an aqueous dispersion of magnetic particles is obtained. It is obtained by separating the body into solid and liquid by centrifugation and / or collecting magnetism, and washing with water, methanol or the like.
Further, if necessary, the magnetic particles obtained by the above operation, (alkyl) alkoxysilane, water, a water-soluble organic solvent, a nonionic surfactant, and a catalyst for hydrolysis of (alkyl) alkoxysilane are further mixed. Then, a hydrolysis reaction and a condensation reaction of (alkyl) alkoxysilane may be carried out to obtain silica particles having a core-shell structure.
In the above and below, the (alkyl) alkoxysilane means an alkylalkoxysilane or an alkoxysilane.

As for the silica particles containing the metal oxide, since the supernormal magnetic metal oxide is contained in the silica and the abundance on the particle surface is relatively small, the substance (D) that specifically binds to many substances to be measured, The substance to be measured (G) and its similar substance (G') can be immobilized on the surface thereof.

In the present invention, the antigen (X) is not particularly limited as long as it is generally measured in this field, and specifically includes the antigen used for the following immunoassay.
Substance chains (oligonucleotide chains, polynucleotide chains) contained in biological samples such as serum, blood, plasma, urine and other biological fluids, lymph fluid, blood cells, various cells, etc .; chromosomes; nucleic acids (deoxyribonucleic acid (DNA)) , Ribonucleic acid (RNA), etc.); Peptide chains (eg, C-peptide, angiotensin I, etc.); Proteins [eg, procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunity Globulin M (IgM), Immunoglobulin D (IgD), β2-microglobulin, albumin, hemoglobulin, myoglobin, transferase, protein A, C reactive protein (CRP), ferritin, troponin T (TnT), human brain sodium diuretic peptide Precursor N-terminal fragment (NT-proBNP), degradation products thereof]; Blood coagulation-related factors (eg, fibrinogen, fibrin degradation products, prothrombin, thrombin, etc.); Enzymes [eg, amylase (eg, pancreatic type, salivary gland type, X type, etc.)) , Alkaline phosphatase (eg, hepatic, osseous, placenta, small bowel, etc.), Acid phosphatase (eg, PAP, etc.), γ-glutamyl transferase (eg, renal, pancreatic, hepatic, etc.), Lipase (eg, pancreatic, gastric) Types, etc.), creatin kinase (eg, CK-1, CK-2, mCK, etc.), lactic acid dehydrogenase (eg, LDH1-LDH5, etc.), glutamate oxaloacetate transaminase (eg, ASTm, ASTs, etc.), glutamate pyruvate transaminase (eg, eg). ALTm, ALTs, etc.), cholineresterase (eg, ChE1 to ChE5, etc.), 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, estradiol, prolactin, etc.); Receptors (eg, receptors for estrogen, TSH, etc.); Ligands (eg, estrogen, TSH, etc.); , Glyco, staphylococcus, lensasphere, enterobacteria, Escherichia coli, helicobacter pylori, etc.); Viruses (eg, rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus, enterovirus, poliovirus) , EB Will Su, HAV, HBV, HCV, HIV, HTLV, etc.); Fungi (eg, Candida, Cryptococcus, etc.); Spiroheta (eg, Leptspira, Pyramid treponema, etc.); Microorganisms such as Chlamydia, Mycoplasma; Proteins or peptides or sugars derived from the microorganisms. Glycan antigens; various allergens that cause allergies such as bronchial asthma, allergic rhinitis, and atopic dermatitis (eg, house dust, for example, mites such as Kona leopard mite, Yake leopard mite, etc. Pollen such as lime cane, for example animals such as cats, dogs, crabs, for example foods such as rice, egg white, allergens derived from fungi, insects, wood, drugs, chemical substances, etc.; lipids (eg, lipoproteins, etc.); proteases (For example, trypsin, plasmin, serine protease, etc.); Tumor marker protein antigen (for example, PSA, PGI, PGII, etc.); Glycan antigen [for example, AFP (for example, L1 to L3, etc.), hCG (hCG family), transferase, IgG, thyroglobulin, etc.) (Tg), Decay-accelerating-factor (DAF), cancer fetal antigens (eg NCA, NCA-2, NFA, etc.), PIVKA-II, CA125, prostate-specific antigens, having special sugar chains produced by cancer cells. Tumor marker sugar chain antigen, ABO sugar chain antigen, etc.]; Sugar chain (for example, hyaluronic acid, β-glucan, sugar chain of the above sugar chain antigen, etc.); Protein that binds to the sugar chain (for example, hyaluronic acid-binding protein, β) Glucan-binding protein, etc.); Phosphorlipids (eg, cardiolipin, etc.); Lipopolysaccharides (eg, endotoxins, etc.); Chemical substances (eg, T3, T4, FT3, FT4, tributyltin, nonylphenol, 4-octylphenol, di-n-butyl phthalate, etc.) Environmental hormones such as dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate); various drugs administered / inoculated to the human body and their metabolites; aptamers; nucleic acid-binding substances and the like.

In the present invention, the antibody (Y) is not particularly limited as long as it is generally measured in this field, and specific examples thereof include antibodies against those exemplified as the antigen (X). The antibody used in the present invention also includes proteolytic enzymes such as papain and pepsin, and degradation products such as Fab and F (ab') ₂ fragments produced by chemical degradation.

In the present invention, the antigen (X) or the antibody (Y) is immobilized on the magnetic particles [fixed as a substance (D) that specifically binds to the substance to be measured or a substance (G) to be measured or a similar substance (G'). As a method of converting into magnetic particles (H), a method of physically adsorbing an antigen (X) or an antibody (Y) to the above-mentioned magnetic particles can be mentioned. At least one organic compound selected from the group consisting of glutaaldehyde, albumin, carbodiimide, streptavidin, biotin and an alkylalkoxysilane having a functional group from the viewpoint of immobilizing an antigen (X) or an antibody (Y). Is preferably bonded to the surface of the magnetic particles, and the antigen (X) or the antibody (Y) is immobilized on the magnetic particles through them, more preferably a functional group (ethylenically unsaturated group, epoxy group, amino). It is a method of immobilization via an alkylalkoxysilane having a group (group, mercapto group, isocyanate group, etc.).
Examples of the alkylalkoxysilane having such a functional group include vinyltrimethoxysilane, vinyltriethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, and 3-. Glycydoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyl Trimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2- (aminoethyl) -3-aminopropylmethyldimethoxysilane, N -2- (Aminoethyl) -3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N- (1,3-dimethyl-butylidene) propyl Amin, N-Phyl-3-aminopropyltrimethoxysilane, Tris- (trimethoxysilylpropyl) isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, 3 -Isocyanate propyltriethoxysilane and the like can be mentioned.

Further, as the magnetic particles (H) having the antigen (X) or the antibody (Y), the magnetic particles (H1) formed by binding to the magnetic particles via the antibody (XA) against the antigen (X) and / Alternatively, it is preferably a magnetic particle (H2) that is bound to the magnetic particle via an antigen (YA) against the antibody (Y).
Examples of the antigen (YA) include those similar to the above-mentioned antigen (X), and preferred ones are also the same.
Moreover, as the antibody (XA), the same thing as the said antibody (Y) can be mentioned, and the preferable one is also the same.
Further, F (ab') ₂ and Fab are preferable as the antibody (XA) in the magnetic particles (H1) and (H2).

As a method for producing the magnetic particles (H1), first, an antibody (XA) is provided by a known method (methods described in JP-A-2014-210680 and JP-A-2013-019889, etc.). A method for producing magnetic particles (H) [magnetic particles on which an antibody (XA) is immobilized], and then binding an antigen (X) to an antibody (XA) on the magnetic particles (H) by a known method or the like. And so on.
Further, as a method for producing the magnetic particles (H2), first, an antigen (YA) is produced by a known method (methods described in JP-A-2014-210680 and JP-A-2013-019889, etc.). The magnetic particles (H) [magnetic particles on which the antigen (YA) is immobilized] are produced, and then the antibody (Y) is bound to the antigen (YA) on the magnetic particles (H) by a known method or the like. There is a method of making the particles.

The content of the solid phase carrier (a1) or (a2) in the solid phase carrier reagent (A) is preferably 0.001 to 10% by weight, more preferably 0.01, from the viewpoint of detergency of the solid phase carrier. ~ 1% by weight.

In addition to the solid phase carrier (a) or (a2), the solid phase carrier reagent (A) contains gelatin, proteins other than gelatin, serum (mouse serum, etc.), saccharides, surfactants, inorganic salts, ethylenediaminetetraacetic acid. And may contain water.

The gelatin includes known gelatin, and the molecular weight and properties are not limited, and gelatin may be obtained from any animal (such as mammals, birds, and fish).
Examples of gelatin include acid-treated gelatin and alkali-treated gelatin produced by chemically treating collagen with an acid or an alkali and then heat-treating the gelatin. Further, a chemically modified gelatin derivative can also be used by introducing this gelatin into a functional group such as an amino group, an imino group, a carboxyl group, a mercapto group and a hydroxyl group by a well-known method.
The gelatin content is preferably 1 to 8% by weight, more preferably 2 to 5% by weight, based on the weight of the solid phase carrier reagent (A) from the viewpoint of storage stability of the solid phase carrier reagent (A). Is.

The protein other than gelatin is not particularly limited as long as it is generally used in the field of immunoassay, and examples thereof include bovine serum albumin (BSA), casein, skim milk and the like. The protein may be used alone or in combination of two or more.
The protein content is preferably 0 to 15% by weight, more preferably 0.1 to 15% by weight, based on the weight of the solid phase carrier reagent (A), from the viewpoint of storage stability of the solid phase carrier reagent (A). It is% by weight.
The serum content is preferably 0 to 15% by weight, more preferably 0.1 to 10% by weight, based on the weight of the solid phase carrier reagent (A).

Sugars include monosaccharides, disaccharides and polysaccharides.
Examples of monosaccharides include triose (ketotriose, etc.), tetrose (ketotetorose, etc.), pentose (ketopentose, aldopentos, deoxysaccharides, etc.), hexose [ketohexose (psicose, fructose, sorbose, tagatose, etc.), ald hexose (allose, alto). Loin, glucose, mannose, growth, idose, galactose, tarose, etc.), deoxy sugar (fucose, fukurosu, ramnorth, etc.), etc.], hexose (sedhepturose, etc.), etc. may be mentioned.
Examples of the disaccharide include those in which two molecules are dehydrated and condensed to form a glycosidic bond among the above monosaccharides, and specific examples thereof include sucrose, lactose, maltose and cellobiose.
Examples of the polysaccharide include those in which three or more of the above monosaccharides are dehydrated and condensed to form a glycosidic bond, and specifically, amylose, amylopectin, glycogen, cellulose, hyaluronic acid, chondroitin sulfate, heparin and the like. Can be mentioned.
As the saccharide, one type may be used alone or two or more types may be used in combination.
As the saccharide, disaccharide is preferable, and sucrose and lactose are more preferable, from the viewpoint of storage stability of the solid phase carrier reagent (A).
The content of the saccharide is preferably 5 to 40% by weight, more preferably 10 to 20% by weight, based on the weight of the solid phase carrier reagent (A) from the viewpoint of storage stability of the solid phase carrier reagent (A). Is.

Examples of the surfactant include the surfactants exemplified in the explanation of (B) described in detail later, and the same applies to preferable ones.
The content of the surfactant is preferably 0 to 2% by weight, more preferably 0.01 to 1% by weight, based on the weight of the solid phase carrier reagent (A).

Inorganic salts include alkali metal salts [halogenates (sodium chloride, potassium chloride, sodium bromide, sodium fluoride, etc.), sulfates (sodium sulfate, potassium sulfate, etc.), nitrates (sodium nitrate, potassium nitrate, etc.) and phosphoric acid. Salts (sodium phosphate, potassium phosphate, etc.)], alkaline earth metal salts [halogenates (calcium chloride, magnesium chloride, etc.) and sulfates (magnesium sulfate, etc.)] and the like can be mentioned.
As the inorganic salt, one kind may be used alone or two or more kinds may be used in combination.
Of these, at least one selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium phosphate, sodium sulfate, potassium sulfate, sodium nitrate and potassium nitrate from the viewpoint of storage stability of the solid phase carrier reagent (A). Seeds are preferred.
The content of the inorganic salt is preferably 0.1 to 2% by weight, more preferably 0, based on the weight of the solid phase carrier reagent (A) from the viewpoint of storage stability of the solid phase carrier reagent (A). It is 5 to 1% by weight.
The content of ethylenediaminetetraacetic acid is preferably 0 to 2% by weight, more preferably 0.01 to 1% by weight, based on the weight of the solid phase carrier reagent (A).

In the immunoassay method of the present invention, as described in the description of the specific immunoassay method (sandwich method, competitive method, etc.), the measurement target substance (F1) and the labeled substance (F1) labeled with the labeling substance (b) Labeling reagent (B) containing a substance (F2) similar to the substance to be measured labeled with b) or a substance (F3) labeled with the labeling substance (b) and specifically binding to the substance to be measured. It is preferable to use.

Examples of the measurement target substance used for the measurement target substance (F1) labeled with the labeling substance (b) include the same substances as those of the above-mentioned measurement target substance (G), and the preferred substances are also the same.
Examples of the substance similar to the substance to be measured used in the substance (F2) similar to the substance to be measured labeled by the labeling substance (b) include the same substances as the substance similar to the substance to be measured (G) described above.
The substance labeled by the labeling substance (b) and specifically bound to the substance to be measured and specifically bound to the substance to be measured (F3) is specific to the above-mentioned substance to be measured. Examples thereof include substances (D) that bind to the substance (D), and the same applies to preferred substances.

As the labeling substance (b) used for labeling,
For example, alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, apple acid dehydrogenase, luciferase, tyrosinase, acid phosphatase and the like used in enzyme immunoassay (EIA). Enzymes;
Radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P used in radioimmunoassay (RIA);
Fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or derivatives thereof, green fluorescent protein (GFP) used in fluorescent immunoassays (FIA);
Luminescent substances such as luciferin, isolminol, luminol, bis (2,4,6-trifluorophenyl) oxalate;
Substances with ultraviolet absorption 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-tetramethylpyrrolidin-1-oxyl, 2,6-di-t-butyl. As a spin labelant typified by a compound having an oxyl group such as -α- (3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-iriden) -p-trioxyl. Examples thereof include substances having properties.
Of these, enzymes and fluorescent substances are preferable from the viewpoint of sensitivity and the like, alkaline phosphatase, peroxidase and glucose oxidase are more preferable, and peroxidase is particularly preferable.

In order to bind the labeling substance (b) to a substance to be measured, a substance similar to the substance to be measured, and a substance that specifically binds to the substance to be measured, a method generally used in the field of immunoassay, for example, a known EIA, is used. Known labeling methods commonly used in RIA, FIA, etc. [For example, Medical Chemistry Experiment Course, Volume 8, Supervision by Yuichi Yamamura, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody, by Akira Kawao, 1st Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Muroi, 2nd Edition, Medical Shoin, 1982, etc.], etc. may be used.

The amount of the labeling substance (b) to be used varies depending on the type of the labeling substance (b) to be used and cannot be unequivocally determined. However, for example, when peroxidase (hereinafter abbreviated as POD) is used as the labeling substance (b). , The 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 and the labeling substance (b) are, for example, preferably 1: 1 to 20 (more preferably 1: 1 to 10, particularly 1: 1 to 10). It may be contained in a buffer solution so as to preferably have a molar ratio of 1: 1 to 2).
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 generally used in the field of immunoassay, and the pH thereof is an antigen. It may be a range that does not suppress the antibody reaction, and 5 to 9 is preferable.
Further, such a buffer solution contains, for example, a stabilizer such as albumin, globulin, water-soluble gelatin, polyethylene glycol, a surfactant, a saccharide and the like as long as it does not inhibit the target antigen-antibody reaction. You may leave it.

Labeled by the substance to be measured (F1) labeled by the labeling substance (b) in the labeling reagent (B), the substance similar to the substance to be measured (F2) labeled by the labeling substance (b), and the labeling substance (b). The content of the substance (F3) that specifically binds to the substance to be measured is preferably 0.01 to 40 μg / mL, more preferably 0.1 to 20 μg / mL, respectively, from the viewpoint of sensitivity. be.

In addition to the above, the labeling reagent (B) may contain a protein, a surfactant and a polymer compound.
The protein is not particularly limited as long as it is generally measured in the field of immunoassay, and examples thereof include bovine serum albumin (BSA), casein, skim milk and the like. The protein may be used alone or in combination of two or more.
The protein content is preferably 0.001 to 8% by weight based on the weight of the labeling reagent (B) from the viewpoint of sensitivity and storage stability of the reagent.

Examples of the surfactant include known nonionic surfactants, amphoteric surfactants, anionic surfactants and the like, and examples of the surfactant include water-soluble nonionic surfactants from the viewpoint of reducing nonspecific adsorption. Surfactants are preferred.
In addition, water-soluble means that 10 g is dissolved in 100 g of water at 25 ° C.
Specific examples of the water-soluble nonionic surfactant include polyoxyethylene nonylphenyl ether having an HLB of 12 or more, polyoxyethylene alkyl ether (polyoxyethylene octyl ether, etc.), polyoxyethylene sorbitan aliphatic ester, and the like. Can be mentioned.
The content of the surfactant is 0.001 to 4% by weight based on the weight of the labeling reagent (B) from the viewpoint of the detergency of the particles.

The polymer compound is not particularly limited as long as it is generally used in the field of immunoassay, and is, for example, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, Blockmaster (manufactured by JSR Co., Ltd.) and Lipidure (NOF CORPORATION). (Made by Oil Co., Ltd.).
As the polymer compound, one kind may be used alone or two or more kinds may be used in combination.
From the viewpoint of suppressing non-specific adsorption, the content of the polymer compound is preferably 0.001 to 3% by weight based on the weight of the labeling reagent (B). More preferably, it is 0.5 to 1% by weight.

In the above steps (1) and (2), as described above, the reaction proceeds in the presence of the compound (C) represented by the general formula (1).
HO-[(A ¹ O) _x / (A ² O) _y ] -H (1)

In the general formula (1), A ¹ is an ethylene group.
In the general formula (1), A ² is a propylene group.
In the general formula (1), x is an integer of 250 to 800, and is preferably an integer of 250 to 600 from the viewpoint of accuracy.
In the general formula (1), y is an integer of 55 to 200, preferably an integer of 55 to 120 from the viewpoint of accuracy, and more preferably an integer of 60 to 100.
Further, in the general formula (1), [(A ¹ O) _x / (A ² O) _y ] is an (A ¹ O) unit which is x divalent groups and y divalent groups. It is shown that the binding order of a certain (A ² O) unit is arbitrary.

From the viewpoint of accuracy, it is preferable that x and y in the general formula (1) have a relationship in which the value of (x / y) is 3.0 to 7.0.
Further, from the viewpoint of accuracy, x and y preferably have a value of (44 × x + 58 × y) of 15,000 to 35,000.

From the viewpoint of accuracy, the compound (C) represented by the general formula (1) is preferably the compound (C1) represented by the following general formula (2).
HO- (A ³ O) _p- (A ⁴ O) _q- (A ⁵ O) _r -H (2)

In the general formula ( ² ), A3 and ^A5 are ethylene groups.
In the general formula ( ² ), A4 is a propylene group.
In the general formula (2), p and r are integers in which the values of (p + r) are 250 to 800, respectively. The value of (p + r) is preferably an integer of 250 to 600 from the viewpoint of accuracy.
In the general formula (2), q is an integer of 55 to 200, preferably an integer of 55 to 120 from the viewpoint of accuracy, and more preferably an integer of 60 to 100.
From the viewpoint of accuracy, p, q and r in the general formula (2) preferably have a relationship in which the value of [(p + r) / q] is 3.0 to 7.0.
Further, from the viewpoint of accuracy, p, q and r preferably have a value of [44 × (p + r) + 58 × q] in a relationship of 15,000 to 35,000.

In the immunoassay method of the present invention, the content of the compound (C) in the reaction solution in the steps (1) and (2) is the weight of the reaction solution [excluding the weight of the solid phase carrier (a)]. As a reference, it is 3 to 10% by weight, preferably 3 to 9% by weight, and more preferably 3 to 6% by weight from the viewpoint of accuracy.
If the content of the compound (C) is less than 3% by weight, there is a problem that the accuracy is low.
It is presumed that this is due to the fact that the reaction promoting effect of compound (C) is not sufficient.
Further, when the content of the compound (C) is larger than 10% by weight, there is a problem that the accuracy is low and the reproducibility is particularly low.
It is presumed that this is because the excessive presence of the compound (C) having a relatively large molecular weight causes the components (including impurities) derived from the substance to be measured (G) to be measured in the reaction system to precipitate, resulting in a decrease in reproducibility. Will be done.

Compound (C) is added when the reaction in the above steps (1) or (2) is carried out, but from the viewpoint of measurement accuracy, water and / or a buffer solution [a buffer solution for immunoassay described later] is added in advance. It is preferable to dissolve it in the buffer solution or the like used in (W)].

Examples of the method for measuring the amount of the labeling substance (b) include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA) and chemiluminescence immunoassay (CLIA and CLEIA). From the viewpoint of sensitivity in immunoassay in a short period of time, EIA, CLIA and CLEIA are preferable, and CLEIIA is more preferable.

For example, when the amount of labeled substance is measured by the chemiluminescence method, the chemiluminescent reagent (E) is used.
The chemiluminescent reagent (E) is selected based on the above-mentioned labeling substance (b), for example, when the labeling substance (b) is peroxidase, the 2,3-dihydro-1,4-phthalazindione compound and chemiluminescence. It contains a first chemiluminescent reagent solution containing an enhancer as an essential component and a second chemiluminescent reagent solution containing an oxidizing agent and water as essential components.

Examples of the 2,3-dihydro-1,4-phthalazinedione compound are known 2, as described in JP-A-2-291299, JP-A-10-31901, JP-A-2000-279196 and the like. 3-Dihydro-1,4-phthalazinedione compounds and mixtures thereof can be used.
Of these, luminol, isolminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof (alkali metal salts, etc.) are preferable. More preferred are luminol and metal salts thereof, and particularly preferred are sodium salts of luminol.

Examples of the chemiluminescence enhancer include known chemiluminescence enhancers described in JP-A-59-500252, JP-A-59-171839, JP-A-2-291299, and the like, and mixtures thereof. Can be used. Of these, phenol is preferable from the viewpoint of chemiluminescence enhancing effect and the like, more preferably p-iodophenol, 4- (cyanomethylthio) phenol and 4-cyanomethylthio-2-chlorophenol, and particularly preferably 4-. (Cyanomethylthio) Phenol.

The first solution of the chemiluminescent reagent 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. in accordance with JIS K0400-12-10: 2000.

Examples of the oxidizing agent contained in the second liquid of the chemical luminescent reagent include known oxidizing agents described in JP-A-8-261943 and JP-A-2000-279196 [Inorganic peroxide (hydrogen peroxide). , Sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxide, acyl peroxide, etc.), peroxic acid compounds (peroxosulfate, peroxolic acid, etc.), etc.].
Of these, hydrogen peroxide, sodium perborate and potassium perborate are preferable, and hydrogen peroxide is more preferable, from the viewpoint of storage stability and the like.

The immunoassay kit of the present invention is an immunoassay kit containing a solid phase carrier reagent (A), a labeling reagent (B), and an immunoassay buffer solution (W).
The above-mentioned solid-phase carrier reagent (A) can be used as the solid-phase carrier reagent (A) in the immunoassay kit of the present invention, and the above-mentioned labeling reagent (B) is used as the labeling reagent (B). be able to.

Further, the immunoassay buffer solution (W) in the immunoassay kit of the present invention contains the above-mentioned compound (C).

The buffer solution contained in the immunoassay buffer solution (W) used in the immunoassay kit of the present invention includes a Tris buffer solution, a phosphate buffer solution, and a veronal buffer solution, which are generally used in the field of immunoassay. , Borate buffer, Good buffer and the like, and the pH thereof may be in a range that does not impair the effects of the present invention, and is preferably 5 to 9.
Further, in such a buffer solution, salts (sodium chloride, ethylenediamine tetraacetic acid, etc.), albumin (bovine serum albumin, etc.), globulin, proteins (casein hydrolyzate, etc.) are contained as long as the effects of the present invention are not impaired. ), Stabilizers such as water-soluble gelatin and polyethylene glycol, surfactants [surfactants exemplified in the description of the labeling reagent (B) above] and saccharides [in the description of the solid phase carrier reagent (A) above. Examples of sugars, etc.] may be contained.

The concentration of the compound (C) contained in the immunoassay buffer solution (W) is preferably 3 to 10% by weight, preferably 3 to 9% by weight, based on the weight of the immunoassay buffer solution (W). It is more preferably present, and particularly preferably 3 to 6% by weight.

The immunoassay kit of the present invention preferably further contains a chemiluminescent reagent (E).
Further, the kit for immunomeasurement of the present invention includes a luminol luminescent reagent (E1) [a chemiluminescent reagent first solution in which the 2,3-dihydro-1,4-phthalazinedione compound is luminol and / or a metal salt thereof] and The chemiluminescent reagent (E) containing the chemiluminescent solution (E2) [chemiluminescent reagent second solution in which the oxidizing agent is hydrogen peroxide] is contained as a constituent, and the labeling substance (b) in the labeling reagent (B) is It is preferably a kit for immunoassay, which is peroxidase.

The composition, content, preferred range of each component of the solid phase carrier reagent (A), labeling reagent (B) and chemiluminescent reagent (E) in the immunoassay kit of the present invention can be determined by the above-mentioned immunoassay method. Similar to the one described.

Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto. In the following, parts indicate parts by weight.

<Example 1>
It is composed of a solid phase carrier reagent (A-1), a labeling reagent (B-1), an immune reaction buffer solution (W-1), a luminol luminescent reagent (E1) and a hydrogen peroxide solution (E2) by the method shown below. The immunoassay kit (S-1) of the present invention was obtained.

Fabrication of magnetic particles (PH-1):
<Manufacturing of magnetic metal oxide particles>
186 parts of iron (III) chloride hexahydrate, 68 parts of iron (II) chloride tetrahydrate and 1288 parts of water are charged in a reaction vessel and dissolved to raise the temperature to 50 ° C., and the temperature under stirring is 50 to 55. While maintaining the temperature at ° C., 280 parts of 25 wt% aqueous ammonia was added dropwise over 1 hour to obtain magnetite particles in the 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, the magnetite particles adsorbed with oleic acid obtained by solid-liquid separation by decantation were washed with 1000 parts of water three times, and further washed with 1000 parts of acetone twice, 40. By drying at ° C. for 2 days, ultranormal magnetic metal oxide particles having a volume average particle diameter of 15 nm were obtained.

<Preparation of core layer>
80 parts of superparamagnetic metal oxide particles were added to 240 parts of tetraethoxysilane and dispersed to prepare a dispersion liquid (1).
Next, add 5050 parts of water, 3500 parts of a 25 wt% ammonia aqueous solution, and 400 parts of a nonionic surfactant (“NSA-17”, manufactured by Sanyo Chemical Industries, Ltd.) to the reaction vessel to clear mix (M-Technique Co., Ltd.). ) Was mixed to obtain a solution (2). After raising the temperature to 50 ° C., the dispersion liquid (1) was added dropwise to the solution (2) over 1 hour while stirring at a rotation speed of 6,000 rpm of the clear mix, 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 in which fine particles were present, and a core layer was obtained.

<Making magnetic particles>
80 parts of the core layer, 2500 parts of deionized water, 260 parts of a 25 wt% ammonia aqueous solution, 2500 parts of ethanol, and 1200 parts of tetraethoxysilane were added to the reaction vessel and mixed using Clearmix (manufactured by M-Technique Co., Ltd.). The reaction was carried out for 2 hours while stirring at a rotation speed of 6,000 rpm of the clear mix. After the reaction, the supernatant containing fine particles was removed by centrifugation at 2,000 rpm for 20 minutes. After centrifugation, 4000 parts of deionized water was added to the precipitated particles to redisperse the particles, and the dispersed particles were collected by using a magnet to remove the supernatant 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 in which the fine particles were present was performed 20 times, and then the obtained solid phase was obtained. Classification was performed by precipitating and removing particles having a large particle size by adding 5000 parts of water to disperse the particles and centrifuging at 300 rpm for 10 minutes.
Further, the particles were collected by using a magnet to remove the supernatant liquid. Then, after adding 5000 parts of water to disperse the core-shell particles, the operation of collecting the particles using a magnet and removing the supernatant was performed 10 times, and the target magnetic particles (PH) having a volume average particle diameter of 2.0 μm were performed. -1) was obtained. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-1), the content was 81% by weight.

<Measuring method of volume average particle size of particles (superparamagnetic metal oxide particles and magnetic particles)>
Using a scanning electron microscope (model number: JSM-7000F, manufacturer name: JEOL Ltd.), observe any 200 superparamagnetic metal oxide particles and measure the particle size to obtain the volume average particle size. rice field.
For magnetic particles, the volume average particle diameter was determined by the same method.

<Measuring method of the content of superparamagnetic metal oxide particles>
Observe any 20 magnetic particles with a scanning electron microscope (model number JSM-7000F, manufacturer name JEOL Ltd.) and use an energy dispersive X-ray spectroscope (model number INCA Wave / Energy, manufacturer name Oxford). The content of the ultranormal magnetic metal oxide particles was measured, and the average value thereof was taken as the content S. Further, the silica content was measured by the same measurement, and the average value thereof was taken as the content T. The content of the 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 anti-Tg monoclonal antibody F (ab') ₂ >
10 mg of an anti-Tg monoclonal antibody (mouse) (manufactured by Hytest) was dissolved in 2 ml of 0.1 M acetate buffer having a pH of 4.0, 0.2 mg of pepsin was added, and the mixture was inked at 37 ° C. for 3 hours. After stopping the reaction by adding an appropriate amount of 1M carbonate buffer (pH 9.0) to neutralize (pH: 7.0), use 0.02M phosphate buffer (pH 7.2) containing 0.2M sodium chloride. Gel filtration was performed on an equilibrated Ultragel AcA-44 (LKB) column (02, OX70 cm, manufactured by Sigma Aldrich), followed by 0.05 M phosphate buffer (pH 6.0) containing 5 mM EDTA.2Na. Dialysis was performed to obtain anti-Tg monoclonal antibody F (ab') ₂ .

Preparation of magnetic particles (H1-1) and solid phase carrier reagent (A-1):
Add 40 mg of the produced magnetic particles (PH-1) to a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane, react at 25 ° C. for 1 hour, and collect the magnetic particles with a neodymium magnet. After magnetism, the liquid was removed by suction with an aspirator. Next, 40 mL of deionized water was added, the lid was closed, the polystyrene bottle was slowly inverted and stirred twice, the magnetic particles were collected with a neodymium magnet, and the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 5 times. Next, the washed magnetic particles were added to a polyethylene bottle with a lid containing 40 mL of a 2 wt% glutaraldehyde-containing aqueous solution, and the mixture was reacted at 25 ° C. for 1 hour. Then, 40 mL of deionized water was added to cover the lid, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 10 times.
Further, the washed magnetic particles were added to a polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-Tg monoclonal antibody F (ab') ₂ at a concentration of 10 μg / mL. The reaction was carried out at 25 ° C. for 1 hour. After the reaction, the magnetic particles were collected with a neodymium magnet, and then the phosphate buffer solution containing anti-Tg monoclonal antibody F (ab') ₂ was removed.
The magnetic particles were then placed in a covered polyethylene bottle containing 40 mL of 0.02 M phosphate buffer (pH 7.0) containing 0.001 wt% Tg (thyroglobulin, Hytest) and 1 wt% bovine serum albumin. In addition, the magnetic particles (H1-1) to which the Tg-anti-Tg monoclonal antibody F (ab') ₂ complex is bound by allowing to stand at 25 ° C. for 12 hours (Tg is the anti-Tg monoclonal antibody F (ab'). ) Those bound to the magnetic particles via ₂ ) were adjusted. After collecting the magnetic particles (H1-1) with a neodymium magnet, the supernatant was removed.
Next, the solid phase carrier reagent (A-1) containing the magnetic particles (H1-1) is diluted with the diluted solution described below so that the concentration of the magnetic particles (H1-1) becomes 0.01% by weight. Was prepared.

Preparation of diluent:
10% by weight BSA, 0.1% by weight Naroacty CL-100 [polyoxyalkylene alkyl ether, manufactured by Sanyo Kasei Kogyo Co., Ltd.], 0.1% by weight EDTA (ethylenediamine-N, N, N', N'-tetraacetic acid disodium salt dihydrate, manufactured by Dojin Chemical Research Institute Co., Ltd., 5 wt% mouse serum [manufactured by Cosmo Bio Co., Ltd.] and 0.02 M sodium phosphate (pH 7.0). The diluted solution containing the above was adjusted and stored in a refrigerator (2 to 10 ° C.).

Preparation of immunoassay buffer solution (W-1):
Each component shown in Table 1 was mixed by weight according to Table 1 to obtain an immunoassay buffer solution (W-1).
The following components were used as the components in Table 1.
Phosphate buffer: 0.02M phosphate buffer (pH 7.0)
Compound (C-1) to Compound (C-6): Used by the method described below Compound (C'-1): PEG-20000 [manufactured by Sanyo Chemical Industries, Ltd., number average molecular weight 20000 Polyethylene glycol]
EDTA: ethylenediaminetetraacetic acid disodium salt BSA: (bovine serum albumin)

Production of compound (C-1):
Polyoxypropylene glycol (repeated number of oxypropylene groups: 55) 3208 parts by weight (1 mol part) in a 2 L autoclave equipped with a stirrer, thermometer, pressure gauge, pressure-resistant dropping cylinder, decompression and nitrogen introduction line, hydroxylation. After adding 40 parts of potassium, stirring was started, the mixture was filled with nitrogen, the temperature was raised to 100 ° C., and the mixture was dehydrated at a pressure of −0.1 MPaG for 1 hour.
Next, the temperature was raised to 130 ° C., 812 parts by weight (14 mol) of propylene oxide was sequentially added dropwise over 7 hours at a pressure of 0.3 MPaG or less, and the mixture was stirred at the same temperature for 2 hours until pressure equilibrium was reached (first step). ..
Next, 12188 parts by weight (277 mol parts) of ethylene oxide was sequentially added dropwise over 5 hours at a pressure of 0.3 MPaG or less, and the mixture was stirred at the same temperature for 1 hour until pressure equilibrium was reached (second step).
Then, the mixture was cooled to 60 ° C. and neutralized with 40 parts by weight of acetic acid to obtain compound (C-1) [a compound having a value of x of 277 and a value of y of 69 in the general formula (1)].

Production of compound (C-2):
Add 76 parts by weight (1 mol) of propylene glycol and 8 parts of potassium hydroxide to a 2L autoclave equipped with a stirrer, thermometer, pressure gauge, pressure-resistant dropping cylinder, decompression and nitrogen introduction line, start stirring and fill with nitrogen. After raising the temperature to 100 ° C., the mixture was dehydrated at a pressure of −0.1 MPaG for 1 hour.
Then, the temperature was raised to 130 ° C., and a mixture of 4118 parts by weight (71 mol) of propylene oxide and 12672 parts by weight (288 mol) of ethylene oxide was added dropwise over 50 hours at a pressure of 0.3 MPaG or less, and pressure equilibrium was performed at the same temperature. It was stirred for 3 hours until it became.
Then, the mixture was cooled to 60 ° C. and neutralized with 8 parts by weight of acetic acid to obtain compound (C-2) [a compound having a value of x of 288 and a value of y of 72 in the general formula (1)].

Production of compound (C-3):
Add 76 parts by weight (1 mol) of propylene glycol and 10 parts of potassium hydroxide to a 2L autoclave equipped with a stirrer, thermometer, pressure gauge, pressure-resistant dropping cylinder, decompression and nitrogen introduction line, start stirring and fill with nitrogen. After raising the temperature to 100 ° C., the mixture was dehydrated at a pressure of −0.1 MPaG for 1 hour.
Then, the temperature was raised to 130 ° C., and a mixture of 5336 parts by weight (92 mol parts) of propylene oxide and 16368 parts by weight (372 mol parts) of ethylene oxide was added dropwise over 50 hours at a pressure of 0.3 MPaG or less, and pressure equilibrium was performed at the same temperature. It was stirred for 3 hours until it became.
Then, the mixture was cooled to 60 ° C. and neutralized with 10 parts by weight of acetic acid to obtain compound (C-3). [Compound in which the value of x in the general formula (1) is 372 and the value of y is 93]

Production of compound (C-4):
Add 76 parts by weight (1 mol) of propylene glycol and 21 parts of potassium hydroxide to a 2L autoclave equipped with a stirrer, thermometer, pressure gauge, pressure-resistant dropping cylinder, decompression and nitrogen introduction line, start stirring and fill with nitrogen. After raising the temperature to 100 ° C., the mixture was dehydrated at a pressure of −0.1 MPaG for 1 hour.
Then, the temperature was raised to 130 ° C., and a mixture of 10382 parts by weight (179 mol parts) of propylene oxide and 31680 parts by weight (720 mol parts) of ethylene oxide was added dropwise over 75 hours at a pressure of 0.3 MPaG or less, and pressure equilibrium was performed at the same temperature. It was stirred for 3 hours until it became.
Then, the mixture was cooled to 60 ° C. and neutralized with 21 parts by weight of acetic acid to obtain compound (C-4) [a compound having a value of x of 720 and a value of y of 180 in the general formula (1)].

Production of compound (C-5):
Polyoxypropylene glycol (repeated number of oxypropylene groups: 55) 3208 parts by weight (1 mol part) in a 2 L autoclave equipped with a stirrer, thermometer, pressure gauge, pressure-resistant dropping cylinder, decompression and nitrogen introduction line, hydroxylation. After adding 36 parts of potassium, stirring was started, the mixture was filled with nitrogen, the temperature was raised to 100 ° C., and the mixture was dehydrated at a pressure of −0.1 MPaG for 1 hour.
Then, the temperature was raised to 130 ° C., and 11220 parts by weight (255 mol parts) of ethylene oxide was added dropwise over 5 hours at a pressure of 0.3 MPaG or less, and the mixture was stirred at the same temperature for 1 hour until pressure equilibrium was reached.
Then, the mixture was cooled to 60 ° C. and neutralized with 36 parts by weight of acetic acid to obtain compound (C-5) [a compound having a value of x of 255 and a value of y of 55 in the general formula (1)].

Production of compound (C-6):
Polyoxypropylene glycol (repeated number of oxypropylene groups: 55) 3208 parts by weight (1 mol part) in a 2 L autoclave equipped with a stirrer, thermometer, pressure gauge, pressure-resistant dropping cylinder, decompression and nitrogen introduction line, hydroxylation. 78 parts of potassium was added, stirring was started, the mixture was filled with nitrogen, the temperature was raised to 100 ° C., and the mixture was dehydrated at a pressure of −0.1 MPaG for 1 hour.
Next, the temperature was raised to 130 ° C., 2378 parts by weight (41 mol parts) of propylene oxide was sequentially added dropwise over 10 hours at a pressure of 0.3 MPaG or less, and the mixture was stirred at the same temperature for 2 hours until pressure equilibrium was reached (first step). ..
Then, 25652 parts by weight (583 mol parts) of ethylene oxide was sequentially added dropwise over 5 hours at a pressure of 0.3 MPaG or less, and the mixture was stirred at the same temperature for 1 hour until pressure equilibrium was reached (second step).
Then, the mixture was cooled to 60 ° C. and neutralized with 78 parts by weight of acetic acid to obtain compound (C-6) [a compound having a value of x of 583 and a value of y of 96 in the general formula (1)].

Preparation of labeling reagent (B-1):
Using anti-human IgG polyclonal antibody (rabbit) (manufactured by Dako Japan Co., Ltd.) and POD derived from western wasabi (manufactured by Toyobo Co., Ltd.), literature (S Yoshitake, M Imagawa, E Ishikawa, Etol; Jay Biochem , Vol. 92, 1982, 1413-1424) prepared POD-labeled anti-human IgG polyclonal antibody (rabbit) (F3). A 0.02M phosphate buffer (pH 7.0) containing 0.5% by weight of bovine serum albumin and 1% by weight of Naroacty CL-100 as a surfactant was used as a POD-labeled anti-human IgG polyclonal antibody (rabbit). ) (F3) was diluted to a concentration of 100 nM to prepare a labeling reagent (B-1), which was stored in a refrigerator (2 to 10 ° C.).

Preparation of Luminol Luminescent Reagent (E1):
0.7 g of luminol sodium salt [manufactured by Sigma-Aldrich Japan Co., Ltd.] and 0.1 g of 4- (cyanomethylthio) phenol were charged into a 1,000 mL volumetric flask. Add 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer (10 mM, pH = 8.6) so that the volume of the solution becomes 1,000 mL, and 25 ° C. Luminol luminescent reagent (E1) was prepared by uniformly mixing with. It was stored refrigerated (2-10 ° C) until used for measurement.

Preparation of hydrogen peroxide solution (E2):
6.6 g of hydrogen peroxide [manufactured by Wako Pure Chemical Industries, Ltd., reagent special grade, concentration 30% by weight] was 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 hydrogen peroxide solution (E2). It was stored refrigerated (2-10 ° C) until used for measurement.

<Examples 2 to 8, 22 to 23 and Comparative Examples 1 to 2>
In the production of the immunoassay kit (S-1) in Example 1, the immunoassay buffer solution (W-2) to be obtained by the following method instead of the immunoassay buffer solution (W-1) used. Immunoassay kits (S-2) to (S-8), in the same manner as in Example 1 except that (W-10) and (W'-1) to (W'-2) were used, respectively. (S-22) to (S-23) and (S'-1) to (S'-2) were obtained.
<Preparation of immunoassay buffers (W-2) to (W-10) and (W'-1) to (W'-2)>
Each component shown in Table 1 is mixed by weight according to Table 1 to obtain immunoassay buffers (W-2) to (W-10) and (W'-1) to (W'-2). rice field.

<Example 9>
The magnetic particles in Example 1 except that the input amount of the superparamagnetic metal oxide particles was changed from 80 parts to 63 parts in <Preparation of the core layer> during the production of the magnetic particles (PH-1) in Example 1. The same procedure as in the production of (PH-1) was carried out to obtain magnetic particles (PH-2) having a volume average particle diameter of 2.0 μm. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-2), the content was 61% by weight.
In the subsequent operations of Example 1, the same procedure was carried out except that the magnetic particles (PH-2) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). -Contains magnetic particles (H1-2) to which the anti-Tg monoclonal antibody F (ab') ₂ complex is bound (Tg is bound to the magnetic particles via the anti-Tg monoclonal antibody F (ab') ₂ ). The same procedure as in Example 1 was carried out except that the solid phase carrier reagent (A-2) was prepared to obtain the immunoassay kit (S-9) of the present invention.

<Example 10>
The magnetic particles in Example 1 except that the input amount of the superparamagnetic metal oxide particles was changed from 80 parts to 92 parts in <Preparation of the core layer> during the production of the magnetic particles (PH-1) in Example 1. The same procedure as in the production of (PH-1) was carried out to obtain magnetic particles (PH-3) having a volume average particle diameter of 2.0 μm. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-2), the content was 90% by weight.
In the subsequent operations of Example 1, the same procedure was carried out except that the magnetic particles (PH-3) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). -Contains magnetic particles (H1-3) (Tg bound to magnetic particles via anti-Tg monoclonal antibody F (ab') ₂ ) formed by binding an anti-Tg monoclonal antibody F (ab') ₂ complex. The same procedure as in Example 1 was carried out except that the solid phase carrier reagent (A-3) was prepared to obtain the immunoassay kit (S-10) of the present invention.

<Example 11>
In <Preparation of Magnetic Particles> during the production of magnetic particles (PH-1) in Example 1, a step of "centrifugation at 1200 rpm for 10 minutes" was carried out instead of the step of "centrifugation at 600 rpm for 10 minutes". Further, the same procedure as the production of the magnetic particles (PH-1) in Example 1 was carried out except that the step of "centrifuging at 600 rpm for 10 minutes" was carried out instead of the step of "centrifuging at 600 rpm for 10 minutes". Then, magnetic particles (PH-4) having a volume average particle diameter of 1.0 μm were obtained. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-4), the content was 81% by weight.
In the subsequent operations of Example 1, the same procedure was carried out except that the magnetic particles (PH-4) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). -Contains magnetic particles (H1-4) (Tg bound to magnetic particles via anti-Tg monoclonal antibody F (ab') ₂ ) formed by binding an anti-Tg monoclonal antibody F (ab') ₂ complex. The same procedure as in Example 1 was carried out except that the solid phase carrier reagent (A-4) was prepared to obtain the immunoassay kit (S-11) of the present invention.

<Example 12>
In <Preparation of Magnetic Particles> during the production of magnetic particles (PH-1) in Example 1, a step of "centrifugation at 200 rpm for 10 minutes" was carried out instead of the step of "centrifugation at 600 rpm for 10 minutes". Further, the same procedure as the production of the magnetic particles (PH-1) in Example 1 was carried out except that the step of "centrifuging at 100 rpm for 10 minutes" was carried out instead of the step of "centrifuging at 300 rpm for 10 minutes". Then, magnetic particles (PH-5) having a volume average particle diameter of 5.0 μm were obtained. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-5), the content was 81% by weight.
In the subsequent operations of Example 1, the same procedure was carried out except that the magnetic particles (PH-5) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). -Contains magnetic particles (H1-5) to which the anti-Tg monoclonal antibody F (ab') ₂ complex is bound (Tg is bound to the magnetic particles via the anti-Tg monoclonal antibody F (ab') ₂ ). The same procedure as in Example 1 was carried out except that the solid phase carrier reagent (A-5) was prepared to obtain the immunoassay kit (S-12) of the present invention.

<Example 13>
In the preparation of the solid phase carrier reagent (H1-1) and the solid phase carrier reagent (A-1) in Example 1, the anti-Tg monoclonal antibody produced by the following method instead of the anti-Tg monoclonal antibody F (ab') ₂ By using Fab, magnetic particles (H1-6) formed by binding a Tg-anti-Tg monoclonal antibody Fab complex instead of magnetic particles (H1-1) (Tg is magnetic via an anti-Tg monoclonal antibody Fab). The same procedure as in Example 1 was carried out except that the solid-solid carrier reagent (A-6) containing the substance bound to the particles was prepared, to obtain the immunoassay kit (S-13) of the present invention. ..
<Preparation of anti-Tg monoclonal antibody Fab>
10 mg of anti-Tg monoclonal antibody (mouse) (manufactured by Hytest) was dissolved in 2 ml of 0.02 M phosphate buffer (pH 7.2) containing 0.2 M sodium chloride, 0.2 mg of papain was added, and the temperature was 37 ° C. for 3 hours. Ink buffered. Iodoacetamide was added to the reaction solution to a final concentration of 0.03 M, completely dissolved to stop the reaction, and then equilibrated with 0.02 M phosphate buffer (pH 7.2) containing 0.2 M sodium chloride. Gel filtration was performed with an Ultragel AcA-44 (LKB) column (02, OX70 cm, manufactured by Sigma Aldrich), followed by application to NAb ^TM Protein A Plus Spin Volume (5 mL, manufactured by Thermo Fisher), and the column was passed through. By recovering the liquid, an anti-Tg monoclonal antibody Fab was obtained.

<Example 14>
In the production of the immunoassay kit (S-1) in Example 1, the solid phase carrier reagent (A-7) obtained by the following method was used instead of the solid phase carrier reagent (A-1) and labeled. An immunoassay kit (S-14) was obtained in the same manner as in Example 1 except that the labeling reagent (B-2) was used instead of the reagent (B-1).
<Preparation of solid phase carrier reagent (A-7)>
To a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane, 40 mg of the magnetic particles (PH-1) produced in Example 1 were added, and the mixture was reacted at 25 ° C. for 1 hour. After collecting the magnetic particles with a neodymium magnet, the liquid was sucked and removed with an aspirator. Next, 40 mL of deionized water was added, the lid was closed, the polystyrene bottle was slowly inverted and stirred twice, the magnetic particles were collected with a neodymium magnet, and the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 5 times. Next, the washed magnetic particles were added to a polyethylene bottle with a lid containing 40 mL of a 2 wt% glutaraldehyde-containing aqueous solution, and the mixture was reacted at 25 ° C. for 1 hour. Then, 40 mL of deionized water was added to cover the lid, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 10 times.
Further, the washed magnetic particles are placed in a polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing an anti-Tg monoclonal antibody (mouse) (manufactured by Hytest) at a concentration of 10 μg / mL. In addition, the reaction was carried out at 25 ° C. for 1 hour. After the reaction, the magnetic particles were collected with a neodium magnet, and then the phosphate buffer containing the anti-Tg monoclonal antibody was removed to obtain magnetic particles (H-1) to which the anti-Tg monoclonal antibody was bound.
Next, the magnetic particles (H-1) were diluted with the "diluting solution" used in the preparation of the solid phase carrier reagent (A-1) so that the concentration of the magnetic particles (H-1) was 0.01% by weight, and the magnetic particles (H-1) were (diluted). A solid phase carrier reagent (A-7) containing H-1) was prepared.
<Preparation of labeling reagent (B-2)>
The labeling reagent (B-1) in Example 1 was prepared in the same manner except that an anti-Tg mouse monoclonal antibody (manufactured by Hytest) was used instead of the anti-human IgG polyclonal antibody (rabbit). (B-2) [Reagent containing 100 nM POD-labeled anti-Tg mouse monoclonal antibody] was prepared and stored in a refrigerator (2 to 10 ° C.).

<Examples 15 to 21>
In the production of the immunoassay kit (S-14) of Example 14, the above-mentioned immunoassay buffers (W-2) to (W-8) are used instead of the immunoassay buffer (W-1). ) Was used, and immunoassay kits (S-15) to (S-21) were obtained in the same manner as in Example 14.

<Examples 24 to 46 and Comparative Examples 3 to 4>
The immunoassay kits (S-1) to (S-23) obtained in Examples 1 to 23 or the immunoassay kits (S'-1) to (S'-2) obtained in Comparative Examples 1 and 2 are used. Using, immunoassay was performed by the following method (sandwich method), and sensitivity and simultaneous reproducibility were evaluated.
In addition, the turbidity of the immunoassay buffer solution (W) contained in each immunoassay kit was evaluated by the following method.

<Evaluation of sensitivity>
Put 0.025 mL each of the solid phase carrier reagent (A) of the immunoassay kit into a test tube, collect magnetic 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, and remove the liquid in the test tube with a neodymium magnet. Was sufficiently separated from the side.
Next, in a test tube, 0.2 mL of the immunoassay buffer (W) and pooled serum [immunosassay kits (S-1) to (S-13) and (S-22) to (S-23). In addition, in the measurement using the immunoassay kits (S'-1) to (S'-2), the immunoassay kit (S-14) using pooled serum containing 5 IU / mL anti-thyroglobulin antibody (TgAb) was used. )-(S-21), using a pooled serum containing 50 ng / mL Tg] 0.05 mL was injected and reacted in a test tube at 37 ° C. for 3 minutes, and the complex [immunity] In the measurement using the measurement kits (S-1) to (S-13) and (S-22) to (S-23) and the immunoassay kits (S'-1) to (S'-2), Magnetic particles / TgAb complex on which Tg is immobilized, magnetic particles / Tg complex on which anti-Tg monoclonal antibody is immobilized in the measurement using immunoassay kits (S-14) to (S-21)] are formed. I let you.
After the reaction, magnetic particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently separated from the side surface. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and after magnetizing, the washing operation of removing the liquid with an aspirator was performed twice.

Subsequently, 0.05 mL of the labeling reagent (B) corresponding to each immunoassay kit was injected into a test tube and reacted at 37 ° C. for 3 minutes in the test tube, and the complex [immunosassay kit (S-1) to In the measurement using (S-13) and (S-22) to (S-23) and the immunoassay kits (S'-1) to (S'-2), Tg-immobilized magnetic particles / TgAb / POD-labeled anti-human IgG polyclonal antibody complex, magnetic particles immobilized with anti-Tg monoclonal antibody / Tg / POD-labeled anti-Tg mouse in the measurement using immunoassay kits (S-14) to (S-21) Monoclonal antibody complex] was formed.
After the reaction, magnetic particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently separated from the side surface. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and after magnetizing, the washing operation of removing the liquid with an aspirator was performed twice.
Finally, 0.07 mL of the chemiluminescent reagent 1st solution (E1) and 0.07 mL of the chemiluminescent reagent 2nd solution (E2) are added at the same time, and the chemiluminescent reaction is carried out at 37 ° C. for 45 seconds. The average amount of chemiluminescence for 45 seconds was measured with a luminometer [“Lumat LB9507” manufactured by Berthold Japan Co., Ltd.]. The average light emission amount at this time was defined as the average light emission amount (Z1).

Further, in the above operation, the average luminescence amount was measured with a luminometer in the same manner except that the "diluted solution" used in the preparation of the solid phase carrier reagent (A-1) was used instead of the "pool serum". It was measured. The average light emission amount at this time was defined as the average light emission amount (Z2).
The value of [average emission amount (Z1)] / [average emission amount (Z2)] was calculated, and this value was used as the sensitivity.
The higher the sensitivity, the better the measurement accuracy.

<Evaluation of turbidity>
0.2 mL of each immunoassay buffer (W) and the above-mentioned pooled serum [immunosassay kits (S-1) to (S-13) and (S-22) to (S-23) and immunoassay kit In the case of (S'-1) to (S'-2), immunoassay kits (S-14) to (S-21) using pooled serum containing 5 IU / mL anti-thyroglobulin antibody (TgAb). In the case of, a pooled serum containing 50 ng / mL Tg was used.] 0.05 mL was mixed at 25 ° C., and a spectrophotometer (manufactured by Shimadzu Corporation, UV-1800, optical path length: 10 mm) was used. It was used to measure the absorbance at 600 nm.
The absorbance that can be measured by the above method is preferably 0.7 or less, and more preferably 0.45 or less.
If it is out of the above range, it means that proteins and the like derived from the measurement sample are precipitated in the process of measurement, and the reproducibility of the measurement may deteriorate.

<Evaluation of simultaneous reproducibility>
In the above <measurement of sensitivity>, the average luminescence amount was measured with a luminometer in the same manner except that the pooled serum was changed as follows.
-Changed "pool serum containing 5 IU / mL anti-thyroglobulin antibody (TgAb)" to "pool serum containing 50 IU / mL anti-thyroglobulin antibody (TgAb)"-"pool serum containing 50 ng / mL Tg" Is changed to "pool serum containing 500 ng / mL Tg". The same pool serum is measured 20 times in total, and the standard deviation of the average luminescence amount in 20 measurements and the average luminescence amount in 20 measurements. The average value was calculated, then the value of "standard deviation / mean value" was calculated, and this was used as the fluctuation coefficient.
The smaller the value of the coefficient of variation, the higher the reproducibility of the measurement, which is preferable.

Since the immunoassay kit and the immunoassay method of the present invention are excellent in accuracy and sensitivity, they can be widely applied to clinical tests such as radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and chemiluminescence immunoassay.

Claims (5)

An immunoassay method for measuring the concentration of the substance (G) to be measured in a sample.
The content of the solid phase carrier (a1) on which the substance (D) specifically bound to the substance to be measured is immobilized and the substance (G) to be measured are the compound (C) represented by the general formula (1). The step (1) of forming a complex (J1) of the substance (D) and the substance to be measured (G) by reacting in a solution containing 3 to 10% by weight, or
The substance (F3) labeled by the labeling substance (b) and specifically bound to the substance to be measured and the substance (G) to be measured are represented by the compound (C) represented by the general formula (1). An immunoassay method comprising a step (2) of reacting in a solution having a content of 3 to 10% by weight to form a complex (J2) of a substance (F3) and a substance to be measured (G).
HO-[(A ¹ O) _x / (A ² O) _y ] -H (1)
[In the general formula (1), A ¹ is an ethylene group; A ² is a propylene group; x is an integer of 250 to 800; y is an integer of 55 to 200; [(A ¹ ). O) _x / (A ² O) _y ] has an arbitrary binding order of (A ¹ O) units having x divalent groups and (A ² O) units having y divalent groups. Indicates that. ] The immunoassay method according to claim 1, wherein the compound (C) represented by the general formula (1) is the compound (C1) represented by the general formula (2).
HO- (A ³ O) _p- (A ⁴ O) _q- (A ⁵ O) _r -H (2)
[In the general formula (2), A ³ and A ⁵ are ethylene groups; A ⁴ is a propylene group; p and r are integers having (p + r) values of 250 to 800, respectively. Yes; q is an integer from 55 to 200. ] The immunoassay method according to claim 1 or 2, wherein the solid phase carrier (a1) is a magnetic particle. An immunoassay kit containing a solid phase carrier reagent (A), a labeling reagent (B), and an immunoassay buffer solution (W) as immunoassay reagents.
The solid phase carrier reagent (A) immobilizes the solid phase carrier (a1) or the substance to be measured (G) or a similar substance (G') on which the substance (D) specifically bound to the substance to be measured is immobilized. Containing the solid phase carrier (a2)
The labeling reagent (B) is a substance labeled with the labeling substance (b), a substance to be measured (F1), a similar substance (F2) or a substance labeled with the labeling substance (b), and is specific to the substance to be measured. Contains the binding substance (F3) and
The immunoassay kit used for the immunoassay method according to any one of claims 1 to 3, wherein the immunoassay buffer solution (W) contains the compound (C) represented by the general formula (1). The immunity according to claim 4, further comprising a chemiluminescent reagent (E) containing a luminol luminescent reagent (E1) and a hydrogen peroxide solution (E2), and the labeling substance (b) in the labeling reagent (B) is peroxidase. Measurement kit.