阅读说明：本技术 试剂盒、测定试剂盒及测定方法 (Kit, assay kit and assay method ) 是由 大内亮 中村和浩 于 2019-03-15 设计创作，主要内容包括：本发明的课题在于提供一种不使用作为消防法中的危险物的醇而用于准确且以高灵敏度对血清淀粉样蛋白A进行免疫学测定的试剂盒、测定试剂盒及测定方法。根据本发明,提供一种用于测定血清淀粉样蛋白A的试剂盒,其包含：用与血清淀粉样蛋白A具有特异性结合性的第一结合物质进行了改性且具有标记的第一粒子；及至少一种分子量1000以下的非离子性表面活性剂。(The present invention addresses the problem of providing a kit, a measurement kit, and a measurement method for immunologically measuring serum amyloid A accurately and with high sensitivity without using alcohol, which is a hazardous substance in the fire-fighting method. According to the present invention, there is provided a kit for measuring serum amyloid a, comprising: first particles modified with a first binding substance having specific binding to serum amyloid A and having a label; and at least one nonionic surfactant having a molecular weight of 1000 or less.)

1. A kit for the determination of serum amyloid a comprising: first particles modified with a first binding substance having specific binding to serum amyloid A and having a label; and at least one nonionic surfactant having a molecular weight of 1000 or less.

2. The kit according to claim 1, wherein,

the surfactant is a compound having a glucosamine skeleton.

3. The kit according to claim 1 or 2, wherein,

the surfactant is a compound represented by the following formula (1),

[ chemical formula No. 1]

In the formula, R¹Represents a hydrocarbon group which may be substituted, R²、R³、R⁴、R⁵And R⁶Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted, wherein R²、R³、R⁴、R⁵And R⁶At least 3 of which are hydrogen atoms, R⁷Represents a hydrocarbon group which may be substituted.

4. The kit according to claim 1, wherein,

the surfactant is a monosaccharide having a hydrophobic group or a disaccharide having a hydrophobic group.

5. The kit according to claim 4, wherein,

the surfactant is a compound having a glucose skeleton or a maltose skeleton.

6. The kit according to any one of claims 1, 4 and 5, wherein,

the surfactant is a compound represented by the following formula (2) or the following formula (3),

[ chemical formula No. 2]

In the formula, R¹¹Represents an alkoxy group which may be substituted, an alkenyloxy group which may be substituted, an alkynyloxy group which may be substituted, an alkylthio group which may be substituted, an alkenylthio group which may be substituted, an alkynylthio group which may be substituted, R¹²、R¹³、R¹⁴And R¹⁵Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted, wherein R¹²、R¹³、R¹⁴And R¹⁵At least 3 of which are hydrogen atoms,

in the formula, R²¹Represents an alkoxy group which may be substituted, an alkenyloxy group which may be substituted, an alkynyloxy group which may be substituted, an alkylthio group which may be substituted, an alkenylthio group which may be substituted, an alkynylthio group which may be substituted, R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷And R²⁸Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted, wherein R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷And R²⁸At least 3 of which are hydrogen atoms.

7. The kit according to any one of claims 1 to 6, wherein,

the first particles are latex particles.

8. The kit according to any one of claims 1 to 7, wherein,

the first particles have an average particle diameter of 70nm to 500 nm.

9. The kit according to any one of claims 1 to 8, wherein,

the label comprises a fluorescent dye.

10. The kit according to any one of claims 1 to 9, wherein,

the first binding substance is an antibody.

11. The kit according to any one of claims 1 to 10,

the kit further comprises second particles that are modified with a second binding substance that does not specifically bind to serum amyloid A and that are not labeled.

12. An assay kit for assaying serum amyloid a, comprising: the kit of any one of claims 1 to 11; and a substrate having a first metal film on which a third binding substance having specific binding to serum amyloid A or the first binding substance is immobilized.

13. The assay kit according to claim 12, wherein,

the substrate is further provided with a second metal film, and a fourth binding substance that does not bind to serum amyloid a and specifically binds to the first binding substance is immobilized on the second metal film.

14. A method for measuring serum amyloid A in a biological sample, comprising the steps of:

a step of preparing a mixed solution containing: a biological sample containing serum amyloid a; first particles modified with a first binding substance having specific binding to serum amyloid A and having a label; and at least one nonionic surfactant having a molecular weight of 1000 or less;

adding the mixed solution to an injection port at one end of a substrate on which a first metal film is formed, the first metal film having immobilized thereon a third binding substance having specific binding to serum amyloid a;

flowing the mixed liquid onto the substrate; and

and a step of acquiring information of the mark on the first metal film.

15. The method for measuring serum amyloid A according to claim 14, wherein,

the concentration of the surfactant in the mixed solution is 0.01 mass% or more and 10 mass% or less.

16. The method for measuring serum amyloid A according to claim 14 or 15, wherein,

the step of preparing the mixed solution is a step of preparing a mixed solution diluted at least 5 times with respect to the biological sample.

Technical Field

The present invention relates to a kit for measuring serum amyloid a, a measurement kit for measuring serum amyloid a, and a method for measuring serum amyloid a in a biological sample.

Background

Conventionally, fluorescence detection methods have been widely used as highly sensitive and easy measurement methods in biological assays and the like. This fluorescence detection method is a method of detecting fluorescence by irradiating a sample, which is considered to contain a substance to be detected that emits fluorescence by excitation with light of a specific wavelength, with excitation light of the specific wavelength, and then confirming the presence of the substance to be detected by detecting the fluorescence. In addition, when the detection target substance is not a fluorescent substance, a method of detecting fluorescence in the same manner as described above after bringing a substance specifically binding to the detection target substance labeled with a fluorescent dye into contact with the sample, thereby confirming the binding, that is, the presence of the detection target substance, is also widely used.

Patent document 1 and the like propose a method of utilizing the effect of electric field enhancement by plasmon resonance in such a fluorescence detection method in order to improve sensitivity. This is because a sensor chip including a metal layer provided in a predetermined region on a transparent support is provided to generate plasmon resonance, excitation light is incident at a predetermined angle equal to or greater than the total reflection angle on the interface between the support and the metal film from the surface opposite to the metal layer formation surface of the support, surface plasmon is generated in the metal layer by irradiation of the excitation light, and fluorescence is increased by the electric field enhancement effect, thereby increasing signal/noise (S/N).

Serum Amyloid A (also referred to as SAA) is a very hydrophobic protein with a molecular weight of about 11,600, and has a property of sharply increasing the concentration in blood when a living body is subjected to various stimuli such as infection, tumor, and trauma, thereby causing inflammation. Therefore, as an inflammation marker for knowing the degree of inflammation in real time, the SAA concentration in blood is measured by an immunological measurement method.

However, SAA is known to be associated with high-density lipoprotein (HDL) mostly in blood, probably due to high hydrophobicity. The antigenic determinant of SAA is hidden in the portion of lipoprotein that is not in contact with the outside, and sometimes cannot directly react with the antibody. Therefore, in immunological assays, pretreatment for exposing hidden epitopes is often required. For example, patent document 2 discloses that SAA can be measured with high sensitivity and high accuracy by adding a hydrophilic alcohol as a diluent component in a dilution step, which is one step of an immunoassay method. Patent document 3 describes an antibody that reacts with SAA contained in a fraction having a molecular weight of 10 to 40kD obtained by fractionating serum containing high-density lipoprotein and SAA by gel filtration under non-modified conditions.

Disclosure of Invention

Technical problem to be solved by the invention

However, as described in patent document 2, when SAA is measured by adding a hydrophilic alcohol, there is a problem that the alcohol concentration is likely to change by volatilization, and thus SAA cannot be accurately quantified. Further, patent document 3 has a problem that a special method for purifying an antibody is required. The present invention addresses the problem of providing a kit, a measurement kit, and a measurement method for immunologically measuring SAA accurately and with high sensitivity without using alcohol, which is a dangerous substance in the fire control method.

Means for solving the technical problem

As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by using a combination of first particles modified with a first binding substance having specific binding to serum amyloid a and having a label, and at least one nonionic surfactant having a molecular weight of 1000 or less, and have completed the present invention.

That is, according to the present invention, the following inventions are provided.

[1] A kit for the determination of serum amyloid a comprising: first particles modified with a first binding substance having specific binding to serum amyloid A and having a label; and at least one nonionic surfactant having a molecular weight of 1000 or less.

[2] The kit according to [1], wherein the surfactant is a compound having a glucosamine skeleton.

[3] The kit according to [1] or [2], wherein the surfactant is a compound represented by the following formula (1).

[ chemical formula No. 1]

In the formula, R¹Represents a hydrocarbon group which may be substituted, R²、R³、R⁴、R⁵And R⁶Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted. Wherein R is²、R³、R⁴、R⁵And R⁶At least 3 of which are hydrogen atoms. R⁷Represents a hydrocarbon group which may be substituted.

[4] The kit according to [1], wherein the surfactant is a monosaccharide having a hydrophobic group or a disaccharide having a hydrophobic group.

[5] The kit according to [4], wherein the surfactant is a compound having a glucose skeleton or a maltose skeleton.

[6] The kit according to any one of [1], [4] and [5], wherein the surfactant is a compound represented by the following formula (2) or the following formula (3).

[ chemical formula No. 2]

In the formula, R¹¹Represents an optionally substituted alkoxy group, an optionally substituted alkenyloxy group, an optionally substituted alkoxy groupAlkynyloxy, alkylthio which may be substituted, alkenylthio which may be substituted, alkynylthio which may be substituted, R¹²、R¹³、R¹⁴And R¹⁵Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted. Wherein R is¹²、R¹³、R¹⁴And R¹⁵At least 3 of which are hydrogen atoms.

In the formula, R²¹Represents an alkoxy group which may be substituted, an alkenyloxy group which may be substituted, an alkynyloxy group which may be substituted, an alkylthio group which may be substituted, an alkenylthio group which may be substituted, an alkynylthio group which may be substituted, R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷And R²⁸Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted. Wherein R is²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷And R²⁸At least 3 of which are hydrogen atoms.

[7] The kit according to any one of [1] to [6], wherein the first particles are latex particles.

[8] The kit according to any one of [1] to [7], wherein the first particles have an average particle diameter of 70nm or more and 500nm or less.

[9] The kit according to any one of [1] to [8], wherein the label contains a fluorescent dye.

[10] The kit according to any one of [1] to [9], wherein the first binding substance is an antibody.

[11] The kit according to any one of [1] to [10], further comprising second particles which are modified with a second binding substance having no specific binding property to serum amyloid A and have no label.

[12] An assay kit for assaying serum amyloid a, comprising: [1] the kit of any one of [1] to [11 ]; and a substrate having a first metal film on which a third binding substance having specific binding to serum amyloid A or the first binding substance is immobilized.

[13] The measurement kit according to [12], wherein a second metal film is further formed on the substrate, and a fourth binding substance that does not bind to serum amyloid A and specifically binds to the first binding substance is immobilized on the second metal film.

[14] A method for measuring serum amyloid A in a biological sample, comprising the steps of:

a step of preparing a mixed solution containing: a biological sample containing serum amyloid a; first particles modified with a first binding substance having specific binding to serum amyloid A and having a label; and at least one nonionic surfactant having a molecular weight of 1000 or less;

adding the mixed solution to an injection port at one end of a substrate on which a first metal film to which a third binding substance having specific binding to serum amyloid a is immobilized is formed;

flowing the mixture onto the substrate; and

and a step of acquiring information of the mark on the first metal film.

[15] The method for measuring serum amyloid A according to [14], wherein the concentration of the surfactant in the mixed solution is 0.01% by mass or more and 10% by mass or less.

[16] The method for measuring serum amyloid A according to [14] or [15], wherein the step of preparing the mixed solution is a step of preparing a mixed solution diluted at least 5 times with respect to the biological sample.

Effects of the invention

According to the kit, the assay kit and the assay method of the present invention, SAA can be immunologically assayed accurately and with high sensitivity.

Drawings

Fig. 1 shows a schematic view of a sensor chip.

Fig. 2 shows an exploded view of the sensor chip.

Detailed Description

The embodiments of the present invention will be described in detail below.

In the present specification, a numerical range represented by "to" means a range in which numerical values before and after "to" are included as a minimum value and a maximum value, respectively.

[ kit ]

The kit of the present invention is for the determination of serum amyloid a, comprising: first particles modified with a first binding substance having specific binding to serum amyloid A and having a label; and at least one nonionic surfactant having a molecular weight of 1000 or less.

The kit of the present invention includes the first particles and the nonionic surfactant as described above, but the first particles and the nonionic surfactant may be included in the kit as a mixture or may be included in the kit separately.

(serum amyloid A)

The test substance in the present invention is Serum Amyloid A (SAA). SAA is useful as a marker of inflammation. Examples of the serum amyloid a include serum amyloid a in cat serum or cat plasma.

(surfactant)

It is known that in serum, most of SAA exists in a state of being associated with high specific gravity lipoprotein (HDL). Therefore, there is a problem that the recognition sites of serum amyloid a for the first binding substance and the third binding substance having binding properties to serum amyloid a are blocked by HDL, and the first binding substance and the third binding substance cannot bind to serum amyloid a. In order to solve such a phenomenon, the present invention can accurately quantify SAA in serum by dissociating serum amyloid a from HDL using a surfactant.

The surfactant used in the present invention is a nonionic surfactant having a molecular weight of 1000 or less. If the molecular weight of the nonionic surfactant exceeds 1000, a site recognized as a sample as in HDL may be masked, and SAA may not be measured accurately in some cases, so that a nonionic surfactant having a molecular weight of 1000 or less is used. The lower limit of the molecular weight of the surfactant is preferably 200 or more, and more preferably 300 or more. The upper limit of the molecular weight of the surfactant is preferably 900 or less, more preferably 800 or less, further preferably 700 or less, and particularly preferably 600 or less.

In the present invention, a nonionic surfactant having a small influence on the binding property of the binding substance is used. In the case of an ionic surfactant, it may be difficult to accurately measure SAA because it affects the measurement by interacting with a group having a charge in a biological sample, and therefore, a nonionic surfactant is used in the present invention. As another name of the nonionic surfactant, a nonionic surfactant is sometimes referred to. Anionic surfactants are sometimes described as anion surfactants, cationic surfactants as cation surfactants, and zwitterionic surfactants as betaines. In the present invention, nonionics, i.e., nonionic surfactants, are used. The specific nonionic surfactant is preferably selected from ester type, ether type, ester ether type or alkanolamide type surfactants, and more preferably ester type or alkanolamide type surfactants.

As the surfactant, a compound having a glucosamine skeleton is more preferable, and particularly, an alkanolamide type surfactant having a glucosamine skeleton is preferable. Preferred examples of the alkanolamide type nonionic surfactant having a glucosamine skeleton include compounds represented by the following formula (1).

[ chemical formula No. 3]

In the formula, R¹Represents a hydrocarbon group which may be substituted, R²、R³、R⁴、R⁵And R⁶Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted. Wherein R is²、R³、R⁴、R⁵And R⁶At least 3 of which are hydrogen atoms. R⁷Represents a hydrocarbon group which may be substituted.

Further preferable examples of the compound represented by the formula (1) include compounds represented by the following formula (1A) or the following formula (1B).

[ chemical formula No. 4]

[ chemical formula No. 5]

In the formula, R¹And R⁷The same as defined in formula (1).

As the compound represented by formula (1), formula (1A) or formula (1B), the following surfactants are preferably used.

N-octanoyl-N-methyl-D-glucamine (MEGA-8, DOJINDO LABORATORIES)

N-nonanoyl-N-methyl-D-glucamine (MEGA-9, DOJINDO LABORATORIES)

As the surfactant, monosaccharides having a hydrophobic group or disaccharides having a hydrophobic group are also preferable. The monosaccharide having a hydrophobic group or the disaccharide having a hydrophobic group is more preferably a compound having a glucose skeleton or a maltose skeleton. Examples of the compound having a glucose skeleton or a maltose skeleton include compounds represented by the following formula (2) or (3).

[ chemical formula No. 6]

In the formula, R¹¹Represents an alkoxy group which may be substituted, an alkenyloxy group which may be substituted, an alkynyloxy group which may be substituted, an alkylthio group which may be substituted, an alkenylthio group which may be substituted, an alkynylthio group which may be substituted, R¹²、R¹³、R¹⁴And R¹⁵Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted. Wherein R is¹²、R¹³、R¹⁴And R¹⁵At least 3 of which are hydrogen atoms.

In the formula, R²¹Represents an alkoxy group which may be substituted, an alkenyloxy group which may be substituted, an alkynyloxy group which may be substituted, an alkylthio group which may be substituted, an alkenylthio group which may be substituted, an alkynylthio group which may be substituted, R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷And R²⁸Each independently represents a hydrogen atom or a hydrocarbon group which may be substituted. Wherein R is²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷And R²⁸At least 3 of which are hydrogen atoms.

Further preferable examples of the compound represented by the formula (2) or (3) include compounds represented by the following formula (2A) or (3A).

[ chemical formula No. 7]

[ chemical formula No. 8]

In the formula, R¹¹And R²¹The same meanings as defined in the formulae (2) and (3).

As the compound represented by the formula (2A), the following surfactants are preferably used. N-octyl- β -D-glucoside (O001 from DOJINDO laboraries). R in the formula (2A)¹¹Is a compound of octyloxy.

As the compound represented by the formula (3A), the following surfactants are preferably used. n-dodecyl-beta-D-maltoside (D316 manufactured by DOJINDO LABORATORIES)

N-decyl-. beta. -D-maltoside (D382 made by DOJINDO LABORATORIES)

The hydrocarbon group, alkoxy group, alkenyloxy group, alkynyloxy group, alkylthio group, alkenylthio group, and alkynylthio group may be substituted, and examples of the substituent include those described in the following substituent group a. The substituents of substituent group a may be further substituted by the substituents of substituent group a.

Substituent group a:

alkyl groups substituted with a sulfamoyl group, a cyano group, an isocyano group, a thiocyano group, an isothiocyanato group, a nitro group, a nitrosyl group, a halogen atom, a hydroxyl group, an amino group, a mercapto group, an amide group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, a carbamoyl group, an acyl group, an aldehyde group, a carbonyl group, an aryl group, an alkyl group, a halogen atom, a vinyl group, an ethynyl group, a silyl group, and a trialkylsilyl group (trimethylsilyl.

Examples of the hydrocarbon group include an alkyl group, an alkenyl group, and an alkynyl group. The number of carbon atoms of the hydrocarbon group is not particularly limited, but is usually 1 to 20, preferably 1 to 16, and more preferably 1 to 12.

The number of carbon atoms of the alkoxy group, alkenyloxy group, alkynyloxy group, alkylthio group, alkenylthio group, and alkynylthio group is not particularly limited, but is usually 1 to 20, preferably 1 to 16, and more preferably 1 to 12.

(first binding substance)

The first binding substance used in the present invention is a substance having specific binding to serum amyloid a. As the first binding substance, an antibody can be used, but the first binding substance is not limited thereto. Preferably, the first binding substance is an antibody. When the first binding substance is an antibody, examples of the antibody having specific binding to serum amyloid A include antiserum prepared from serum of an animal immunized with serum amyloid A, an immunoglobulin fraction purified from the antiserum, a monoclonal antibody obtained by cell fusion of spleen cells of an animal immunized with serum amyloid A, and fragments thereof [ e.g., F (ab')₂Fab, Fab' or Fv]And the like. The production of these antibodies can be carried out by a conventional method. Further, when the antibody is a chimeric antibody or the like, it may be a modified antibody, and a commercially available antibody or an antibody prepared from animal serum or culture supernatant by a known method may also be used.

The antibody can be used without being limited to the animal species or subclass thereof. For example, the antibody that can be used in the present invention is an antibody derived from an organism that can cause an immune reaction, such as a mouse, a hamster, a goat, a rabbit, a sheep, a cow, a chicken, or the like, specifically, a mouse IgG, a mouse IgM, a mouse IgG, a white mouse IgM, a hamster IgG, a hamster IgM, a rabbit IgG, a rabbit IgM, a goat IgM, a sheep IgG, a sheep IgM, a cow IgG, a cow IgM, a chicken IgY, or the like, and any of polyclonal antibodies or monoclonal antibodies can be used.

In particular, as the first binding substance having specific binding to serum amyloid a by electrostatic interaction, an anti-serum amyloid a antibody is preferably used. In the present invention, the sandwich method is preferably selected, and in this case, it is necessary to coat a substrate with a pair of antibodies, and these anti-SAA monoclonal antibodies can be used on the substrate and the first particles.

(first particles)

The first particles used in the present invention are particles modified with the first binding substance and having a label.

The first particles are preferably dry particles, but are not particularly limited. As the first particles, for example, polymer particles such as polystyrene beads and glass particles such as glass beads can be used as particles that can be generally used in immunoassay. Specific examples of the material of the first particles include polymers using monomers such as styrene, methacrylic acid, glycidyl (meth) acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, or butyl methacrylate, or synthetic polymers such as copolymers using 2 or more monomers, and preferably latexes in which these are uniformly suspended. Further, other organic polymer powder or inorganic substance powder, microorganism, blood cell or cell membrane, liposome, and the like can be mentioned.

When latex particles are used, specific examples of the material of the latex include polystyrene, styrene-acrylic acid copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, polyvinyl acetate acrylate, and the like. As the latex, a copolymer containing at least styrene as a monomer is preferable, and a copolymer of styrene and acrylic acid or methacrylic acid is particularly preferable. The method for producing the latex is not particularly limited, and the latex can be produced by any polymerization method. Among them, if a surfactant is present when an antibody is labeled, it becomes difficult to immobilize the antibody, and in the production of a latex, emulsifier-free emulsion polymerization, that is, emulsion polymerization without using an emulsifier such as a surfactant is preferable.

The first particles have a label. Preferably the label fluoresces. When the latex obtained by polymerization is itself fluorescent, it can be used directly as fluorescent latex particles. When the latex obtained by polymerization is non-fluorescent, fluorescent latex particles can be produced by adding a fluorescent substance (fluorescent dye or the like) to the latex. That is, the fluorescent latex particles can be produced by adding the fluorescent dye to a solution of latex particles containing water and a water-soluble organic solvent, stirring the mixture, and immersing the fluorescent dye in the latex particles.

As the first particles having a label, liposomes, microcapsules, or the like containing a fluorescent dye can also be used as the fluorescent particles. The fluorescent color is not particularly limited as long as it absorbs ultraviolet light and the like to be excited and is released when returning to the background state, and examples of the fluorescent color include yellow-green (excitation wavelength: 505 nm/release wavelength: 515nm, the same applies hereinafter), blue (350 to 356nm/415 to 440nm), red (535 to 580nm/575 to 605nm), orange (540nm/560nm), red-orange (565nm/580nm), deep red (625nm/645nm), and dark red (660nm/680 nm). These fluorescent particles emitting fluorescence can be purchased from, for example, Invitrogen corporation, and sold under the trade name FluoSpheres (registered trademark).

The particle diameter of the first particles having the label is defined as an average particle diameter. The preferable range of the average particle size of the first particles is not particularly limited, and varies depending on the material of the particles, the concentration range for quantifying serum amyloid a, the measurement apparatus, and the like, and is preferably 70nm to 500nm, more preferably 70nm to 300nm, still more preferably 80nm to 250nm, and particularly preferably 90nm to 200 nm.

The average particle diameter of the particles used in the present invention can be measured by a commercially available particle size distribution analyzer or the like. As a method for measuring the particle size distribution, an optical microscopy, a confocal laser microscopy, an electron microscopy, an atomic force microscopy, a static light scattering method, a laser diffraction method, a dynamic light scattering method, a centrifugal precipitation method, an electric pulse measurement method, a chromatography method, an ultrasonic attenuation method, and the like are known, and apparatuses corresponding to the respective principles are sold, and among these methods, it is preferable to measure the particle size by the dynamic light scattering method, and in the present invention, the average particle size is determined as a viscosity at 25 ℃ of 0.8872_CP (0.8872 mPas) and a water refractive index of 1.330, and a median particle diameter (50% diameter, d 50).

The first particles used in the present invention are modified by the above-described first binding substance. The method for binding the first binding substance to the first particles having the label is not particularly limited. For example, any known method for producing a reagent for immunoagglutination can be used, such as the instructions added to FluoSpheres (registered trademark) polystyrene microspheres F8813 described in Japanese patent application laid-open No. 2000-206115 or Molecular Probes. As a method for immobilizing a binding substance such as an antibody on particles, any of a method using physical adsorption and a method using a chemical bond by a covalent bond can be used. As the binding agent for coating the surface of the particles not coated with the binding substance after the binding substance such as an antibody is immobilized on the particles, known substances such as BSA (bovine serum albumin), skimmed milk, casein, a soybean-derived component, a fish-derived component, polyethylene glycol, and the like, and substances containing these substances or commercially available blocking agents for immunoreaction containing the same in properties as these substances can be used. These blocking agents may be subjected to pretreatment such as partial modification by heat, acid/alkali, or the like, as necessary.

(second binding substance)

The kit of the present invention may further comprise second particles which are modified with a second binding substance having no specific binding property to serum amyloid a and have no label.

There is a test sample which is positive by reacting not only a test sample which is positive and contains serum amyloid a but also a test sample which is negative and does not contain serum amyloid a, and it is considered as a problem to solve false positive. The reason for this false positive is not clear, but it is considered that the presence of a certain factor contained in serum may cause a nonspecific reaction. In the present invention, it is preferable to solve such a problem by using second particles which are modified with a second binding substance having no specific binding property to serum amyloid a and have no label simultaneously.

As the second binding substance, a substance having no specific binding property to serum amyloid a can be used, and preferably a substance that is likely to bind to the causative substance that indicates false positive as described above, and more preferably a compound having no affinity for the first binding substance is used. As the second binding substance, an antibody, a Protein (Protein a, Protein G) that binds to an antibody, or the like can be used, and an antibody is preferable. For example, when the second binding substance is an antibody, for example, antiserum prepared from serum of an animal immunized with the antigen, an immunoglobulin fraction purified from the antiserum, a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with serum amyloid A, or a fragment thereof [ e.g., F (ab')₂Fab, Fab' or Fv]And the like. The production of these antibodies can be carried out by a conventional method. Further, when the antibody is a chimeric antibody or the like, it may be a modified antibody, and a commercially available antibody or an antibody prepared from animal serum or culture supernatant by a known method may also be used. In the present invention, as the second binding substance, a mode of using an anti-CRP antibody is particularly preferable.

(second particle)

The second particles have no label. The second particles are preferably dry particles, but are not particularly limited.

As the second particles, for example, polymer particles such as polystyrene beads, glass particles such as glass beads, and the like can be used as particles that can be generally used in immunoassay. Specific examples of the material of the second particles include polymers using monomers such as styrene, methacrylic acid, glycidyl (meth) acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, or butyl methacrylate, or synthetic polymers such as copolymers using 2 or more monomers, and preferably latexes in which these are uniformly suspended. Further, other organic polymer powder or inorganic substance powder, microorganism, blood cell or cell membrane, liposome, and the like can be mentioned.

When latex particles are used, specific examples of the material of the latex include polystyrene, styrene-acrylic acid copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, polyvinyl acetate acrylate, and the like. As the latex, a copolymer containing at least styrene as a monomer is preferable, and a copolymer of styrene and acrylic acid or methacrylic acid is particularly preferable. The method for producing the latex is not particularly limited, and the latex can be produced by any polymerization method. Among them, if a surfactant is present when an antibody is labeled, it becomes difficult to immobilize the antibody, and in the production of a latex, emulsifier-free emulsion polymerization, that is, emulsion polymerization without using an emulsifier such as a surfactant is preferable.

The particle size of the second particles bound with the second binding substance is defined as an average particle size. The average particle size of the second particles varies depending on the material of the particles, the concentration range for quantifying serum amyloid a, the measurement apparatus, and the like, and is preferably 70nm to 500nm, more preferably 100nm to 200nm, still more preferably 120nm to 180nm, and particularly preferably 130nm to 170 nm.

The ratio of the first particles to the second particles is preferably 1 to 10, more preferably 1 to 6, and still more preferably 2 to 6 by mass of the second particles to the first particles.

[ measurement kit ]

The assay kit of the present invention comprises: the kit of the present invention described above; and a substrate having a first metal film on which a third binding substance having a specific binding property to serum amyloid a or the first binding substance (that is, a third binding substance having a specific binding property to serum amyloid a or a third binding substance having a specific binding property to the first binding substance) is immobilized. The substrate may be further provided with a second metal film, and a fourth binding substance that has no binding property to serum amyloid a and has a specific binding property to the first binding substance may be immobilized on the second metal film.

(third binding substance)

The third binding substance is not particularly limited as long as it has specific binding properties to serum amyloid a or the first binding substance, and preferred examples thereof include an antigen, an antibody, or a complex thereof, and an antibody is preferably used. When the third binding substance is an antibody, examples of the antibody having specificity for serum amyloid a include antiserum prepared from serum of an animal that has passed through serum amyloid a, an immunoglobulin fraction purified from the antiserum, a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with serum amyloid a, and fragments [ e.g., F (ab ') 2, Fab', or Fv ] of these. The production of these antibodies can be carried out by a conventional method. Further, when the antibody is a chimeric antibody or the like, it may be a modified antibody, and a commercially available antibody or an antibody prepared from animal serum or culture supernatant by a known method may also be used.

The antibody can be used without being limited to the animal species or subclass thereof. For example, the antibody that can be used in the present invention is an antibody derived from an organism that can cause an immune reaction, such as mouse, white mouse, hamster, goat, rabbit, sheep, cow, chicken, and the like, and specifically, is mouse IgG, mouse IgM, white mouse IgG, white mouse IgM, hamster IgG, hamster IgMAny of polyclonal and monoclonal antibodies can be used, for example, rabbit IgG, rabbit IgM, goat IgG, goat IgM, sheep IgG, sheep IgM, bovine IgG, bovine IgM, and chicken IgY. The fragmented antibody is a molecule derived from a full-length antibody having at least 1 antigen-binding site, specifically Fab, F (ab')₂And the like. These fragmented antibodies are molecules obtained by enzymatic or chemical treatment, or using genetic engineering methods.

The third binding substance having specific binding to the first binding substance is not particularly limited, but preferred examples thereof include an antigen, an antibody, and a complex thereof. The method for producing the antibody and the kind of the antibody are the same as described above.

(fourth binding substance)

The fourth binding substance has no binding property to serum amyloid a and has specific binding property to the first binding substance. As the fourth binding substance, for example, a compound having affinity for the first binding substance, such as an antibody that binds to the first binding substance (antibody), or a Protein (Protein a, Protein G) that binds to the binding substance (antibody), is preferably used, and among these, an antibody can be more preferably used. The method for producing the antibody and the kind of the antibody are the same as those described for the third binding substance. Further, a compound that is in a ligand-non-ligand relationship with the fourth binding substance can be preferably used as a part of the first binding substance bound to the first particles having labels.

(method of immobilizing binding substance on substrate)

The method of immobilizing the third binding substance such as an antibody and the fourth binding substance on the substrate is described in Tech Notes Vol.2-12 and the like, which are provided by Nunc, and any known method of preparing a reagent for general ELISA (enzyme-linked immunosorbent assay) can be used. Further, surface modification can be performed by disposing a self-assembled monolayer (SAM) on the substrate, and as a method for immobilizing an antibody as a binding substance on the substrate, any of a method using physical adsorption and a method using a chemical bond by a covalent bond can be used. As the blocking agent for covering the surface of the substrate, on which the antibody is not coated, after the antibody is immobilized on the substrate, a known substance, for example, BSA (bovine serum albumin), skimmed milk, casein, a soybean-derived component, a fish-derived component, polyethylene glycol, or the like, a commercially available blocking agent for immunoreaction containing these substances or a substance having the same property as these substances, or the like can be used. These blocking agents may be subjected to pretreatment such as partial modification by heat, acid/alkali, or the like, as necessary.

(substrate)

The substrate used in the present invention is a substrate on which a metal film is formed, and the form thereof is not particularly limited, and when a fluorescence detection method (SPF method) based on surface plasmon excitation described later is performed, it is preferable to use a flow path described later and a substrate having a metal film as a reaction site on the surface. As the metal constituting the metal film, a material that can generate surface plasmon resonance, for example, can be used. Preferably, metals such as gold, silver, copper, aluminum, and platinum are used, and gold is particularly preferable. The above metals can be used alone or in combination. In consideration of the adhesion to the substrate, an intervening layer of chromium or the like may be provided between the substrate and the metal layer. The thickness of the metal film is not particularly limited, and is, for example, preferably 0.1nm or more and 500nm or less, more preferably 1nm or more and 200nm or less, and particularly preferably 1nm or more and 100nm or less. If it exceeds 500nm, the surface plasmon phenomenon of the mediator cannot be sufficiently detected. When an interlayer made of chromium or the like is provided, the thickness of the interlayer is preferably 0.1nm to 10 nm.

The metal film may be formed by a general method, and may be formed by, for example, sputtering, magnetron sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like, and in order to achieve good adhesion of the metal film to the substrate, it is preferable to form the metal film by sputtering.

The metal film is preferably disposed on the substrate. Here, "disposed on the substrate" means that the metal film is disposed on the substrate so as to be in direct contact with the substrate, and includes a case where the metal film is not in direct contact with the substrate but is disposed via another layer. As the substrate that can be used in the present invention, for example, optical glass such as BK7 (borosilicate glass), which is one of general optical glasses, or a synthetic resin, specifically, a material made of a material transparent to laser light, such as polymethyl methacrylate, polyethylene terephthalate, polycarbonate, or cycloolefin polymer, can be used. It is desirable that the material of such a substrate is preferably a material which exhibits no anisotropy with respect to polarization and is excellent in processability. Examples of a substrate used for fluorescence detection by the SPF method include a substrate formed by sputtering a gold film on polymethyl methacrylate.

[ method for measuring serum amyloid A ]

The method for measuring serum amyloid A in a biological sample according to the present invention comprises the steps of:

a step of preparing a mixed solution containing: a biological sample containing serum amyloid a; first particles modified with a first binding substance having specific binding to serum amyloid A and having a label; and at least one nonionic surfactant having a molecular weight of 1000 or less;

adding the mixed solution to an injection port at one end of a substrate on which a first metal film to which a third binding substance having specific binding to serum amyloid a is immobilized is formed;

flowing the mixture onto the substrate; and

and a step of acquiring information of the mark on the first metal film.

The biological sample is not particularly limited as long as it is a sample that may contain serum amyloid a as a test substance, and examples thereof include a biological sample, particularly a body fluid (e.g., blood, serum, plasma, cerebrospinal fluid, tear fluid, sweat, urine, pus, nasal discharge, or sputum) or an excretion (e.g., feces) of an animal (particularly, a cat, a dog, or a human), an organ, a tissue, a mucous membrane, and skin.

The nonionic surfactant is used in the pretreatment of biological samples containing SAA. As the pretreatment liquid, a solution containing a surfactant is prepared in advance and mixed with the test body fluid to be used, or a dried surfactant as a solid component is directly added to the biological sample fluid and melted in the test body fluid to be used. In this case, the concentration of the surfactant in the mixed solution in which the biological sample solution and the surfactant are mixed is preferably 0.01 mass% or more and 10 mass% or less, more preferably 0.05 mass% or more and 5 mass% or less, still more preferably 0.1 mass% or more and 3 mass% or less, and particularly preferably 0.1 mass% or more and 2 mass% or less. When the concentration is 0.01% by mass or more, SAA can be efficiently separated from HDL, and when the concentration is 10% by mass or less, SAA can react with the first binding substance on the first particles having the label or the third binding substance immobilized on the first metal film on the substrate in a state in which the SAA is not easily affected by the surfactant.

The mixture may further contain second particles modified with a second binding substance that does not specifically bind to serum amyloid a.

The method for measuring serum amyloid a according to the present invention may further include: bringing the mixed solution into contact with a second metal film to which a fourth binding substance that does not bind to serum amyloid a and binds to the first binding substance is immobilized; detecting a signal corresponding to the mark from the second metal film; and correcting a signal detected from the first metal film using a signal detected from the second metal film.

As the mixed solution of the biological sample and the surfactant, a mixed solution in which the biological sample is diluted can be used. The solution used for dilution is not particularly limited as long as the effect of the present invention can be exhibited, but it is preferable to use physiological saline having a composition close to that of the biological sample. The dilution can be performed at any ratio with respect to the ratio of the biological sample diluted with physiological saline, but in order to provide a kit, a measurement kit, and a measurement method for immunologically measuring SAA, which is the subject of the present invention, accurately and with high sensitivity, it is preferable to perform the measurement of serum amyloid a in a state of being diluted at 5 times or more. SAA can be accurately and highly sensitively measured by diluting a biological sample, but if the dilution rate of the biological sample is very high, the signal itself with respect to the serum amyloid a concentration decreases, so it is preferable to suppress the dilution rate to 200 times or less.

(measurement method)

The measurement method of the present invention can be understood as the broadest concept including detection of the presence or absence of serum amyloid a, measurement (i.e., quantification) of the amount of serum amyloid a, and the like. Specific embodiments of the measurement method of the present invention include a sandwich method and a competition method, and the sandwich method is preferred.

< Sandwich method >

The sandwich method is not particularly limited, and serum amyloid a can be measured, for example, in the following order. First, a first binding substance having specific binding to serum amyloid a and a third binding substance having specific binding to serum amyloid a are prepared in advance. First particles modified with a first binding substance having specific binding properties to serum amyloid A are produced by binding the first binding substance to first particles having a label. Next, a third binding substance is prepared and immobilized on the substrate as a reaction site (test region). Then, a fourth binding substance is prepared and fixed on the substrate as a control region. In addition, a second binding substance that does not specifically bind to serum amyloid a is prepared and bound to second particles that do not have a label, and second particles that are modified with the second binding substance that does not specifically bind to serum amyloid a and that do not have a label are produced. The first particles and the second particles are mixed and contained in a container and dried. A test sample (or an extract thereof) that may contain serum amyloid A, a mixture of first particles and second particles, and a surfactant are mixed, and the mixed solution is applied to a substrate, developed on a flow path on the substrate, and brought into contact with a reaction site. When serum amyloid a is present in the test sample, a reaction (antigen-antibody reaction when an antigen and an antibody are used) is caused between the serum amyloid a and a first binding substance that binds to the first particle and between the serum amyloid a and a third binding substance on the reaction site at the reaction site, and the first particle corresponding to the amount of serum amyloid a is fixed to the reaction site. In the sandwich method, after the reaction between the third binding substance immobilized on the reaction site and serum amyloid a and the reaction between serum amyloid a and the first binding substance bound to the first particles are completed, washing can be performed for the purpose of removing the first particles that are not bound to the test region and the control region on the substrate. By detecting the intensity of the signal from the first particles bound to the reaction site, the concentration of serum amyloid a can be accurately measured. In addition, the fluorescence intensity has a positive correlation with the concentration of serum amyloid a.

< Competition method >

The competition method is not particularly limited, and serum amyloid a can be measured, for example, in the following order. In this technical field, a competition method is known as a method for detecting an antigen of a low-molecular compound that cannot be assayed by the sandwich method. First, a first binding substance having specific binding to serum amyloid a and a second binding substance having no specific binding to serum amyloid a are prepared in advance. The first binding substance is then bound to the first particles, and the second binding substance is bound to the second particles. Further, serum amyloid a itself having binding property to the first binding substance or a compound having an epitope on the first binding substance similarly to serum amyloid a is immobilized on the substrate as a reaction site. Next, the first particles and the second particles are mixed and contained in a container and dried. A test sample (or an extract thereof) that may contain serum amyloid A, a mixture of first particles and second particles, and a surfactant are mixed, spread on a flow path on a substrate, and brought into contact with a reaction site. When serum amyloid a is not present in the test sample, a reaction is caused on the substrate by the first binding substance that binds to the first particles, the serum amyloid a itself that has binding to the first binding substance immobilized on the reaction site, or a similar compound that has an epitope on the first binding substance antibody, as in serum amyloid a. On the other hand, when serum amyloid a is present, the first binding substance binds to serum amyloid a, and therefore, the reaction of serum amyloid a itself having binding to the first binding substance or a compound having a site similar to serum amyloid a and having an epitope with respect to the antibody to the first binding substance is inhibited at the reaction site, and the immobilization of the first labeled particle at the reaction site is inhibited. In the competitive method, a plurality of samples having different serum amyloid a concentrations and known amounts of serum amyloid a are prepared in advance, and fluorescent signals from the reaction sites are measured at different timings while bringing the samples and the binding substance-labeled fluorescent particles into contact with each other at the reaction sites. From these measurement results, the time change (slope) of the fluorescence amount in each serum amyloid a concentration was obtained. The time change is plotted as a Y-axis and the serum amyloid a concentration is plotted as an X-axis, and a calibration curve of the serum amyloid a concentration with respect to the time change of the fluorescence amount is obtained by a positive fitting method such as a least square method. From the calibration curve thus obtained, the amount of serum amyloid a contained in the test sample can be quantified from the result of using the temporal change in the fluorescence amount of the target test sample.

(flow path)

In a preferred embodiment of the present invention, a test sample (or an extract thereof) that may contain serum amyloid a, first particles having a label, and a surfactant are further mixed with second particles as required to prepare a mixture. The mixture can be applied to a substrate and spread along a flow path. The flow path is not particularly limited as long as it is a path through which the test sample and the labeled first particles (and, if necessary, the second particles) flow to the reaction site. The preferred flow path configuration has the following structure: a flow path exists across a dropping port for dropping a test sample liquid containing first particles (and second particles as required) having labels, a metal thin film as a reaction site for immobilizing a third binding substance, and the metal thin film, and the test sample can pass over the metal thin film. It is preferable that the suction port be provided on the opposite side of the dripping port with respect to the metal film.

(method of detecting a signal corresponding to a marker)

In the present invention, a signal corresponding to a mark is detected. As mentioned above, it is preferable that the label emits fluorescence, and in this case, a signal corresponding to the label can be detected by detecting the fluorescence. As a method for detecting fluorescence, for example, a device capable of detecting fluorescence intensity, specifically, a microplate reader, a biosensor for performing fluorescence detection method by surface plasmon excitation (SPF method), or the like is preferably used to detect fluorescence intensity. The detection of the fluorescence intensity is usually completed after a predetermined time, for example, several minutes to several hours after the antigen-antibody reaction. By detecting the degree of immunocomplex formation as the fluorescence intensity, the concentration of serum amyloid a can be quantified from the relationship between the fluorescence intensity and the concentration of serum amyloid a. The fluorescence measurement method may be a microplate reader measurement or a flow rate measurement. The SPF method can measure the SPF with higher sensitivity than a fluorescence detection method based on epi-excitation (epi-fluorescence method).

As the Surface Plasmon Fluorescence (SPF) biosensor, for example, a sensor provided with the following components as described in japanese patent application laid-open No. 2008-249361 can be used: a light guide made of a material that transmits excitation light of a predetermined wavelength; a metal film formed on one surface of the optical waveguide; a light source generating a light beam; an optical system for allowing the light beam to pass through an optical waveguide and to enter an interface between the optical waveguide and the metal film at an incident angle at which surface plasmon is generated; and a fluorescence detection unit that detects fluorescence generated by excitation with an evanescent wave that is enhanced by the surface plasmon polariton.

(method of measuring amount of serum amyloid A)

As an example of the method for quantifying serum amyloid a in the SPF method of the present invention, serum amyloid a can be quantified by the following method. Specifically, a sample containing serum amyloid a at each known concentration is prepared, and fluorescence signals from a fluorescence detection site are measured at different times while flowing to the fluorescence detection site is detected. From these measurement results, the time change (slope) of the fluorescence amount in each serum amyloid a concentration was obtained. The time change is plotted as a Y-axis and the serum amyloid a concentration is plotted as an X-axis, and a calibration curve of the serum amyloid a concentration with respect to the time change of the fluorescence amount is obtained by a positive fitting method such as a least square method. As the optical signal system, the amount of serum amyloid a in the target test sample can be specified from a calibration curve corresponding to each serum amyloid a.

The fluorescence detection (SPF) system based on surface plasmon excitation in the present invention is an assay method for detecting fluorescence from a fluorescent substance depending on the amount of serum amyloid a immobilized on a metal thin film on a substrate, and is a method for detecting a change in optical transparency, for example, as turbidity, by the progress of a reaction in a solution, unlike the so-called latex agglutination method. In the latex agglutination method, an antibody-sensitized latex in a latex reagent is bound and agglutinated with an antigen in a subject by an antibody reaction. The method of quantifying the antigen concentration based on the change in absorbance per unit time obtained by irradiating the aggregates with near infrared light is a latex agglutination method. In the present invention, a very simple method for detecting serum amyloid a can be provided as compared with the latex agglutination method.

The present invention will be described in more detail below with reference to examples of the present invention. The materials, the amounts used, the ratios, the contents of the treatments, the procedures of the treatments, and the like shown in the following examples can be appropriately changed without departing from the gist of the present invention. Therefore, the scope of the present invention is not to be construed as being limited to the specific examples shown below. In the following chemical formula Et represents an ethyl group.