Source: http://www.google.com/patents/US4792527?dq=inassignee:integral+inassignee:peripherals
Timestamp: 2014-09-18 06:52:22
Document Index: 566568438

Matched Legal Cases: ['Application No. 43618', 'in fine', 'in fine', 'Application No. 43618', 'in fine', 'in fine', 'in fine']

Patent US4792527 - Method of assaying biologically active substances and labelling agents therefor - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsA method of assaying biologically active substances by the competitive method or by the sandwich technique, characterized in that fine particles having a diameter of 0.03 to 3 μm are used in the labelling agent....http://www.google.com/patents/US4792527?utm_source=gb-gplus-sharePatent US4792527 - Method of assaying biologically active substances and labelling agents thereforAdvanced Patent SearchPublication numberUS4792527 APublication typeGrantApplication numberUS 06/707,171Publication dateDec 20, 1988Filing dateFeb 28, 1985Priority dateJul 17, 1981Fee statusLapsedPublication number06707171, 707171, US 4792527 A, US 4792527A, US-A-4792527, US4792527 A, US4792527AInventorsTakafumi Uchida, Shuntaro HosakaOriginal AssigneeToray Industries, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (12), Referenced by (7), Classifications (23), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetMethod of assaying biologically active substances and labelling agents thereforUS 4792527 AAbstract A method of assaying biologically active substances by the competitive method or by the sandwich technique, characterized in that fine particles having a diameter of 0.03 to 3 μm are used in the labelling agent.
We claim: 1. A competitive method of assaying a biologically active substance wherein amounts of a biologically active substance to be assayed are previously calibrated against amouts of a labelled biologically active substance remaining in solution by competitively reacting (i) known amounts of an unlabelled biologically active substance, (ii) a predetermined amount of the same biologically active substance labelled with a labelling agent, and (iii) a predetermined amount of a hydrophilic solid phase having fixed thereon a known number of bonding partner(s) capable of specifically bonding with the biologically active substance; separating the hydrophilic solid phase with the labelled and non-labelled substance bonded thereto; and measuring the amounts of labelled biologically active substance remaining in solution comprising the steps of:a. competitively reacting:(i) the biologically active substance to be assayed in a sample solution; (ii) the predetermined amount of the same biologically active substance labelled with a labelling agent, the labelling agent comprising hydrophilic fine particles having a diameter of about 0.03-3 μm; and (iii) the predetermined amount of the hydrophilic solid phase; and b. measuring the amount of labelled biologically active substance remaining in the solution, said amount being quantitatively related to the concentration of said biologically active substance to be assayed. 2. The method as defined in claim 1, wherein the labelled biologically active substance is bonded to the fine particles by chemical bonding.
Preparation of a Solid Phase Having Anti-swine Insulin Antiserum Fixed Thereon The aminated and hydrolyzed fine particles were activated with glutaraldehyde according to the method of Japanses Patent Application No. 43618/1980. The thus treated fine particles were dispersed in 0.15 mol/l physiological phosphate buffer saline solution (PBS) of pH 7.2 to obtain a 1% dispersion. The dispersion was mixed with an equal volume of anti-swine insulin antiserum (Miles) and the mixture was allowed to react at 30� C. for 3 hours. Bovine serum albumin (hereinafter referred to a BSA) was added to the particle dispersion to attain a concentration of 1%. After carrying out the reaction for an additional 1 hour, the fine particles were washed by repeated centrifugation (3000 rpm) and resuspension. The particles were dispersed (1% dispersion) in PBS containing 0.1% of BSA to obtain solid phase fine particles having anti-swine insulin antiserum fixed thereon.
Preparation of Fine Particles Used as Active Fine Particles Glycidyl methacrylate, methacrylic acid and ethylene glycol dimethacrylate were mixed at a molar ratio of 85:10:5. The mixture was added to an aqueous solution containing 0.1% of sodium dodecylsulfate and 0.01 mol/l of ammonium persulfate. The resulting emulsion having a monomer concentration of 10% (W/V) was allowed to react at 60� C. in argon gas atmosphere for 22 hours. The resulting fine particles were aminated and hydrolyzed in the same manner as in the above-described treatment of the solid phase fine particles to obtain fine particles having a uniform diameter of 0.27 μm.
Preparation of Active Fine Particles Having Insulin Fixed Thereon Fixation of insulin was effected according to the above-described method of fixing anti-swine insulin antiserum on the solid phase fine particles. That is, a 1% dispersion of fine particles treated with glutaraldehyde was mixed with an equal volume of 40 U/ml of swine insulin (NOVO) solution. After carrying out the reaction at 30� C. for 2 hours, BSA was added to the reaction mixture to attain a concentration of 1%. The reaction was continued at 30� C. for 1 hour. The fine particles were washed by repeated centrifugation (10,000 rpm for 30 min) and resuspension. The particles were dispersed in PBS containing 0.1% of BSA to obtain a dispersion having a particle concentration of 1%. Thus, active fine particles having swine insulin fixed thereon were prepared.
Determination of Insulin 100 μl of 1% dispersion of solid phase fine particles having anti-swine insulin antiserum fixed thereon were added to 100 μl of PBS solution containing 25, 12.5 or 6.25 μU/ml of swine insulin. The mixture was allowed to react at 25� C. for 2 hours. Further, 10 μl of the active fine particles having insulin fixed thereon (0.01% dispersion) were added to the reaction mixture and the reaction was carried out at 25� C. overnight. 2.5 ml of PBS were added to the reaction liquid and the mixture was centrifuged at 3000 rpm for 5 minutes to sediment the solid phase and the active fine particles reacted with the solid phase. The dispersion of fine particles unreacted with the solid phase was obtained as a supernatant. The light-scattering intensity of the dispersion of the active fine particles was measured by irradiation with 400 nm light using an Aminco-Bowman spectrofluorometer. The number of fine particles was determined from the light-scattering intensity according to a previously prepared calibration curve, since the light-scattering intensity was approximately proportional to the number of fine particles. As shown in FIG. 1, insulin can be determined quantitatively in the range of 25 to 6.25 μU/ml.
EXAMPLE 2 DETERMINATION OF BSA (Competitive Method) Preparation of a Solid Phase Having Anti-BSA Antiserum Fixed Thereon A 1% dispersion of solid phase fine particles prepared in the same manner as in Example 1 and treated with glutaraldehyde was mixed with an equal volume of anti-BSA antiserum (Miles). They were reacted at 30� C. for 3 hours and then human serum albumin (hereinafter referred to as HSA) was added thereto to attain an HSA concentration in the particle dispersion of 1%. The reaction was continued for an additional 1 hour and then the product was washed by repeated centrifugation (3000 rpm) and resuspension. The product was dispersed in PBS containing 0.1% of HSA to obtain fine particles having anti-BSA antiserum fixed thereon.
Preparation of Active Fine Particles Having BSA Fixed Thereon BSA was added in an amount of 10 mg/ml to 1% dispersion of active fine particles having a diameter of 0.27 μm prepared in the same manner as in Example 1 and treated with glutaraldehye. The reaction was carried out at 30� C. for 3 hours. After washing by repeated centrifugation (10,000 rpm) and resuspension, the product was dispersed in PBS containing 0.1% of HSA to obtain active fine particles having BSA fixed thereon.
Determination of BSA 100 μl of 1% dispersion of solid phase fine particles having anti-BSA antiserum fixed thereon were added to 90 μl of a PBS solution containing 10 μg/ml, 1 μg/ml, 100 ng/ml or 10 ng/ml of BSA and they were allowed to react at 30� C. for 1 hour. Then, the active fine particles having BSA fixed thereon were added to the reaction mixture and the reaction was carried out at 30� C. overnight. 2.5 ml of PBS were added to the reaction liquid. The active fine particles not bonded with the solid phase were separated from the solid phase and the active fine particles bonded with the solid phase by means of a membrane (Millipore Filter RA) having a pore size of 1.2 μm. Scattered light intensity was measured in the same manner as in Example 1 and the number of the fine particles was determined from the intensity.
EXAMPLE 3 Determination of Human Chorionic Gonadotropin (hereinafter referred to as HCG) (Competitive Method) Preparation of a Solid Phase Having Anti-HOG Antiserum Fixed Thereon A 1% dispersion of solid phase fine particles prepared in the same manner as in Example 1 and treated with glutaraldehyde was mixed with an equal volume of anti-HCG antiserum (16,000 IU/ml) (Miles). They were reacted at 30� C. for 3 hours and then BSA was added thereto to attain a BSA concentration in the particle dispersion of 1%. The reaction was continued for an additional 1 hour and then the product was washed by repeated centrifugation (3,000 rpm) and resuspension. The product was dispersed in PBS containing 0.1% of BSA to obtain a solid phase having anti-HCG antiserum fixed thereon.
Preparation of Fine Particles Used as Active Fine Particles Glycidyl methacrylate, ethylene glycol dimethacrylate and sodium sulfopropyl methacrylate were mixed in a molar ratio of 88:10:2. The mixture was added to an solution containing 0.125% of sodium dodecylsulfate and 0.01 mol/l of ammonium persulfate to obtain an emulsion having a monomer concentration of 10% (W/V). After carrying out the reaction at 60� C. for 3 hours in argon gas atmosphere, the resulting fine particles were washed by repeated centrifugation (10,000 rpm) and resuspension. After the amination and hydrolysis carried out in the same manner as in the treatment of the solid phase fine particles, uniform fine particles having a diameter of 0.1 μm were obtained.
Preparation of Active Fine Particles Having HCG Fixed Thereon HCG was fixed in the same manner as in the fixation of BSA on the solid phase fine particles. That is, 3200 IU/ml HCG (Sigma) solution in PBS was mixed with an equal volume of a 0.1% dispersion of fine particles treated with glutaraldehyde and they were allowed to react at 30� C. for 3 hours. Then, BSA was added thereto to attain a BSA concentration in the particle dispersion of 1%. The reaction was carried out for an additional 1 hour. After washing by repeated centrifugation (15,000 rpm for 30 min) and resuspension, the product was dispersed in PBS containing 0.1% of BSA to obtain active fine particles having HCG fixed thereon.
Determination of HCG A 1/10-fold dilution series (each 90 μl) of HCG ranging from 104 to 102 U/l was prepared. Each sample was reacted with 50 μl of a 1% dispersion of solid phase fine particles having anti-HCG antiserum fixed thereon and 10 μl of a 0.01% HCG fine particle dispersion at 25� C. overnight in a glass test tube. 2.5 ml of PBS were added to the reaction liquid. The active fine particles remaining not bonded with the solid phase were separated out by means of a membrane in the same manner as in Example 2.
Fixation of BSA on Fine Particles Used as Solid Phase The aminated and hydrolyzed fine particles were activated with glutaraldehyde according to the method of Japanese Patent Application No. 43618/1980. The thus-treated fine particles were dispersed in 0.15 mol/l physiological phosphate buffer saline solution (PBS) of pH 7.2 containing 10 mg/ml of BSA (Miles) to obtain a 1% dispersion. The dispersion was allowed to react at 30� C. for 3 hours. The fine particles were washed by repeated centrifugation (3000 rpm) and resuspension. The particles were dispersed in 0.1% human serum albumin (hereinafter referred to as HSA) solution in PBS to obtain fine particles having BSA fixed thereon.
Preparation of Fine Particles Used for Fixing the Bonding Substance Glycidyl methacrylate, methacrylic acid and ethyleneglycol dimethacrylate were mixed at a molar ratio of 85:10:5. The mixture was added to an aqueous solution containing 0.1% of sodium dodecylsulfate and 0.01M of ammonium persulfate. The resulting emulsion having a monomer concentration of 10% (W/V) was allowed to react at 60� C. in argon gs atmosphere for 22 hours. The resulting fine particles were aminated and hydrolyzed in the same manner as in the above-described treatment of solid phase fine particles to obtain fine particles having a uniform diameter of 0.27 μM.
Fixation of Anti-Rabbit Immunogloblin G (hereinafter referred to as anti-rabbit Ig G) Antibody on Fine Particles Used for Fixing the Bonding Substance The fine particles were activated with glutaraldehyde according to the above-mentioned method of fixing BSA on the solid phase fine particles. The activated particles were dispsered in PBS in which 1 mg/ml of anti-rabbit IgG antibody (prepared using goat) had been dissolved. After carrying out the reaction at 30� C. for 2 hours, HSA was added to the reaction mixture to attain a concentration of 1%. The reaction was continued at 30� C. for 1 hour. The fine particles were washed by repeated centrifugation (10,000 rpm) and resuspension. The particles were dispersed in PBS containing 0.1% of HSA to obtain a dispersion having a particle concentration of 1%. Thus, fine particles having anti-rabbit IgG antibody fixed thereon were prepared.
Quantitative Determination of Anti-BSA Antibody 50 μl of 1% BSA-fixed solid phase fine particle dispersion were added to 200 μl of PBS solution containing 10 μl/ml, 1 μg/ml, 100 ng/ml 10 ng/ml or 1 ng/ml of anti-BSA antibody prepared by immunizing rabbit with BSA in a glass test tube. The mixture was allowed to react at 37� C. under shaking for 1.5 hours. The particles were washed with PBS by repeated centrifugation (3000 rpm) and resuspension. The solid phase was dispersed in 50 μl of PBS. 25 μl of anti-rabbit IgG antibody-fixed fine particles (0.01% dispersion) was added to the resulting dispersion and the mixture was allowed to react at 30� C. for 2 hours. After standing at 4� C. overnight, 2.5 ml of PBS were added to the reaction mixture. The solid phase fine particles were separated from the anti-rabbit IgG antibody-fixed fine particles by means of a membrane having pores of 1.2 μm diameter. (Millipore Filter RA). The light-scattering intensity of the dispersion of the anti-rabbit IgG antibody-fixed fine particles passed through the membrane was measured by the irradiation with 400 nm light using an Aminco-Bowman spectrofluorometer. The number of fine particles was determined from the light-scattering intensity according to a previously prepared calibration curve, since the light-scattering intensity was approximately proportional to the number of the fine particles. As shown in FIG. 4, anti-BSA antibody can be determined quantitatively in the range of 1 ng/ml to 10 μg/ml.
EXAMPLE 5 DETERMINATION OF HUMAN CHLORIONIC GONADOTROPIN (hereinafter refered to as HCG)(Sandwich Technique) Fixation of Anti-HCG Antibody On Solid Phase Fine Particles A 1% dispersion of solid phase fine particles prepared in the same manner as in Example 1 and treated with glutaraldehyde was mixed with an equal volume of anti-HCG antiserum (16,000 IU/ml) [Miles]. They were reacted at 30� C. for 3 hours and then BSA was added thereto to attain a BSA concentration in the particle dispersion of 1%. The reaction was continued for an additional 1 hour and then the product was washed by repeated centrifugation (3,000 rpm) and resuspension. The product was dispersed in PBS containing 0.1% of BSA to obtain anti-HCG antibody-fixed solid phase fine particles.
Determination of HCG A 1/10-fold dilution series (each 90 μl) of HCG ranging from 104 U/l to 1 U/l was prepared. Each sample was reacted with 100 μl of 1% dispersion of anti-HCG antibody-fixed solid phase fine particles at 30� C. under agitation for 2 hours in a glass test tube. The product was washed three times with PBS by centrifugation at 3000 rpm to obtain a solid phase bonded with HCG via anti-HCG antibody. With 100 μl of a 1% dispersion of these solid phase fine particles, 10 μl of 0.02% dispersion of anti-HCG antibody-fixed fine particles (0.27 μm) were mixed in a glass test tube and they were reacted at 30� C. for 2 hours. After completion of the reaction, 2.5 ml of PBS were added to the reaction mixture and the whole was centrifuged at 3,000 rpm for 10 min to sediment the solid phase. The supernatant fine particle dispersion was withdrawn. A scattered light intensity was measured in the same manner as in Example 1 and the number of the fine particles was determined from the intensity.
EXAMPLE 6 DETERMINATION OF HCG (Sandwich Technique) Adsorption of Anti-HCG Antibody on Microplates 0.05M Tris-HCl (pH 8.0) wad added to the anti-HCG antiserum used in Example 3 in an equal vouume. 200 μl of the resulting liquid was poured in each well of a polystyrene microplate. After standing at 25� C. for 3 hours, the plates were washed with PBS to obtain anti-HCG antibody-adsorbed microplates.
Reaction of HCG with Solid Phase 150 μl of a PBS solution containing 1% rabbit serum and 104, 103, 102 or 0 U/l of HCG were poured in each well of the microplates coated with anti-HCG antiserum. They were reacted at 25� C. for 3 hours. After completion of the reaction, the product was washed with PBS containing 0.1% Triton X-100 and then washed twice with PBS.
Determination of HCG The anti-HCG antibody-fixed fine particles used were the same as those used in Example 2 (0.27 μm). 10 μl of a 0.02% fine particle dispersion was placed in each well of the solid phase microplate containing 140 μl of PBS. They were reacted at 25� C. for 3 hours and then left to stand at 4� C. overnight. The fine particle dispersion was sucked from each well by means of a pipette and added to 2.5 ml of PBS. Intensity of light scattered by the fine particles was measured in the same manner as in Example 1. The number of fine particles was determined from the intensity. A calibration curve in the range of HCG 104 to 102 U/l prepared as described above in shown in FIG. 6.
EXAMPLE 7 DETERMINATION OF HCG (Sandwich Technique) Anti-HCG antibody-bonded fine particles (4 μm) were used as the solid phase. A 1/10-fold dilution series (each 90 μl) of HCG ranging from 105 to U/l was prepared. Each sample was reacted with 100 μl of 1% dispersion of anti-HCG antibody-fixed solid phase fine particles at 25� C. under agitation for 3 hours in a glass test tube. The product was washed three times with PBS by repeated centrifugation (3000 rpm) and resuspension to obtain fine particles bonded with HCG via anti-HCG antibody.
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