WO2014051098A1 - 粒子凝集測定用ラテックス粒子 - Google Patents
粒子凝集測定用ラテックス粒子 Download PDFInfo
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- WO2014051098A1 WO2014051098A1 PCT/JP2013/076389 JP2013076389W WO2014051098A1 WO 2014051098 A1 WO2014051098 A1 WO 2014051098A1 JP 2013076389 W JP2013076389 W JP 2013076389W WO 2014051098 A1 WO2014051098 A1 WO 2014051098A1
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- latex particles
- particle
- measurement
- polymerizable monomer
- particle aggregation
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- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F290/00—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups
- C08F290/02—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups on to polymers modified by introduction of unsaturated end groups
- C08F290/06—Polymers provided for in subclass C08G
- C08F290/061—Polyesters; Polycarbonates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
- C08L33/04—Homopolymers or copolymers of esters
- C08L33/14—Homopolymers or copolymers of esters of esters containing halogen, nitrogen, sulfur, or oxygen atoms in addition to the carboxy oxygen
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Definitions
- the present invention relates to latex particles for particle aggregation measurement capable of highly sensitive measurement while highly suppressing non-specific reaction and a particle aggregation measurement reagent using the particles.
- the latex immunoturbidimetric method using latex particles carrying antibodies or the like (hereinafter sometimes referred to as sensitized latex particles) is widely used in laboratories because it is simple and has a short measurement time. Quantification of the antigen or antibody in the specimen by latex immunoturbidimetry is performed by optically detecting a change in absorbance due to aggregation of sensitized latex particles accompanying immune complex formation. This change in absorbance is based on the apparent change in particle size due to aggregation of the sensitized latex particles.
- Patent Document 1 it is easy to immobilize (sensitize) an antigen or antibody that specifically reacts with a detection target substance, is relatively inexpensive, and polymerizes. Since the reaction is easy to control, polystyrene latex particles mainly composed of polystyrene have been used. However, polystyrene latex particles have the merit of physical adsorption (sensitization) of antigens and antibodies, but also adsorb non-detection target proteins in the specimen, so they are not based on specific reactions due to antigen-antibody reactions. The agglutination reaction of the sensitized latex particles, which may cause a so-called non-specific reaction, has been a conventional problem to suppress this non-specific reaction.
- Patent Document 1 an attempt is made to block latex particles sensitized with an antigen or antibody with bovine serum albumin (BSA) or the like in order to suppress nonspecific reaction, but the effect cannot be sufficiently obtained. Since many give a high background value, it was a big subject in producing the reagent which can perform a highly sensitive measurement.
- BSA bovine serum albumin
- Patent Document 2 styrene, a compound represented by the following general formula, a salt of styrene sulfonic acid, and a water-soluble radical polymerization initiator are used for copolymerization in water to form polymer particles.
- styrene a compound represented by the following general formula
- a salt of styrene sulfonic acid and a water-soluble radical polymerization initiator
- a compound other than styrene a compound represented by the following general formula, a salt of styrene sulfonic acid, and a water-soluble radical polymerization initiator is used in the production of carrier particles.
- Patent Document 3 and Patent Document 4 immunological measurement reagents using polymers composed of a compound represented by the following general formula as a main component monomer as an aggregation promoter are shown.
- the polymer may be a copolymer, but it is preferable that the polymer has a structural unit of the following general formula in the polymer at 20% or more. It is described that may be used.
- the copolymer is present in the reaction solution in a free state as a chain polymer and is not shown to be used in the form of latex particles.
- Patent Document 4 when a coagulation accelerator such as polyethylene glycol is added to the reaction solution, non-specific turbidity occurs due to salting out and the blank value increases, but the compound represented by the following general formula is the main component. It is described that when a polymer constituted as a monomer is used as an aggregation accelerator, non-specific turbidity due to salting out hardly occurs. However, there is no indication that these polymers can suppress excessive aggregation such as dissociated specimens that cause nonspecific aggregation reactions.
- a coagulation accelerator such as polyethylene glycol
- R 1 to R 3 are each independently an alkyl group optionally having a hydrogen atom or a hydroxyl group
- R 4 is an alkylene group
- R 5 may have a substituent
- R 6 represents a hydrogen atom or a methyl group
- X represents an oxygen atom or a —NH— group
- the present invention has been made to solve the problems in the conventional latex immunoturbidimetric method as described above, and is capable of highly sensitive measurement while highly suppressing non-specific reaction, and can be easily diagnosed.
- An object of the present invention is to provide latex particles for particle aggregation measurement capable of producing a drug and a particle aggregation measurement reagent using the particles.
- the present inventors have prepared latex particles for particle aggregation measurement of the present invention by making the composition specific during latex particle polymerization. As a result, the present invention has been completed. That is, the present invention is as follows. *
- It contains a polymerizable monomer having a phenyl group, a polymerizable monomer having a phenyl group and a sulfonate, and a polymerizable monomer having the formula (1) as components, and has the formula (1)
- the above-mentioned polymerizable monomer having a phenyl group is at least one selected from the group consisting of styrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, and 4-vinylbenzoic acid.
- the polymerizable monomer having a phenyl group and a sulfonate is selected from the group consisting of styrene sulfonate, divinylbenzene sulfonate, o-methyl styrene sulfonate, and p-methyl styrene sulfonate.
- a biologically-derived substance such as a protein or a specific component in a test reagent is involved in an immune reaction, and a target specific reaction (for example, Since it is possible to suppress reactions that are not suitable for measurement purposes other than antigen-antibody reactions (for example, excessive agglutination reactions that occur in dissociated specimens), only the desired specific agglutination reactions can be measured with higher sensitivity than before. It is possible to produce a medicine.
- the latex particles of the present invention are prepared by a one-step polymerization reaction, and the latex particles are designed to be sensitized with antigens or antibodies by physical adsorption. Thus, it is possible to obtain highly sensitive latex particles for particle aggregation measurement in a very simple process.
- FIG. 1 is a calibration curve obtained by measuring D-dimer standard antigen using latex particles for particle aggregation measurement of Examples and Comparative Examples sensitized with anti-D-dimer antibody.
- FIG. 2 shows the results of measuring D-dimer detachment sample 1 and D-dimer detachment sample 2 using the latex particles for particle aggregation measurement of Examples and Comparative Examples sensitized with anti-D dimer antibody, and based on the calibration curve of FIG. It is the graph converted into concentration.
- the present invention relates to a polymerizable monomer having a phenyl group, a polymerizable monomer having a phenyl group and a sulfonate, and a monomer represented by formula (1) (hereinafter referred to as “MPC monomer”).
- MPC monomer a monomer represented by formula (1)
- the present invention provides latex particles for particle aggregation measurement which are contained as monomer components and have a density (functional group density) of MPC monomers on the particle surface of 0.003 to 0.05 ⁇ mol / m 2 .
- the polymerizable monomer having a phenyl group used in the present invention is not particularly limited, and examples thereof include styrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, and 4-vinylbenzoic acid. . These may be used alone or in combination of two or more. Of these, styrene is preferably used.
- the polymerizable monomer having a phenyl group and a sulfonate is not particularly limited as long as it is a monomer that can contain a sulfonic acid group on the surface of the polymerized particles.
- styrene sulfonate divinyl
- examples thereof include benzene sulfonate, o-methyl styrene sulfonate, and p-methyl styrene sulfonate.
- it does not specifically limit as a salt in this case Sodium salt, potassium salt, lithium salt, ammonium salt etc. are mentioned. These may be used independently and 2 or more types may be used together. Of these, styrene sulfonate is preferable, and sodium styrene sulfonate is more preferably used.
- the MPC monomer is a monomer represented by the following general formula (1).
- CH 2 CR 1 —COOCH 2 CH 2 O (PO 2 ) OCH 2 CH 2 —N (CH 3 ) 3
- R 1 represents a hydrogen atom or a methyl group
- Examples include ethyl phosphate (manufactured by Tokyo Chemical Industry).
- the polar group portion of the MPC monomer of the present invention is the same as the polar group structure of the phospholipid constituting the cell membrane, and the polymer polymerized with the MPC monomer as the main component is generally the same as the biological cell membrane component.
- this polymer has high biocompatibility, and it is known that when it is coated on plastic equipment, it hardly adsorbs protein in blood and exhibits the property of suppressing non-specific adsorption. Application is under consideration.
- Patent Document 3 and Patent Document 4 As an application to a diagnostic agent, as shown in Patent Document 3 and Patent Document 4, a role as an aggregation promoter is known.
- a specific constituent component in a biological substance such as a protein or a test reagent is involved in an immune reaction, and is not suitable for a measurement purpose other than a target specific reaction (for example, an antigen-antibody reaction).
- a target specific reaction for example, an antigen-antibody reaction.
- the latex particles of the present invention can be obtained by soap-free emulsion polymerization in an aqueous medium containing a polymerizable monomer having a phenyl group, a polymerizable monomer having a phenyl group and a sulfonate, and an MPC monomer. It is done.
- a conventionally known soap-free emulsion polymerization method can be used. For example, a polymerizable monomer having a phenyl group, a polymerizable monomer having a phenyl group and a sulfonate, an MPC monomer, and a polymerization initiator are added to a reaction vessel filled with water as a medium. And a method of heating with stirring in a nitrogen atmosphere.
- the polymerization temperature is preferably 50 to 100 ° C, more preferably 60 to 85 ° C.
- the polymerization time is usually 5 to 50 hours, although it depends on conditions such as the composition and concentration of the polymerizable monomer and the polymerization initiator.
- aqueous medium water (deionized water) alone or a mixed solvent of water and a water-soluble solvent is preferably used, for example, a mixed solvent of water and alcohol such as ethanol. Among these, it is preferable to use water alone.
- a known radical initiator can be used.
- persulfates such as potassium persulfate, sodium persulfate, ammonium persulfate, 2,2′-azobisisobutyronitrile, 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile),
- Organic compounds such as azo compounds such as 2,2′-azobis-2,4-dimethylvaleronitrile, benzoyl peroxide, di-t-butyl peroxide, lauroyl peroxide, t-butylperoxy-2-ethylhexanoate Mention may be made of peroxides.
- persulfate is preferable, and potassium persulfate is more preferably used.
- the amount of the polymerization initiator used is not particularly limited, but it is preferably used in the range of 0.01 to 5% by weight based on the amount of the polymerizable monomer.
- a polymerizable unsaturated monomer copolymerizable with the above monomer may be further added during the above polymerization.
- the polymerizable unsaturated monomer is not particularly limited as long as it can be used for ordinary radical polymerization.
- (meth) acrylic acid means acrylic acid or methacrylic acid.
- the styrene derivative is a styrene derivative other than the polymerizable monomer having a phenyl group and the polymerizable monomer having a phenyl group and a sulfonate used in the present invention.
- the amount of the MPC monomer of the present invention used for the polymerization is not particularly limited as long as the density (functional group density) of the MPC monomer on the particle surface is 0.003 to 0.05 ⁇ mol / m 2. Absent. If the density of functional groups on the particle surface is increased, the effect of suppressing non-specific reaction is high, but the sensitivity is significantly lowered, which is not preferable. In addition, if the density of the functional group on the particle surface is too low, the effect of suppressing nonspecific reaction of the MPC monomer is reduced, and there is no difference from particles that do not use the MPC monomer. Absent.
- the functional group density of the MPC monomer is set to 0.003 to 0.05 ⁇ mol / m 2 , preferably 0.004 to 0.02 ⁇ mol / m 2 . This is very important.
- the functional group density can be calculated in detail by the following method.
- r average particle diameter (nm) of produced latex particles
- V Total volume of latex particles (cm 3 )
- S Total surface area of latex particles (cm 2 )
- x Total number of latexes
- Z Functional group density ( ⁇ mol / m 2 )
- K Total weight (g) of polymerizable monomers having a phenyl group, polymerizable monomers having a phenyl group and a sulfonate, polymerization initiator, and MPC monomer used in the polymerization reaction
- V 4 / 3 ⁇ (r / 2 ⁇ 10 ⁇ 7 ) 3 ⁇ x
- S 4 ⁇ (r / 2 ⁇ 10
- the average particle size of the latex particles for particle aggregation measurement of the present invention is preferably 0.05 to 1.0 ⁇ m. If it is less than 0.05 ⁇ m, the amount of optical change due to aggregation is too small to obtain the sensitivity required for measurement, and much time is required for centrifugation during reagent preparation, and the reagent cost increases. If it exceeds 1.0 ⁇ m, the amount of optical change due to particle aggregation exceeds the measurable region when the concentration of the detection target substance is high, and an optical change amount corresponding to the amount of detection target substance cannot be obtained. . Although it varies depending on the measuring method and measuring instrument using latex particles for particle aggregation measurement, it is preferably 0.05 to 0.7 ⁇ m, more preferably 0.05 to 0.4 ⁇ m.
- the particle diameter variation coefficient (CV value) of the particles is preferably 20% or less. If it exceeds 20%, the lot reproducibility at the time of reagent preparation may be poor, and the reproducibility of the measurement reagent may be lowered. More preferably, it is 15% or less.
- the functional group density which is the density occupied by the MPC monomer, on the surface of the latex particle for particle aggregation measurement of the present invention can be controlled by the molar amount of the MPC monomer used for producing the latex particle. That is, the amount of functional groups on the surface of the latex particles per unit area is determined not by the weight of the MPC monomer used for the production of the latex particles but by the molar amount, so that MPC monomers having different molecular weights can be used. As long as the following general formula (1) is satisfied, the functional group density can be determined by a simple operation, and the effects of the present invention can be obtained.
- CH 2 CR 1 —COOCH 2 CH 2 O (PO 2 ) OCH 2 CH 2 —N (CH 3 ) 3 (1) (Wherein R 1 represents a hydrogen atom or a methyl group)
- the latex particles for particle aggregation measurement of the present invention are obtained in a state suspended in water or an aqueous solvent.
- the particle concentration is not particularly limited, but it is usually preferably 1 to 20% by weight. If it is less than 1% by weight, it needs to be concentrated during preparation of the reagent, and if it exceeds 20% by weight, it may aggregate.
- a latex particle for particle aggregation measurement in which a substance that specifically binds to a detection target substance is physically adsorbed and supported is also one aspect of the present invention.
- Substances that specifically bind to the substance to be detected include immunoserologic test reagents (used in immunological agglutination and agglutination inhibition reactions) and physiologically active substances usually used as biochemical measurement methods. There is no particular limitation as long as it is present, but among these, those that can be used for antigen-antibody reaction are preferred.
- antigen-antibody reaction examples include antigens such as proteins, nucleic acids, nucleoproteins, estrogens, lipids, and antibodies.
- antigen examples include various antigens, receptors, enzymes, and the like. ⁇ 2 microglobulin, C-reactive protein (CRP), insulin, human fibrinogen, ferritin, rheumatoid factor (RA), ⁇ -fetoprotein (AFP), Examples include mycoplasma antigen and HBs antigen.
- CRP C-reactive protein
- RA rheumatoid factor
- AFP ⁇ -fetoprotein
- mycoplasma antigen and HBs antigen examples include mycoplasma antigen and HBs antigen.
- the antibody examples include antibodies against various toxins and pathogens, such as anti-streptolysin O antibody, anti-estrogen antibody, anti- ⁇ 2 microglobulin antibody, anti-syphilis treponema antibody, antibody against syphilis lipid antigen, anti-HBs antibody.
- the antibody may be a fragment such as F (ab ′) 2 .
- either a polyclonal antibody or a monoclonal antibody may be used as the antibody.
- the method of supporting (sensitizing) a substance that specifically binds to the detection target substance on the latex particles is not particularly limited as long as it is a physical adsorption method, and may be supported by a conventionally known method.
- sensitized latex dispersion After loading, if necessary, it can be blocked with bovine serum albumin or the like and dispersed in an appropriate buffer solution to prepare a sensitized latex dispersion.
- a buffer solution and a standard substance used for measurement are attached to this sensitized latex dispersion, and can be used as a kit for a reagent for measuring particle agglutination.
- the amount of the substance that specifically reacts with the detection target substance supported on the latex particles varies depending on the type of the substance that specifically reacts with the detection target substance used, and is not particularly limited.
- sensitizers When using a measurement reagent containing the above-mentioned latex particles for particle aggregation measurement carrying an antigen or an antibody, various sensitizers may be contained in order to improve measurement sensitivity or promote antigen-antibody reaction.
- the sensitizer include alkylated polysaccharides such as methylcellulose and ethylcellulose, pullulan, and polyvinylpyrrolidone.
- a protein such as albumin (bovine serum albumin, egg albumin), casein, gelatin or the like, a decomposition product thereof, an amino acid or a surfactant may be contained.
- the detection target substance may be diluted with an appropriate diluent.
- an appropriate diluent any buffer solution having a pH of 5.0 to 9.0 can be used.
- phosphate buffer, glycine buffer, Tris buffer, borate buffer, citric acid examples include a buffer solution.
- reaction amount of the detection target substance in the specimen can be measured.
- any optical bioanalyzer represented by an optical instrument capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc., or an optical instrument provided with a plurality of these detection methods, etc. can also be used.
- a conventionally known method is used. For example, an integrating sphere turbidity method in which a change in forward scattered light due to the formation of aggregates is measured using an integrating sphere, and the ratio to the transmitted light intensity is compared.
- a speed test (obtaining at least two measurement values at different time points, and determining the degree of aggregation based on the increment (increase rate) of the measurement values between these time points ( Rate assay)
- an end point test (end point assay) in which one measurement value is obtained at a certain time point (usually considered as an end point of the reaction), and the degree of aggregation is determined based on this measurement value.
- the end point test by the turbidimetric method is preferable from the viewpoint of simplicity of measurement and rapidity.
- the particle diameter of the obtained latex particles for particle aggregation measurement was measured as follows.
- Particle size measurement of latex particles for particle aggregation measurement The latex particles for particle aggregation measurement are placed on a collodion film according to a conventional method, a particle image is taken with a transmission electron microscope, and the average particle size and standard deviation are obtained by measuring the particle size (100 or more) on the image. It was.
- Example 1 In a glass reaction vessel (capacity 2 L) equipped with a stirrer, reflux condenser, temperature detector, nitrogen inlet tube and jacket, 1000 g of ultrapure water, 135 g of styrene monomer, 2- (methacryloyloxy) ethyl-2- (trimethyl) (Ammonium) 0.021 g of ethyl phosphate (manufactured by Tokyo Chemical Industry), 1.2 g of sodium styrenesulfonate, 0.7 g of potassium persulfate were added, the inside of the container was replaced with nitrogen gas, and the mixture was stirred at 70 ° C. and 210 rpm. The polymerization was carried out for 24 hours.
- the solution was filtered with a filter paper, and latex particles were taken out. Thereafter, a dialysis treatment was performed for 48 hours with a dialysis membrane to obtain purified latex particles.
- the obtained latex particles had a particle size of 0.108 ⁇ m (CV 9.7%) and an MPC density of 0.010 ⁇ mol / m 2 .
- Latex particles were obtained in the same manner as in Example 1 except that 0.021 g of 2- (methacryloyloxy) ethyl-2- (trimethylammonium) ethyl phosphate (manufactured by Tokyo Chemical Industry) was changed to 0.009 g.
- the obtained latex particles had a particle size of 0.102 ⁇ m (CV 10.4%) and an MPC density of 0.004 ⁇ mol / m 2 .
- Latex particles were obtained in the same manner as in Example 1 except that 0.021 g of 2- (methacryloyloxy) ethyl-2- (trimethylammonium) ethyl phosphate (manufactured by Tokyo Chemical Industry) was changed to 0.042 g.
- the obtained latex particles had a particle size of 0.105 ⁇ m (CV 10.1%) and an MPC density of 0.019 ⁇ mol / m 2 .
- Latex particles were obtained in the same manner as in Example 1 except that 0.021 g of 2- (methacryloyloxy) ethyl-2- (trimethylammonium) ethyl phosphate (manufactured by Tokyo Chemical Industry) was not used.
- the particle size of the obtained latex particles was 0.107 ⁇ m (CV 10.3%).
- the MPC density is 0 because no MPC monomer is used.
- Latex particles were obtained in the same manner as in Example 1 except that 0.021 g of 2- (methacryloyloxy) ethyl-2- (trimethylammonium) ethyl phosphate (manufactured by Tokyo Chemical Industry) was changed to 0.005 g.
- the obtained latex particles had a particle size of 0.105 ⁇ m (CV 9.8%) and an MPC density of 0.002 ⁇ mol / m 2 .
- Latex particles were obtained in the same manner as in Example 1 except that 0.021 g of 2- (methacryloyloxy) ethyl-2- (trimethylammonium) ethyl phosphate (manufactured by Tokyo Chemical Industry) was changed to 0.120 g.
- the obtained latex particles had a particle size of 0.110 ⁇ m (CV 9.1%) and an MPC density of 0.058 ⁇ mol / m 2 .
- the particle agglutination measurement reagent for D-dimer obtained by collecting this was adjusted to 0.5% (w / v) using a buffer solution to obtain an antibody-sensitized latex dispersion.
- a calibration curve was prepared using a D-dimer antigen standard solution.
- Substance to be detected (0-58 ⁇ g / mL D-dimer standard solution): 12 ⁇ L First reagent (sample dilution buffer: 0.15% (w / v) BSA, 30 mM Tris-HCl (pH 8.5): 100 ⁇ L Second reagent (0.5% (w / v) dispersion of antibody-sensitized latex particles): 100 ⁇ L Photometric point: 18-34
- the latex particles for particle aggregation measurement of the present invention in order to induce only the target specific aggregation reaction without adsorbing the non-detection target protein in the sample causing the nonspecific aggregation reaction, It is possible to obtain a diagnostic agent with higher sensitivity than before. It is useful as an immunoserologic test reagent (used in immunological agglutination and agglutination inhibition reactions) and a test reagent for physiologically active substances usually used as biochemical measurement methods.
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Abstract
Description
CH2=CR3-COO(CH2CH2O)X(CH(CH3)CH2O)y(CH2CH2O)ZR4
(式中、R3はHまたはCH3、R4はHまたはCH3を示し、x、yおよびzはそれぞれ0または100以下の整数であって、x、y、zは1≦x+y+z≦100を満たす)
CH2=CR6-CO-X-R5-O(PO2)OR4-N-(R1)(R2)(R3)
(式中、R1からR3はそれぞれ独立して水素原子または水酸基を有していてもよいアルキル基、R4はアルキレン基、R5は置換基を有していてもよく、かつ鎖中に酸素原子を有していても良いアルキレン基、R6は水素原子またはメチル基、Xは酸素原子または-NH-基を示す)
CH2=CR1-COOCH2CH2O(PO2)OCH2CH2-N(CH3)3・・・(1)
(式中、R1は水素原子またはメチル基を示す)
[2]上記フェニル基を有する重合性単量体が、スチレン、o-メチルスチレン、p-メチルスチレン、p-クロロスチレン、4-ビニル安息香酸からなる群から選ばれる少なくとも1つである上記[1]に記載の粒子凝集測定用ラテックス粒子。
[3]上記フェニル基とスルホン酸塩を有する重合性単量体が、スチレンスルホン酸塩、ジビニルベンゼンスルホン酸塩、o-メチルスチレンスルホン酸塩、p-メチルスチレンスルホン酸塩からなる群から選ばれる少なくとも1つである上記[1]または[2]に記載の粒子凝集測定用ラテックス粒子。
[4]上記フェニル基を有する重合性単量体がスチレン、フェニル基とスルホン酸塩を有する重合性単量体がスチレンスルホン酸ナトリウムであることを特徴とする上記[1]に記載の粒子凝集測定用ラテックス粒子。
[5]抗原または抗体を物理吸着により担持することを特徴とする上記[1]~[4]のいずれかに記載の粒子凝集測定用ラテックス粒子。
[6]上記[1]~[5]のいずれかに記載の粒子凝集測定用ラテックス粒子を用いる粒子凝集測定試薬。
CH2=CR1-COOCH2CH2O(PO2)OCH2CH2-N(CH3)3・・・(1)
(式中、R1は水素原子またはメチル基を示す)
具体的には、R1が水素原子である2-(アクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート、R1がメチル基である2-(メタクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート(東京化成製)が挙げられる。
尚、MPC単量体の官能基密度は、以下の計算式により算出する。
官能基密度(μmol/m2)=(ラテックス粒子製造に用いたMPC単量体のモル数)/(生成したラテックスの全表面積)
r:生成したラテックス粒子の平均粒径(nm)
Y:ラテックス粒子の製造に用いたMPC単量体のモル数(μmol)
すなわち、
Y=(製造に用いたMPC単量体の重量(g))/(製造に用いたMPC単量体の平均分子量)
V:ラテックス粒子の総体積(cm3)
S:ラテックス粒子の総表面積(cm2)
x:ラテックスの総数(個)
Z:官能基密度(μmol/m2)
K:重合反応に用いられる、フェニル基を有する重合性単量体、フェニル基及びスルホン酸塩を有する重合性単量体、重合開始剤、MPC単量体の重量の総和(g)
としたとき、
V=4/3π(r/2×10-7)3×x
S=4π(r/2×10-7)2×x
より、S=6V/(r×10-7)
よって、
Z=Y/(S×10-4)
=Y×r×10-3/6V
ここで、重合したポリスチレンの比重が1.06g/cm3より、
V=K/1.06
Z=1.766×10-4×Y×r/K
粒子径の変動係数(CV値)=粒子径の標準偏差/平均粒子径
CH2=CR1-COOCH2CH2O(PO2)OCH2CH2-N(CH3)3・・・(1)
(式中、R1は水素原子またはメチル基を示す)
粒子凝集測定用ラテックス粒子の粒径測定:
粒子凝集測定用ラテックス粒子を常法に従ってコロジオン膜状に載せ、透過型電子顕微鏡により粒子画像を撮影し、画像上の粒子径(100個以上)を計測することにより平均粒径及び標準偏差を求めた。
攪拌機、還流用冷却器、温度検出器、窒素導入管及びジャケットを備えたガラス製反応容器(容量2L)に、超純水1000g、スチレンモノマー135g、2-(メタクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート(東京化成製)0.021g、スチレンスルホン酸ナトリウム1.2g、過硫酸カリウム0.7gを添加し、容器内を窒素ガスで置換した後、70℃、210rpmの速度で攪拌しながら24時間重合を実施した。重合終了後、上記溶液をろ紙でろ過処理し、ラテックス粒子を取り出した。その後、透析膜にて48時間透析処理を行い、精製されたラテックス粒子を得た。得られたラテックス粒子の粒径は0.108μm(CV9.7%)、MPC密度は0.010μmol/m2であった。
2-(メタクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート(東京化成製)0.021gを0.009gに変更したこと以外は実施例1と同様にしてラテックス粒子を得た。得られたラテックス粒子の粒径は0.102μm(CV10.4%)、MPC密度は0.004μmol/m2であった。
2-(メタクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート(東京化成製)0.021gを0.042gに変更したこと以外は実施例1と同様にしてラテックス粒子を得た。得られたラテックス粒子の粒径は0.105μm(CV10.1%)、MPC密度は0.019μmol/m2であった。
2-(メタクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート(東京化成製)0.021gを用いなかったこと以外は実施例1と同様にしてラテックス粒子を得た。得られたラテックス粒子の粒径は0.107μm(CV10.3%)であった。尚、MPC単量体を用いていないため、MPC密度は0である。
2-(メタクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート(東京化成製)0.021gを0.005gに変更したこと以外は実施例1と同様にしてラテックス粒子を得た。得られたラテックス粒子の粒径は0.105μm(CV9.8%)、MPC密度は0.002μmol/m2であった。
2-(メタクリロイルオキシ)エチル-2-(トリメチルアンモニウム)エチルホスフェート(東京化成製)0.021gを0.120gに変更したこと以外は実施例1と同様にしてラテックス粒子を得た。得られたラテックス粒子の粒径は0.110μm(CV9.1%)、MPC密度は0.058μmol/m2であった。
実施例及び比較例のラテックス粒子を用いてDダイマー用粒子凝集測定試薬の評価を実施した。用いた試薬及び材料は以下の通りである。
<試薬及び材料>
・抗Dダイマー抗体
・抗体担持ラテックス調製用緩衝液:20mM Tris-HCl(pH8.0)を用いた。
・ブロッキング用緩衝液:2%(w/v)BSA 、20mM Tris-HCl(pH8.0)を用いた。
・検体希釈用緩衝液:0.15%(w/v)BSA、30mM Tris-HCl (pH8.5)を用いた。
実施例1~3、比較例1~3で得られたラテックス粒子を遠心分離で精製した後、5%(w/v)となるよう抗体担持ラテックス調製用緩衝液にて希釈して、希釈ラテックス液を調製した。
一方、抗Dダイマー抗体を濃度1mg/mLとなるよう抗体担持ラテックス調製用緩衝液にて希釈して、希釈抗体溶液を調製した。
上記希釈ラテックス液1容を攪拌しながら上記希釈抗体溶液1容を添加・混合し、更に攪拌した。その後、ブロッキング用緩衝液2容を追加添加し、攪拌を続けた。その後、これを回収して得られたDダイマー用粒子凝集測定試薬を、緩衝液を用いて0.5%(w/v)に調整して、抗体感作ラテックス分散液とした。この抗体感作ラテックス分散液を用い、Dダイマー抗原標準液にて検量線を作成した。
装置:日立 7170型自動分析装置
使用波長:570/800nm、測定温度:37℃
検出対象物質(0~58μg/mLのDダイマー標準液):12μL
第1試薬(検体希釈用緩衝液:0.15%(w/v)BSA、30mM Tris-HCl (pH8.5):100μL
第2試薬(抗体感作ラテックス粒子の0.5%(w/v)分散液):100μL 測光ポイント:18-34
抗Dダイマー抗体を感作した実施例1~3および比較例1~3の抗体感作ラテックス粒子(0.5%(w/v))を用いて、上記測定方法に従い測定し、検量線を作成した(図1)。実施例1~3の抗体感作ラテックス粒子を用いた場合は比較的高感度の測定が可能であった。一方、比較例1~3の抗体感作ラテックス粒子を用いた場合は、実施例に比較して低い感度しか得られなかった。
抗Dダイマー抗体を感作した実施例1~3および比較例1~3の抗体感作ラテックス粒子(0.5%(w/v))とDダイマー乖離検体(2種)を用いて、上記測定方法に従い測定し、その値を測定例1によって求めた検量線からそれぞれ濃度換算を行った。ここで、Dダイマー乖離検体とは、従来のDダイマー免疫比濁測定系で非特異的凝集反応が観察された検体をいう。その結果を図2に示す。実施例1~3の抗体感作ラテックス粒子を用いた場合は、MPC単量体を用いなかった比較例1に対して、乖離検体の非特異凝集反応を高度に抑制することが認められた。また、比較例3の抗体感作ラテックス粒子を用いた場合は、乖離検体の非特異的な凝集反応を実施例1~3と同様に抑制することが認められたが、測定例1で示されたように感度が大幅に低下した。この理由としては、MPC単量体の密度が高すぎた為に、ラテックス粒子表面に抗体が十分に吸着(感作)しなかったことが考えられる。
Claims (6)
- フェニル基を有する重合性単量体、フェニル基とスルホン酸塩を有する重合性単量体、及び式(1)を有する重合性単量体を成分として含み、式(1)を有する重合性単量体の粒子表面に占める密度(官能基密度)が0.003~0.05μmol/m2であることを特徴とする粒子凝集測定用ラテックス粒子。
CH2=CR1-COOCH2CH2O(PO2)OCH2CH2-N(CH3)3・・・(1)
(式中、R1は水素原子またはメチル基を示す) - 上記フェニル基を有する重合性単量体が、スチレン、o-メチルスチレン、p-メチルスチレン、p-クロロスチレン、4-ビニル安息香酸からなる群から選ばれる少なくとも1つである請求項1に記載の粒子凝集測定用ラテックス粒子。
- 上記フェニル基とスルホン酸塩を有する重合性単量体が、スチレンスルホン酸塩、ジビニルベンゼンスルホン酸塩、o-メチルスチレンスルホン酸塩、p-メチルスチレンスルホン酸塩からなる群から選ばれる少なくとも1つである請求項1または2に記載の粒子凝集測定用ラテックス粒子。
- 上記フェニル基を有する重合性単量体がスチレン、フェニル基とスルホン酸塩を有する重合性単量体がスチレンスルホン酸ナトリウムであることを特徴とする請求項1に記載の粒子凝集測定用ラテックス粒子。
- 抗原または抗体を物理吸着により担持することを特徴とする請求項1~4のいずれかに記載の粒子凝集測定用ラテックス粒子。
- 上記請求項1~5のいずれかに記載の粒子凝集測定用ラテックス粒子を用いる粒子凝集測定試薬。
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