JP2000345052A - Fluorescent polymer microparticle, its production, and reagent and method for fluoro immunoassay utilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain fluorescent polymer microparticles having a long fluorescence lifetime and improved luminance by uniformly dispersing a fluorescent lanthanoid complex in a polymer. SOLUTION: A fluorescent lanthanoid complex (1-30 wt.%) is dissolved in a liquid containing a starting monomer constituting a styrene resin or a (meth)acrylic resin, and the solution is polymerized in the presence of a radical initiator to obtain fluorescent polymer minute particles each of which is composed of the complex uniformly dispersed in the polymer, and which has a particle diameter dispersion coefficient δof at most 1.0 and a volume-average median of particle diameters of 0.05-2 μm. The particle diameter dispersion coefficient δ is a quantity defined by the equation: δ=(D90-D10)/D50 (wherein DA is the particle diameter at which the cumulative volume from the side of the smaller particle diameters of a particle diameter distribution is A%, and A is 10, 50, or 90). The fluorescent lanthanoid complex is one which has ligands each having a carboxylate group (-COO) on the focal point, and comprises aromatic rings in repeating units, and the dendron is one having a 3,4-oxybenzoate structure on the focal point.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a high-brightness fluorescent polymer particle having a long fluorescence lifetime, a method for producing the same, and a fluorescent immunoassay reagent and a fluorescent immunoassay using the same. Since the fluorescent polymer fine particles of the present invention contain the fluorescent lanthanoid complex uniformly at a high concentration, they exhibit high brightness and a long fluorescence lifetime that are not present in conventional polymer fine particles.
For example, by binding an appropriate antibody or antigen to the surface of the polymer microparticles of the present invention, a fluorescent immunoassay reagent for analyzing an antigen or an antibody that selectively binds thereto, particularly a raw material of a reagent for a time-resolved fluorescence immunoassay method Very useful as.

[0002]

2. Description of the Related Art In a lanthanoid trivalent cation (hereinafter abbreviated as Ln ³⁺ ) complex having a dendrimer as a ligand, the dendrimer sensitizes Ln ³⁺ to greatly improve its fluorescence ability. Kawa et al .; Chem. Mate
r. 10, pp. 286-296 (1998). This effect is due to the fact that in a complex of Ln ³⁺ with a polybenzyl ether dendrimer that absorbs ultraviolet light, the spatially widely spread dendrimer first absorbs the energy of ultraviolet light as a “light-collecting antenna,” and then converts this energy to the dendrimer. Focal Point
(point: the starting point of the branched structure of the dendrimer) is understood as the “antenna effect” that is transmitted to the lanthanoid cation located at the point where the lanthanoid cation emits light. It is reported in the above-mentioned literature that such an "antenna effect" is observed even when the complex is dispersed in a matrix polymer (for example, the dendrimer itself). But,
Fine particles of a composition containing a dendrimer complex exhibiting such an "antenna effect" and using an amorphous resin as a matrix, a method for obtaining the same, and the usefulness of such fine particles have not been known.

On the other hand, a fluorescent immunoassay, that is, a fluoroimmunoassay (hereinafter abbreviated as FIA) method is a radioimmunoassay (RIA).
This method is a practical analysis method for trace amounts of biologically active substances utilizing an immune reaction, along with the A) method, enzyme immunoassay (EIA) method, and the like. In recent years, an automatic analyzer has been developed and widely used for clinical tests and the like. Among them, the RIA method has been rapidly developed because of its extremely high sensitivity and high specificity and its analytical operation is simple, and is used in many fields including the clinical diagnostic field. However, there is a problem in handling safety, such as the use of radioactive substances as labels or disposal problems after their use.Recently, non-radioactive immunoassays that use non-radioactive substances instead of radioactive substances for labels Laws (FIA, EIA, etc.) are becoming mainstream.

Conventional FIAs include organic fluorescent substances such as, for example, fluorescein, and europium trivalent cations (E
A label reagent in which a complex of Ln ³⁺ represented by u ³⁺ ) and a low molecular weight organic compound such as ethylenediaminetetraacetic acid (EDTA) is bound to an antibody or an antigen has been used. However, since the fluorescence intensity of the organic fluorescent substance itself is not so strong, it is said that the sensitivity is reduced due to the large background fluorescence emitted from the structure having a fluorescent ability (chromophore) contained in the physiologically active substance to be measured. There were drawbacks. Further, even if the intensity of the excitation light is increased to attempt to improve the fluorescence intensity of the fluorescent label substance, the background fluorescence also increases at the same time, so that the S / N ratio is not significantly improved and the sensitivity is not improved.

In order to solve these drawbacks, a more sensitive time-resolved fluorescence immunoassay (Time Resol)
ved Fluoroimmunoassay, ie T
The R-FIA method has been developed (for example,
546, JP-A-61-128168, JP-A-3-188374, and German Offenlegungsschrift No. 2628158). The TR-FIA method is Eu
^Exciting a fluorescent substance that emits a relatively long fluorescence such as ³⁺ with a continuous pulsed light source, and after quenching the background fluorescence with a short lifetime due to organic substances, time-resolved measurement of only the Ln ³⁺ specific fluorescence signal This is a method based on the principle of integrating for a certain period of time. For this purpose, for example, a substance in which a β-diketonate-type complex of Eu ³⁺ is supported on polymer fine particles is used as an assay reagent. However, the detection sensitivity is still not sufficiently high as compared with the RIA method. There is a demand for improved fluorescent brightness.

There is also a limit on the type of Ln ^{3+ that} can be used. For example, compared to the fluorescence intensity of Eu ³⁺ β-diketonate complex, that of a similar complex of Tb ³⁺ was extremely low, and it was difficult to put to practical use. Therefore, there is also a strong demand for the development of an efficient measurement method for simultaneously measuring several types of specimens at different wavelengths by using a plurality of emission bands having a unique wavelength of Ln ³⁺ .

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel high-brightness heavy-duty compound containing a lanthanoid cation represented by Tb ³⁺ or Eu ^3+. To provide a combined microparticle and a method for producing the same, and to provide a fluorescent immunoassay reagent using these polymer microparticles and a fluorescence immunoassay capable of simultaneously measuring at various different wavelengths using different polymer microparticles in combination Is to do.

[0008]

Means for Solving the Problems In order to achieve the above object, the present inventor has been diligently and particularly concerned with the synthesis of Ln ³⁺ and a coordinating organic compound, and a method for producing polymer fine particles containing the same. As a result of extensive studies, the present inventors have found that dispersing a fluorescent lanthanoid complex inside a polymer results in a fluorescent material having extremely high luminance and a long fluorescence lifetime.

That is, the gist of the present invention is as follows: (1) fluorescent polymer fine particles in which a fluorescent lanthanoid complex is uniformly dispersed in a polymer; (2) raw material monomer or (meth) (3) A method for producing fluorescent polymer fine particles, comprising dissolving a fluorescent lanthanoid complex in a liquid containing a raw material monomer constituting an acrylic resin and then polymerizing the fluorescent lanthanoid complex; A fluorescent immunoassay reagent using polymer fine particles, (4) a fluorescent immunoassay reagent containing Tb ³⁺ and described in (3) above in combination with a fluorescent immunoassay reagent containing any Eu ³⁺ complex Fluorescent immunoassay, characterized by:

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. <Fluorescent Lanthanoid Complex> The fluorescent lanthanoid complex used in the present invention comprises a lanthanoid cation and an organic ligand as components, and sensitizes the ligand (energy of light that excites the ligand). (A phenomenon in which lanthanoid cations emit light).

The lanthanoid cations include C
e³⁺, Pr³⁺, Nd³⁺, Nd⁴⁺, Sm²⁺, Sm³⁺, Eu
²⁺, Eu³⁺, Tb³⁺, Dy³⁺, Dy⁴⁺, Ho³⁺, E
r³⁺, Tm²⁺, Tm³⁺, Yb²⁺, Yb³⁺And the like,
Among them, Pr³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Tb³⁺, D
y³⁺, Ho³⁺, Er³⁺, Tm³⁺, Yb³⁺Trivalent cations such as
Has a long wavelength, a short lifetime,
It is preferable because it emits fluorescent light having a characteristic.
³⁺, Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺, And Tm ³⁺But
More preferred is Eu³⁺And Tb³⁺In terms of emission intensity
Most preferred.

On the other hand, the chemical structure of the organic ligand which can be used in the fluorescent lanthanoid complex used in the present invention is not particularly limited as long as it exhibits a sensitizing effect on a lanthanoid cation. And those having a β-diketonate group or a carboxylate group as a coordination structure are preferred. There is no particular limitation on the combination of these ligands and lanthanoid cations, but in the present invention, preferred combinations in terms of luminescence properties include ligands having a carboxylate group for Tb ³⁺ and Eu ³⁺ , and Ligand having a β-diketonate group for Eu ³⁺ .

As will be described later in the description of the polymer fine particles, the fluorescent lanthanoid complex used in the present invention is converted into an organic liquid such as a polymerizable monomer (eg, styrene, acrylate, methacrylate, etc.). Has good dispersibility, desirably solubility,
This is important for efficiently producing polymer fine particles in which the complex is uniformly dispersed. <Preferred Carboxylate Complex> As the fluorescent lanthanoid complex composed of a ligand having a carboxylate group suitable for the present invention, a dendron having a carboxylate group at a focal point and an aromatic ring as a repeating unit may be used. Can be exemplified as a ligand.

In the present invention, the dendron (Dendro) is used.
n) is a dendrimer (D
endrimer: a generic term for polymer structures having dendritic ordered branches), which is an agreement with a term widely used in the meaning of a molecular building component having such a structural unit,
For example, G. R. Newcome, Newcome; Dend
ric Molecules, Concepts
Synthesis Perspectives (VC
H VerlagsgesellschaftmbH;
Weinheim, Germany; 1996, ISB
N: 3-527-29325-6). The starting point of the branch structure (corresponding to the main point of the fan when the dendron is schematically considered to be a fan shape) is called a focal point, and the degree of the branch is referred to as “generation”.
on)) (see FIG. 1). Although the number of branches at the branch point of the dendron in the present invention is not limited,
Usually two (in the case of FIG. 1) or three, preferably two
It is a book. In the present invention, a structure having one branch point (that is, the first generation) is also regarded as a dendron.

The dendron used in the present invention preferably has an aromatic ring in a repeating unit of its chemical structure. This is because the dendron exhibits the above-mentioned "antenna effect" by absorbing ultraviolet light or visible light. Here, the aromatic ring means, for example, a hydrocarbon aromatic ring such as a benzene ring, a naphthalene ring, and an anthracene ring, and a nitrogen-containing aromatic ring such as a pyridine ring and a quinoline ring. As a structural example of the dendron suitable for the present invention, specifically, aromatic polyethers such as polybenzyl ether, aromatic polyesters such as polyhydroxybenzoic acid,
Aromatic polysulfides such as aromatic polysulfides such as aromatic or semi-aromatic polyamides, aromatic polycarbonates, aromatic polyester carbonates, and polyphenylene sulfides, aromatic polyimides, and aromatic polyamideimides, which contain elements other than carbon in the polymer main chain. Molecular structure, polyphenylene, polyphenylene ethynylene, aromatic conjugated polymer structure whose main chain is composed of carbon-carbon bonds such as polyphenylene ethylene, etc., among which aromatic polyethers such as polybenzyl ether, Aromatic polyesters such as hydroxybenzoic acid and the like are preferable, and aromatic polyethers such as polybenzyl ether are more preferable, and a structure having a 3,5-dioxybenzyl group represented by the following formula (1) as a repeating unit ( C. J. Hawker et al .;
hem. Soc. Chem. Commun. , 101
(See pages 0-1013 (1990)). In addition, as long as the luminescence properties of the complex are not significantly impaired,
These plural kinds of structures may coexist in one dendron.

[0016]

Embedded imageIn addition, Dendron uses the following formula
3,5-dioxybenzoate structure represented by (2a)
Has Tb in terms of emission characteristics.³⁺And Eu ³⁺To
And 3,4-di represented by the following formula (2b)
When having an oxybenzoate structure, Tb³⁺Against
Is particularly suitable.

[0017]

Embedded image The generation of the dendron used in the present invention is not particularly limited, but is usually 1 to 6, preferably 1 to 4 because of the ease of synthesis.
Most preferably, it is 1 to 3, while on the other hand, it is more preferably 2 to 4, most preferably 3 or 4 from the viewpoint of the light emitting effect.

Accordingly, specific structural examples of the carboxylate complex suitable for the present invention include the following structural formulas (3) to (3).
Ln ³⁺ complexes represented by the structural formula (10) (in the following formulas (3) to (6), Ln ³⁺ is Tb ³⁺ or Eb
u3 ⁺ ). Among them, a complex having a third-generation polybenzyl ether dendron carboxylate represented by the following structural formula (5) or (9) as a ligand, and a fourth-generation poly represented by the following structural formula (6) or (10) A complex having benzyl ether dendron carboxylate as a ligand is very preferable in terms of the fluorescence ability of one complex molecule itself. on the other hand,
A complex having a first-generation polybenzyl ether dendron carboxylate represented by the following structural formula (3) or (7) as a ligand, or a second-generation poly represented by the following structural formula (4) or (8) A complex having benzyl ether dendron carboxylate as a ligand is practically very useful in terms of luminance per unit weight of the complex. In particular, the most important points in terms of luminance, fluorescence wavelength, and economy are the first and second structures represented by the following structural formulas (7) and (8).
This is a Tb ³⁺ complex having a generation polybenzyl ether dendron carboxylate as a ligand.

[0019]

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[0020]

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[0021]

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[0022]

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[0023]

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[0024]

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[0025]

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[0026]

Embedded image <Suitable β-diketonate-type Eu ³⁺ complex> A fluorescent lanthanoid complex composed of a ligand having Eu ³⁺ and a β-diketonate group suitable for the present invention is represented by the following general formula (1)
This is represented by 1).

[0027]

Embedded image However, in the general formula (11), R represents an alkyl group having 6 or less carbon atoms or a fluorinated alkyl group having 6 or less carbon atoms,
R ′ represents an aromatic group. Suitable R is, for example, a linear alkyl group such as a methyl group, an ethyl group, an n-butyl group, an n-hexyl group, an alkyl group having a branch such as an isopropyl group or an isobutyl group, a trifluoromethyl group,
Pentafluoroethyl group, heptafluoropropyl group,
A linear perfluoroalkyl group such as a nonafluorobutyl group, a heptafluoroisopropyl group, a perfluoroalkyl group having a branch such as a nonafluoroisobutyl group, and the like, and more preferred are a trifluoromethyl group, a pentafluoroethyl group, Heptafluoropropyl group, linear perfluoroalkyl group such as nonafluorobutyl group,
And a perfluoroalkyl group having 4 or less carbon atoms such as heptafluoroisopropyl group, more preferably a perfluoroalkyl group having 3 or less carbon atoms such as trifluoromethyl group, pentafluoroethyl group and heptafluoropropyl group; It is a fluoromethyl group. On the other hand, suitable R's include aromatic hydrocarbon groups such as phenyl group, naphthyl group, anthracenyl group and pyrenyl group; sulfur atoms such as thienyl group (or tenoyl group) and furanyl group (or furoyl group); And the like. Among them, aromatic groups containing a hetero atom such as phenyl group and naphthyl group are more preferable, and naphthyl group is most preferable. Therefore, specific structural examples suitable for the present invention include a structure having a naphthyl group represented by the following formula (12), a structure in which the naphthyl group of the formula (12) is substituted with a phenyl group, and the like. Among them, a compound of the formula (12) (hereinafter Eu)
(NTA) is abbreviated as ₃ . ) Is optimal.

[0028]

Embedded image <Polymer Fine Particles> The polymer fine particles of the present invention are characterized in that the above-mentioned fluorescent lanthanoid complex is uniformly dispersed inside the polymer. Here, as the polymer, a styrene resin or a (meth) acrylic resin described later is preferable, and a (meth) acrylic resin is particularly preferable from the viewpoint of transparency in an ultraviolet region. Further, when the (meth) acrylic resin is used, when the polymer fine particles of the present invention are used as a fluorescent immunoassay reagent to be described later, the polymer fine particles in the emulsion are unlikely to adhere and remain on the inner wall of the synthetic resin container. In some cases, such secondary advantageous effects as described above can be seen. Note that a polymer obtained by copolymerizing monomers of these plural kinds of polymers may be used.

"Uniform dispersion" in the present invention means that the concentration of the fluorescent lanthanoid complex is constant at an arbitrary position inside the fine particles. For example, according to a conventional technique in which polymer fine particles are first produced and then the fluorescent lanthanoid complex is absorbed from the surface of the fine particles by adsorption or swelling with a solution, the concentration of the complex near the surface of the fine particles is increased in principle. Such a state is not called "uniform dispersion" in the present invention. Therefore, the "uniform dispersion" defined by the present invention
Can be achieved, for example, by dissolving or uniformly dispersing the fluorescent lanthanoid complex at the stage of the raw material (polymerizable monomer) of the polymer fine particles.

The state of “uniform dispersion” of the present invention includes:
Ideally, the fluorescent lanthanoid complex is dissolved in the molecular phase in the matrix phase of the polymer fine particles, but the complex phase may be dispersed as particle domains by phase separation, and the `` uniform As a condition to satisfy "dispersion",
For example, the average particle size (the molecular dispersion state is considered to be zero) of the particle domain phase confirmed by electron microscope observation is 300 nm or less, preferably 100 nm or less, more preferably 80 nm or less, and most preferably 50 nm or less. A state can be illustrated.

The distribution of the complex in the fine particles also needs to be uniform. The condition of the uniform distribution is as follows: EDX (Energy Dispersion) which is an elemental analysis means of a very small area by a transmission electron microscope.
ve X-ray) analysis, the signal intensity ratio between the lanthanoid element contained in the complex and the appropriate element in the background is substantially the same in an arbitrary extremely small area (for example, an observation screen several tens of nm square). Some examples can be given. More specifically, the standard deviation of the signal intensity ratio in an arbitrary observation region of 30 nm square is 2.0 or less, preferably 1.5 or less, more preferably 1.0 or less, further preferably 0.8 or less, Most preferably, it is 0.5 or less. Since the background element used in the EDX analysis is generally preferable to have an atomic number of 11 or more from the viewpoint of measurement accuracy, a transition metal dye (4) generally used for preparing a transmission electron microscope sample of a resin material is used. Ruthenium oxide RuO ₄ and osmium tetroxide OsO ₄
Etc.), the transition metal element can be used as a suitable background element by dyeing the polymer fine particles of the present invention.

As another definition of the “uniform dispersion” state of the present invention, for any polymer fine particle, the fluorescent lanthanoid complex dispersed inside the polymer shows a unique transition point derived from the complex structure. What is not shown in the thermal analysis (DSC analysis) can be exemplified. In other words, when the complex forms a coarse phase separation domain and is dispersed, a unique transition point such as a melting point and a glass transition point of the complex is observed by DSC analysis. This is exemplified as a condition satisfying the “uniform dispersion” of the present invention. Specifically, for example, a glass transition point of a polybenzyl ether dendrimer structure having a 3,5-dioxybenzyl group as a repeating unit (about 38 ° C., Wooley, KL et al .; Ma)
cromolecules, 26, 1514 (19
93) is not observed in the polymer fine particles of the present invention in which the lanthanoid complex having the dendrimer structure (for example, the complex of the structural formula 3 to 10) is uniformly dispersed.

The concentration of the fluorescent lanthanoid complex in the polymer fine particles of the present invention is not limited, but is usually 0.01 to 50.
%, Preferably 0.1 to 50% by weight in terms of luminance, and more preferably 0.1 to 4% in terms of controlling the particle size of the polymer fine particles.
0% by weight, more preferably 1 to 30% by weight, most preferably 2 to 25% by weight. The particle size of the polymer fine particles of the present invention is not particularly limited, but is usually in the range of 0.01 to 20 μm as a volume average median. In particular, when used as a fluorescent immunoassay reagent, the volume average median is preferably 0.05 to 2 μm. If this value is too large, it is not preferable because of sedimentation, etc. Conversely, if this value is too small, it is not preferable in light emission characteristics. For these reasons, the volume average median of the polymer fine particles of the present invention is more preferably 0.1 to 1 μm, and still more preferably 0.2 to 0.8 μm.
m, most preferably 0.2 to 0.6 μm.

The particle size distribution of the polymer fine particles of the present invention is not particularly limited, but is usually set to 2 or less as the particle size dispersion coefficient δ. Here, the particle diameter dispersion coefficient δ is a particle diameter D at which the integrated volume from the smaller particle diameter side becomes A% in the particle diameter distribution measurement result of the polymerized fine particles measured by a general method such as laser light scattering.
_The amount is defined by the following formula (I) using _A. Therefore, in an ideal case where the particle diameters of all the particles are the same (that is, there is no particle diameter distribution), the value of δ becomes zero.

[0035]

Δ = (D ₉₀ −D ₁₀ ) / D ₅₀ (I) In particular, when the polymer fine particles of the present invention are used as a fluorescent immunoassay reagent, the smaller the value of δ, the higher the analysis accuracy. , Preferably 1.0 or less, more preferably 0.8
Or less, more preferably 0.7 or less, and most preferably 0.
6 or less.

The lifetime of the fluorescence generated by the polymer fine particles of the present invention is not particularly limited, but when used as a fluorescent immunoassay reagent, the fluorescence intensity by the pulse excitation light is 1 / e.
(Where e is the base of the natural logarithm) It is desirable that the fluorescence lifetime defined by the time until it becomes 0.005 milliseconds or more. This is because after the background fluorescence (often derived from organic matter, usually having a lifetime of several to several tens of nanoseconds) due to impurities contained in the measurement sample has disappeared, it has a sufficient fluorescence intensity. This is because the principle of TR-FIA measurement is necessary. The fluorescence lifetime depends on the fluorescent species, but is preferably 0.01 ms or more, more preferably 0.1 ms or more, further preferably 0.5 ms or more, and most preferably 1 ms or more. . Particularly when Tb ³⁺ is a fluorescent species, the present invention can make the fluorescence lifetime 1 millisecond or more, which is very useful.

When the polymer fine particles of the present invention are used as a fluorescent immunoassay reagent, it is necessary that the surface of the polymer fine particles has a functional group that binds to an antibody or antigen that binds to an antigen or antibody to be measured. Although there is no limitation on the mode of such bonding, the functional groups provided on the surface of the polymer fine particles are preferably a carboxyl group, an acid halide group such as an acid chloride group, an acid anhydride group, an ester group, an amide group, a maleimide group. , A thiol group, a hydroxyl group, and an amino group, among which a carboxyl group, an acid chloride group, an acid anhydride group, a maleimide group, a thiol group, an amino group, and the like are more preferable, and a carboxyl group or an acid chloride group is most preferable. It is. Since the ester group can be easily converted to a carboxyl group by hydrolysis, polymer fine particles having an ester group on the surface are useful intermediates. The amount of the functional group for binding these antibodies, when expressed as a functional group equivalent per unit weight of the polymer fine particles of the present invention, is usually 0.001 to 0.5 meq / gram, preferably 0.005 meq / g. -0.2 meq / gram, more preferably 0.01-0.1 meq / gram, even more preferably 0.02-0.07 meq / gram, and most preferably 0.03-0.05 meq. Equivalent / gram. <Production method of polymer fine particles> The polymer fine particles of the present invention are:
Usually, it is produced by dispersing a radically polymerizable monomer such as a styrene derivative or an acrylic acid derivative in water or a water-containing organic solvent, and subjecting it to radical polymerization in the presence of a suitable dispersion stabilizer and a radical generator. Either emulsion polymerization, which is a method of adding monomers to a system in which micelles of a dispersion stabilizer is first formed, or monomer droplets stabilized with a dispersion stabilizer, as a general-purpose method that conforms to such a production format, is first used. Solid emulsion such as mini-emulsion polymerization or micro-emulsion polymerization, which is a method of polymerizing after forming a polymer, suspension polymerization, which is a method of polymerizing a solution obtained by adding a radical generator to monomers, or polymer ultra-fine particles Seed polymerization, which is a method of adding monomers to a system in which nuclei are dispersed and polymerizing the same, may be used. Of these methods, preferred are miniemulsion polymerization, microemulsion polymerization or seed polymerization, and particularly preferred are miniemulsion polymerization and microemulsion polymerization.

Suitable examples of the water-containing organic solvent include, for example, a mixture of water and methanol, ethanol, acetone, N, N-dimethylformamide, tetrahydrofuran, etc., and water is most suitable as a dispersion medium. Suitable dispersion stabilizers include, for example, sulfonates such as sodium dodecylbenzenesulfonate and sodium dodecylsulfonate, cationic surfactants such as carboxylate such as sodium palmitate and sodium stearate, and tetraethylammonium. Anionic surfactants having a quaternary onium salt bonded thereto, nonionic surfactants having a water-soluble structure bonded such as polyethylene glycol chains, and water-soluble polymers such as polyethylene glycol, polyvinylpyrrolidone, polyvinylpyridine and polyvinyl alcohol In the case of a type that requires an emulsified state such as mini-emulsion polymerization, micro-emulsion polymerization, emulsion polymerization, and seed polymerization, sodium dodecylbenzenesulfonate, sodium dodecylsulfonate, sodium dihexylsulfate, etc. Cationic surfactants such as sulphonates such as succinate, carboxylate such as sodium palmitate and sodium stearate are preferred, especially in the case of miniemulsion polymerization or microemulsion polymerization, where decyl methacrylate and methacrylic acid are used. Co-stabilizers such as dodecyl acid, hexadecyl methacrylate, and octadecyl methacrylate may be used in combination. In the case of suspension polymerization, water-soluble polymers such as polyethylene glycol, polyvinylpyrrolidone, polyvinylpyridine, and polyvinyl alcohol are used.

A general radical generator is a water-soluble radical generator, such as persulfate, in the case of a type requiring an emulsified state such as miniemulsion polymerization, microemulsion polymerization, emulsion polymerization, or seed polymerization. In the case of suspension polymerization, such as persulfates such as sodium, potassium persulfate, lithium persulfate and ammonium persulfate, which are soluble in radically polymerizable monomers, for example, N, N-azobisisobutyronitrile (AIBN) ), Peroxides such as benzoyl peroxide, etc., but different types of radical initiators may be used in combination.

A preferred method for producing the polymer fine particles of the present invention will be specifically described below. Since the polymer fine particles of the present invention uniformly contain a fluorescent lanthanoid complex therein, it is desirable to disperse (preferably dissolve) the complex in a radically polymerizable monomer in advance. As the radical polymerizable monomer, for example, a raw material monomer constituting a styrene-based resin such as styrene, α-methylstyrene, chloromethylstyrene, 4-acetoxystyrene, or methyl acrylate, ethyl acrylate, isopropyl acrylate, or acrylic Acrylates such as n-butyl acrylate, hexyl acrylate, phenyl acrylate, benzyl acrylate, adamantyl acrylate, 2-hydroxyethyl acrylate, methyl methacrylate, ethyl methacrylate, isopropyl methacrylate, n-methacrylate Methacrylates such as butyl, hexyl methacrylate, phenyl methacrylate, benzyl methacrylate, adamantyl methacrylate, 2-hydroxyethyl methacrylate, acrylamide, methacrylamide, etc. Raw material monomers can be mentioned that constitutes the (meth) acrylic resins such as acrylamide derivatives, dissolved fluorescent lanthanoid complex in a liquid containing these monomers (hereinafter referred to as monomer solution), and then polymerized.

In this case, a method of dispersing the bulk polymer solution of the monomer solution in a poor solvent for the polymer to form fine particles,
A suspension polymerization method in which a radical initiator is added to a monomer solution and dispersed and polymerized in the above-mentioned water or water-containing organic solvent, and a monomer solution is emulsified in the above water or water-containing organic solvent containing a radical initiator to carry out polymerization. Specific examples thereof include a miniemulsion polymerization method and a microemulsion polymerization method, and an emulsion polymerization method in which a monomer solution is added to the above-mentioned water or water-containing organic solvent containing micelles of a dispersion stabilizer and a radical initiator. Alternatively, a method including a membrane emulsification step of passing the monomer solution through a porous membrane (eg, a glass microporous membrane such as SPG membrane manufactured by SPG Techno Co., Ltd.) and emulsifying the same in water or a water-containing organic solvent. Also available.

In practical use of the polymer fine particles of the present invention, any additives such as organic phosphorus compounds such as trioctyl phosphine oxide and tributyl phosphine oxide can be used as long as the purpose of the present invention is not significantly impaired. In addition, it is also possible to use an additive capable of suppressing a decrease in fluorescence intensity due to hydration or the like by coordinating with a lanthanoid cation, and such an additive may be added in advance to the monomer solution. It is suitable. <Fluorescent immunoassay reagent> As described above, the polymer fine particles of the present invention can be used as an immunoassay reagent having fluorescence ability (fluorescent immunoassay reagent) by binding to an antibody or an antigen.

The anti-immunoassay reagent used in the present invention
Polyclonal antibodies such as rabbits and goats,
Mouse monoclonal antibody IgG, IgM, or
F (a) obtained by treating them with an enzyme or a reducing agent.
b ')_Two, Fab, Fab 'fractionation and the like.
Originally, proteins, polypeptides, steroids,
Various things such as polysaccharides, lipids, drugs, pollen, etc.
You. As a method for binding such an antibody or antigen, a polymer microparticle is used.
Antibody or antigen amino group to carboxyl group of particle
By using a condensing agent such as carbodiimide,
A sugar chain of an antibody or an antigen is added to the amino group of the polymer fine particles.
Method of bonding using periodic acid,
Glutar al amino group of antibody or antigen to amino group
Method of bonding using aldehyde, polymer fine particles
React thiol group of antibody or antigen to mid group
Method and the like, and the amount of the binding may be a polymer fine particle.
Usually 50 nanograms as weight per milligram
~ 500 micrograms, preferably 500 nanograms
~ 200 micrograms. <Fluorescent immunoassay> The present invention is based on the conventional TR-FIA method and the like.
Tb was difficult to use for fluorescent immunoassay ³⁺Use complex
To provide an analytical method. That is, Tb³⁺Contains complex
A fluorescent immunoassay reagent using the above-mentioned polymer microparticles (hereinafter referred to as “reagent”)
Referred to as the first reagent) can be any Eu, including known ones³⁺
A fluorescent immunoassay reagent containing a complex (hereinafter referred to as a second reagent)
Fluorescence immunoassay, which is used in combination with
You. This is because the wavelength of the main fluorescence band generated by the first reagent (540)
545 nm) and the wavelength of the main fluorescence band generated by the second reagent.
(Around 610 to 615 nm),
It is possible to measure these fluorescence intensities at the same time.
And use. That is, the analysis target of the first reagent
Simultaneous quantitative analysis of substance and analyte of second reagent is possible
Become.

There is no particular limitation on the ratio of the first and second reagents used. There is no limitation on the fluorescent band of each reagent to be used. Fluorescent bands other than the above-mentioned main fluorescent band (for example, for the first reagent, around 490 nm given by Tb ³⁺ complex or 585 n
The fluorescent band around m or the like, and the second reagent may use the fluorescent band around 580 nm or 592 nm provided by the Eu ³⁺ complex.

[0045]

EXAMPLES Specific examples of the present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the invention. [Measurement device and conditions, etc.] (1) NMR JNM-EX270 type FT-NMR manufactured by JEOL Ltd.
( ¹ H: 270 MHz, ¹³ C: 67.8 MHz), solvent: CDCl ₃ . (2) Fluorescence: F-3000 type fluorometer manufactured by Hitachi, Ltd. (3) Fluorescence lifetime: The sample is irradiated with pulsed light having a wavelength of 337 nm (pulse width 5 nanoseconds, frequency 10 Hz) obtained from a pulsed nitrogen laser manufactured by Laser Photonics, and the fluorescence is passed through a scattered light filter to Chromex.
The light was condensed with a lens on a 250is spectrometer (250 mm focal length, equipped with a C4334 streak scope manufactured by Hamamatsu Photonics) and measured. The fluorescence lifetime was the time until the fluorescence intensity of the pulsed light became 1 / e (where e is the base of natural logarithm). (4) Differential thermal analysis (DSC): DuPont Inst
thermal Analyst2
The measurement was performed by changing the temperature at a rate of 5 ° C./min under a nitrogen gas flow using a Model 000 Differential Thermal Analyzer. <Synthesis of Fluorescent Dendrimer Complex> (1) Having a 3,5-dioxybenzoate structure at the focal point According to the literature by Kawa et al., First-, third-, and fourth-generation polybenzyl ether dendritic carboxylic acids were synthesized, and first- and fourth-generation Tb ³⁺ complexes using these as ligands ( In the literature, [G-1] ₃ -Tb and [G
-4] ₃ -Tb, abbreviated hereinafter in the present invention), and a third-generation Eu ³⁺ complex ([G-
3] Abbreviated as _3- Eu, and in the present invention, similarly abbreviated below). That is, benzyl bromide (2.05 equivalents) and 3,5-dihydroxybenzyl alcohol (1.
0 equivalents) with 18-crown-6 ether (0.2 equivalents) and freshly ground anhydrous potassium carbonate (2.5 equivalents)
Reaction in which acetone is condensed in acetone at 60 ° C. to give dendritic benzyl alcohol, and carbon tetrabromide (1.25 equivalent) and triphenylphosphine (1.25 equivalent) are added to tetrahydrofuran. (Hereinafter abbreviated as THF) to obtain a dendritic bromide of any generation by repeating the bromination reaction for converting the compound into the corresponding dendritic benzyl bromide. This is condensed with ethyl 3,5-dihydroxybenzoate in the same etherification reaction as described above, and then an ester hydrolysis reaction in which an excess equivalent of potassium hydroxide is allowed to act in a mixed solution of aqueous methanol / THF results in a dendritic reaction of one generation. A carboxylic acid is obtained. The carboxylic acid (3 equivalents) thus obtained is converted to Tb
Reaction with ³⁺ or Eu ³⁺ acetate anhydride (1 equivalent) in refluxing chlorobenzene gives the desired complex by ligand exchange reaction by deacetic acid. Note that [G-1] ₃ -Ln, [G
-3] ₃ -Ln and [G-4] ₃ -Ln (provided that Ln
Represents Tb or Eu), respectively, corresponding to structural formulas 3, 5, and 6 herein. The two types of T
The fluorescence lifetime of the b ³⁺ complex was about 1.1 to 1.7 milliseconds in a 100 μM THF solution. On the other hand, the fluorescence lifetime of the above Eu ³⁺ complex was in the range of 0.6 to 0.8 millisecond in a 100 μM THF solution. (2) Those having a 3,4-dioxybenzoate structure at the focal point. In the literature by Kawa et al., 3,5-
A similar synthesis was performed using ethyl 3,4-dihydroxybenzoate instead of ethyl dihydroxybenzoate to synthesize first and third generation dendritic carboxylic acids, and to use these as ligands for Tb ³⁺ Complexes (each [34
G-1] ₃ -Tb and [34G-3] ₃ -Tb)
Was similarly prepared. Note that these two types of structures respectively correspond to Structural Formulas 7 and 9 in this specification. The fluorescence lifetimes of the two Tb ³⁺ complexes were about 1.0 to 1.4 milliseconds in a 100 μM THF solution. <Preparation of Fluorescent Polymer Fine Particles> Hereinafter, preparation examples of polymer fine particles will be described. Table 1 summarizes the average particle size (volume average median), particle size distribution (particle size dispersion coefficient δ), wavelength of the main fluorescent band, and fluorescence lifetime of the obtained polymer fine particles.

Example 1 [Emulsion polymerization] The inside of a glass vessel equipped with a stirring blade and a reflux tube was purged with nitrogen.
An aqueous solution in which sodium dodecylbenzenesulfonate (0.58 parts by weight) and ammonium persulfate (0.23 parts by weight) were dissolved in pure water (100 parts by weight) was added. While stirring at 40 ° C. under a gentle nitrogen stream, styrene (7.1 parts by weight), ethyl acrylate (2.1 parts by weight), divinylbenzene (0.08 parts by weight), Eu (N
TA) ₃ (see formula 12 for the structure; 0.48 parts by weight), and about 20% of a solution of a mixture of trioctylphosphine oxide (0.39 parts by weight) was added dropwise in about 10 minutes.
The temperature was raised to 0 ° C. When a translucent emulsion was obtained, the remainder was added dropwise over 2 hours, the temperature was further raised to 85 ° C., and stirring was continued for 8 hours. The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.38 μm and a particle size distribution coefficient δ defined by the following formula (I) of 1.1.

[0047]

Δ = (D₉₀-D_Ten) / D₅₀ (I) Ultraviolet light having a wavelength of 365 nm is applied to the polymer fine particles.
When irradiated, Eu ³⁺Fireflies around 615 nm
Gave a light belt. The fluorescence lifetime was 0.7 millisecond. This
The fine particles contained in the suspension have ester groups on the surface,
Carboxyl groups required due to hydrolysis of ester groups
Control between the carboxyl group and the antibody molecule.
In some cases, it can be used as a fluorescent immunoassay reagent.

Example 2 [Emulsion polymerization] In Example 1, instead of Eu (NTA) ₃ , the fluorescent dendrimer complex [G-1] ₃ -Tb synthesized above was used.
(0.30 parts by weight) was replaced with tributylphosphine oxide (0.1%) instead of trioctylphosphine oxide.
(11 parts by weight) to perform the same emulsion polymerization operation. The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.31 μm and a particle size distribution coefficient δ defined by the formula (I) of 1.0.
When this polymer fine particle was irradiated with ultraviolet light having a wavelength of 365 nm, a fluorescent band around 545 nm characteristic of Tb ³⁺ was given. The fluorescence lifetime was 1.8 milliseconds. The fine particles contained in this suspension have ester groups on the surface,
Since the amount of carboxyl group can be controlled as required by hydrolysis of the ester group, it can be used as a fluorescent immunoassay reagent by binding the carboxyl group to the antibody molecule.

Example 3 [Emulsion polymerization] In Example 2, instead of [G-1] ₃ -Tb, the fluorescent dendrimer complex [G-4] ₃ -Tb synthesized above was used.
(0.40 parts by weight) and 0.04 parts by weight of tributylphosphine oxide were used to carry out the same emulsion polymerization operation. The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.41 μm and the above formula (1)
Was 0.9. When this polymer fine particle was irradiated with ultraviolet light having a wavelength of 365 nm, a fluorescent band around 545 nm characteristic of Tb ³⁺ was given. The fluorescence lifetime was 1.3 milliseconds. The fine particles contained in this suspension have ester groups on the surface, and the amount of carboxyl groups can be controlled as necessary by hydrolysis of the ester groups. Therefore, the suspension can be used as a fluorescent immunoassay reagent by binding the carboxyl groups to antibody molecules. It is.

Example 4 [Emulsion polymerization] In Example 2, instead of [G-1] ₃ -Tb, the fluorescent dendrimer complex [G-3] ₃ -Eu synthesized above was used.
(0.35 parts by weight) and 0.05 parts by weight of tributylphosphine oxide were used to carry out the same emulsion polymerization operation. The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.49 μm and the above formula (1)
Was 1.2. When this polymer fine particle was irradiated with ultraviolet light having a wavelength of 365 nm, a fluorescent band around 615 nm characteristic of Eu ³⁺ was given. The fluorescence lifetime was 0.7 millisecond. The fine particles contained in this suspension have ester groups on the surface, and the amount of carboxyl groups can be controlled as necessary by hydrolysis of the ester groups. Therefore, the suspension can be used as a fluorescent immunoassay reagent by binding the carboxyl groups to antibody molecules. It is.

Example 5 [Emulsion polymerization] In Example 2, instead of [G-1] ₃ -Tb, the fluorescent dendrimer complex [34G-1] ₃ -synthesized above was used.
The same emulsion polymerization operation was performed using Tb (0.30 parts by weight) and 0.11 part by weight of tributylphosphine oxide. The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.32 μm and a particle size distribution coefficient δ defined by the above formula (1) of 1.0.
When this polymer fine particle was irradiated with ultraviolet light having a wavelength of 365 nm, a fluorescent band around 545 nm characteristic of Tb ³⁺ was given. The fluorescence lifetime was 1.1 millisecond. The fine particles contained in this suspension have ester groups on the surface,
Since the amount of carboxyl group can be controlled as required by hydrolysis of the ester group, it can be used as a fluorescent immunoassay reagent by binding the carboxyl group to the antibody molecule.

Example 6 [Emulsion polymerization] In Example 2, instead of [G-1] ₃ -Tb, the fluorescent dendrimer complex [34G-3] ₃ -synthesized above was used.
The same emulsion polymerization operation was carried out using Tb (0.35 parts by weight) and tributylphosphine oxide at 0.05 parts by weight. The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.40 μm and a particle size distribution coefficient δ defined by the above formula (1) of 0.9.
When this polymer fine particle was irradiated with ultraviolet light having a wavelength of 365 nm, a fluorescent band around 545 nm characteristic of Tb ³⁺ was given. The fluorescence lifetime was 1.1 millisecond. The fine particles contained in this suspension have ester groups on the surface,
Since the amount of carboxyl group can be controlled as required by hydrolysis of the ester group, it can be used as a fluorescent immunoassay reagent by binding the carboxyl group to the antibody molecule.

Example 7 [Mini-emulsion polymerization] Water (80 g) was placed in a glass flask whose inside was replaced with nitrogen.
And sodium hydrogen carbonate (0.0179 g) and sodium dodecylsulfonate (0.115 g) were dissolved. While stirring this, methyl methacrylate (20
g), Eu ³⁺ complex Eu (NTA) ₃ used in Example 1
(0.5 g), and stearyl methacrylate (0.42
The monomer solution mixed with g) was dropped and suspended. Then, the reaction vessel was placed in an ice bath and irradiated with ultrasonic waves for about 1 hour until oil droplets disappeared (UH-600 ultrasonic disperser supplied by Shonan Science Co., Ltd .; output 600 W, frequency 20)
Hz, maximum output). Thereafter, the mixture was placed in a warm water bath, the temperature of the reaction solution was adjusted to 50 ° C., an aqueous solution in which potassium persulfate (0.053 g) was dissolved in water (5 g) was added with stirring, and stirring was continued for about 6 hours. Removed and allowed to cool to room temperature. The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.19 μm and a particle size distribution coefficient δ defined by the above formula (1) of 0.6.
When this polymer fine particle was irradiated with ultraviolet light having a wavelength of 365 nm, a fluorescent band around 615 nm characteristic of Eu ³⁺ was given. The fluorescence lifetime was 0.7 millisecond. The fine particles contained in this suspension have ester groups on the surface,
Since the amount of carboxyl group can be controlled as required by hydrolysis of the ester group, it can be used as a fluorescent immunoassay reagent by binding the carboxyl group to the antibody molecule.

Example 8 [Mini-emulsion polymerization] In Example 7, instead of Eu (NTA) ₃ , the first-generation fluorescent dendrimer Tb ³⁺ complex [34] synthesized above.
G-1] The same operation was performed using _3- Tb (0.5 g). The polymer fine particles contained in the suspension thus obtained had a volume average median of 0.15 μm and a particle size distribution coefficient δ defined by the above formula (1) of 0.5.
When this polymer fine particle was irradiated with ultraviolet light having a wavelength of 365 nm, a fluorescent band around 545 nm characteristic of Tb ³⁺ was given. The fluorescence lifetime was 1.1 millisecond. The fine particles contained in this suspension have ester groups on the surface,
Since the amount of carboxyl group can be controlled as required by hydrolysis of the ester group, it can be used as a fluorescent immunoassay reagent by binding the carboxyl group to the antibody molecule.

[0055]

Table 1 Properties of polymer fine particles No. Fluorescent complex Volume average median Particle diameter dispersion coefficient Main fluorescence band (μm) Wavelength (nm) Lifetime (millisecond) Example 1 Eu (NTA) 3 0.38 1.1 615 0.7 Example 2 [G-1] ₃ -Tb 0.31 1.0 545 1.8 Example 3 [G-4] ₃ -Tb 0.41 0.9 545 1.3 Example 4 [G-3] ₃ -Eu 0.49 1.2 615 0.7 Example 5 [34G -1] ₃ -Tb 0.32 1.0 545 1.1 Example 6 [34G-3] ₃ -Tb 0.40 0.9 545 1.1 Example 7 Eu (NTA) 3 0.19 0.6 615 0.7 Example 8 [34G-1] ₃ -Tb 0.15 0.5 545 1.1 <Evaluation of uniformity of dispersion of fluorescent complex in polymer fine particles> The glass transition point of the fluorescent dendrimer complex used in Examples 2 to 6 and 8 was measured by the differential thermal analysis described above. Also showed a glass transition point of 36 to 40 ° C.
These results are based on Wooley et al.
Reported value of glass transition point of polybenzyl ether dendrimer having dioxybenzyl group as a repeating unit (about 38
° C). On the other hand, as a result of performing the same differential thermal analysis on the residue obtained by evaporating the emulsion of the polymer fine particles of each Example to any of the residues, for any polymer fine particles, 36 to
No glass transition point was observed at 40 ° C. Therefore, it is concluded that the dispersion of the fluorescent complex in these polymer fine particles is uniform. <Combined Use of Tb ³⁺ Complex-Containing Polymer Fine Particles and Eu ³⁺ Complex-Containing Polymer Fine Particles> The polymer fine particles of Example 11 were treated with an aqueous sodium hydroxide solution and then acidified with hydrochloric acid.
The surface of the fine particles has a carboxyl group corresponding to the equivalent of sodium hydroxide used. The carboxyl group content is
It is determined by titration with an aqueous solution of sodium hydroxide of known concentration.

The thus obtained polymer fine particles having a carboxyl group on the surface and a modified polymer fine particle in which an arbitrary antibody molecule is bound by a known method are used as a first reagent, and commercially available Eu (N
TA) When the second reagent is a TR-FIA method reagent in which an anti-thyroid stimulating hormone antibody is bound to a latex impregnated with ₃ (Ceradyne Co., Ltd.), the main fluorescent band wavelength (around 545 nm) generated by the first reagent and the second reagent Since the main fluorescence band wavelength (around 615 nm) generated by the two reagents is sufficiently separated, the fluorescence intensities of these can be measured simultaneously. Accordingly, simultaneous quantitative analysis of an antigen for any antibody bound to the first reagent and two antigens to be analyzed by the second reagent can be performed. <Fluorescent immunoassay> Example 9 [Preparation of fluorescent immunoassay reagent] First-generation fluorescent dendrimer Tb3 + obtained in Example 8
Polymer fine particles mainly containing methyl methacrylate uniformly containing the complex [34G-1] 3-Tb (hereinafter referred to as Tb-p
Antibody (abbreviated as MMA). That is, Tb-p
MMA is diluted with a 0.05M concentration of 2-morpholinoethanesulfonic acid buffer (hereinafter referred to as MES buffer, pH is adjusted by adding sodium hydroxide, in this case, pH 6.0), and 1% suspension is added. 2 mL was prepared. 1-ethyl-3-
(3-Dimethylaminopropyl) -carbodiimide hydrochloride (hereinafter abbreviated as EDC, 0.63 mg) was added, and 1
Allowed to react for hours. After centrifugation to remove unreacted EDC and washing, 0.05 M MES buffer (pH 7.0)
After dispersing Tb-pMMA in the above, an anti-thyroid stimulating hormone-a antibody (hereinafter abbreviated as TSH-a antibody; MedixB)
0.8mg of iochemicaOy Ab)
The reaction was performed at room temperature for 1 hour. Centrifugation is again performed to remove unreacted antibodies, and a 0.1 M concentration of tris (hydroxymethyl) aminomethane buffer (hereinafter abbreviated as TRIS buffer; pH is adjusted by adding hydrochloric acid; in this case, pH 8.0). A solution in which 0.3% of bovine serum albumin (hereinafter abbreviated as BSA) was dissolved was added to the mixture to stabilize the particles. After stirring at room temperature for 30 minutes, the mixture was centrifuged and washed with purified water. After washing, 0.
It was dispersed in a 05% NaN ₃ aqueous solution to prepare anti-TSH-a antibody-immobilized Tb-pMMA. The unbound antibody concentration at the time of immobilizing the antibody was 0.07 mg / mL. Therefore,
The antibody immobilization ratio was (0.40-0.07) /0.40×
100 = 82.5 (%)

Example 10 [Tb immobilized with anti-TSH-a antibody]
-Sandwich Assay Using pMMA] This experimental operation is schematically shown in FIG. Anti-TSH antibody (mouse IgG, Medix Biochemical)
After adjusting Oy Ab) to 10 mg / mL, dispensing 100 mL per well into a 96-well microtiter well, shaking at room temperature for 60 minutes, and immobilizing the primary antibody on the plate (FIG. 2-1) After washing with pure water, 0.3% B
0.1 M TRIS buffer (pH 8.
0) was dispensed in 330 mL portions, and the plate was blocked by shaking at room temperature for 60 minutes, and TRIS containing Tween 20 (a nonionic surfactant supplied from Sigma) was added.
The plate was washed three times with a buffer solution (pH 7.5) and twice with pure water to prepare a primary antibody-immobilized plate. TSH standard product (Zym
ed) was diluted with BSA-containing TRIS buffer to give concentrations of 0, 62.5, 125, 250, and 1000 μIU /
Prepare 5 standard solutions of 5 mL and add 100 mL to each well.
The mixture was shaken at room temperature for 120 minutes to carry out a primary reaction (FIG. 2-2). After reaction, Tween 20-containing TR
Washing was performed three times with an IS buffer (pH 7.5) and twice with pure water to remove excess components and unreacted TSH. The anti-TSH-a antibody-immobilized Tb-pMMA prepared in Example 9 was compared with 0.6 M
Buffer containing 0.05% MSA, 0.5% BSA, and 0.05% NaN ₃ (pH
6.0), and Tb-pMM immobilized with anti-TSH-a antibody
The concentration was adjusted so that A became 0.01%. This reagent solution was dispensed into wells at 100 mL each and shaken at room temperature for 120 minutes to cause a secondary reaction (FIG. 2-3). After the secondary reaction, Tween 20-containing TRIS buffer (pH 7.5)
And washed twice with pure water to remove unreacted Tb-pMMA. The fluorescence intensity of each well was measured using SpectraMaxG
EMINI (Molecular Devices)
Was measured at an excitation wavelength of 290 nm and a fluorescence wavelength of 550 nm. The results are shown in Table-2. As the TSH concentration increased, the measured value of the fluorescence intensity increased, indicating a good result.

[0058]

[Table 2] Example 11 Dot Blot Using Polymer Fine Particle Tb-pMMA This experimental operation is schematically shown in FIG. In the same manner as in Example 9, an anti-human IgM antibody (rabbit IgG, self-made) was immobilized on the polymer fine particle Tb-pMMA obtained in Example 8,
Anti-human IgM antibody-immobilized Tb-pMMA was prepared. 1 mL of a 4.4 mg / mL IgM solution was dropped on a nitrocellulose membrane (Bio-Rad), and 3 mL at room temperature.
After drying for 0 minutes, IgM was immobilized on the membrane (FIG. 3-1). 0.1 M concentration TRI with 0.3% BSA
Immerse the membrane in S buffer (pH 8.0).
Shake for 0 minutes to block the membrane. 0.
6M NaCl, 0.05M MES, 0.5
Anti-human IgM adjusted to 0.1% in a buffer (pH 6.0) containing 0.1% BSA and 0.05% NaN ₃
200 mL of a liquid in which the antibody-immobilized Tb-pMMA was dispersed was added, and reacted at room temperature for 120 minutes (FIG. 3-2). Twe
After washing with TRIS buffer (pH 7.5) containing en20, unreacted anti-human IgM antibody-immobilized Tb-pMM
A was removed. Clean the membrane with UV lamp DT
Irradiation was performed at -10MP (ATTO), and fluorescence was observed.
At the position where the IgM was immobilized on the nitrocellulose membrane, yellow-green Tb-pMMA-derived fluorescent color was observed. No fluorescence was observed in a control experiment in which IgM was not immobilized (FIG. 3-3).

[0059]

Industrial Applicability The polymer fine particles of the present invention uniformly contain a fluorescent lanthanoid complex having a long fluorescence lifetime, and thus can be used as a high-luminance phosphor. Further, a functional group such as a carboxyl group is introduced on the surface of the polymer fine particles of the present invention,
By binding an appropriate antibody or antigen, it is very useful as a fluorescent immunoassay reagent for analyzing an antigen or antibody that selectively binds thereto.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing dendrimer generations and focal points.

FIG. 2 is a schematic diagram showing a sandwich assay.

FIG. 3 is a schematic diagram showing a dot blot.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C08L 33/04 C08L 33/04 101/12 101/12 C09K 11/06 660 C09K 11/06 660 690 690 G01N 33/533 G01N 33/533 33/543 575 33/543 575 (72) Inventor Takaaki Sobayashi 1000 Kamoshita-cho, Aoba-ku, Aoba-ku, Yokohama-shi, Kanagawa Prefecture Inside Mitsubishi Research Institute of Yokohama, Ltd. 1000 Kamoshita-cho, Aoba-ku, Yokohama-shi Yokohama Research Laboratory

Claims (17)

[Claims] 1. Fluorescent polymer particles in which a fluorescent lanthanoid complex is uniformly dispersed in a polymer. 2. The fluorescent substance according to claim 1, wherein the fluorescent lanthanoid complex dispersed in the polymer has a uniform dispersibility that does not show a unique transition point derived from the complex structure in a differential thermal analysis. Polymer fine particles. 3. The polymer is a styrene resin or (meth)
3. The fluorescent polymer fine particles according to claim 1, which is an acrylic resin. 4. The fluorescent polymer fine particles according to claim 1, wherein the concentration of the fluorescent lanthanoid complex in the polymer fine particles is 1 to 30% by weight. 5. The fluorescent lanthanoid complex according to claim 1, wherein the ligand is a dendron having a carboxylate group (—COO ⁻ ) at a focal point and an aromatic ring as a repeating unit. The fluorescent polymer fine particles according to any one of the above. 6. The fluorescent polymer fine particles according to claim 5, wherein the dendron has a polybenzyl ether structure. 7. The fluorescent lanthanoid complex is terbium-3
7. The fluorescent polymer fine particles according to claim 5, which is a cation (Tb ³⁺ ) complex, wherein the dendron has a 3,4-dioxybenzoate structure at its focal point. 8. The fluorescent lanthanoid complex is a europium trivalent cation (Eu ³⁺ ) represented by the following general formula (11).
The fluorescent polymer fine particles according to any one of claims 1 to 4, which is a β-diketonate complex. Embedded image (In the general formula (11), R represents an alkyl group having 6 or less carbon atoms or a fluorinated alkyl group having 6 or less carbon atoms, and R ′ represents an aromatic group.) 9. The fluorescent polymer particles according to claim 8, wherein the β-diketonate complex contains a naphthalene ring. 10. The fluorescence intensity by the pulse excitation light is 1 / e
The fluorescent polymer fine particles according to any one of claims 1 to 9, wherein a fluorescence lifetime defined by a time until e becomes the base of natural logarithm is 0.1 millisecond or more. 11. The fluorescent polymer fine particles according to claim 10, having a fluorescence lifetime of 1 millisecond or more. 12. The fluorescent polymer particles according to claim 1, wherein the volume average median of the particle diameter of the polymer particles is 0.05 to 2 μm. 13. The fluorescent polymer fine particles according to claim 12, having a particle diameter dispersion coefficient δ of 1.0 or less. (Particle size dispersion coefficient [delta], is a quantity defined by the following formula (I) with a particle diameter D _A of the integrated volume of the smaller particle size side of the particle size distribution is A%.) Equation 1] δ = (D ₉₀ −D ₁₀ ) / D ₅₀ (I) 14. A fluorescent lanthanoid complex is dissolved in a liquid containing a raw material monomer constituting a styrene resin or a raw material monomer constituting a (meth) acrylic resin,
The method for producing fluorescent polymer fine particles according to any one of claims 1 to 13, wherein the polymerization is performed to obtain emulsified fine particles. 15. A fluorescent immunoassay reagent using the fluorescent polymer fine particles according to any one of claims 1 to 13. 16. The reagent according to claim 15, wherein the fluorescent lanthanoid complex contains a Tb ³⁺ complex. 17. A fluorescent immunoassay method comprising using the fluorescent immunoassay reagent containing the Tb ³⁺ complex according to claim 16 in combination with a fluorescent immunoassay reagent containing any Eu ³⁺ complex.