WO2018110709A1 - Particules de latex colorées et réactif d'immunoessai utilisant ces dernières - Google Patents

Particules de latex colorées et réactif d'immunoessai utilisant ces dernières Download PDF

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WO2018110709A1
WO2018110709A1 PCT/JP2017/045223 JP2017045223W WO2018110709A1 WO 2018110709 A1 WO2018110709 A1 WO 2018110709A1 JP 2017045223 W JP2017045223 W JP 2017045223W WO 2018110709 A1 WO2018110709 A1 WO 2018110709A1
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colored latex
latex particles
dye
particles
colored
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PCT/JP2017/045223
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Japanese (ja)
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理 杉本
脇屋　武司
匡志 岩本
北原　慎一郎
真亜紗 池上
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積水化学工業株式会社
積水メディカル株式会社
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Priority to JP2018556767A priority Critical patent/JP7034089B2/ja
Publication of WO2018110709A1 publication Critical patent/WO2018110709A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/20—Compounding polymers with additives, e.g. colouring
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the present invention relates to a colored latex particle and an immunoassay reagent using the same.
  • immunoassays using antigen-antibody reactions are widely used as methods for measuring trace substances in specimens.
  • Various methods classified into immunoassays have a common point that the measurement principle is based on an antigen-antibody reaction (immune reaction), but are classified into various categories depending on the detection method.
  • Examples of various immunoassay methods include immunoagglutination methods typified by latex immunoturbidimetry, radioimmunoassay methods, enzyme immunoassay methods, immunochromatographic methods, flow-through membrane immunoassay methods, and the like.
  • the immunochromatographic method having both the immune reaction and the chromatographic principle is widely used in the field of clinical tests as a simple rapid test method.
  • the immunochromatography method will be described below by taking the sandwich method using the substance to be detected as an antigen as an example.
  • Particles sensitized by antibodies that bind to the substance to be detected (hereinafter sometimes referred to as sensitized particles) and the substance to be detected are mixed to form a complex of the substance to be detected and the sensitized particle, and this is detected.
  • the immobilized antibody captures the sensitized particle, the substance to be detected, and the immobilized antibody.
  • a ternary complex (sandwich) is formed and the ternary complex is detected.
  • the complex of the substance to be detected and the sensitizing particle binds to and is captured by the antibody immobilized on the stationary phase, and accumulates at the position of the immobilized antibody on the chromatographic medium.
  • colored particles are sometimes used as particles used for detection of a substance to be detected in order to facilitate visual determination.
  • the colored particles include colloidal particles such as colloidal metal particles or colloidal metal oxide particles that naturally color depending on the particle diameter (hereinafter sometimes referred to as particle diameter) and preparation conditions, and polymer compounds.
  • examples thereof include colored particles obtained by coloring (polymer) -based latex particles, or by chemically bonding a reactive dye (dye) and a functional group derived from a monomer.
  • the above colloidal particles are (1) the color tone is determined by the particle size and the preparation conditions, so that it is difficult to obtain a desired vivid dark color tone, and (2) the coefficient of variation of the average particle size (hereinafter, CV value is sometimes large) and the particle sizes are not uniform, which may cause clogging in the pores of chromatographic media such as membrane filters, and poor capture on the stationary phase. There was a problem that the reproducibility of the measurement results was poor.
  • Patent Document 1 discloses cellulose-derived organic colored fine particles having an average particle diameter of 10 nm to 1000 nm and a color development intensity of 1.0 to 5.0. Since the fine particles have a large amount of hydroxyl groups on the surface, a large amount of reactive dyes can be supported by chemical bonds (covalent bonds) with the hydroxyl groups, so that the dye content can be increased. However, since the CV value of the average particle size becomes large as in the case of the colloidal particles described above, clogging in the pores of a chromatographic medium such as a membrane filter, poor capture on the stationary phase, etc., result of measurement The reproducibility of was low.
  • the hydrophobicity of the pigment to be contained (bonded) gives the particle surface hydrophobicity, enabling physical adsorption of antigens or antibodies to the particle surface.
  • the amount of physical adsorption on the surface of the antigen or antibody also varies due to variations in the amount of dye contained (bound).
  • the method of stably carrying the antigen or antibody on the particles is substantially limited to only a specific chemical bond, and there is a problem to be improved in terms of usability.
  • in order to increase the content (bonding) of the dye it is necessary to repeat the coloring step, and there is a problem that the manufacturing work becomes complicated.
  • the CV value of the average particle size is smaller than the CV value of the average particle size, the method of supporting the antigen or antibody on the surface, and the cellulose-based particles that have problems in increasing the pigment content. Therefore, it has been demanded to increase the pigment content in polymer compound-based particles having good usability.
  • the CV value of the average particle diameter is 10% or less, the average particle diameter is 0.05 to 3.0 ⁇ m, and the average specific gravity is 1.02 to 3.
  • colored latex particles made of a polymer compound such as polystyrene, which is 0, are disclosed, in order to dye latex particles in an organic solvent, a solvent in which the latex particles are insoluble and the dye is soluble is selected. Or the latex particles need to be given solvent resistance, but the pigment content of the colored latex particles is as low as 15.6% or less, and there is a problem that the visual judgment and detection sensitivity are low during immunoassay. there were.
  • Non-patent Document 1 a polymerizable dye having a radical polymerizable group introduced into the dye skeleton, and introduces the dye into the resulting resin (copolymer: polymer) by copolymerizing with a polymerizable monomer. It is supposed to be possible.
  • polymer particles colored with a polymerizable dye for an electrophoretic device have been reported, there is only a description that the content of the polymerizable dye is 30% at the maximum (Patent Document 3).
  • Patent Document 3 there has been no report of colored particles containing a high degree of polymerizable dye (for example, 40% or more), and there have been no reports of using them as immunoassay reagents, particularly labeled particles for immunochromatography.
  • the present invention is a colored latex particle having high pigment content and low particle dispersity (small CV value of average particle diameter) and good usability, although it is a polymer compound particle, and immunoassay using the same
  • the object is to provide forensic reagents.
  • the present invention relates to the following contents.
  • Colored latex particles which contain a radical polymerizable dye and a polymer compound, the content of the dye per particle weight is 40% or more and 98% or less, and the average particle size is 0.05 ⁇ m to Colored latex particles having a mean particle size CV value of 20% or less of 3.0 ⁇ m.
  • the polymer compound comprises styrene, a styrene sulfonate, and a hydrophilic carboxy monomer.
  • colored latex particles having high pigment content, low particle dispersity (small CV value of average particle diameter) and good usability, although they are polymer compound particles, and the use thereof Provided are immunoassay reagents.
  • FIG. 1A shows a specific example of a polymerizable dye monomer.
  • FIG. 1B shows a specific example of a polymerizable dye monomer.
  • FIG. 1C shows a specific example of a polymerizable dye monomer.
  • FIG. 1D shows a specific example of a polymerizable dye monomer.
  • the present inventor uses a method such as a soap-free emulsion polymerization method or a seed polymerization method to color latex particles in water, so that the CV value of the average particle size is 20% or less. It was found that a colored latex having a pigment content of 40% or more can be obtained. That is, the present invention is a colored latex particle, which contains a radical polymerizable dye and a polymer compound, the content of the dye per particle weight is 40% or more and 98% or less, and the average particle diameter is 0.00. The present invention relates to colored latex particles having a mean particle size CV value of from 20 ⁇ m to 3.0 ⁇ m.
  • the colored latex particles are excellent in visual judgment and detection sensitivity in immunoassay due to a high dye content, and the reproducibility of the reagent measurement results is good because the CV value of the average particle diameter is low.
  • the surface is provided with a shell of a hydrophobic polymer compound, it is excellent in carrying antigens or antibodies.
  • the polymer compound is not particularly limited.
  • styrene homopolymer styrene-methacrylic acid copolymer or styrene-itaconic acid copolymer, styrene and styrene sulfonate copolymer are preferable.
  • a polymer composed of a hydrophilic carboxyl monomer such as a styrene homopolymer, a styrene and styrenesulfonate copolymer, or methacrylic acid is particularly preferable.
  • styrene sulfonic acid exists on the surface of each polymer latex particle, it contributes to dispersion stability by the electrostatic repulsive force of sulfonic acid groups.
  • a carboxy group is preferably present on the particle surface because it can serve as a chemical binding site with an antibody while contributing to dispersion stability.
  • the styrene sulfonate salt is not particularly limited, and examples thereof include a sodium salt, a potassium salt, a lithium salt, and an ammonium salt. These may be used independently and 2 or more types may be used together. Of these, sodium styrenesulfonate is preferably used.
  • As the hydrophilic carboxyl monomer used in the present invention methacrylic acid, acrylic acid, itaconic acid, maleic acid, fumaric acid and the like can be used. Preferably, methacrylic acid and acrylic acid can be used.
  • the content of the polymer compound is preferably 2% or more, and more preferably 5% or more. This is because when the content of the polymer compound is lower than the lower limit, it becomes difficult to maintain the shape of the particles and prevent the elution of the dye. Moreover, it is preferable to set it as the said range also from a viewpoint of controlling protein carrying amount so that it may mention later. From the viewpoint of increasing the pigment content, 60% or less is preferable.
  • the average particle size of the particles is not particularly limited, and any average particle size may be used as long as it can be used as a colored latex particle for diagnostic agents. In particular, there is no particular problem if it is developed by chromatography, but if small particles are used, sufficient detection sensitivity may not be obtained, so an average particle size of 0.05 ⁇ m to 3.0 ⁇ m is preferable. 1 ⁇ m to 1 ⁇ m is more preferable.
  • the “average particle diameter (particle diameter)” can be obtained by observing and measuring 500 arbitrary particles with a transmission electron microscope (TEM) and calculating the average value of the particle diameter.
  • TEM transmission electron microscope
  • the coefficient of variation (CV value) of the average particle diameter of the particles is limited to 20% or less in order to improve the reproducibility of the reagent measurement results. If it exceeds 20%, lot reproducibility at the time of reagent preparation may be poor, and reproducibility of measurement results may be reduced.
  • the method for preparing the suspension of polymer compound particles is not particularly limited, and a known method can be used, but a soap-free emulsion polymerization method without using an emulsifier (surfactant) is preferable.
  • the polymerization initiator used in the soap-free emulsion polymerization method include potassium persulfate and ammonium persulfate, and potassium persulfate is preferable.
  • ion-exchanged water such as a monomer and a polymerization initiator is charged into a reaction vessel, and the reaction vessel is purged with nitrogen while stirring, and then reacted at 65 ° C. to 80 ° C. for 24 to 42 hours. be able to.
  • the resulting particles have a uniform particle size distribution (ie a low CV value) and excellent dispersion stability.
  • the amount of dye contained in the colored latex particles that have been colored is defined by the dye content (%) of the following formula (1).
  • the pigment content is 40% or more. This is because, when the dye content is less than 40%, satisfactory visual determination and detection sensitivity cannot be obtained when the measurement reagent is used. Preferably it is 50% or more, More preferably, it is 70% or more, More preferably, it is 90% or more.
  • the upper limit of the content of the radical polymerizable dye is not particularly limited, but is 98%.
  • Dye content (%) radically polymerizable dye amount (g) / ⁇ radically polymerizable dye amount (g) + latex dry mass (g) ⁇ ⁇ 100 (1)
  • the actual pigment content is calculated as follows.
  • the absorption spectrum of the dye solution is measured, and the maximum absorption wavelength of the dye is measured.
  • the dye and the powdered latex particle powder before coloring are dissolved in an organic solvent at an arbitrary ratio, and the absorbance at the maximum absorption wavelength of the dye is measured.
  • a calibration curve can be created from the dye concentration and absorbance measurement.
  • the produced colored latex is pulverized, weighed, dissolved in an organic solvent, and the absorbance at the maximum absorption wavelength is measured, whereby the amount of the dye contained in the colored latex particles is calculated from the calibration curve to obtain the dye content.
  • the radical polymerizable dye a radical polymerizable dye soluble in a hydrophobic organic solvent can be used.
  • the radical polymerizable dye dissolved in the hydrophobic organic solvent is present as droplets in water, the dye solution dissolved in a small amount in water permeates into the polymer compound particles, and the particles are swollen. That is, by performing the particle synthesis and coloring process in water, a more hydrophilic polymer compound composition is distributed outside the particle, and a more hydrophobic dye component is easily taken into the particle.
  • the polymer compound-based particles used are in a state of being dispersed in water, so that it is not necessary to impart solvent resistance to the particles, and high-density coloring is possible.
  • the hydrophobic organic solvent include hexane, benzene, toluene, diethyl ether, chloroform, ethyl acetate, and methylene chloride.
  • the term “pigment” includes the concept of “dye”
  • the term “coloring” includes the concept of “dye”. Therefore, the radical polymerizable dye that can be used in the present invention is not affected by the name of the trade.
  • the radical polymerizable dye a dye having a radical polymerizable group can be used. It is possible to prepare radical copolymerizable monomers such as radically polymerizable dyes and polymerizable monomers by mixing radically polymerizable monomers and polymerizable monomers such as styrene. By allowing the radical polymerizable dye to coexist, in the latex particles, the polymerizable group of the radical polymerizable dye is polymerized to form a dye polymer, thereby preventing elution of the dye from the particles. Further, since the particles can be stoichiometrically colored, the color intensity of the colored latex particles can be maintained high, the color intensity can be controlled, and the degree of dispersion of the particles can be kept low.
  • a copolymer of a monomer having a methacryl group as a polymerizable group and a pigment sold as “polymerizable dye RDW series” from Wako Pure Chemical Industries, Ltd. can be used. More specifically, RDW-R13 (dye skeleton: xanthene series, color: purple, maximum absorption wavelength: 559 nm), RDW-R60 (dye skeleton: xanthene series, color: red, maximum absorption wavelength: 499 nm), RDW- Y03 (dye skeleton: cyanine series, color: yellow, maximum absorption wavelength: 419 nm), RDW-G01 (dye skeleton: triarylmethane series, color: green, maximum absorption wavelength: 643 nm), RDW-B01 (dye skeleton: tria Reel methane, color: blue, maximum absorption wavelength: 624 nm), RDW-K01 (dye skeleton: x
  • (meth) acrylate means acrylic acid or methacrylic acid
  • (meth) acrylamide means acrylamide or methacrylamide.
  • latex particles In order to apply latex particles to diagnostic drug applications, it is necessary to be able to carry proteins such as antigens and antibodies on the particle surface.
  • the protein carrying capacity of latex particles depends on the surface state of the particles. For example, when styrene is used as the polymer compound, the latex particle surface is composed of styrene, and any protein can be adsorbed using a hydrophobic interaction (physical adsorption). At this time, the higher the styrene content, the easier the adsorption.
  • the protein when a monomer having a hydrophilic carboxy group (also referred to as a hydrophilic carboxy monomer) is used for the polymer compound, the protein can be chemically bonded, and the amount of binding increases as the amount of the hydrophilic carboxy monomer increases.
  • the protein loading rate is determined by measuring the amount of protein adsorbed (or not adsorbed) on the particle before and after physical adsorption treatment using an appropriate protein, or bound (or bound) to the particle before and after chemical bonding treatment. It can be determined by measuring the amount of protein).
  • a method for calculating the protein loading rate (%) will be described with reference to an example in which protein is supported on particles by physical adsorption.
  • the protein adsorption rate by physical adsorption and the protein binding rate by chemical bonding are collectively referred to as protein loading rate.
  • the colored latex particle suspension is mixed with an arbitrary protein solution, and the protein is adsorbed on the particles while gently mixing.
  • An appropriate electrolyte aqueous solution is added to the mixed solution, and only particles are aggregated and settled by salting out, and then the particles are removed from the mixed solution with a filter paper.
  • the absorbance at 280 nm of each of the filtrate and the protein solution is measured, and after correcting the liquid volume, the protein loading is calculated according to the following formula.
  • Loading ratio (%) ⁇ 1 ⁇ (absorbance of filtrate / absorbance of protein solution (without latex)) ⁇ ⁇ 100
  • the protein loading rate is preferably 30% or more, more preferably 40% or more. If the protein loading is less than 30%, the antibody-antigen reaction is not sufficiently carried out, and there is a possibility that the visual judgment property and detection sensitivity at the time of immunoassay are lowered.
  • the method for producing colored latex particles containing a radical polymerizable dye is not particularly limited, and conventionally known polymerization methods such as a suspension polymerization method, an emulsion polymerization method, a soap-free polymerization method, a seed polymerization method, and a dispersion polymerization method can be used. Any polymerization method may be used, but an emulsion polymerization method, a soap-free polymerization method, and a seed polymerization method, which can be performed in water and can obtain particles having a uniform particle diameter, are preferable. The following method is mentioned as an example.
  • a dye solution dissolved in a hydrophobic organic solvent is added to water in which polymer compound particles are dispersed, and the mixture is stirred to incorporate oil-soluble dye molecules in the latex particles. Thereafter, heating is performed to evaporate the hydrophobic organic solvent to obtain colored latex particles (seed polymerization method).
  • seed polymerization method In the case of a polymerizable pigment (dye), it can be polymerized after encapsulating the pigment by coexisting an oil-soluble radical polymerization initiator.
  • colored latex particles may be obtained by adding a dye solution dissolved in styrene and a hydrophobic organic solvent in water, heating and stirring, and polymerizing (soap-free polymerization method).
  • the colored latex particles of the present invention have an antigen (or antibody) supported on the surface, thereby allowing biological immunology such as enzyme immunoassay, fluorescence immunoassay, latex agglutination, and immunochromatography using an antigen-antibody reaction. It can use suitably for the various methods using reaction.
  • an immunoassay reagent colored latex particles containing a radical polymerizable dye carrying an antigen or an antibody using the colored latex particles described above.
  • the method for supporting the antigen (or antibody) on the surface of the colored latex particles is not particularly limited, and a conventionally known method can be used.
  • a loading method by physical adsorption such as immersing colored latex particles in a buffer solution containing an antigen (or antibody) and incubating at a constant temperature for a certain time
  • a loading method using chemical bonds both of which are suitable.
  • the loading method by physical adsorption is easy to operate, and the loading method using a chemical bond that supports the antigen (or antibody) by crosslinking the carboxyl group of the colored latex particles and the amino group in the antibody molecule is a three-dimensional structure. It is preferably used for carrying an antigen (or antibody) whose properties change with changes.
  • sufficiently dark colored latex particles can be produced. Moreover, when this colored latex particle is used as a reagent for immunoassay, the visual judgment property is remarkably improved, and the detection sensitivity can be improved. In addition, since the CV value of the average particle size is low, the lot reproducibility at the time of reagent preparation and the reproducibility of the measurement results are improved. In this specification, since it is easy to enjoy the benefits of the present invention, the description is focused on “visual determination”, but it is not intended to exclude measurement by equipment, and can be applied to measurement by equipment. It goes without saying that those skilled in the art can naturally understand that the benefits of the present invention can be enjoyed.
  • the seed particles used for producing the colored latex particles of the present invention were prepared by a soap-free emulsion polymerization method. First, 1200 mL of ion exchange water and 120 mL of styrene as a latex monomer were added to the reaction vessel and stirred, and then the inside of the reaction vessel was purged with nitrogen. After the temperature in the reaction vessel reached 70 ° C., 13 mL of 3% (w / v) aqueous potassium persulfate solution was added dropwise. After 24 hours from the dropwise addition of the 3% (w / v) aqueous potassium persulfate solution, the reaction was stopped and filtered to obtain a seed particle suspension.
  • the particle diameter of the seed particles obtained by the soap-free emulsion polymerization method was measured with a transmission electron microscope JEM-1010 (manufactured by JEOL Ltd.). As a result, the average particle size was 254 nm, and the CV value of the particle size was 5.3%.
  • the colored latex particles of the present invention were produced using a seed polymerization method. Dissolve 1.5 g of radical polymerizable dye (RDW-R60) and 0.01 g of oil-soluble polymerization initiator (benzoyl peroxide) in 70.0 g of ethyl acetate, stir overnight at room temperature and filter to obtain a dye solution. It was. At this time, there was no sediment on the filter paper (pore diameter 5 ⁇ m), and all the dye was dissolved. The dye solution was mixed with 100.0 g of a suspension of seed particles obtained by the soap-free emulsion polymerization method comprising styrene, and the seed particles were encapsulated by stirring at room temperature for 5 hours.
  • RW-R60 radical polymerizable dye
  • oil-soluble polymerization initiator benzoyl peroxide
  • the mixture was heated and stirred at 70 ° C. for 10 hours, and the colored latex particles of the present invention were obtained by polymerization of the radical polymerization dye and evaporation removal of ethyl acetate.
  • the average particle size of the obtained colored latex particles was measured with a transmission electron microscope JEM-1010 (manufactured by JEOL Ltd.). As a result, the average particle size was 293 nm, and the CV value of the average particle size was 3.6%.
  • the pigment content (%) of the colored latex particles obtained was calculated.
  • the colored latex particles were heated at 95 ° C. for 2 hours and dried, and then ground in a menor mortar to obtain uniform colored latex particle powder.
  • the powder was weighed and dissolved in toluene.
  • the absorbance was measured at ⁇ max (499 nm) of RDW-R60, and the dye content (%) was calculated by a calibration curve prepared from a dilution series prepared by dissolving RDW-R60 and seed particle powder in toluene. % (See Table 1).
  • the protein loading (%) of the obtained colored latex particles was calculated.
  • a colored latex particle suspension (0.03 g / mL) (1.0 mL) is mixed with bovine serum albumin solution (0.05 g / mL) 0.05 mL, and gently stirred at 4 ° C. for 3 hours.
  • Bovine serum albumin was supported by physical adsorption.
  • an electrolyte solution 500 mmol / L phosphate buffer (pH 7.4)
  • the electrolyte-containing mixed solution was filtered through a filter paper (pore size: 0.22 ⁇ m) to remove the aggregated colored latex particles, and the filtrate (7 mL) was collected.
  • the absorbance at 280 nm of a colored latex-free bovine serum albumin solution (colored latex-free solution) obtained by diluting the filtrate and bovine serum albumin solution 140-fold with an electrolyte solution was measured. (%) was calculated.
  • the calculation result of the protein loading of the colored latex particles was 52.8% (see Table 1).
  • Example 2 The seed particles used in the production of the colored latex particles of the present invention were produced using a soap-free emulsion polymerization method. First, 1200 mL of ion exchange water and 120 mL of styrene as a latex monomer and 16 mL of methacrylic acid were added to the reaction vessel and stirred, and then the inside of the reaction vessel was replaced with nitrogen. After the temperature in the reaction vessel reached 70 ° C., 13 mL of 3% (w / v) aqueous potassium persulfate solution was added dropwise.
  • the colored latex particles of the present invention were produced using a seed polymerization method.
  • a radical polymerizable dye (disperse red 1 acrylate) 3.0 g and an oil-soluble polymerization initiator (benzoyl peroxide) 0.01 g were dissolved in ethyl acetate 100.0 g (at this time, there was no sediment on the filter paper, Colored latex particles were obtained in the same manner as in Example 1 except that all the dyes were dissolved. Moreover, it was 73% when the pigment
  • Example 3 2.5 g of radical polymerizable dye (RDW-R60) and 0.01 g of an oil-soluble polymerization initiator (benzoyl peroxide) were dissolved in 90.0 g of ethyl acetate (at this time, there was no sediment on the filter paper, the dye Except for the above, the same procedure as in Example 2 was carried out, and a radical polymerizable dye was contained in the seed particles by a soap-free polymerization method to obtain colored latex particles. Similarly, the pigment content (%) was calculated, and the average particle size and the CV value of the average particle size were measured (see Table 1).
  • Example 4 A radical polymerizable dye (methylene blue VBC) 2.5 g and an oil-soluble polymerization initiator (benzoyl peroxide) 0.01 g were dissolved in ethyl acetate 90.0 g (At this time, there was no sediment on the filter paper, the dye was Colored latex particles were obtained in the same manner as in Example 1 except that all were dissolved. Similarly, the pigment content (%) was calculated, and the average particle diameter and the CV value of the particle diameter were measured (see Table 1).
  • Example 5 2.5 g of radical polymerizable dye (solvent red 49-VBC) and 0.01 g of oil-soluble polymerization initiator (benzoyl peroxide) were dissolved in 150.0 g of ethyl acetate (at this time, there was no sediment on the filter paper) All the dyes were dissolved.) Colored latex particles were obtained in the same manner as in Example 2 except that. Similarly, the pigment content (%) was calculated, and the average particle size and the CV value of the average particle size were measured (see Table 1).
  • Example 6 A radical polymerizable dye (dispersed MOI) (3.0 g) and an oil-soluble initiator (benzoyl peroxide) (0.01 g) were dissolved in ethyl acetate (0.00.0 g of ethyl acetate). Colored latex particles were obtained in the same manner as in Example 2 except that all were dissolved. Similarly, the pigment content (%) and protein loading (%) were calculated, and the average particle size and the CV value of the average particle size were measured (see Table 1).
  • Example 2 except that 0.5 g of a non-polymerizable dye (Solvent Red 197) was dissolved in 40.0 g of ethyl acetate (at this time, there was no sediment on the filter paper and all the dye was dissolved). Similarly, colored latex particles were obtained. Similarly, the pigment content (%) and protein loading (%) were calculated, and the average particle size and the CV value of the average particle size were measured (see Table 1).
  • a non-polymerizable dye Solvent Red 197
  • the colored latex of this study was prepared using a suspension polymerization method. First, 1200 mL of ion exchange water in a reaction vessel, 120 mL of styrene as a monomer, 0.01 g of an oil-soluble polymerization initiator (benzoyl peroxide), 0.5 g of polyvinylpyrrolidone as a dispersant, and Solvent Red 197 as a non-polymerizable dye 5 g was added and the mixture was stirred for 5 minutes using a homogenizer (disperser, IKA homogenizer ULTRA-TURRAX) to obtain an emulsion. Heating was performed at 70 ° C.
  • Example 1 When the particle diameter was measured in the same manner as in Example 1, the particle diameter was 532 nm (CV value 25.0%). Similarly, the pigment content (%) and protein loading (%) were calculated, and the average particle size and the CV value of the average particle size were measured (see Table 1).
  • the organic fine particles to be colored in this study were prepared by dissolving cellulose in a good solvent and using a coagulation liquid in which water, an organic solvent, ammonia or the like was mixed.
  • cellulose linter was dissolved in a good solvent for cellulose (copper ammonia solution), and the above-mentioned copper ammonia cellulose solution was added and coagulated while stirring the coagulation liquid (organic solvent + water + ammonia mixed system). Further, sulfuric acid was added to neutralize and regenerate to obtain a cellulose particle suspension.
  • the cellulose particles were dyed using a reactive dye. Since fine particles made of cellulose have a large amount of hydroxyl groups, many reactive dyes can be held by covalent bonds.
  • the method described in Japanese Patent No. 5788330 was used. The pigment content rate (%) and the protein loading rate (%) were calculated, and the average particle size and the CV value of the average particle size were measured (see Table 1).
  • the pigment particles in this study were prepared using a suspension polymerization method.
  • a dye solution in which 1200 mL of ion exchange water, 2.5 g of an oil-soluble dye (RDW-R60) and 0.01 g of an oil-soluble initiator (benzoyl peroxide) are dissolved in 90.0 g of ethyl acetate in a reaction vessel, as a dispersant 0.5 g of polyvinylpyrrolidone was added, and the mixture was stirred for 5 minutes using a homogenizer (disperser, IKA homogenizer ULTRA-TURRAX) to obtain an emulsion. The mixture was heated at 70 ° C.
  • the particle diameter was measured in the same manner as in Example 1, the particle diameter was 712 nm (CV value 60.0%). Similarly, the pigment content (%) was calculated, and the average particle diameter and the CV value of the particle diameter were measured (see Table 1).
  • test strip The above-mentioned anti-influenza virus antibody-immobilized membrane is affixed to a plastic adhesive sheet, and the conjugate application pad prepared in 3. above is placed and mounted, and an absorption pad (manufactured by Whatman, 740-E) was installed. Finally, a polyester film was placed on the upper surface so as to cover the antibody-immobilized membrane and the absorption pad, and laminated. Thus, the structure which piled up each component was cut
  • Sample preparation a In the case of a nasal aspirate sample A cotton swab soaked in a nasal aspirated solution, put the swab soaked in the sample into 320 uL of PBS, dissolve the sample components in PBS, did. b. Nasal Wipe Sample The nasal cavity was wiped with two cotton swabs, and the cotton swabs were dissolved in 320 uL of PBS to prepare a sample for reagent performance evaluation.
  • radical polymerizable dye colored latex particles having a dye content of 40% or more were used.
  • the reagent performance evaluation values were all 3.5 or more
  • the reproducibility of Examples 1 to 6 using radical polymerizable dye-colored latex particles having an average particle size CV value of 20% or less is all ⁇ or more, and the CV value of the average particle size exceeds 20%.
  • the colored latex particles of the present invention are used as colored latex particles in an immunoassay method such as an immunochromatography method, they are excellent in visual judgment and detection sensitivity, thereby contributing to early diagnosis of diseases and prevention of misjudgment.
  • the measurement sensitivity is about the conventional level, the amount of antibody used for reagent preparation can be reduced, which is useful for cost reduction.

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  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Polymers & Plastics (AREA)
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  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
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Abstract

L'invention concerne des particules de latex colorées, un colorant polymérisable par voie radicalaire et un composé polymère étant contenus à l'intérieur de ces dernières, la teneur fractionnaire du colorant par poids de particule unitaire étant de 40 à 98 %, la taille moyenne des particules étant de 0,05 à 3,0 µm et la valeur CV de la taille moyenne des particules étant inférieure ou égale à 20 %. Les particules de latex colorées présentent une teneur élevée en colorant bien que constituant des particules à base de composé polymère, présentent un faible degré de dispersion de particules et présentent également une facilité d'utilisation élevée, ce qui permet de servir de supports supérieurs à des antigènes ou des anticorps.
PCT/JP2017/045223 2016-12-15 2017-12-15 Particules de latex colorées et réactif d'immunoessai utilisant ces dernières WO2018110709A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020196434A1 (fr) * 2019-03-28 2020-10-01 日東電工株式会社 Composition adhésive sensible à la pression, couche adhésive sensible à la pression et feuille adhésive sensible à la pression

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5918711A (ja) * 1982-07-21 1984-01-31 Konishiroku Photo Ind Co Ltd 有色ポリマ−ラテツクス
JP2012517486A (ja) * 2009-02-09 2012-08-02 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 電気泳動ディスプレイのための着色粒子
JP2012517487A (ja) * 2009-02-09 2012-08-02 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 電気泳動ディスプレイのための粒子
JP2014163758A (ja) * 2013-02-22 2014-09-08 Asahi Kasei Fibers Corp 蛍光色素化合物を含むセルロース微粒子

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5918711A (ja) * 1982-07-21 1984-01-31 Konishiroku Photo Ind Co Ltd 有色ポリマ−ラテツクス
JP2012517486A (ja) * 2009-02-09 2012-08-02 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 電気泳動ディスプレイのための着色粒子
JP2012517487A (ja) * 2009-02-09 2012-08-02 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 電気泳動ディスプレイのための粒子
JP2014163758A (ja) * 2013-02-22 2014-09-08 Asahi Kasei Fibers Corp 蛍光色素化合物を含むセルロース微粒子

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SUENAGA, YUSAKU ET AL.: "Preparation and Characterization of Core-shell Polystyrene Particles Containing Red Pigments", THE SOCIETY OF POLYMER SCIENCE , JAPAN, vol. 67, no. 9, 24 September 2010 (2010-09-24), pages 530 - 536, XP055492898 *
TSE, A. S. ET AL.: "SYNTHESIS OF DYED MONODISPERSE POLY (METHYL METHACRYLATE) COLLOIDS FOR THE PREPARATION OF SUBMICRON PERIODIC LIGHT-ABSORBING ARRAYS", MACROMOLECULES, vol. 28, no. 19, 1 September 1995 (1995-09-01), pages 6533 - 6538, XP009074073 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020196434A1 (fr) * 2019-03-28 2020-10-01 日東電工株式会社 Composition adhésive sensible à la pression, couche adhésive sensible à la pression et feuille adhésive sensible à la pression
JP2020164609A (ja) * 2019-03-28 2020-10-08 日東電工株式会社 粘着剤組成物、粘着剤層、及び粘着シート
CN113646401A (zh) * 2019-03-28 2021-11-12 日东电工株式会社 粘合剂组合物、粘合剂层、及粘合片
JP7193401B2 (ja) 2019-03-28 2022-12-20 日東電工株式会社 粘着剤組成物、粘着剤層、及び粘着シート

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