WO2012169453A1 - 凝集促進剤 - Google Patents
凝集促進剤 Download PDFInfo
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- WO2012169453A1 WO2012169453A1 PCT/JP2012/064355 JP2012064355W WO2012169453A1 WO 2012169453 A1 WO2012169453 A1 WO 2012169453A1 JP 2012064355 W JP2012064355 W JP 2012064355W WO 2012169453 A1 WO2012169453 A1 WO 2012169453A1
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- general formula
- aggregation
- polymer
- reagent
- measurement
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Classifications
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- 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
- C08F20/00—Homopolymers and 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 a salt, anhydride, ester, amide, imide or nitrile thereof
- C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
- C08F20/10—Esters
- C08F20/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
- C08F20/36—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate containing oxygen in addition to the carboxy oxygen, e.g. 2-N-morpholinoethyl (meth)acrylate or 2-isocyanatoethyl (meth)acrylate
-
- 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
- C08F220/00—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 a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
-
- 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
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
Definitions
- the present invention relates to an aggregation promoter used in an immunoagglutination measurement method and an immunoaggregation measurement method using the same.
- aggregation is caused by using an insoluble carrier such as latex in which an antigen or antibody that reacts with the substance to be measured is immobilized.
- an insoluble carrier such as latex in which an antigen or antibody that reacts with the substance to be measured is immobilized.
- an immunoaggregation promoter that makes it easier to cause aggregation based on an antigen-antibody reaction is usually used for the purpose of improving sensitivity.
- Polyethylene glycol (PEG) or the like is well known as the immunoaggregation promoter, but PEG is salted out in a solution having a high salt concentration, so that there is a problem that the blank value increases and the measurement accuracy deteriorates. there were.
- an object of the present invention is to provide an aggregation promoter that exhibits an aggregation promoting effect superior to conventional immune aggregation promoters, which enables highly sensitive immunoagglutination measurement.
- an aggregation accelerator for immunoagglutination measurement comprising a polymer having a monomer unit represented by:
- the polymer has a monomer unit represented by the general formula [1] and the following general formula [2]
- R 4 represents a hydrogen atom or a methyl group
- k represents an integer of 1 to 10
- a reagent for an immunoagglutination measurement method comprising an aggregation promoter that is a copolymer having a monomer unit
- An immunoagglutination measurement method characterized in that an antigen-antibody reaction is carried out by bringing an antibody or antigen against a measurement target substance into contact with the measurement target substance in the presence of the above-described aggregation promoter for immunoagglutination measurement method.
- an antigen-antibody reaction is carried out by bringing an antibody or antigen against a measurement target substance into contact with the measurement target substance in the presence of the above-described aggregation promoter for immunoagglutination measurement method.
- the immune aggregation promoter of the present invention has a stronger aggregation promoting effect than conventional immune aggregation promoters. Therefore, in an immunoagglutination measurement method such as an immunoturbidimetric method or an immunotrophic method using an agglutination of an antigen-antibody reaction product generated using a carrier such as a latex particle on which an antigen or antibody is immobilized, By using an immune aggregation promoter, aggregation due to an antigen-antibody reaction is likely to occur. As a result, highly sensitive measurement is possible.
- the polymer used in the aggregation accelerator for immunoagglutination measurement of the present invention is any polymer having a monomer unit represented by the general formula [1]. It may be a homopolymer or a copolymer. Specific examples of the copolymer include those comprising a monomer unit represented by the general formula [1] and a monomer unit represented by the general formula [2].
- the weight average molecular weight of the polymer used in the aggregation accelerator of the present invention is usually 50,000 to 3,000,000, preferably 100,000 to 3,000,000, more preferably 200,000 to 3,000,000.
- the aggregation accelerator is a copolymer
- its weight average molecular weight is usually 50,000 to 3,000,000, preferably 100,000 to 3,000,000, more preferably 200,000 to 3,000,000.
- R 1 in the homopolymer general formula [1] having a monomer unit represented by the general formula [1] includes a hydrogen atom, a methyl group and the like, and a methyl group is preferable.
- R 2 in the general formula [1] examples include a methyl group and an ethyl group, and a methyl group is preferable.
- R 3 in the general formula [1] examples include a methyl group and an ethyl group, and a methyl group is preferable.
- X in the general formula [1] represents —NH— or an oxygen atom, preferably an oxygen atom.
- N in the general formula [1] usually represents an integer of 1 to 6, preferably an integer of 2 to 4, and more preferably an integer of 2 to 3.
- M in the general formula [1] usually represents an integer of 1 to 3, preferably an integer of 1 to 2, and more preferably 1.
- Preferred specific examples of the monomer unit represented by the general formula [1] include, for example, the following general formulas [1-1] to [1-8].
- n an integer of 1 to 6
- n an integer of 1 to 6
- n an integer of 1 to 6
- n an integer of 1 to 6
- n an integer of 1 to 6
- n an integer of 1 to 6
- n an integer of 1 to 6
- n an integer of 1 to 6
- general formulas [1-1] to [1-4] are preferable, general formula [1-1], general formula [1-3], and general formula [1-4] are more preferable, and general formula [1- 1] is particularly preferable. More specifically, those represented by the following [1-1-1], [1-1-2], [1-3-1], and [1-4-1] are preferable.
- (meth) acrylic acid derivative represented by the general formula [3] include N- [2- (dimethylamino) ethyl] methacrylic acid and N- [3- (dimethylamino) propyl] methacrylic acid.
- N- [2- (dimethylamino) ethyl] methacrylic acid N Particularly preferred are-[4- (dimethylamino) butyl] methacrylic acid, N- [3- (dimethylamino) propyl] acrylamide, N- [3- (dimethylamino) propyl] methacrylamide and the like.
- these (meth) acrylic acid derivatives commercially available products may be used, or those synthesized appropriately from (meth) acrylic acid by a conventional method or the like may be used.
- halogen atom represented by X in the general formula [4] examples include fluorine, chlorine, bromine and iodine, and chlorine and bromine are particularly preferable.
- carboxylic acid compound represented by the general formula [4] include, for example, chloroacetic acid, fluorinated acetic acid, bromoacetic acid, iodoacetic acid, chloropropionic acid, fluorinated propionic acid, bromopropionic acid, iodopropionic acid, chlorobutanoic acid. Fluorobutanoic acid, bromobutanoic acid, iodobutanoic acid and the like, bromoacetic acid, chloropropionic acid and chloroacetic acid are preferable, and chloroacetic acid is particularly preferable.
- the amount of the carboxylic acid compound represented by the general formula [4] is usually 0.5 to 3 times mol, preferably 1 to 2 times mol of the (meth) acrylic acid derivative represented by the general formula [3]. Such an amount is sufficient.
- reaction solvent at the time of reaction of the (meth) acrylic acid derivative represented by the general formula [3] and the carboxylic acid compound represented by the general formula [4] or a salt thereof for example, toluene, xylene, benzene, cyclohexane, n -Hydrocarbons such as hexane and n-octane, for example, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isotanol, tert-butanol, dimethylformamide (DMF), water, etc.
- DMF dimethylformamide
- Alcohols are preferable, and methanol, ethanol, n-propanol, isopropanol and the like are particularly preferable.
- the amount of the reaction solvent used is usually 100 to 300 mL with respect to a total amount of 50 g of the (meth) acrylic acid derivative represented by the general formula [3] and the carboxylic acid compound represented by the general formula [4] or a salt thereof. .
- the reaction temperature between the (meth) acrylic acid derivative represented by the general formula [3] and the carboxylic acid compound represented by the general formula [4] or a salt thereof may be appropriately set according to the reaction solvent, etc.
- the reaction time is 20 to 120 ° C, preferably 40 to 80 ° C, and the reaction time is usually 1 to 20 hours, preferably 5 to 12 hours.
- the polymerization reaction in the method for producing a polymer having a monomer unit represented by the general formula [1] can be carried out by a method known per se such as solution polymerization, bulk polymerization, emulsion polymerization, suspension polymerization and the like.
- the polymerization initiator include azoisobutyronitrile, 2,2′-azobis (2,4-dimethylvaleronitrile), 2,2′-azobis (methyl 2-methylpropionate), 2,2′-azobis.
- Azo compounds such as (2-methylbutyronitrile), peroxides such as benzoyl peroxide, lauroyl peroxide, potassium peroxodisulfate, and ammonium peroxodisulfate can be used.
- the amount of the polymerization initiator used is usually 0.1 to 3% by weight based on the total weight of all monomers.
- the polymerization reaction is preferably carried out in an inert gas atmosphere such as nitrogen or argon, the polymerization temperature is usually 40 to 120 ° C., preferably 50 to 70 ° C., and the polymerization time is 1 to 20 hours, preferably It may be performed for 0.5 to 5 hours.
- the solvent used here in addition to the specific examples of the reaction solvent at the time of the reaction between the (meth) acrylic acid derivative represented by the general formula [3] and the carboxylic acid compound represented by the general formula [4] or a salt thereof, And water, and water is preferred.
- the amount of the solvent used is usually 30 to 360 mL with respect to a total weight of 20 g of the polymer.
- a homopolymer having a monomer unit represented by the general formula [1] for example, first, 1 mol of a (meth) acrylic acid derivative represented by the above general formula [3] and the above general formula [4]
- the monomer is obtained by reacting 1 to 2 mol of the carboxylic acid compound or its salt in 500 to 1000 mL of ethanol at 40 to 80 ° C. for 5 to 12 hours. Thereafter, 10 g of the obtained monomer is dissolved in 50 to 100 mL of water, 1 to 30 mg of peroxide is added to the solution, and polymerization reaction is performed at 50 to 70 ° C. for 1 to 20 hours in an argon atmosphere. .
- Copolymer having a monomer unit represented by the general formula [1] and a monomer unit represented by the general formula [2]
- the content of the monomer unit represented by the general formula [1] in the copolymer is usually 50 mol%.
- the amount is less than 100 mol%, preferably 50 to 95 mol%, more preferably 50 to 60 mol%.
- the content of the monomer unit represented by the general formula [2] in the copolymer is usually more than 0 mol% and 50 mol% or less, preferably 5 to 50 mol%, more preferably 40 to 50 mol%.
- R 4 in the general formula [2] represents a hydrogen atom or a methyl group, and is preferably a hydrogen atom.
- k is usually 1 to 10, preferably 4 to 8, and more preferably 6 to 8.
- Specific examples of the monomer unit represented by the general formula [2] include, for example, the general formulas [2-1] to [2-2], and the general formula [2-1] is preferable.
- the monomer unit represented by the general formula [1] is represented by [1-1-1], [1-1-2], [1-3-1] or [1-4-1].
- a combination in which the monomer unit represented by the general formula [2] is [2-1-1] is more preferable, and the monomer unit represented by the general formula [1] is [1-1-1]
- a combination in which the monomer unit represented by the general formula [2] is [2-1-1] is particularly preferable.
- Vinyl compounds can be produced by polymerizing by a polymerization reaction according to a method known per se, for example, as described in JP-A-52-27713.
- the monomer represented by the general formula [1 ′] include those according to the monomer unit represented by the general formula [1]. Further, the monomer represented by the general formula [1 ′] is a (meth) acrylic acid derivative represented by the general formula [3] in the method for producing a homopolymer having the monomer unit represented by the general formula [1]. And a carboxylic acid compound represented by the general formula [4] or a salt thereof.
- vinyl compound represented by the general formula [2 ′] include, for example, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenoic acid, 8-nonenoic acid, 9-decenoic acid, 10- Undecenoic acid, 4-methyl-4-pentenoic acid, 5-methyl-5-hexenoic acid, 6-methyl-6-heptenoic acid, 7-methyl-7-octenoic acid, 8-methyl-8-nonenoic acid, 9- Examples include methyl-9-decenoic acid, 10-methyl-10-undecenoic acid and the like, and 7-octenoic acid, 8-nonenoic acid, 9-decenoic acid, 10-undecenoic acid and the like are preferable. Among them, 10-undecenoic acid is preferable. preferable.
- these vinyl compounds commercially available products may be used, or those synthesized appropriately from halogenated alkyl compounds by a conventional method or the like may
- the amount of the vinyl compound represented by the general formula [2 ′] is usually 1 to 100 mol%, preferably 3 to 100 mol%, more preferably 50 to 100 mol based on the monomer represented by the general formula [1 ′]. %.
- the aggregation promoter of the present invention comprises a polymer having a monomer unit represented by the general formula [1] as described above.
- a homopolymer having a monomer unit represented by the general formula [1], or a monomer unit represented by the general formula [1] and a monomer unit represented by the general formula [2] which comprises a copolymer having
- the aggregation promoter is preferably used by being dissolved in a reagent used in an immunoturbidimetric method among immunoagglutination measurement methods, and among them, it is preferably used by being dissolved in a reagent for latex aggregation method using latex as a carrier. Particularly preferred.
- the above-mentioned aggregation promoter of the present invention is usually 0.1 to 8 w / v%, preferably 0.1 to 4 w / v%, more preferably 0.1 to 2 w / v as the concentration in the reaction solution. It is added and used so that it may become v%. The concentration is preferably changed depending on the substance to be measured.
- the substance to be measured is CRP or Fer
- it is usually 0.1 to 8 w / v%, preferably 0.1 to 4 w / v%, more preferably 0.1 to
- the substance to be measured is PSA
- it is usually 0.1-7 w / v%, preferably 0.1-4 w / v%, more preferably 0.1-2 w / v%. It is preferable to be added and used.
- the measurement target substance is measured based on aggregation derived from the antigen-antibody reaction, except that the aggregation promoter of the present invention coexists. What is necessary is just to carry out according to the operation method known per se, using various reagents used in the immunoagglutination measurement method known per se (immunoturbidimetric method, immunotrophic method, etc.).
- the antibody or antigen against the measurement target substance may be brought into contact with the measurement target substance to cause an antigen-antibody reaction.
- an antibody against the measurement target substance or a carrier carrying the antibody, or an antigen against the measurement target substance or a carrier carrying the antigen is brought into contact with the measurement target substance to carry out an antigen-antibody reaction.
- the aggregation promoter of the present invention in the above method may be present at the concentration in the reaction solution as described above.
- a specific method other than the coexistence of the aggregation promoter of the present invention for example, when using a method of measuring scattered light (a comparative method), for example, Kanbara Publishing Co., Ltd.
- a method for measuring transmitted light for example, It may be performed according to the method described in Kanbara Publishing Co., Ltd., Clinical Laboratory Law Recommendation, 30th edition, 2nd edition, p.853-854 (1993).
- a latex agglutination method for measuring the degree of agglutination of latex sensitized with an antibody or antigen against a substance to be measured based on changes in scattered light, transmitted light, etc., and measuring the substance to be measured based on the result when used, for example, it may be carried out in accordance with the method described in, for example, a new application example of immunoassay and its application to the development of diagnostic reagents and therapeutic agents (Management Education Publishers) p.103-187.
- the buffer used during the reaction of the immunoaggregation measurement method of the present invention include, for example, conventional immunoturbidimetric methods such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, etc.
- the buffer used in the immuno-ratio brazing method include all, and the pH during the measurement reaction is not particularly limited as long as it does not suppress the antigen-antibody reaction, but is usually preferably selected from the range of 6 to 10.
- the measurement in the immunoagglutination measurement method of the present invention is performed by measuring scattered light or transmitted light, and the measurement is performed by a biochemical general-purpose machine such as an automatic analyzer or a spectrophotometer, a laser neferometer, or the like. What is necessary is just to make it using a dedicated machine for the measurement of bran and the like.
- the measurement target component that can be measured by the immunoagglutination measurement method of the present invention may be any component that can be measured using an antigen-antibody reaction.
- a biological sample such as serum, plasma, urine, lymph, and cerebrospinal fluid
- CRP C-reactive protein
- immunoglobulin G IgG
- immunoglobulin A IgA
- immunoglobulin M IgM
- immunoglobulin E IgE
- ASO anti-streptridine O value
- CRP CRP
- Fer CMP
- PSA CPP
- CK-MB CRP
- a monomer unit represented by the following general formula [2] may be used.
- an aggregation accelerator comprising a copolymer having a monomer unit represented by the general formula [1] and a monomer unit represented by the following general formula [2] may be used.
- a copolymer having a monomer unit represented by the general formula [1] and a monomer unit represented by the following general formula [2] may be used. preferable.
- the reagent for the immunoagglutination measurement method of the present invention may be any as long as it contains the aggregation promoter of the present invention, and the content thereof is usually 0.1 to 10 w / vW / V%, preferably as the concentration in the reagent, 0.1 to 5 w / v%, more preferably 0.1 to 2 w / v%.
- the concentration is preferably changed depending on the substance to be measured. For example, when the substance to be measured is CRP or Fer, it is usually 0.1 to 10 w / v%, preferably 0.1 to 5 w / v%, more preferably 0.1 to 1 w / v.
- the substance to be measured is PSA, it is usually 0.1 to 10 w / v%, preferably 0.1 to 5 w / v%, more preferably 0.1 to 2 w / v%.
- the measurement target component is an antigen
- a suitable carrier for example, latex or the like
- a suitable carrier for example, latex or the like
- a buffer for example, Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, etc.
- stabilizer for example, albumin, globulin, water-soluble gelatin, Surfactants, saccharides, etc.
- preservatives eg salicylic acid, benzoic acid, sodium azide, etc.
- concentration may be used in a concentration range usually used in this field.
- Examples of the measurement target in the reagent for immunoagglutination measurement method of the present invention include the same as those described in the section of the immunoagglutination measurement method of the present invention, and preferred ones are also the same.
- Synthesis Example 1 Synthesis of polymers 1-3
- Synthesis of N- [2- (carboxymethyldimethylamino) ethyl] methacrylic acid (monomer A) 297 g (4.5 mol) of potassium hydroxide was dissolved in 1.6 L of ethanol, and 426 g (4.5 mol) was added to the solution.
- Chloroacetic acid manufactured by Wako Pure Chemical Industries, Ltd. was added, followed by stirring at room temperature for 2 hours. Next, crystals precipitated by the reaction were collected by filtration and washed with isopropanol.
- the washed crystal was suspended in 1 L of isopropanol, and 475 g (3.0 mol) of N- [2- (dimethylamino) ethyl] methacrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the suspension. Then, the reaction was stirred for 12 hours under reflux. After completion of the reaction, insoluble matters were filtered off, the insoluble matters were washed with isopropanol, and the washing liquid was recovered. The filtrate and washing solution were mixed and dried under reduced pressure, and then acetone was added to the resulting residue to precipitate crystals.
- Synthesis Example 2 Synthesis of Polymer 4 (1) Synthesis of N- (4-dimethylamino) butylmethacrylic acid 23 g (0.2 mol) of N- (4-dimethylamino) butanol (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 200 mL of chloroform, 25 g (0.24 mol) of methacrylic acid chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the solution under ice-cooling, and then reacted at room temperature with stirring for 1.5 hours. Next, the obtained reaction solution was washed with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over magnesium sulfate. Thereafter, magnesium sulfate was filtered off, and the obtained filtrate was concentrated under reduced pressure to obtain 38 g of N- [4- (dimethylamino) butyl] methacrylic acid.
- polymer 4 For polymer 4, the presence of methacrylic acid segment (0.84 ppm to 1.45 ppm) was confirmed by 1 H-NMR spectrum analysis, and the presence of carbonyl group (—C ⁇ O) (1725 cm ⁇ 1 ) was confirmed by IR spectrum analysis. did.
- the physical properties of Polymer 4 were measured by GPC (SB-806M-HQ, manufactured by Shodex). As a result, the weight average molecular weight was 807,920 and the molecular weight distribution was 3.895.
- Synthesis Example 3 Synthesis of polymer 5 (1) Synthesis of N- [3- (carboxymethyldimethylamino) propyl] acrylamide (monomer C) 198 g (3.0 mol) of potassium hydroxide was dissolved in 1 L of ethanol, and 284 g (3.0 mol) of chloroacetic acid (sum) After adding Koyo Pure Chemical Industries, Ltd., the mixture was stirred at room temperature for 2 hours. Next, crystals precipitated by the reaction were collected by filtration and washed with isopropanol.
- Synthesis Example 4 Synthesis of polymer 6 (1) Synthesis of N- [3- (carboxymethyldimethylamino) propyl] methacrylamide (monomer D) 198 g (3.0 mol) of potassium hydroxide was dissolved in 1 L of ethanol and 284 g (3.0 mol) of chloroacetic acid ( After adding Wako Pure Chemical Industries, Ltd., the mixture was stirred at room temperature for 2 hours. Next, crystals precipitated by the reaction were collected by filtration and washed with isopropanol.
- the polymer 6, 1 by H-NMR spectroscopy, the presence of methacrylic acid segment (1.10 ppm ⁇ 2.00 ppm), the IR spectrum analysis, an amide group (-NH-C O) ( 1640cm -1, 1540cm -1 ) was confirmed.
- the physical properties of the polymer 6 were measured by GPC (SB-806M-HQ, manufactured by Shodex). As a result, the weight average molecular weight was 407,359 and the molecular weight distribution was 2.375.
- Synthesis Example 5 Synthesis of Copolymer 1 22 g of monomer A obtained in Synthesis Example 1 and 1.8 g of 10-undecenoic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in a mixed solvent of 50 mL of ion-exchanged water and 40 mL of DMF. Then, argon gas replacement for 30 minutes was performed. Next, 2 mL of 10% ammonium peroxodisulfate solution was added to this solution, and the mixture was stirred at 50 ° C. for 2 hours.
- the reaction solution was purified with a dialysis tube [Spectpore 2 (fractionated molecular weight: 12K to 14K, manufactured by Spectrum), ion-exchanged water; 5 L ⁇ 3 times]. After purification, the resulting polymer solution was freeze-dried to obtain 16.2 g of copolymer 1.
- the copolymer 1 contains 40 mol% of monomer units derived from undecenoic acid (hereinafter abbreviated as monomer unit E).
- Synthesis Example 6 Synthesis of Copolymer 2 22 g of monomer A obtained in Synthesis Example 1 and 7.4 g of 10-undecenoic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in a mixed solvent of 50 mL of ion-exchanged water and 40 mL of DMF. Then, argon gas replacement for 30 minutes was performed. Next, 2 mL of 10% ammonium peroxodisulfate solution was added to this solution, and the mixture was stirred at 50 ° C. for 2 hours.
- the reaction solution was purified with a dialysis tube [Spectpore 2 (fractionated molecular weight: 12K to 14K, manufactured by Spectrum), ion-exchanged water; 5 L ⁇ 3 times]. After purification, the polymer solution obtained was lyophilized to obtain 19.4 g of copolymer 2.
- Copolymer 2 contains 5 mol% of monomer unit E.
- copolymer 2 For copolymer 2, the presence of methacrylic acid segments (1.10 ppm to 2.00 ppm) and undecenoic acid segments (1.03 ppm) was determined by 1 H-NMR spectral analysis, and carbonyl groups (—C ⁇ O) (1725 cm) were determined by IR spectral analysis. -1 )) was confirmed.
- the physical properties of Copolymer 2 were measured by GPC (SB-806M-HQ, manufactured by Shodex). As a result, the weight average molecular weight was 957,680 and the molecular weight distribution was 2.041.
- Example 1 Measurement of CRP by latex immunoagglutination assay using polymers with different molecular weights as immune aggregation promoters
- Preparation of anti-human CRP antibody sensitized (immobilized) latex reagent 4.5 mL of 50 mM borate buffer (pH 7.5) containing 1 mg / mL of anti-human CRP goat polyclonal antibody (produced by Oriental Yeast Co., Ltd.) Then, 0.5 mL of a 10 w / v% aqueous solution of polystyrene latex (manufactured by Nippon Paint Co., Ltd.) having a particle size of 0.12 ⁇ m was mixed and reacted at 7 ° C. overnight.
- an anti-human CRP antibody-sensitized latex solution 2 was prepared in the same manner as described above using polystyrene latex (manufactured by Nippon Paint Co., Ltd.) having a particle size of 0.2 ⁇ m.
- Sample Saline (0.85% NaCl) was used as a reagent blind measurement sample (blank).
- Samples include LT / CRP-HS calibrator set HO (manufactured by Wako Pure Chemical Industries, Ltd., CRP concentrations of 0.2 mg / dL, 1.0 mg / dL, 4.0 mg / dL, 18.0 mg / dL, 35.0 mg / dL, respectively) Used).
- Reagent first reagent 0.1 M Tris buffer solution containing 0.1% BSA and 1% NaCl, containing 0.4 w / v% of the polymer 1, polymer 2 or polymer 3 as an aggregation accelerator synthesized in Synthesis Example 1 ( pH 8.0) was prepared, and three kinds of first reagents were prepared.
- the CRP concentration in the sample was measured using the BM-8 type automatic analyzer (manufactured by JEOL Ltd.) for the sample, the first reagent, and the second reagent under the following conditions.
- Comparative Example 1 Measurement of CRP by Latex Immunoaggregation Measurement Method Using Conventional Immunoaggregation Promoter Instead of Polymers 1 to 3 in Example 1, polyethylene glycol 6,000 (PEG 6,000, manufactured by Wako Pure Chemical Industries, Ltd.) or A 0.1M Tris buffer solution (pH 8.0) containing 0.1% BSA and 1% NaCl containing MPC polymer (manufactured by NOF Corporation) as a flocculation promoter was used as the first reagent. Measured CRP by the same method as in Example 1. The results are shown in Table 1 together with the results of Example 1.
- Example 2 Fer measurement by latex immunoagglutination assay using polymers with different molecular weights as immune aggregation promoters
- Preparation of anti-human Fer antibody sensitized (immobilized) latex reagent 1 ml of 50 mM borate buffer (pH 7.5) containing 0.6 mg / mL of anti-human FerF polyclonal antibody (manufactured by Dako) and particle size of 0.3 1 ml of 50 mM borate buffer (pH 7.5) suspended so as to contain 2 w / v% of ⁇ m polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) was mixed and reacted at 25 ° C. for 2 hours.
- Example 3 Measurement of PSA by latex immunoagglutination assay using polymers with different molecular weights as immune aggregation promoters
- PSA10 manufactured by Wako Pure Chemical Industries, Ltd.
- polystyrene latex (Sekisui Chemical Co., Ltd.) with a particle size of 0.28 ⁇ m 1 ml of 50 mM borate buffer solution (pH 7.1) suspended so as to contain 2 w / v%, and reacted at 25 ° C. for 2 hours. Thereafter, the entire amount of latex was separated from the suspension by centrifugation (45,000 g, 30 minutes), and washed with 2 mL of 50 mM borate buffer (pH 7.1).
- an anti-human PSA antibody-sensitized latex solution 1 was suspended in 2 mL of 50 mM borate buffer solution (pH 7.3) containing 0.5 w / v% BSA.
- 1 ml of 50 mM borate buffer (pH 7.1) containing 1.4 mg of anti-human PSA monoclonal antibody (clone No.
- Anti-human PSA antibody-sensitized latex prepared by mixing 1 ml of 50 mM borate buffer (pH 7.1) suspended in latex (made by Sekisui Chemical Co., Ltd.) to contain 2 w / v% Solution 2) was obtained.
- Sample A phosphate buffer (1 w / v% BSA, 10 mM phosphate buffer containing 0.85% NaCl) was used as a reagent blind measurement sample (blank).
- the sample is a PSA calibrator set (manufactured by Wako Pure Chemical Industries, Ltd., with PSA concentrations of 5.0 ng / mL, 10.0 ng / mL, 39.8 ng / mL, 69.3 ng / mL, and 98.6 ng / mL, respectively) used.
- Second reagent Suspend the anti-human PSA antibody-sensitized latex 1) and 2) prepared in (1) above in 2 mL each and 50 mL borate buffer (pH 7.5) containing 0.5 w / v% BSA in 16 mL. The mixture was used as the second reagent.
- Example 4 Measurement of CRP by Latex Immunoaggregation Measurement Method Using Various Polymers as Immune Aggregation Accelerator 0.1% BSA containing 0.4 to 4% of polymer 4-6 as an aggregation accelerator instead of polymers 1-3 CRP was measured by the same method as in Example 1 except that 0.1 M Tris buffer (pH 8.0) containing 1% NaCl was used as the first reagent. The results are shown in Table 4.
- Example 5 Measurement of Fer by Latex Immunoaggregation Measurement Method Using Various Polymers as Immune Aggregation Accelerator 0.1% BSA containing 0.4 to 4% of polymer 4-6 as an aggregation accelerator instead of polymers 1-3 Fer was measured by the same method as in Example 2 except that 0.1 M Tris buffer (pH 8.0) containing 1% NaCl was used as the first reagent. The results are shown in Table 5.
- Example 6 Measurement of PSA by Latex Immunoaggregation Measurement Method Using Various Polymers as Immune Aggregation Accelerator 0.1% BSA containing 0.4 to 4% of polymer 4-6 as an aggregation accelerator instead of polymers 1-3 PSA was measured by the same method as in Example 3 except that 0.1 M HEPES-NaOH buffer (pH 7.0) containing 1% NaCl was used as the first reagent. The results are shown in Table 6.
- Example 7 Measurement of CRP by latex immunoagglutination assay using copolymer as an immune aggregation promoter 0.1% BSA and 1 containing 0.4 w / v% of copolymer 1 or 2 as an aggregation promoter instead of polymers 1 to 3 CRP was measured by the same method as in Example 1 except that 0.1 M Tris buffer (pH 8.0) containing% NaCl was used as the first reagent. The results are shown in Table 7.
- Example 8 Measurement of Fer by Latex Immunoaggregation Method Using Copolymer as Immune Aggregation Promoter 0.1% BSA and 1 containing 0.4 w / v% of copolymer 1 or 2 as an aggregation promoter instead of polymers 1 to 3 Fer was measured by the same method as in Example 2 except that 0.1MHEPES-NaOH buffer (pH 7.0) containing% NaCl was used as the first reagent. The results are shown in Table 8.
- Example 9 Measurement of PSA by latex immunoagglutination assay using copolymer as an immune aggregation promoter 0.1% BSA and 1 containing 0.75 w / v% of copolymer 1 or 2 as an aggregation promoter instead of polymers 1 to 3 PSA was measured by the same method as in Example 3 except that 0.1 MHEPES-NaOH buffer (pH 7.0) containing% NaCl was used as the first reagent. The results are shown in Table 9.
- Example 10 Measurement of CK-MB by latex immunoagglutination measurement using copolymer as an immune aggregation promoter
- Anti-human CK-MB monoclonal antibody (clone MAK ⁇ CK-MB> M-7.4.5-IgG, manufactured by Roche) 0.8 mg / mL 50 mM borate buffer solution (pH 7.5) containing 1 ml and 50 mM borate buffer solution (pH 7.5) suspended in polystyrene latex (manufactured by Fujikura Kasei Co., Ltd.) having a particle size of 0.4 ⁇ m so as to contain 2 w / v%.
- 50 mM borate buffer (pH 7.5) containing 0.8 mg of anti-human CK-MB monoclonal antibody (clone MAK ⁇ CK-MB> M-6.12.47-IgG, manufactured by Roche)
- Sample Saline (0.85% NaCl) was used as a reagent blind measurement sample (blank).
- Samples were diluted with CK-MB antigen (human-derived CK-MB, manufactured by Cliniqa) with phosphate buffer (containing 10 mM phosphate, 1 w / v% BSA, 0.85% NaCl), and each concentration was 5.2 ng / mL. 19.0 ng / mL, 47.9 ng / mL, 98.7 ng / mL and 204.3 ng / mL were used.
- Reagent First Reagent A 0.1M HEPES-NaOH buffer solution (pH 7.0) containing 0.1% BSA and 1% NaCl containing 0.75 w / v% of polymer 1, copolymer 1 or copolymer 2 as an aggregation accelerator. 3 types of first reagents were prepared. Second reagent 2 mL each of the anti-human CK-MB antibody-sensitized latex solutions 1) and 2) prepared in (1) above, and 50 mL borate buffer solution (pH 7.5) containing 0.5 w / v% BSA 16 mL The second reagent was suspended and mixed in the solution.
- CK-MB concentration in the sample was measured using the BM-8 type automatic analyzer (manufactured by JEOL Ltd.) with the sample, the first reagent and the second reagent under the following conditions. .
- Comparative Example 4 Measurement of CK-MB by Latex Immunoaggregation Assay Using Conventional Immunoaggregation Accelerator Polyethylene glycol 6,000 (PEG6,000, manufactured by Wako Pure Chemical Industries, Ltd.) instead of polymer 1, copolymer 1 and copolymer 2 Or 0.1M HEPES-NaOH buffer (pH 7.0) containing 0.1% BSA and 1% NaCl containing 0.75% of MPC polymer (manufactured by NOF Corporation) as an aggregation accelerator. Except for the above, CK-MB was measured by the same method as in Example 10. The results are shown in Table 10 together with the results of Example 10.
- Example 11 Measurement of CRP by Latex Immunoaggregation Measurement Method with Different Contents of Immunoaggregation Promoter Content 0.12 containing 0.24 w / v%, 0.32 w / v%, 0.56 w / v% or 0.72 w / v% of polymer 1 CRP was measured by the same method as in Example 1 except that 0.1 M Tris buffer (pH 8.0) containing% BSA and 1% NaCl was used as the first reagent. The results are shown in Table 11.
- Example 12 Measurement of Fer by Latex Immunoaggregation Measurement Method with Different Content of Immunoaggregation Promoter
- polymer 1 was 0.25 w / v%, 0.31 w / v%, 0.49 w / v% or 0.61 w / v Fer was measured by the same method as in Example 2 except that 0.1 M HEPES-NaOH buffer (pH 7.0) containing 0.1% BSA and 1% NaCl was used. The results are shown in Table 12.
- Example 13 Measurement of PSA by latex immunoagglutination measurement method with different content of immune aggregation promoter Polymer 1 was 0.60 w / v%, 0.90 w / v%, 1.35 w / v% or 1.50 w / v as the first reagent.
- % PSA was measured by the same method as in Example 3 except that 0.1 M HEPES-NaOH buffer (pH 7.0) containing 0.1% BSA and 1% NaCl was used. The results are shown in Table 13.
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Abstract
Description
(式中、R1は水素原子又はメチル基を表し、R2及びR3はそれぞれ独立してメチル基又はエチル基を表し、Xは-NH-又は酸素原子を表し、nは1~6の整数を表し、mは1~3の整数を表す)で示されるモノマー単位を有するポリマーが、従来の免疫凝集促進剤よりも優れた凝集促進効果を示すことを見出し、本発明を完成するに至った。
「下記一般式[1]
(式中、R1、R2、R3、X、n及びmは上記と同じ)で示されるモノマー単位を有するポリマーを含んでなる免疫凝集測定法用凝集促進剤、
上記ポリマーが、上記一般式[1]で示されるモノマー単位と下記一般式[2]
一般式[1]におけるR1としては、水素原子、メチル基等が挙げられるが、メチル基が好ましい。
X-(CH 2)m-COOH [4]
(式中、Xはハロゲン原子を表し、mは上記と同じ)で示されるカルボン酸化合物とを、適当な溶媒中で反応させ、得られたモノマーを、更に自体公知の重合反応により重合することにより製造することができる。
該コポリマー中の一般式[1]で示されるモノマー単位の含有量は、通常50モル%以上100モル%未満であり、50~95モル%が好ましく、50~60モル%がより好ましい。また、該コポリマー中の一般式[2]で示されるモノマー単位の含有量は、通常0モル%超50モル%以下であり、5~50モル%が好ましく、40~50モル%がより好ましい。
一般式[2]におけるkとしては、通常1~10であり、4~8が好ましく、6~8がより好ましい。
一般式[2]で示されるモノマー単位の具体例としては、例えば一般式[2-1]~[2-2]が挙げられ、一般式[2-1]が好ましい。
一般式[1]で示されるモノマー単位と一般式[2]で示されるモノマー単位を有するコポリマーの製造方法としては、例えば下記一般式[1’]
(式中、R1、R2、R3、X、n及びmは上記と同じ)で示されるモノマーと
一般式[2’]で示される
これらビニル化合物は、市販品を用いてもよいし、ハロゲン化アルキル化合物から常法等により適宜合成したものを用いてもよい。
本発明の凝集促進剤は、上記の如き、一般式[1]で示されるモノマー単位を有するポリマーを含んでなるものであり、より具体的には、上記一般式[1]で示されるモノマー単位を有するホモポリマー、或いは、上記一般式[1]で示されるモノマー単位と一般式[2]で示されるモノマー単位を有するコポリマーを含んでなるものである。該凝集促進剤は、免疫凝集測定方法の中でも免疫比濁法で用いられる試薬に溶解させて用いられるのが好ましく、その中でもラテックスを担体に用いるラテックス凝集法用試薬に溶解させて用いられるのが特に好ましい。
(1)N-〔2-(カルボキシメチルジメチルアミノ)エチル〕メタアクリル酸(モノマーA)の合成
297g(4.5mol)の水酸化カリウムを1.6Lのエタノールに溶解し、該溶液に426g(4.5mol)のクロロ酢酸(和光純薬工業(株)製)を添加した後、室温で2時間攪拌反応させた。次いで、反応により析出した結晶を濾取し、イソプロパノールで洗浄した。更に、洗浄した結晶を1Lのイソプロパノールに懸濁し、該懸濁液に475g(3.0mol)のN-〔2-(ジメチルアミノ)エチル〕メタアクリル酸(和光純薬工業(株)製)を添加した後、還流下で12時間攪拌反応させた。反応終了後、不溶物を濾別し、該不溶物をイソプロパノールで洗浄し、該洗浄液を回収した。濾液と洗浄液を混合して減圧乾燥した後、得られた残渣にアセトンを添加して結晶を析出させた。その後、析出した結晶を濾取し、減圧乾燥して420gのN-〔2-(カルボキシメチルジメチルアミノ)エチル〕メタアクリル酸(以下、モノマーAと略記する)を得た。
上記(1)で得たモノマーA(11g~22g)をイオン交換水(90mL~180mL)に溶解した後、5~30分間のアルゴンガス置換を行った。この溶液に2mLの10%-ペルオキソ二硫酸アンモニウム溶液を添加した後、50℃で1~2時間攪拌反応させた。反応終了後、該反応液を透析チューブ[スペクトラポア2(分画分子量12K~14K、Spectrum社製),イオン交換水;5L×3回]で精製した。上記操作を3回行い、得られたポリマー溶液をそれぞれ凍結乾燥し、9.4g~14.8gのポリマー1~3を得た。
(1)N-(4-ジメチルアミノ)ブチルメタアクリル酸の合成
23g(0.2mol)のN-(4-ジメチルアミノ)ブタノール(和光純薬工業(株)製)を200mLのクロロホルムに溶解し、氷冷下で該溶液に25g(0.24mol)のメタアクリル酸クロリド(和光純薬工業(株)製)を添加した後、室温で1.5時間攪拌しながら反応させた。次いで、得られた反応溶液を飽和重曹水及び飽和食塩水で洗浄した後、硫酸マグネシウムで乾燥した。その後、硫酸マグネシウムを濾別し、得られた濾液を減圧濃縮して38gのN-〔4-(ジメチルアミノ)ブチル〕メタアクリル酸を得た。
12g(0.21mol)の水酸化カリウムを150mLのエタノールに溶解し、該溶液に20g(0.21mol)のクロロ酢酸(和光純薬工業(株)製)を添加した後、室温で2時間攪拌して反応させた。次いで、反応により析出した結晶を濾取し、エタノールで洗浄した。その後、洗浄した結晶を50mLのエタノールに懸濁し、上記(1)で得た38g(0.2mol)のN-(4-ジメチルアミノ)ブチルメタアクリル酸を添加した後、還流下で12時間攪拌反応させた。反応終了後、不溶物を濾別し、エタノールで洗浄し、該洗浄液を回収した。濾液と洗浄液とを混合して減圧乾燥した後、得られた残渣にアセトンを添加して結晶を析出させた。その後、析出した不溶物を濾別し、減圧濃縮して26gのN-〔4-(カルボキシメチルジメチルアミノ)ブチル〕メタアクリル酸(以下モノマーBと略記する)を得た。
上記(2)で得た25gのモノマーBを90mLのイオン交換水に溶解し、30分間のアルゴンガス置換を行った。この溶液に2mLの10%-ペルオキソ二硫酸アンモニウム溶液を添加した後、50℃で2時間攪拌反応させた。反応終了後、反応液を透析チューブ[スペクトラポア2(分画分子量12K~14K、Spectrum社製),イオン交換水;5L×3回]で精製した。精製後、得られたポリマー溶液を凍結乾燥し、17.2gのポリマー4を得た。
ポリマー4の物性をGPC(SB-806M-HQ、Shodex社製)により測定した結果、重量平均分子量は807,920,分子量分布は3.895であった。
(1)N-〔3-(カルボキシメチルジメチルアミノ)プロピル〕アクリルアミド(モノマーC)の合成
198g(3.0mol)の水酸化カリウムを1Lのエタノールに溶解し、284g(3.0mol)のクロロ酢酸(和光純薬工業(株)製)を添加した後、室温で2時間攪拌反応させた。次いで、反応により析出した結晶を濾取し、イソプロパノールで洗浄した。その後、洗浄した結晶を400mLのイソプロパノールに懸濁し、該懸濁液に511g(3.0mol)のN-〔3-(ジメチルアミノ)プロピル〕アクリルアミド(和光純薬工業(株)製)を添加した後、還流下で12時間攪拌反応させた。反応終了後、不溶物を濾別し、イソプロパノールで洗浄し、該洗浄液を回収した。濾液と洗浄液とを混合して減圧乾燥した後、得られた残渣にアセトンを添加して結晶を析出させた。その後、析出した結晶を濾取し、減圧乾燥して565gのN-〔3-(カルボキシメチルジメチルアミノ)プロピル〕アクリルアミド〔以下モノマーCと略記する〕を得た。
上記(1)で得た46gのモノマーCを90mLのイオン交換水に溶解し、30分間のアルゴンガス置換を行った。次いで、この溶液に2mLの10%-ペルオキソ二硫酸アンモニウム溶液を添加した後、50℃で2時間攪拌反応させた。反応終了後、反応液を透析チューブ[スペクトラポア2(分画分子量12K~14K、Spectrum社製),イオン交換水;5L×3回]で精製した。精製後、得られたポリマー溶液を凍結乾燥することで26.2gのポリマー5を得た。
ポリマー5の物性をGPC(SB-806M-HQ、Shodex社製)により測定した結果、重量平均分子量は262,065、分子量分布は4.332であった。
(1)N-〔3-(カルボキシメチルジメチルアミノ)プロピル〕メタアクリルアミド(モノマーD)の合成
198g(3.0mol)の水酸化カリウムを1Lのエタノールに溶解し、284g(3.0mol)のクロロ酢酸(和光純薬工業(株)製)を添加した後、室温で2時間攪拌反応させた。次いで、反応により析出した結晶を濾取し、イソプロパノールで洗浄した。その後、洗浄した結晶を400mLのイソプロパノールに懸濁し、該懸濁液に511g(3.0mol)のN-〔3-(ジメチルアミノ)プロピル〕メタアクリルアミド(和光純薬工業(株)製)を添加した後、還流下で12時間攪拌反応させた。反応終了後、不溶物を濾別し、イソプロパノールで洗浄し、該洗浄液を回収した。濾液と洗浄液とを混合して減圧乾燥した後、得られた残渣にアセトンを添加して結晶を析出させた。その後、析出した結晶を濾取し、減圧乾燥して565gのN-〔3-(カルボキシメチルジメチルアミノ)プロピル〕メタアクリルアミド〔以下モノマーDと略記する〕を得た。
上記(1)で得た21gのモノマーDを90mLのイオン交換水に溶解し、30分間のアルゴンガス置換を行った。次いで、この溶液に2mLの10%-ペルオキソ二硫酸アンモニウム溶液を添加した後、50℃で2時間攪拌反応させた。反応終了後、反応液を透析チューブ[スペクトラポア2(分画分子量12K~14K、Spectrum社製),イオン交換水;5L×3回]で精製した。精製後、得られたポリマー溶液を凍結乾燥することで。13.5gのポリマー6を得た。
ポリマー6の物性をGPC(SB-806M-HQ、Shodex社製)により測定した結果、重量平均分子量は407,359、分子量分布は2.375であった。
上記合成例1で得た22gのモノマーAと1.8gの10-ウンデセン酸(和光純薬工業(株)製)を50mLのイオン交換水と40mLのDMFとの混合溶媒に溶解し、30分間のアルゴンガス置換を行った。次いで、この溶液に2mLの10%-ペルオキソ二硫酸アンモニウム溶液を添加した後、50℃で2時間攪拌反応させた。反応終了後、反応液を透析チューブ[スペクトラポア2(分画分子量12K~14K、Spectrum社製),イオン交換水;5L×3回]で精製した。精製後、得られたポリマー溶液を凍結乾燥することで16.2gのコポリマー1を得た。尚、コポリマー1はウンデセン酸由来のモノマー単位(以下、モノマー単位Eと略記する)を40モル%含む。
コポリマー1の物性をGPC(SB-806M-HQ、Shodex社製)により測定した結果、重量平均分子量は658,206,分子量分布は2.216であった。
上記合成例1で得た22gのモノマーAと7.4gの10-ウンデセン酸(和光純薬工業(株)製)を50mLのイオン交換水と40mLのDMFとの混合溶媒に溶解し、30分間のアルゴンガス置換を行った。次いで、この溶液に2mLの10%-ペルオキソ二硫酸アンモニウム溶液を添加した後、50℃で2時間攪拌反応させた。反応終了後、反応液を透析チューブ[スペクトラポア2(分画分子量12K~14K、Spectrum社製),イオン交換水;5L×3回]で精製した。精製後、得られたポリマー溶液を凍結乾燥することで19.4gのコポリマー2を得た。尚、コポリマー2は、モノマー単位Eを5モル%含む。
コポリマー2の物性をGPC(SB-806M-HQ、Shodex社製)により測定した結果、重量平均分子量は957,680,分子量分布は2.041であった。
(1)抗ヒトCRP抗体感作(固定化)ラテックス試薬の調製
抗ヒトCRP山羊ポリクローナル抗体(オリエンタル酵母工業(株)製)1mg/mLを含む50mMホウ酸緩衝液(pH7.5) 4.5mLと、粒径0.12μmのポリスチレンラテックス(日本ペイント(株)製)の10w/v%水溶液0.5mLとを混合し、7℃で一晩反応させた。その後、得られた懸濁液 5mLとウシ血清アルブミン(BSA)を2.5w/v%含有する50mMホウ酸緩衝液(pH7.5) 5mLとを混合し、7℃で2時間反応させた。次いで、遠心分離(45,000g、30分)により分離したラテックス全量を50mMホウ酸緩衝液(pH7.5) 5mLで洗浄し、BSAを0.5W/V%含有する50mMホウ酸緩衝液(pH7.5) 12.5mLに懸濁させ、該懸濁液を抗ヒトCRP抗体感作ラテックス溶液1)とした。また、上記と同様にして粒径0.2μmのポリスチレンラテックス(日本ペイント(株)製)を用いて調製したものを、抗ヒトCRP抗体感作ラテックス溶液2)とした。
試薬盲検測定用試料(ブランク)には、生理食塩水(0.85%NaCl)を使用した。試料には、LT・CRP-HSキャリブレーターセットHO(和光純薬工業(株)製、CRP濃度がそれぞれ0.2 mg/dL、1.0 mg/dL、4.0 mg/dL、18.0 mg/dL、35.0mg/dLのもの)を使用した。
第一試薬
合成例1で合成した、ポリマー1、ポリマー2又はポリマー3を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1Mトリス緩衝液(pH8.0)を調製し、3種類の第一試薬を準備した。
第二試薬
上記(1)で調製した抗ヒトCRP抗体感作ラテックス1) 12mL及び抗ヒトCRP抗体感作ラテックス2) 8mLを混合したものを第二試薬とした。
上記試料、上記第一試薬及び上記第二試薬を下記条件でBM-8形自動分析装置(日本電子(株)製)を用いて、試料中のCRP濃度を測定した。
第一試薬 : 100μL
第二試薬 : 25μL
測定方法 : 2ポイントエンド法(34-65)
主波長 : 596nm
得られた結果を表1に示す。
実施例1のポリマー1~3の代わりにポリエチレングリコール6,000(PEG6,000、和光純薬工業(株)製)又はMPCポリマー(日油(株)製)を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1Mトリス緩衝液(pH8.0)を第一試薬として用いた以外は、実施例1と同様の方法によりCRPを測定した。その結果を、実施例1の結果と併せて表1に示す。
(1)抗ヒトFer抗体感作(固定化)ラテックス試薬の調製
抗ヒトFer兎ポリクローナル抗体(Dako社製)0.6mg/mLを含む50mMホウ酸緩衝液(pH7.5) 1mlと、粒径0.3μmのポリスチレンラテックス(積水化学工業(株)製)を2w/v%含むように懸濁させた50mMホウ酸緩衝液(pH7.5) 1mlとを混合し、25℃で2時間反応させた。その後、得られた懸濁液 2mLとBSAを1.25w/v%含有する50mMホウ酸緩衝液(pH7.5) 2mLとを混合し、7℃で2時間反応させた。次いで、遠心分離(45,000g、30分)により分離したラテックス全量を50mMホウ酸緩衝液(pH7.5) 4mLで洗浄し、BSAを0.5w/v%含有する50mMホウ酸緩衝液(pH7.5) 20mLに懸濁したものを、抗ヒトFer抗体感作ラテックス溶液とした。
試薬盲検測定試料(ブランク)には、生理食塩水(0.85%NaCl)を使用した。試料には、フェリチンキャリブレーターセット(和光純薬工業(株)製、Fer濃度が、それぞれ30 ng/mL、100 ng/mL 、200 ng/mL、500 ng/mL、1000 ng/mLのもの)を使用した。
第一試薬
合成例1で合成した、ポリマー1、ポリマー2又はポリマー3を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1MHEPES-NaOH緩衝液(pH7.0)を調製し、3種類の第一試薬を準備した。
第二試薬
上記(1)で調製した抗ヒトFer抗体感作ラテックス溶液を第二試薬とした。
上記試料、上記第一試薬及び上記第二試薬を下記条件でBM-8形自動分析装置(日本電子(株)製)を用いて、試料中のFer濃度を測定した。
試料 : 12.0μL(生理食塩水で2倍希釈)
第一試薬 : 90μL
第二試薬 : 30μL
測定方法 : 2ポイントエンド法(35-59)
主波長 : 694nm
得られた結果を表2に示す。
実施例2のポリマー1~3の代わりにポリエチレングリコール6,000(PEG6,000, 和光純薬工業(株)製)又はMPCポリマー(日油(株)製)を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1MHEPES-NaOH緩衝液(pH7.0)を第一試薬として用いた以外は、実施例2と同様の方法によりFerを測定した。
その結果を、実施例2の結果と併せて表2に示す。
(1)抗ヒトPSA抗体感作(固定化)ラテックス試薬の調製
抗ヒトPSAモノクローナル抗体(クローンNo.PSA10、和光純薬工業(株)製)0.6mg/mLを含む50mMホウ酸緩衝液(pH7.1) 1mlと、粒径0.28μmのポリスチレンラテックス(積水化学工業(株)製)を2w/v%含むように懸濁させた50mMホウ酸緩衝液(pH7.1) 1mlとを混合し、25℃で2時間反応させた。その後、該懸濁液から遠心分離(45,000g、30分)によりラテックス全量を分離し、50mMホウ酸緩衝液(pH7.1) 2mLで洗浄した。次いで、BSAを0.5w/v%含有する50mMホウ酸緩衝液(pH7.3) 2mLに懸濁したものを抗ヒトPSA抗体感作ラテックス溶液1)とした。
また、上記と同様にして抗ヒトPSAモノクローナル抗体(クローンNo.PSA14、和光純薬工業(株)製)1.4mgを含む50mMホウ酸緩衝液(pH7.1) 1mlと、粒径0.15μmのポリスチレンラテックス(積水化学工業(株)製)を2w/v%含むように懸濁させた50mMホウ酸緩衝液(pH7.1) 1mlとを混合して調製したものを、抗ヒトPSA抗体感作ラテックス溶液2)とした。
試薬盲検測定試料(ブランク)には、リン酸緩衝液(1w/v%BSA、0.85%NaCl含有10mMリン酸緩衝液)を使用した。試料には、PSAキャリブレーターセット(和光純薬工業(株)製、PSA濃度が、それぞれ5.0ng/mL、10.0ng/mL、39.8 ng/mL、69.3ng/mL、98.6ng/mLのもの)を使用した。
第一試薬
合成例1で合成した、ポリマー1、ポリマー2又はポリマー3を0.75w/v%含有した、0.1%BSA及び1%NaClを含む0.1MHEPES-NaOH緩衝液(pH7.0)を調製し、3種類の第一試薬を準備した。
第二試薬
上記(1)で調製した抗ヒトPSA抗体感作ラテックス1)および2)をそれぞれ2mLずつ、BSAを0.5w/v%含有する50mMホウ酸緩衝液(pH7.5) 16mLに懸濁し混合したものを第二試薬とした。
上記試料、上記第一試薬及び上記第二試薬を下記条件でBM-8形自動分析装置(日本電子(株)製)を用いて、試料中のPSA濃度を測定した。得られた結果を表3に示す。
第一試薬 : 90μL
第二試薬 : 30μL
測定方法 : 2ポイントエンド法(37-65)
主波長 : 694nm
実施例3のポリマー1~3の代わりにポリエチレングリコール6,000(PEG6,000, 和光純薬工業(株)製)又はMPCポリマー(日油(株)製)を凝集促進剤として0.75w/v%含有した、0.1%BSA及び1%NaClを含む0.1MHEPES-NaOH緩衝液(pH7.0)を第一試薬として用いた以外は、実施例3と同様の方法によりPSAを測定した。
その結果を、実施例3の結果と併せて表3に示す。
ポリマー1~3の代わりにポリマー4~6を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1Mトリス緩衝液(pH8.0)を第一試薬として用いた以外は、実施例1と同様の方法によりCRPを測定した。その結果を表4に示す。
ポリマー1~3の代わりにポリマー4~6を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1Mトリス緩衝液(pH8.0)を第一試薬として用いた以外は、実施例2と同様の方法によりFerを測定した。その結果を表5に示す。
ポリマー1~3の代わりにポリマー4~6を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1M HEPES-NaOH緩衝液(pH7.0)を第一試薬として用いた以外は、実施例3と同様の方法によりPSAを測定した。その結果を表6に示す。
ポリマー1~3の代わりにコポリマー1又は2を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1Mトリス緩衝液(pH8.0)を第一試薬として用いた以外は、実施例1と同様の方法によりCRPを測定した。その結果を表7に示す。
ポリマー1~3の代わりにコポリマー1又は2を凝集促進剤として0.4w/v%含有した、0.1%BSA及び1%NaClを含む0.1MHEPES-NaOH緩衝液(pH7.0)を第一試薬として用いた以外は、実施例2と同様の方法によりFerを測定した。その結果を表8に示す。
ポリマー1~3の代わりにコポリマー1又は2を凝集促進剤として0.75w/v%含有した、0.1%BSA及び1%NaClを含む0.1MHEPES-NaOH緩衝液(pH7.0)を第一試薬として用いた以外は、実施例3と同様の方法によりPSAを測定した。その結果を表9に示す。
(1)抗ヒトCK-MB抗体感作(固定化)ラテックス試薬の調製
抗ヒトCK-MBモノクローナル抗体(クローンMAK〈CK-MB〉M-7.4.5-IgG、Roche社製)0.8mg/mLを含む50mMホウ酸緩衝液(pH7.5) 1mlと、粒径0.4μmのポリスチレンラテックス(藤倉化成(株)製)を2w/v%含むように懸濁させた50mMホウ酸緩衝液(pH7.5) 1mlとを混合し、25℃で2時間反応させた。その後、遠心分離(45,000g、30分)により分離したラテックス全量を50mMホウ酸緩衝液(pH7.5) 2mLで洗浄した。次いで、BSAを0.5w/v%含有する50mMホウ酸緩衝液(pH7.5) 2mLに懸濁させ、抗ヒトCK-MB抗体感作ラテックス溶液1)とした。
また、上記と同様の方法により、抗ヒトCK-MBモノクローナル抗体(クローンMAK〈CK-MB〉M-6.12.47-IgG、Roche社製)0.8mgを含む50mMホウ酸緩衝液(pH7.5) 1mlと、粒径0.4μmのポリスチレンラテックス(藤倉化成(株)製)を2w/v%含むように懸濁させた50mMホウ酸緩衝液(pH7.5) 1mlとを混合して調製したものを、抗ヒトCK-MB抗体感作ラテックス溶液2)とした。
試薬盲検測定用試料(ブランク)には、生理食塩水(0.85%NaCl)を使用した。試料は、CK-MB抗原(ヒト由来CK-MB、Cliniqa社製)をリン酸緩衝液(10mMリン酸、1w/v%BSA、0.85%NaCl含有)で希釈し、濃度がそれぞれ5.2ng/mL、19.0ng/mL、47.9ng/mL、98.7ng/mL、204.3ng/mLとなるように調製したものを使用した。
第一試薬
ポリマー1、コポリマー1又はコポリマー2を凝集促進剤として0.75w/v%含有した、0.1%BSA及び1%NaClを含む0.1M HEPES-NaOH緩衝液(pH7.0)を調製し、3種類の第一試薬を準備した。
第二試薬
上記(1)で調製した抗ヒトCK-MB抗体感作ラテックス溶液1)および2)をそれぞれ2mLずつ、BSAを0.5w/v%含有する50mMホウ酸緩衝液(pH7.5) 16mLに懸濁し混合したものを第二試薬とした。
上記試料、上記第一試薬及び上記第二試薬を下記条件でBM-8形自動分析装置(日本電子(株)製)を用いて、試料中のCK-MB濃度を測定した。
第一試薬 : 90μL
第二試薬 : 30μL
測定方法 : 2ポイントエンド法(34-65)
主波長 : 596nm
得られた結果を表10に示す。
ポリマー1、コポリマー1及びコポリマー2の代わりにポリエチレングリコール6,000(PEG6,000, 和光純薬工業(株)製)又はMPCポリマー(日油(株)製)を凝集促進剤として0.75%含有した、0.1%BSA及び1%NaClを含む0.1M HEPES-NaOH緩衝液(pH7.0)を第一試薬として用いた以外は、実施例10と同様の方法によりCK-MBを測定した。その結果を、実施例10の結果と併せて表10に示す。
ポリマー1を0.24w/v%、0.32w/v%、0.56w/v%又は0.72w/v%含有した、0.1%BSA及び1%NaClを含む0.1Mトリス緩衝液(pH8.0)を第一試薬として用いた以外は、実施例1と同様の方法によりCRPを測定した。その結果を表11に示す。
第一試薬として、ポリマー1を0.25w/v%、0.31w/v%、0.49w/v%又は0.61w/v%含有した、0.1%BSA及び1%NaClを含む0.1M HEPES-NaOH緩衝液(pH7.0)を用いた以外は、実施例2と同様の方法によりFerを測定した。その結果を表12に示す。
第一試薬として、ポリマー1を0.60w/v%、0.90w/v%、1.35w/v%又は1.50w/v%含有した、0.1%BSA及び1%NaClを含む0.1M HEPES-NaOH緩衝液(pH7.0)を用いた以外は、実施例3と同様の方法によりPSAを測定した。その結果を表13に示す。
Claims (11)
- コポリマー中の一般式[1]で示されるモノマー単位の含有量が50モル%以上100モル%未満である、請求項2記載の凝集促進剤。
- ポリマーの重量平均分子量が、50,000~3,000,000である、請求項1記載の凝集促進剤。
- 一般式[1]におけるR2及びR3がいずれもメチル基である、請求項1記載の凝集促進剤。
- 一般式[1]におけるnが2~4であり、mが1である、請求項1記載の凝集促進剤。
- 一般式[2]におけるR4が水素原子であり、kが4~8である、請求項2記載の凝集促進剤。
- 請求項1記載の凝集促進剤を含んでなる免疫凝集測定法用試薬。
- 試薬が、C反応性タンパク質(CRP)、血清フェリチン(Fer)、前立腺特異抗原(PSA)又はクレアチンキナーゼ-MB(CK-MB)測定用である、請求項8記載の免疫凝集測定法用試薬。
- 請求項1記載の免疫凝集測定法用凝集促進剤の共存下で、測定対象物質に対する抗体又は抗原を、測定対象物質と接触させて抗原抗体反応を行わせることを特徴とする、免疫凝集測定方法。
- 測定対象物質が、C反応性タンパク質(CRP)、血清フェリチン(Fer)、前立腺特異抗原(PSA)又はクレアチンキナーゼ-MB(CK-MB)測定用である、請求項9記載の免疫凝集測定方法。
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WO2022154121A1 (ja) | 2021-01-18 | 2022-07-21 | 富士フイルム和光純薬株式会社 | ヘプシジンの吸着抑制剤、吸着抑制方法、標準品、試薬、キットおよび測定方法 |
CN114716603A (zh) * | 2022-04-15 | 2022-07-08 | 深圳可孚生物科技有限公司 | 一种用于葡萄糖传感器的高分子膜的制备方法 |
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- 2012-06-04 US US14/123,899 patent/US9797886B2/en active Active
- 2012-06-04 KR KR1020137032151A patent/KR20140043371A/ko not_active Application Discontinuation
- 2012-06-04 EP EP12796593.7A patent/EP2720041B1/en active Active
- 2012-06-04 CN CN201280027997.9A patent/CN103597352B/zh active Active
- 2012-06-04 JP JP2013519479A patent/JP6107653B2/ja active Active
- 2012-06-04 WO PCT/JP2012/064355 patent/WO2012169453A1/ja active Application Filing
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WO2019031581A1 (ja) * | 2017-08-10 | 2019-02-14 | Jsr株式会社 | 免疫凝集の検出又は測定方法 |
Also Published As
Publication number | Publication date |
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EP2720041A1 (en) | 2014-04-16 |
JP6107653B2 (ja) | 2017-04-05 |
US20140113311A1 (en) | 2014-04-24 |
US9797886B2 (en) | 2017-10-24 |
KR20140043371A (ko) | 2014-04-09 |
CN103597352B (zh) | 2015-10-21 |
JPWO2012169453A1 (ja) | 2015-02-23 |
EP2720041A4 (en) | 2015-01-14 |
CN103597352A (zh) | 2014-02-19 |
EP2720041B1 (en) | 2016-09-07 |
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