JP2014153102A - Agglutination accelerator for latex agglutination reaction, method for detecting target substance, and kit used for detection of target substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agglutination accelerator for latex agglutination reaction having excellent effect in accelerating agglutination, a method for detecting a target substance, and a kit used for the detection of the target substance.SOLUTION: The agglutination accelerator for latex agglutination reaction contains a polymer having a recurring unit, the recurring unit having a sulfinyl group in its side chain.

Description

  The present invention relates to an aggregation accelerator for latex aggregation reaction, a method for detecting a target substance, and a kit for use in detecting a target substance.

  An immunolatex agglutination measurement method that utilizes an agglutination reaction that occurs between an antigen (antibody) that is a target substance and an antibody (antigen) against it is widely used in areas such as clinical tests and biochemical research. This measurement method may point to a lack of detection sensitivity compared to other measurement methods (enzyme amplification method, chemiluminescence measurement method, etc.).

  Therefore, an aggregation accelerator for latex aggregation reaction has been proposed. As such an aggregation accelerator, water-soluble polymers such as polyethylene glycol, polyhydric alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose, saccharose, dextran, polyamino acid, alginic acid, aminoethanesulfonic acid derivative, aminopropane sulfonic acid derivative are used. (Patent Documents 1 to 4).

JP 2006-266970 A JP-A-8-278308 JP 2003-294753 A JP 2006-105910 A

  The problem to be solved by the present invention is to provide an aggregation accelerator for latex aggregation reaction that is excellent in the aggregation promotion effect, a method for detecting a target substance, and a kit for use in detecting a target substance.

  Thus, the present inventors have found that a polymer having a repeating unit having a sulfinyl group in the side chain is excellent in the aggregation promoting effect of latex aggregation reaction, and completed the present invention.

  That is, the present invention provides an aggregation accelerator for latex aggregation reaction, which contains a polymer having a repeating unit having a sulfinyl group in the side chain.

  The present invention also relates to a method for detecting a target substance in a specimen by a latex agglutination method, wherein an antigen or antibody against the target substance is immobilized in the presence of a polymer having a repeating unit having a sulfinyl group in the side chain. The detection method is characterized by mixing the prepared particles and a specimen that may contain a target substance.

  Furthermore, the present invention provides a kit for use in detecting a target substance in a specimen by a latex agglutination method, comprising an aggregation accelerator for latex agglutination reaction containing a polymer having a repeating unit having a sulfinyl group in the side chain. A kit characterized by comprising at least the above is provided.

The aggregation accelerator for latex aggregation reaction of the present invention is excellent in the aggregation promotion effect of latex aggregation reaction.
In addition, the target substance detection method and the kit for use in detection of the target substance of the present invention have high detection sensitivity.

[Aggregation accelerator for latex agglutination reaction]
The aggregation accelerator for latex aggregation reaction of the present invention contains a polymer having a repeating unit having a sulfinyl group in the side chain (hereinafter also referred to as a repeating unit (A)).

<Repeating unit (A)>
As said repeating unit (A), what shows hydrophilic property is preferable. Here, in this specification, hydrophilic means that it has a property with strong affinity with water. Specifically, a homopolymer consisting of only one type of repeating unit (having a number average molecular weight of about 10,000 to 100,000 by the measurement method of the Examples) is 1 g or more per 100 g of pure water at room temperature (25 ° C.). When dissolved, the repeating unit is hydrophilic.
Moreover, as said repeating unit (A), the thing of Hydrophile-Lipophile Balance (HLB value) which shows the scale of hydrophilicity hydrophobicity is 10 or more is preferable. In the case of obtaining high hydrophilicity, the HLB value is more preferably 15 or more, further preferably 20 to 40, and particularly preferably 20 to 30.
Further, in this specification, the HLB value means a value calculated from the ratio of the organic value and the inorganic value of the compound (Oda equation), and “Formation Design with Organic Concept Diagram” [1998, NIHON]. EMULSION CO. , LTD]. For example, the HLB value of the repeating unit (A) contained in the copolymer of N-1-1 described in Examples described later is (60 × 1 + 140 × 1 + 100 × 3) / (20 × 10 + 40 × 1 + (− 10 ) × 3) × 10 = 23.8.

The repeating unit (A) is not particularly limited, but nonionic ones are preferred.
In addition to the sulfinyl group, the repeating unit (A) may have a hydrophilic group such as a hydroxy group, a carboxy group, an amino group, a sulfo group, a thiol group, a phosphoric acid group, or an aldehyde group. Further, the position and the number of such hydrophilic groups are arbitrary, but the position is preferably a side chain of the polymer. On the other hand, the number of hydrophilic groups other than sulfinyl groups is preferably 0 to 12, more preferably 0 to 10, more preferably 0 to 5 in one repeating unit from the viewpoint of obtaining appropriate hydrophilicity. More preferably, 0 to 3 is more preferable, 1 to 3 is more preferable, and 2 or 3 is particularly preferable. Among the hydrophilic groups, a hydroxy group is preferable from the viewpoint of obtaining appropriate hydrophilicity. In addition, as long as the effect of this invention is not lost, some sulfinyl groups contained in a polymer may be sulfide groups or sulfonyl groups.

  Specific examples of the repeating unit (A) include a repeating unit containing at least one structure represented by the following formula (1) in the side chain. As the polymer species to be a repeating unit having the structure represented by the formula (1) in the side chain, known polymers can be used, among which (meth) acrylate polymer species and (meth) acrylamide polymer species Styrene-based polymer species are preferred. More specifically, a repeating unit represented by the following formula (2) is exemplified.

[In Formula (1), R < ³ > shows a direct bond or a C1-C24 bivalent organic group, and R < ⁴ > shows a C1-C10 organic group. ]

[In formula (2), R ¹ represents a hydrogen atom or a methyl group, R ² is a group -O-, a group * - (C = O) -O- , group * - (C = O) -NR ⁵ -, group * -NR ⁵ - (C = O) - (R ⁵ is a hydrogen atom or an organic group having 1 to 10 carbon atoms, and *, R ¹ in the formula (2) is attached R represents a position bonded to a carbon atom) or a phenylene group, and R ³ and R ⁴ are as defined above. ]
Here, each symbol in the formulas (1) and (2) will be described in detail.

R ¹ represents a hydrogen atom or a methyl group, preferably a methyl group.

R ² represents a group —O—, a group * — (C═O) —O—, a group * — (C═O) —NR ⁵ —, a group * —NR ⁵ — (C═O) — or phenylene. Indicates a group. Examples of such a phenylene group include a 1,2-phenylene group, a 1,3-phenylene group, and a 1,4-phenylene group.

The number of carbon atoms of the organic group represented by the R ⁵ is preferably 1 to 10, more preferably from 2 to 8, more preferably from 2 to 6. Examples of the organic group include hydrocarbon groups. Such a hydrocarbon group is a concept including an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, and an aromatic hydrocarbon group.

The aliphatic hydrocarbon group in R ⁵ may be linear or branched, and specifically includes a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a sec-butyl group. Alkyl groups such as tert-butyl group, pentyl group, hexyl group, heptyl group and octyl group.
The alicyclic hydrocarbon group is roughly classified into a monocyclic alicyclic hydrocarbon group and a bridged ring hydrocarbon group. Examples of the monocyclic alicyclic hydrocarbon group include cycloalkyl groups such as a cyclopropyl group and a cyclohexyl group. Examples of the bridged ring hydrocarbon group include an isobornyl group.
Examples of the aromatic hydrocarbon group include aryl groups such as a phenyl group.

Among R ² as described above, the group * — (C═O) —O— and the phenylene group are preferable, and the group * — (C═O) —O— is particularly preferable from the viewpoint of the aggregation promoting effect.

R ³ represents a direct bond or a divalent organic group having 1 to 24 carbon atoms. Such direct bonds include single bonds.

Among such R ^3, a divalent organic group having 1 to 24 carbon atoms is preferable. The carbon number of such a divalent organic group is preferably 2 to 18, more preferably 2 to 10, still more preferably 2 to 9, and particularly preferably 3 to 6.

  Examples of the divalent organic group include a divalent hydrocarbon group. The divalent hydrocarbon group is preferably a divalent aliphatic hydrocarbon group and may be linear or branched. Specifically, methane-1,1-diyl group, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2-diyl group, Propane-1,3-diyl group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3-diyl group, butane-1,4-diyl group, pentane-1,4 -Diyl group, pentane-1,5-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, octane-1,8-diyl group, etc. An alkanediyl group may be mentioned.

The divalent hydrocarbon group may have a substituent and may contain an ether bond between carbon-carbon bonds.
Examples of the substituent that the divalent hydrocarbon group may have include the hydrophilic group. The number of the substituents is preferably 1 to 5, more preferably 1 to 3, and further preferably 1 or 2.
Moreover, as the number of the ether bonds which the said bivalent hydrocarbon group may contain, 0-5 are preferable and 0-3 are more preferable.

  Specific examples of the divalent organic group include a linking group represented by the following formula (3) and an alkanediyl group having 1 to 24 carbon atoms, and more preferably represented by the formula (3). A linking group.

[In Formula (3), R ⁶ is a single bond, a group —R ⁸ —O— (R ⁸ represents an alkanediyl group having 1 to 4 carbon atoms) or a linking group represented by the following Formula (4). R ⁷ represents an alkanediyl group having 1 to 4 carbon atoms, n represents 1 or 2, and ** represents a position bonded to a sulfur atom in the formulas (1) and (2). ]

[In the formula (4), R ⁹ represents an alkanediyl group having 1 to 4 carbon atoms, R ¹⁰ represents an alkanediyl group having 2 or 3 carbon atoms, m ¹ represents 1 or 2, m ² Represents an integer of 1 to 3. ]

R ⁶ is preferably a single bond or a group —R ⁸ —O—, and particularly preferably a single bond.

The alkanediyl group represented by R ⁷ , R ⁸ and R ⁹ has 1 to 4 carbon atoms, but 1 or 2 is preferable.
The alkanediyl group may be linear or branched, and examples thereof include the same alkanediyl groups as described above.

The carbon number of the alkanediyl group represented by R ¹⁰ is preferably 2. Examples of the alkanediyl group include the same as those represented by R ⁷ . When m ² is 2 or 3, m ² R ¹⁰ may be the same or different.
Further, 1 is preferable as n and m ^1, 1 or 2 is preferable as m ^2.

R ⁴ represents an organic group having 1 to 10 carbon atoms. Examples of such an organic group include the same groups as those represented by R ⁵ . In addition, when R ⁴ is a hydrocarbon group, such a hydrocarbon group may have a substituent, and the substituent and the number thereof may be the divalent hydrocarbon group. The thing similar to a good thing is mentioned. From the viewpoint of hydrophilicity, R ⁴ preferably does not contain a ring structure such as a cycloalkyl group, an aryl group, or an aralkyl group.

Preferable specific examples of R ⁴ as described above include organic groups having 1 to 10 carbon atoms having the hydrophilic group, more preferably a monovalent group represented by the following formula (5), carbon. It is a C1-C10 alkyl group, More preferably, it is a monovalent group represented by Formula (5).

Wherein (5), k ¹ represents an integer of 1 to 4, k ² is an integer of 0 to 4, *** the formula (1), combined with the sulfur atom in (2) Indicates the position to perform. ]

In formula (5), k ¹ is preferably 1 or 2. As the k ^2, preferably an integer of 0 to 2, 0 or 1 are more preferable.

In addition, the lower limit of the total content of the repeating unit (A) is preferably 10 mol% or more, more preferably 20 mol% or more, more preferably 30 mol in all repeating units from the viewpoint of imparting water solubility and an effect of promoting aggregation. % Or more is more preferable, and 40 mol% or more is more preferable.
On the other hand, the upper limit is preferably 99 mol% or less, more preferably 90 mol% or less, still more preferably 85 mol% or less, from the viewpoint of hydrophobic-hydrophobic interaction between the polymer and latex particles. The mol% or less is more preferable, and the 70 mol% or less is more preferable.
The content of the repeating unit (A) can be measured by ¹³ C-NMR or the like.

<Repeating unit (B)>
The polymer used in the present invention preferably further has a hydrophobic repeating unit (hereinafter also referred to as a repeating unit (B)). Here, “hydrophobic” means having a property of low affinity with water. Specifically, a homopolymer consisting of only one type of repeating unit (having a number average molecular weight of about 10,000 to 100,000 according to the measurement method in Examples) is dissolved in 100 g of pure water at room temperature (25 ° C.). If the amount is less than 1 g, the repeating unit is hydrophobic.
In addition, the HLB value of the repeating unit (B) is preferably less than 20, more preferably less than 15, more preferably less than 10 and less than 10, more preferably 0.1 to 5 in order to obtain high hydrophobicity. Particularly preferred.

  Examples of the repeating unit (B) include those known to exhibit hydrophobicity, and are not particularly limited. However, from the viewpoint of the hydrophobic-hydrophobic interaction between the polymer and latex particles, styrenes, (meth) acrylates, and (meta ) Those derived from one or more monomers selected from acrylamides are preferred, and those derived from styrenes are more preferred.

  As the repeating unit derived from the styrenes, a repeating unit represented by the following formula (6) is preferable.

[In Formula (6), R < ¹¹ > shows a hydrogen atom or a methyl group, R < ¹² > shows a C1-C10 organic group, and p shows the integer of 0-5. ]

In formula (6), examples of the organic group represented by R ¹² include the same groups as those represented by R ⁵ , but the carbon number thereof is preferably 1 to 6, and more preferably 1 to 3. It is. Moreover, the thing without a hydrophilic group is preferable. Such an organic group may be substituted with an alkoxy group having 1 to 3 carbon atoms. Also, when p is integer of 2 to 5, p number of R ¹² may be the same or different.

  Moreover, although p shows the integer of 0-5, 0-3 are preferable and 0 is more preferable.

  Specific examples of the repeating unit derived from styrenes include styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, 4-ethylstyrene, 4-isopropylstyrene, 4-tert- Examples thereof include repeating units derived from butylstyrene, α-methylstyrene and the like.

Examples of the (meth) acrylates include (meth) acrylic acid C such as methyl (meth) acrylate, ethyl (meth) acrylate, isobutyl (meth) acrylate, and 2-ethylhexyl (meth) acrylate. _1-10 alkyl; (meth) acrylic acid C _6-10 cycloalkyl such as methacrylic cyclohexyl; (meth) acrylic such as 1-methoxyethyl (meth) acrylate and 2-methoxyethyl (meth) acrylate Acid C _1-10 alkoxy C _1-10 alkyl; (meth) acrylate 1-adamantyl, (meth) acrylate 1-methyl- (1-adamantylethyl), (meth) acrylate tricyclo [5.2.1. And (meth) acrylic acid ester having a bridged cyclic hydrocarbon group having 8 to 16 carbon atoms such as 0 ^2,6 ] decan-8-yl. In these (meth) acrylates, the C _1-10 alkyl group is preferably a C _1-8 alkyl group, and the C _6-10 cycloalkyl group is preferably a C _6-8 cycloalkyl group. _{The 1-10} alkoxy group is preferably a C _1-6 alkoxy group, and the bridged ring hydrocarbon group having 8 to 16 carbon atoms is preferably a bridged ring hydrocarbon group having 8 to 12 carbon atoms.

Examples of the (meth) acrylamides include N, N-diC _1-10 alkyl (meth) acrylamide; N-C _1-10 alkyl (meth) acrylamide such as N-isopropyl (meth) acrylamide; N In addition to N—C _2-10 alkanoyl C _1-10 alkyl (meth) acrylamide such as — (1,1-dimethyl-2-acetylethyl) (meth) acrylamide, (meth) acryloylpiperidine and the like can be mentioned. In these (meth) acrylamides, the C _1-10 alkyl group is preferably a C _3-10 alkyl group, and the C _2-10 alkanoyl group is preferably a C _2-6 alkanoyl group.

Further, the lower limit of the total content of the repeating unit (B) is preferably 1 mol% or more, more preferably 10 mol% or more in all repeating units from the viewpoint of the hydrophobic-hydrophobic interaction between the polymer and latex particles. 15 mol% or more is more preferable, 20 mol% or more is more preferable, and 30 mol% or more is more preferable.
On the other hand, the upper limit is preferably 90 mol% or less, more preferably 80 mol% or less, still more preferably 70 mol% or less, and even more preferably 60 mol% or less in all repeating units from the viewpoint of imparting water solubility and promoting aggregation. Is more preferable.
In addition, what is necessary is just to measure content of a repeating unit (B) similarly to content of a repeating unit (A).

  Further, the molar ratio [(A) :( B)] of the repeating unit (A) and the repeating unit (B) contained in the polymer is preferably 10: 1 to 10:30 from the viewpoint of the aggregation promoting effect. 10: 2 to 10:20 are more preferable, and 10: 3 to 10:15 are still more preferable.

  The polymer used in the present invention may have a hydrophilic repeating unit (C) other than the repeating units (A) and (B). Such hydrophilic repeating unit (C) includes an anionic monomer (anionic monomer), a cationic monomer (cationic monomer), or a nonionic monomer (nonionic monomer). The thing which originates is mentioned, These 1 type or 2 types or more may be included.

  Examples of the anionic monomer include unsaturated carboxylic acid monomers such as vinyl benzoic acid and (meth) acrylic acid; unsaturated sulfonic acid monomers such as styrene sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid, and isoprene sulfonic acid. Can be mentioned.

  Examples of the cationic monomer include those having an unsaturated bond with a primary to quaternary amino group such as allylamine, aminostyrene, N, N-dimethylaminopropyl (meth) acrylamide methyl chloride quaternary salt and the like.

  In addition, examples of nonionic monomers include unsaturated carboxylic acid ester monomers having a hydroxy group such as hydroxyethyl (meth) acrylate, glyceryl (meth) acrylate, and polyoxyethylene (meth) acrylate; N- (2-hydroxy And (meth) acrylamide monomers having a hydroxy group such as ethyl) (meth) acrylamide.

  The total content of the repeating unit (C) is preferably 0 to 49 mol%, more preferably 0 to 20 mol%, still more preferably 0 to 10 mol%, and 0 to 1 mol% in all repeating units. Particularly preferred.

  In addition, when the polymer used in the present invention is a copolymer, the arrangement of the repeating units is not particularly limited, and the copolymer may be a block copolymer, a graft copolymer, a random copolymer, an alternating copolymer, or the like. Any of copolymers may be used.

Moreover, as a number average molecular weight ( _Mn ) of the polymer used by this invention, 5000-1 million are preferable, 7000-200000 are more preferable, and 10,000-150,000 are especially preferable. By setting the number average molecular weight to 5000 or more, the aggregation promoting effect is improved. On the other hand, by setting the number average molecular weight to 1 million or less, the viscosity is suppressed and handling becomes easy.
Moreover, as a weight average molecular weight ( _Mw ) of the polymer used by this invention, 5000-5 million are preferable, 7000-1 million are more preferable, 7000-200,000 are still more preferable.
Moreover, as molecular weight distribution ( _Mw / _Mn ), 1.0-5.0 are preferable, 1.0-4.0 are more preferable, 1.0-3.0 are still more preferable, 1.5- 2.5 is particularly preferred.
In addition, what is necessary is just to measure the said number average molecular weight and a weight average molecular weight according to the method as described in the Example mentioned later.

Moreover, as a polymer used by this invention, a water-soluble thing is preferable from a viewpoint of the aggregation promotion effect. Here, in the present specification, water-soluble means that the polymer becomes transparent when added to and mixed with water (25 ° C.) so that the polymer solid content is 1% by mass.
Moreover, as a polymer used by this invention, a nonionic thing is preferable.

  Moreover, as a polymer used by this invention, the thing whose HLB value is the range of 10-22 is preferable from a viewpoint of provision of water solubility and an aggregation promotion effect.

Next, the manufacturing method of the polymer used by this invention is demonstrated.
The polymer used in the present invention is (1) introducing a sulfide group into the side chain of a known polymer and converting the sulfide group to a sulfinyl group, and (2) forming a side chain when polymerized. A monomer having a sulfide group in a portion thereof is polymerized or copolymerized with another monomer, and the sulfide group of the obtained (co) polymer is converted into a sulfinyl group, (3) or when the side chain is polymerized A monomer having a sulfinyl group in the portion can be produced by polymerization or copolymerization with other monomers.
The said manufacturing method is concretely demonstrated taking the example of the manufacturing method of the following copolymer (N-1).
That is, a copolymer (S-1) is obtained by the step 1-A-1 and the step 1-A-2, or by the step 1-B or the step 1-C, and the copolymer (G- A copolymer (N-1) is obtained through 1).

  (Each symbol in the formula is as defined above.)

<Step 1-A-1>
Step 1-A-1 is a step of obtaining a copolymer (M-1) by polymerizing the compound (A-1-1) and the compound (B-1) in the presence of a polymerization initiator.
Examples of the compound (A-1-1) include (meth) acrylic acid and the like, and these can be used alone or in combination of two or more.
Moreover, the said styrene is mentioned as a compound (B-1), As the total usage-amount, 0.001-1.5 molar equivalent is preferable with respect to a compound (A-1-1).

Examples of the polymerization initiator include 2,2′-azobis (isobutyronitrile), dimethyl 2,2′-azobis (2-methylpropionate), 2,2′-azobis (4-methoxy). Azo initiators such as -2,4-dimethylvaleronitrile; peroxides such as di (3,5,5-trimethylhexanoyl) peroxide and benzoyl peroxide. These polymerization initiators include one kind. It can be used alone or in combination of two or more.
The total amount of the polymerization initiator used is usually about 0.0002 to 0.2 times by mass with respect to the compound (A-1-1).

Moreover, you may use a solvent and a chain transfer agent for process 1-A-1. Solvents include amide solvents such as dimethylformamide, dimethylacetamide and N-methylpyrrolidone; sulfoxide solvents such as dimethyl sulfoxide; ester solvents such as ethyl acetate, butyl acetate and γ-butyrolactone; aromatics such as toluene and benzene Type | system | group solvent; Ether type | system | group solvents, such as 1, 4- dioxane, diethyl ether, are mentioned, These solvents can be used individually by 1 type or in combination of 2 or more types. The total amount of these solvents used is usually about 0.5 to 15 times by mass relative to the compound (A-1-1).
Examples of the chain transfer agent include mercaptoethanol, thioglycerol, tert-dodecyl mercaptan, and the like.

  The reaction time in step 1-A-1 is not particularly limited, but is usually about 0.5 to 24 hours, and the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 0 to 120 ° C. It is.

<Step 1-A-2>
Step 1-A-2, the copolymer obtained in Step 1-A-1 a -R ² of (M-1), is ring-opening addition to a glycidyl group or an oxetanyl group of the compound (C-1), In this step, a copolymer (S-1) is obtained.
Examples of the compound (C-1) used in Step 1-A-2 include ethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, and the total use amount thereof is in the copolymer (M-1). 1.5-10 molar equivalent is preferable with respect to the repeating unit derived from a compound (A-1-1).

Step 1-A-2 is preferably performed in the presence of a catalyst. Examples of the catalyst include quaternary ammonium salts such as tetrabutylammonium bromide; quaternary phosphonium salts such as tetrabutylphosphonium bromide and tetrabutylphosphonium chloride. These catalysts are used singly or in combination of two or more. it can.
The total amount of the catalyst used is usually about 0.01 to 0.2 molar equivalents with respect to the repeating unit derived from the compound (A-1-1) in the copolymer (M-1).
Moreover, as a solvent used suitably at process 1-A-2, the thing similar to process 1-A-1 is mentioned.

  The reaction time in step 1-A-2 is not particularly limited, but is usually about 1 to 24 hours, and the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 40 to 200 ° C. .

<Step 1-B and Step 1-C>
In step 1-B and step 1-C, compound (A-1-2) or compound (A-1-3) and compound (B-1) are polymerized in the presence of a polymerization initiator, This is a step of obtaining a coalescence (S-1).
Examples of the compound (A-1-2) include glycidyl (meth) acrylate and oxetanyl (meth) acrylate, and examples of the compound (A-1-3) include vinylbenzyl glycidyl ether, 4-hydroxybutyl (meta ) Acrylate glycidyl ether and the like. In addition, these can be used individually by 1 type or in combination of 2 or more types.
Step 1-B and step 1-C may be performed in the same manner as in step 1-A-1.

<Process 2>
Step 2 includes ring-opening addition of —SR ⁴ to the glycidyl group or oxetanyl group of the copolymer (S-1) obtained in Step 1-A-2, Step 1-B or Step 1-C. In this step, a polymer (G-1) is obtained.
Examples of the compound represented by R ⁴ SH used in Step 2 include thioglycerol and mercaptoethanol, and thioglycerol is preferable from the viewpoint of improving hydrophilicity.
The total amount of the compound used is usually 0.1 to 20 molar equivalents relative to the repeating unit derived from the compound (A-1-1), (A-1-2) or (A-1-3). , Preferably 1 to 10 molar equivalents.

Step 2 is preferably performed in the presence of a catalyst. Examples of the catalyst include basic catalysts such as triethylamine and N, N-dimethyl-4-aminopyridine, and these catalysts can be used alone or in combination of two or more.
The total amount of the catalyst used is usually 0.01 to 40 molar equivalents with respect to the repeating unit derived from the compound (A-1-1), (A-1-2) or (A-1-3). .

  Step 2 is preferably performed in the presence of a solvent. As the solvent, in addition to the solvent that can be used in Step 1, an alcohol solvent such as ethanol and methanol, or a mixed solvent thereof can be used, and the total amount used is usually 0 with respect to the copolymer (S-1). About 5 to 20 times the mass.

  The reaction time in step 2 is not particularly limited, but is usually about 1 to 8 hours, and the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 40 to 100 ° C.

  Step 2 may be performed before step 1-B or step 1-C, and then polymerization of step 1-B or step 1-C may be performed.

<Step 3>
Step 3 is a step of obtaining a copolymer (N-1) by converting the sulfide group of the copolymer (G-1) obtained in Step 2 into a sulfinyl group using an oxidizing agent. In addition, as long as the effect of this invention is not lost, some sulfinyl groups contained in a copolymer may become a sulfide group or a sulfonyl group.

The oxidizing agent is roughly classified into an organic oxidizing agent and an inorganic oxidizing agent, and examples of the organic oxidizing agent include peracetic acid, perbenzoic acid, and metachloroperbenzoic acid. On the other hand, examples of the inorganic oxidizing agent include hydrogen peroxide, chromic acid, permanganate and the like. In addition, these oxidizing agents can be used individually by 1 type or in combination of 2 or more types.
Moreover, the usage-amount of an oxidizing agent is 1.0-10.0 mol normally with respect to the repeating unit derived from a compound (A-1-1), (A-1-2) or (A-1-3). It is about equivalent.

Step 3 is preferably performed in the presence of a solvent. Examples of such a solvent include water; amide solvents such as dimethylformamide and dimethylacetamide; alcohol solvents such as methanol and ethanol, and the like. These solvents can be used alone or in combination of two or more. Water and alcohol solvents are preferred.
The total amount of the solvent used is usually about 1 to 20 times by mass with respect to the copolymer (G-1).

  The reaction time in step 3 is not particularly limited, but is usually about 1 to 24 hours, and the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 25 to 70 ° C.

  In each step, isolation of each reaction product may be performed by filtration, washing, drying, recrystallization, reprecipitation, dialysis, centrifugation, extraction with various solvents, neutralization, chromatography, etc., if necessary. Ordinary means may be combined as appropriate.

Then, as shown in the examples below, the polymer having a repeating unit having a sulfinyl group in the side chain obtained as described above may contain a particle to which an antigen or antibody against the target substance is immobilized and the target substance. Incorporation into a reaction system that has a characteristic analyte promotes latex agglutination and improves the detection sensitivity of the target substance. Therefore, even when the target substance is at a low concentration, measurement reproduction is possible. Performance and accuracy can be satisfied.
Therefore, the polymer is useful as an aggregation accelerator for latex aggregation reaction (sensitizer for measuring latex aggregation). Moreover, the said polymer can be used for the aggregation promotion method of latex aggregation reaction.
And such a polymer may be used as it is as an aggregation accelerator for latex aggregation reaction, and can also be used as a raw material for producing an aggregation accelerator for latex aggregation reaction. In addition, the aggregation accelerator for latex aggregation reaction of the present invention is easy to handle because of its good solubility in a solvent, hardly deteriorates with time, and has excellent storage stability.
In this specification, the latex agglutination reaction means that latex particles to which an antibody (or antigen) is bound and an antigen (or antibody) as a target substance bind to form an aggregate of two or more particles. Say.

  Moreover, 0.001-50 mass% is preferable, as for content of the said polymer in the aggregation accelerator for latex aggregation reaction of this invention, 0.01-10 mass% is more preferable, 0.01-5 mass% Is more preferable. Even at such a low concentration, an excellent aggregation promoting effect is exhibited.

Moreover, the concentration of the polymer with respect to the measurement reaction system when using the aggregation accelerator for latex aggregation reaction of the present invention is preferably 0.01 to 20% by mass, more preferably 0.01 to 5% by mass. is there. Even at such a low concentration, an excellent aggregation promoting effect is exhibited.
Here, the above measurement reaction system refers to a system that mixes particles and a specimen and optically measures a latex agglutination reaction.

The aggregation accelerator for latex aggregation reaction of the present invention may contain a solvent in addition to the above polymer, and examples of the solvent include an aqueous medium. Examples of the aqueous medium include various buffers such as a phosphate buffer, a glycine buffer, a Good buffer, a Tris buffer, and an ammonia buffer in addition to water, and may contain one or more of these. Good.
In addition, the aggregation accelerator for latex aggregation reaction of the present invention may contain a pH buffer, a surfactant, a blocking agent and the like in addition to the polymer and the solvent. Examples of the blocking agent include biologically derived water-soluble polymers such as bovine serum albumin (BSA), chemically synthesized water-soluble polymers, and the like.

[Target substance detection method]
In the method for detecting a target substance in a specimen by the latex agglutination method of the present invention, particles in which an antigen or antibody against the target substance is immobilized in the presence of a polymer having a repeating unit having a sulfinyl group in its side chain, It mixes with the sample which may contain a target substance. This detection method may be carried out in the same manner as the conventional immunolatex aggregation measurement.

Examples of the specimen usually include various biological fluid samples such as serum, plasma, urine, saliva, and crushed specimens of feces and foods. As a measurement sample, a sample diluent obtained by diluting a sample with a pH buffer solution, protein, amino acid, or the like may be used.
In addition, target substances contained in such specimens usually include antigens such as receptors, enzymes, blood proteins, infectious disease-related antigens, microorganisms, viruses, hormones, environmental substances (for example, environmental hormones), and the like. It is an antibody against an antigen, and the antibody only needs to have a binding property to a specific antigen, and includes an antibody fragment.

Further, examples of the antigen or antibody against the target substance used in the present detection method include the same antigens and antibodies as those described above, but these antigens or antibodies are immobilized on particles.
Although the said particle | grain is not specifically limited if it is a thing (latex particle) used for an immune latex aggregation measurement, A polymer particle is mentioned. Examples of monomers constituting such polymer particles include (meth) acrylamide, N-hydroxyethyl (meth) acrylamide, N, N-dimethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, and (meth) acryloylmorpholine. (Meth) acrylamide compounds such as N, N-dimethylaminopropylacrylamide, methyl chloride quaternary salt of N, N-dimethylaminopropylacrylamide, diacetone acrylamide and N-vinylacetamide; methoxyethyl (meth) acrylate, ( Methyl) methacrylate, ethyl (meth) acrylate, propyl (meth) acrylate, lauryl (meth) acrylate, cyclohexyl (meth) acrylate, isobornyl (meth) acrylate, benzyl (meth) acrylate , (Meth) acrylate compounds such as glycerol (meth) acrylate, polyethylene glycol (meth) acrylate, 2-hydroxyethyl (meth) acrylate; unsaturated aldehyde compounds such as acrolein; (meth) acrylic acid, itaconic acid, maleic anhydride , Unsaturated carboxylic acid compounds such as crotonic acid or unsaturated carboxylic acid compounds; unsaturated sulfonic acid compounds such as styrene sulfonic acid, isoprene sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid; styrene, aminostyrene, etc. In addition to styrene compounds, allylamine, N-vinyl-2-pyrrolidone and the like can be mentioned.
Among such polymer particles, those which are a copolymer of a styrene compound and an unsaturated carboxylic acid compound are preferred, and a copolymer of styrene and an unsaturated carboxylic acid compound selected from acrylic acid, methacrylic acid and itaconic acid. What is a coalescence is more preferable.

The average particle size of the particles is preferably 0.01 to 1 μm.
Immobilization of the antigen or antibody to the target substance on the particles may be performed according to conventional methods such as physical adsorption by hydrophobic-hydrophobic interaction, chemical bonding using a water-soluble carbodiimide condensing agent, and the like.
The particles may be coated with a blocking agent such as a water-soluble polymer derived from a living body such as bovine serum albumin (BSA) or a chemically synthesized water-soluble polymer. The coating may be carried out according to a conventional method such as dispersing particles in a solution containing the blocking agent, then centrifuging, removing the supernatant, and redispersing the particles again with water or a buffer solution.

Moreover, it is preferable to mix the said particle | grain and a test substance in the range of pH 4.0-10. The mixing temperature is usually in the range of 25 to 45 ° C., and the mixing time is usually 1 to 20 minutes.
The mixing of the particles and the specimen is performed in the presence of a polymer having a repeating unit having a sulfinyl group in the side chain. It is preferable to use a solvent together with the polymer. Examples of the solvent include those exemplified as an aqueous medium, lower alcohols such as ethanol, methanol, and isopropanol; ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; acetate esters such as ethyl acetate and butyl acetate; amides such as dimethylformamide; dimethyl Examples thereof include sulfoxides such as sulfoxide, and one of these may be used alone or two or more of them may be combined. When using a solvent, as the usage-amount of the said polymer, 0.01-20 mass parts is preferable with respect to 100 mass parts of solvents, and 0.01-5 mass parts is more preferable.

  The target substance in the specimen can be detected by optically detecting the agglutination reaction resulting from the mixing of the particles and the specimen, and the concentration of the target substance can also be measured. The optical detection of the agglutination reaction may be performed according to a conventional method using an optical instrument that can detect scattered light intensity, transmitted light intensity, absorbance, and the like.

[Kit for use in target substance detection]
A kit for use in detecting a target substance in a specimen by the latex agglutination method of the present invention comprises at least an aggregation accelerator for latex agglutination reaction containing a polymer having a repeating unit having a sulfinyl group in the side chain. It is a feature.
The kit of the present invention includes a reagent (also referred to as a second reagent) containing particles in which an antigen or antibody against the target substance is immobilized, in addition to the aggregation accelerator for latex aggregation reaction (also referred to as a first reagent). What is further provided is preferable.
The second reagent may contain a solvent in addition to the particles on which the antigen or antibody against the target substance is immobilized. Examples of the solvent include the above aqueous media such as various buffer solutions.
Moreover, the kit of this invention may be equipped with positive control, negative control, serum dilution liquid, etc. other than the said 1st reagent and 2nd reagent. The medium for the positive control and negative control may be a serum or physiological saline containing no measurable target substance, or a solvent. Examples of the solvent include the above aqueous media such as various buffer solutions.

  The kit of the present invention can be used in the method for detecting a target substance of the present invention in the same manner as a kit for use in detecting a target substance in a specimen by a usual latex agglutination method. In addition, the concentration of the target substance can be measured according to a conventional method.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

Each analysis condition in the examples is as follows.
<Molecular weight measurement>
Weight average molecular weight (Mw) and number average molecular weight (Mn) were measured using a TSKgel α-M column manufactured by Tosoh Corporation, flow rate: 0.5 ml / min, elution solvent: NMP solvent (H ₃ PO ₄ : 0.016M, LiBr: 0.030M), column temperature: Measured by gel permeation chromatography (GPC) using polystyrene as a standard under analysis conditions of 40 ° C.
<NMR spectrum>
^{The 13} C-NMR spectrum was measured by a model AVANCE 500 (500 MHz) manufactured by BRUKER using d6-DMSO as a solvent and an internal standard substance.

Example 1 Synthesis of Copolymer (N-1-1) A copolymer (N-1-1) was obtained according to the following synthesis route.

113 g of glycidyl methacrylate and 113 g of styrene, 6.8 g of 2,2′-azobis (isobutyronitrile) as a polymerization initiator, and 475 g of N, N-dimethylformamide were mixed and placed in a flask. Nitrogen was blown into this, the temperature was raised to 70 ° C., polymerized for 6 hours, and then cooled to room temperature. This solution was purified by reprecipitation with methanol and dried under reduced pressure to obtain a copolymer (S-1-1).
In the obtained copolymer (S-1-1), the content of repeating units derived from glycidyl methacrylate was 48 mol%, and the content of repeating units derived from styrene was 52 mol%. These contents were measured by ¹³ C-NMR.

Next, 10 g of the obtained copolymer (S-1-1), 32.1 g of thioglycerol, and 95 g of N, N-dimethylformamide were mixed and placed in a flask. The temperature was raised to 60 ° C. while blowing nitrogen, and 120 g of triethylamine was added as a catalyst, followed by reaction for 4 hours, and then cooled to room temperature. This solution was purified by reprecipitation with water and freeze-dried to obtain a copolymer (G-1-1).
Next, 10 g of the obtained copolymer (G-1-1) was dispersed in 85.5 g of water and put into a flask. To this, 4.5 g of 30% aqueous hydrogen peroxide solution was added and reacted at room temperature for 18 hours. The aqueous solution obtained was dialyzed to obtain a copolymer (N-1-1) (yield: 13%). When this copolymer (N-1-1) and water were mixed and the concentration was adjusted to 1% by mass, the copolymer (N-1-1) was dissolved in water.
Moreover, the number average molecular weight of the obtained copolymer (N-1-1) was 19000, the weight average molecular weight was 30000, and molecular weight distribution was 1.59.
The structure of the copolymer (N-1-1) was confirmed by ¹³ C-NMR.

Example 2 Synthesis of Copolymer (N-1-2) A copolymer (N-1-2) was obtained by the same synthesis route as in Example 1 above.
170 g of glycidyl methacrylate and 56.8 g of styrene, 6.8 g of 2,2′-azobis (isobutyronitrile) as a polymerization initiator, and 475 g of N, N-dimethylformamide were mixed and placed in a flask. Nitrogen was blown into this, the temperature was raised to 70 ° C., polymerized for 6 hours, and then cooled to room temperature. This solution was purified by reprecipitation with methanol and dried under reduced pressure to obtain a copolymer (S-1-2).
In the obtained copolymer (S-1-2), the content of repeating units derived from glycidyl methacrylate was 67 mol%, and the content of repeating units derived from styrene was 33 mol%. These contents were measured in the same manner as in Example 1.

  Next, 10 g of the obtained copolymer (S-1-2) and 44.7 g of thioglycerol and 95 g of N, N-dimethylformamide were mixed and placed in a flask. The temperature was raised to 60 ° C. while blowing nitrogen, and 167 g of triethylamine was added as a catalyst, followed by reaction for 4 hours, and then cooled to room temperature. This solution was purified by reprecipitation with water and freeze-dried to obtain a copolymer (G-1-2).

Subsequently, 10 g of the obtained copolymer (G-1-2) was dispersed in 84.4 g of water and put into a flask. To this, 5.6 g of 30% aqueous hydrogen peroxide solution was added and reacted at room temperature for 18 hours. The aqueous solution obtained was dialyzed to obtain a copolymer (N-1-2) (yield: 18%). When the copolymer (N-1-2) and water were mixed and the concentration was adjusted to 1% by mass, the copolymer (N-1-2) was dissolved in water.
Moreover, the number average molecular weight of the obtained copolymer (N-1-2) was 31000, the weight average molecular weight was 56000, and molecular weight distribution was 1.81.
The structure of the copolymer (N-1-2) was confirmed by ¹³ C-NMR.

Example 3 Synthesis of Copolymer (N-1-3) A copolymer (N-1-3) was obtained by the same synthesis route as in Example 1.
170 g of glycidyl methacrylate and 56.8 g of styrene, 2.27 g of 2,2′-azobis (isobutyronitrile) as a polymerization initiator and 450 g of ethyl acetate were mixed and placed in a flask. Nitrogen was blown into this, the temperature was raised to 70 ° C., polymerized for 8 hours, and then cooled to room temperature. This solution was purified by reprecipitation with methanol and dried under reduced pressure to obtain a copolymer (S-1-3).
In the obtained copolymer (S-1-3), the content of repeating units derived from glycidyl methacrylate was 67 mol%, and the content of repeating units derived from styrene was 33 mol%. These contents were measured in the same manner as in Example 1.

  Next, 10 g of the obtained copolymer (S-1-3) and 44.7 g of thioglycerol and 95 g of N, N-dimethylformamide were mixed and placed in a flask. The temperature was raised to 60 ° C. while blowing nitrogen, and 167 g of triethylamine was added as a catalyst, followed by reaction for 4 hours, and then cooled to room temperature. This solution was purified by reprecipitation with water and freeze-dried to obtain a copolymer (G-1-3).

Next, 10 g of the obtained copolymer (G-1-3) was dispersed in 84.4 g of water and put into a flask. To this, 5.6 g of 30% aqueous hydrogen peroxide solution was added and reacted at room temperature for 18 hours. The aqueous solution obtained was dialyzed to obtain a copolymer (N-1-3) (yield: 15%). When the copolymer (N-1-3) and water were mixed and the concentration was adjusted to 1% by mass, the copolymer (N-1-3) was dissolved in water.
Moreover, the number average molecular weight of the obtained copolymer (N-1-3) was 33000, the weight average molecular weight was 60000, and molecular weight distribution was 1.82.
The structure of the copolymer (N-1-3) was confirmed by ¹³ C-NMR.

  The HLB values (Oda formula) of the copolymers (N-1-1) to (N-1-3) obtained in Examples 1 to 3 are shown in Table 1 below. A homopolymer composed of the repeating units (A) contained in the copolymers (N-1-1) to (N-1-3) was synthesized, and 1 g was added to 100 g of pure water at room temperature (25 ° C.). Dissolved. Moreover, when the homopolymer which consists of the repeating unit (B) which a copolymer (N-1-1)-(N-1-3) has was synthesize | combined and 1 g was added to 100 g of pure water, it was normal temperature (25 degreeC). It did not dissolve completely.

Test Example (1) Preparation of First Reagent Copolymer N-1-1, N-1-2 or N- in 0.1% bovine serum albumin (BSA) -containing 10 mM HEPES buffer (pH 7.5) 1-3 were added so that the final concentration would be the concentration shown in Table 2, respectively. It served as 2 to 7 “first reagents”. In addition, Test No. In No. 1, a 10 mM HEPES buffer solution (pH 7.5) containing 0.1% by mass BSA was used as the “first reagent”.
(2) Preparation of second reagent 10 mass of latex particle for immunodiagnostic (Product No .: G0305 (average particle size: 0.31 μm, content of surface COOH group: 0.21 mmol / g-LTx, manufactured by JSR Corporation)) 1 mL of a 1% aqueous solution and 9 mL of HEPES buffer (0.05 M, pH 7.5) were mixed (particle concentration = 1% by mass), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added thereto. (Dojin Chemical Co., Ltd.) was added so that the final concentration was 0.05% by mass. Furthermore, 0.5 mL of a 10 mg / mL aqueous solution of an anti-CRP (C-reactive protein) antibody (rabbit) was added to this particle dispersion and stirred at room temperature for 3 hours to immobilize the antibody on the surface of the particles.
Thereafter, 0.5 mL of a 1% by mass BSA aqueous solution was added to the particle dispersion, and the mixture was rotated and stirred at room temperature for 10 hours, and centrifuged at 15,000 rpm for 15 minutes to collect particles as a precipitate.
The particles were then resuspended in 10 mL of HEPES buffer (0.05 M, pH 7.5) and dispersed with ultrasound for 10 minutes. After repeating this resuspension and dispersion operation once more, 200 mL of 10 mM HEPES buffer (pH 7.5) containing 0.02 mass% BSA is added to the particle dispersion so that the particle solid content becomes 0.05 mass%. And dispersed with an ultrasonic wave for 10 minutes, and passed through a 0.8 μm disk filter.
Through a series of these operations, a dispersion of antibody-immobilized latex particles coated with BSA as a blocking agent was obtained. This dispersion was designated as Test No. It was used as 1 to 7 “second reagents”.
(3) Evaluation of agglutination accelerator for latex agglutination reaction In the measurement of immune latex agglutination, Hitachi 7180 type automatic analyzer was used, and 3 μL of sample (target substance (CRP antigen) was analyzed at a wavelength of 570 nm and a measurement temperature of 37 ° C. (Diluted solution of physiological saline) and 150 μL of the first reagent prepared in (1) above were added to the cell and stirred uniformly for 5 minutes.
Thereafter, 150 μL of the second reagent prepared in (2) above was added to the cell and stirred uniformly, and then the difference between the absorbance after 50 seconds and the absorbance after 200 seconds was measured. The results are shown in Table 2.

  From the above results, it is understood that the target substance can be detected even at a low antigen concentration by using the aggregation accelerator for latex aggregation reaction containing the copolymers obtained in Examples 1 to 3.

Claims (8)

  An aggregation accelerator for latex aggregation reaction, comprising a polymer having a repeating unit having a sulfinyl group in the side chain.   The aggregation accelerator for latex aggregation reaction according to claim 1, wherein the repeating unit having a sulfinyl group in the side chain is hydrophilic. The aggregation accelerator for latex aggregation reaction according to claim 1 or 2, wherein the repeating unit having a sulfinyl group in the side chain is represented by the following formula (2).

[In formula (2), R ¹ represents a hydrogen atom or a methyl group, R ² is a group -O-, a group * - (C = O) -O- , group * - (C = O) -NR ⁵ -, group * -NR ⁵ - (C = O) - (R ⁵ is a hydrogen atom or an organic group having 1 to 10 carbon atoms, and *, R ¹ in the formula (2) is attached Or a phenylene group, R ³ represents a direct bond or a divalent organic group having 1 to 24 carbon atoms, and R ⁴ represents an organic group having 1 to 10 carbon atoms. . ]   The aggregation accelerator for latex aggregation reaction according to any one of claims 1 to 3, wherein the polymer further has a hydrophobic repeating unit.   The aggregation accelerator for latex aggregation reaction according to claim 4, wherein the hydrophobic repeating unit is a repeating unit derived from one or more monomers selected from styrenes, (meth) acrylates and (meth) acrylamides. .   The HLB value of a polymer is 10-22, The aggregation promoter for latex aggregation reaction of any one of Claims 1-5.   A method for detecting a target substance in a specimen by a latex agglutination method, comprising a particle having an antigen or antibody immobilized on a target substance in the presence of a polymer having a repeating unit having a sulfinyl group in the side chain, and a target substance A detection method comprising mixing a specimen that may contain   A kit for use in detection of a target substance in a specimen by a latex agglutination method, comprising at least an agglutination accelerator for latex agglutination reaction containing a polymer having a repeating unit having a sulfinyl group in the side chain. Kit to do.