JPS6315554B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing immunologically active microparticles that specifically bind to components in human or animal cells or body fluids. When using the reaction between an antigen and an antibody to immunologically detect or quantify either one of them, the substance that binds to the substance to be measured is immobilized on particles of an appropriate size, and the A highly sensitive measurement method that utilizes the phenomenon in which particles agglomerate in the presence of a substance to be measured has become an important means for immunological clinical testing. Conversely, when the substance to be measured is immobilized on particles, aggregation of particles immobilized with the substance to be measured due to the presence of antigens or antibodies that specifically react with the substance to be measured occurs due to the presence of the substance to be measured in the test liquid. Methods for detecting or quantifying analyte substances through inhibition are also widely used in immunological clinical tests. Another method for immunological or cytological testing is to immobilize a substance that selectively binds to specific cells on the surface of microparticles and label cells depending on whether the particles bind to the cells or not. Often used as a means. Particles widely used as carriers for agglutination reactions include red blood cells of mammals including humans and birds;
Particles of inorganic substances such as kaolin and carbon, and latexes of organic polymer compounds such as natural rubber latex and polystyrene are known. Red blood cells can immobilize many types of antigens and antibodies and have the widest range of applications. However, there are differences depending on the individual animal collected, stability is difficult, and preservation is difficult.
Further, there are problems such as non-specific agglutination due to human serum. Polystyrene particles, which are most widely used as particles of non-biological origin, are synthetic polymer compounds that can be of constant quality and are stable in themselves. Since polystyrene is hydrophobic and has the property of adsorbing various proteins, antigens or antibodies are usually immobilized on polystyrene by physical adsorption. However, when an antigen or antibody is immobilized by physical adsorption, an equilibrium exists between the immobilized antigen (or antibody) and the free antigen (or antibody), so the corresponding target substance of measurement A competitive reaction occurs between the antigen (or antibody) immobilized on particles and the free antigen (or antibody), and this competitive reaction acts to suppress aggregation. As a result, a lack of sensitivity and stability has been noted in many cases. Also, as a matter of course, substances that are difficult to physically adsorb to polystyrene cannot be immobilized. Due to these problems, the agglutination reaction of polystyrene particles has only been put to practical use to a limited extent compared to when red blood cells are used as a carrier. Therefore, attempts have been made to solve the above problems by immobilizing antigens or antibodies on particles through covalent bonds. for example
DT2649218 describes the use of carbodiimide to bind human chorionic gonadotropin to a styrene-methacrylic acid copolymer latex. In addition, in Japanese Patent Publication No. 53-12966, carboxylated styrene-butadiene, carboxylated polystyrene, carboxylated polystyrene with amino groups, acrylic acid polymer, acrylonitrile polymer, methacrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate acrylate, polyvinyl Human chorionic gonadotropin, human serum albumin, or modified gamma globulin are condensed with amide bonds to various latex polymers such as pyridine and vinyl chloride-acrylate copolymers, and the particle size is 0.01~
It is stated that 0.9 micron particles can be used as immunological diagnostic reagents. Furthermore, clinical pathology 27;
Supplementary volume, p. 522 (1978) describes the method of adding treponemal antigen to a methyl methacrylate latex containing hydroxyl and carboxyl groups produced by copolymerizing methacrylic acid, 2-hydroxyethyl methacrylate and methyl methacrylate using the cyanogen bromide or carbodiimide method. describes how to combine them. In addition, in Japanese Patent Publication No. 55-110118, polystyrene particles are used as cores and styrene particles are used as cores.
Reagents have been proposed in which free epoxy groups of a latex coated with a glycidyl methacrylate copolymer coat are reacted with human chorionic gonadotropin or insulin to bind them to the latex. In these prior art methods, a method of binding an immunoactive substance to particles using carbodiimide is often used, but when carbodiimide is used, condensation reactions occur between and within molecules of the immunoactive substance. This is an undesirable side reaction that impairs the activity of the immunologically active substance. By using cyanogen bromide, it is possible to avoid condensation reactions between and within molecules of the immunoactive substance, but in this case, it is difficult to obtain reproducibility of the reaction between a polymer having a hydroxyl group and cyanogen bromide. As a result, the immunoactivity of particles immobilized with immunoactive substances tends to fluctuate. Compared to these methods of immobilizing immunoactive substances, the method of reacting a free epoxy group introduced into a polymer with a protein or polypeptide causes less deactivation of immune activity and has good reproducibility. However, in the above-mentioned prior art that utilizes epoxy groups, epoxy group-containing monomers and hydrophobic monomers such as styrene are copolymerized in the presence of pre-prepared hard polymer particles such as polystyrene. , employs a method for producing fine carrier particles in which a core made of a hard polymer is coated with a shell made of a copolymer of an epoxy group-containing monomer. Therefore, even after immobilization of an immunoactive substance, hydrophobic portions are exposed on the particle surface and tend to nonspecifically adsorb proteins. In general, human or animal body fluids contain many types of proteins, and plasma or serum in particular contains these proteins at high concentrations. When proteins from a sample body fluid are adsorbed onto carrier particles, they are absorbed into the target antigen.
It may interfere with immunological reactions such as antibody reactions, leading to a decrease in the selectivity and sensitivity of agglutination reactions.
The present inventors conducted studies aimed at solving these problems, and as a result, they arrived at the present invention. The present invention precipitates fine particulate polymers with an average diameter of 0.03 to 10 μm by polymerizing glycidyl acrylate or glycidyl methacrylate in a medium that dissolves the monomer but precipitates the resulting polymer. and then reacting the particulate polymer with an immunoactive substance having an amino group. According to the method of the present invention, free epoxy group-containing carrier microparticles having a diameter suitable for immunological testing and a narrow particle size distribution can be produced in one step.
The immunoactive substance is then immobilized on the carrier microparticles by a covalent bond generated by the reaction between the amino groups of the immunoactive substance and the free epoxy groups on the surface of the microparticles. If there is a possibility that the free epoxy groups will not be completely consumed and remain active due to the reaction with the immunologically active substance, use hydrophilic proteins such as serum albumin and gelatin that will not interfere with the target immunological test. By reacting with free epoxy groups, the reactivity of the epoxy groups can be lost. In this case, the hydrophilic protein for eliminating epoxy groups may be mixed with the immunoactive substance to be immobilized and reacted simultaneously, or the immunoactive substance may be reacted alone first and then reacted. It is also possible to use amino acids such as glycine and alanine in place of the hydrophilic proteins such as albumin and gelatin. The mixing ratio of glycidyl acrylate and glycidyl methacrylate is arbitrary, and either one may be used alone. Although it is not essential to add a crosslinking agent to the polymerization system, it is usually possible to actively polymerize by adding a polyfunctional monomer containing two or more polymerizable carbon-carbon double bonds in the molecule during polymerization. It is desirable to crosslink.
There are many polyfunctional monomers suitable for adding to the polymerization system for such purposes, to name a few: divinylbenzene, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, N, These include N'-methylenebisacrylamide, divinyl succinate, diallyl succinate, vinyl methacrylate, allyl methacrylate, triallyl cyanurate, triallyl isocyanurate, and the like. The amount of crosslinking agent added is usually 30 mol% of the total monomers.
It is as follows. Further, crosslinking can also be introduced by utilizing the reactivity of the polymer produced after the polymerization reaction and reacting it with a polyfunctional compound. For example, a polymer can be crosslinked by reacting an epoxy group contained in the produced polymer with a diamine such as ethylenediamine. Addition of other copolymerization components during polymerization often brings about favorable results. The effect of adding a copolymer component is to adjust the particle size. Hydrophilic monomers are preferred as copolymerization components, and water-soluble monomers are particularly preferred. Suitable water-soluble monomers used in the copolymerization include, for example, 2-oxyethyl acrylate, 2-oxyethyl methacrylate, 2-oxypropyl acrylate, and 2-oxyethyl acrylate.
-Oxypropyl methacrylate, acrylic or methacrylic ester of polyethylene glycol monoalkyl ether with a degree of polymerization of 2 to 25, acrylamide, methacrylamide, N-
These include vinylpyrrolidone and glycerol methacrylate. Two or more of these water-soluble monomers may be used in combination. When these water-soluble monomers are copolymerized, after immobilizing the immunoactive substance, the water-soluble monomers are exposed on the surface of the polymer fine particles without being covered with any bond. It is a hydrophilic moiety derived from polymers. Since proteins are difficult to adsorb to hydrophilic polymers in aqueous media, the immunoactive substance-immobilized microparticles according to the present invention are stable to sample body fluids, do not easily cause non-specific aggregation, and do not adhere non-specifically to cells. There is no. The molar ratio of the sum of the copolymer components to the sum of glycidyl acrylate and glycidyl methacrylate can be varied within the range of 100:0 to 5:95. The polymerization reaction is carried out in a medium in which the monomer is easily dissolved, but the polymer produced by the polymerization is not dissolved but precipitated in the form of fine particles. Examples of such a medium include esters such as ethyl acetate, n-propyl acetate, isopropyl acetate, butyl acetate isomers and the above-mentioned equivalent esters of propionic acid, methyl ethyl ketone, methyl n-propyl ketone, methyl isopropyl ketone, methyl butyl Ketones such as ketone isomers, benzene, toluene, o-xylene, m-xylene, p-xylene, etc. The particle size of the polymer fine particles obtained by this method is excellent in uniformity, and the average diameter is within the range of 0.03 μm to 10 μm. The average diameter can be adjusted by monomer composition and choice of polymerization medium. Another advantage of this method, that is, precipitation polymerization, is that unlike emulsion polymerization or suspension polymerization, it does not use emulsifiers or suspension stabilizers, so there is no need to remove these additives after polymerization. be. As the polymerization initiator, common radical polymerization initiators such as 2,2'-azobisisobutyronitrile, 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2, Azo compounds such as 4-dimethyl-4-methoxyvaleronitrile), peroxides such as benzoyl peroxide, dilauroyl peroxide, and di-tert-butyl peroxide can be used. The polymerization temperature may be within the normal radical polymerization temperature range, from 20℃ to 80℃.
C is particularly preferred. The concentration of the polymerization initiator in the polymerization mixture is usually about 0.001 to 0.03 mol/. The concentration of monomer in the polymerization mixture is usually 5 to 5.
A range of 50% by weight is preferred. When the monomer concentration exceeds 50% by weight, the resulting polymer particles tend to aggregate. Although the present invention can be carried out even if the monomer concentration is less than 5% by weight, productivity will be lower because fewer polymer particles will be obtained. Note that the polymerization is preferably carried out by replacing the atmosphere with an inert gas such as nitrogen or argon. In many cases, the shape of the particles is not necessarily spherical, and may be irregularly shaped. The diameter of irregularly shaped particles is 1/2 of the sum of the maximum and minimum diameters. The average diameter is defined by equation (1). = _N 〓 ⁱ⁼¹ d _i /N (1) where d _i is the diameter of the i-th particle, and N is the total number of particles. Empirically, it is easy to determine the agglutination reaction when the average diameter is 0.1 μm or more and 10 μm or less.
In addition, for the purpose of cell labeling, the average diameter is 0.03 μm or more.
A range of 5 μm or less is preferable. Furthermore, particles suitably colored with dyes or pigments are convenient for both aggregation reactions and cell labeling purposes. Particles with fluorescent light are also preferred for cell labeling. The immobilization reaction of immunologically active substances onto microparticles is carried out in an aqueous medium, with a pH of 7.0 to 9.0 and a temperature of 0 to 40 degrees Celsius.
A range of is appropriate. The concentration of the immunoactive substance in the reaction solution needs to be increased or decreased depending on the properties of the individual immunoactive substance and cannot be determined uniformly. In this case, as already mentioned, adding hydrophilic proteins such as serum albumin and gelatin to the reaction solution is often effective in improving the dispersion stability of the immunoactive substance-immobilized microparticles. Furthermore, treatment with amino acids such as glycine and alanine after the immobilization reaction often brings about similar favorable effects. The immunologically active substance used in the present invention is required to have an amino group, and most of the immunologically active substances are proteins or contain polypeptide moieties and therefore meet this condition. Here, the term "immune active substance" refers not only to antigens and antibodies, but also to substances that specifically bind to substances involved in humoral or cellular immune reactions, such as complement, Fc receptors, and C3 receptors. do. To give a few specific examples, Treponema pallidum antigen, B
Hepatitis surface antigen (HBs antigen), antibody against hepatitis B surface antigen (anti-HBs antibody), rubella antigen, toxoplasma antigen, streptolysin O, anti-streptolysin O antibody, mycoplasma antigen, human chorionic gonadotropin (HCG), anti-HCG Antibody, heat-aggregated human IgG, rheumatoid factor, nuclear protein, DNA, anti-
DNA antibody, C-reactive protein (CRP), anti-CRP antibody,
Anti-estrogen antibodies, α-fetoprotein (α
-FP), anti-α-FP antibody, carcinoembryonic antigen (CEA),
anti-CEA antibody, C1q, anti-C1q antibody, C3, anti-C3 antibody,
These include anti-C3b antibody, anti- _C3bi antibody, C4, anti-C4 antibody, protein-A, conglutinin, and immunoconglutinin. Example 1 Glycidyl methacrylate, 2-oxyethyl methacrylate, and ethylene glycol dimethacrylate were mixed in a molar ratio of 85.7:9.5:4.8, and 24 parts of the monomer mixture (weight, same hereinafter),
A mixture of 76 parts of ethyl propionate and 0.13 parts (4.7 mmol/) of 2,2'-azobis(2,4-dimethyl-4-methoxyvaleronitrile) was polymerized at 40 DEG C. for 3 hours under a nitrogen gas atmosphere. The cloudy polymerization mixture was poured into acetone and centrifuged at 1500×g for 10 minutes, and the precipitated particles were redispersed and washed with ethanol, followed by centrifugation again. It was dried under reduced pressure to obtain 11.3 parts of a particulate polymer. This particulate polymer was spherical and had a diameter between 1.8 μm and 4.2 μm, with an average of 3.3 μm and a standard deviation of 0.45 μm. The polymeric fine particles prepared as above
TP antigen was immobilized as described below. First, the syphilis pathogen Treponema pallidum (hereinafter abbreviated as TP)
Nichols strain bacterial components were added to phosphate buffered saline (disodium hydrogen phosphate + monopotassium hydrogen phosphate).
0.01mol/, Sodium chloride 0.14mol/, PH
7.2, hereafter abbreviated as PBS) at a rate of 10 ⁹ cells/ml was heated at 10KHz while cooling with ice water.
The cells were treated with ultrasonic waves for 20 minutes to destroy the bacterial cells.
It was used as a TP antigen solution. One volume of this TP antigen solution was mixed with three volumes of a solution of bovine serum glycoprotein dissolved in PBS at a concentration of 1 mg/ml, and 1 ml of this mixture was mixed with a dispersion of 50 mg of the polymer in 1 ml of PSB. The mixture was stirred at 30°C for 2 hours. Next, the fine particles were separated by centrifugation, washed three times with PBS by centrifugation, and then dispersed in 20 ml of PBS supplemented with 1% bovine serum albumin (hereinafter abbreviated as BSA). After this TP antigen-immobilized particle dispersion was left in a refrigerator at 4°C for 3 days, the activity was assayed as follows. A diluted syphilis-positive serum solution with a TPHA titer of 640 was added to a polystyrene microtiter plate with a U-shaped tube bottom in a ²ⁿ dilution series starting from a dilution factor of 10 times, in an amount of 50 μm each. However, as a diluting solution, 1% BSA in PBS, 5% Reiter antigen solution for syphilis complement fixation reaction KW (Japan Lyophilization Research Institute),
A solution to which ammonium chloride was added at a concentration of 0.73 mol/ was used. A similar dilution of syphilis-negative serum was also added to a microtiter plate as a control. Next, 50μ of the TP antigen-immobilized microparticle dispersion was added to each well of the microtiter plate containing the serum dilution solution, shaken for 3 minutes to mix both solutions, and then allowed to stand at room temperature for 2 hours. The degree of aggregation was determined by The results are shown in Table 1, and it can be seen that syphilis antibodies in serum can be detected with a sensitivity higher than that of TPHA.

[Table] Example 2 Human IgG with a concentration of 1 mg/ml/0.4 ml of PBS solution and bovine serum glycoprotein with a concentration of 1 mg/ml/
1.6 ml of PBS solution was mixed, and 50 mg of the same polymer fine particles used in Example 1 were dispersed in the mixed solution, followed by stirring at 30° C. for 3 hours. The reaction mixture was left in a refrigerator at 4°C overnight, and then centrifuged.
After washing with PBS, the microparticles were redispersed in 4 ml of PBS containing 1% BSA, stirred at 30°C for 2 hours, and then
It was left in the refrigerator at ℃ overnight. In this way, humans
Polymer microparticles on which IgG was immobilized were reacted with an anti-human IgG antibody in the following manner. i.e. anti-human IgG antiserum (goat) on a microscope slide.
10μ of the PBS solution of the IgG fraction and 10μ of the human IgG-immobilized fine particle dispersion were mixed, and the state of agglutination after 3 minutes was visually determined. The results are shown in Table 2. It can be seen that the detection limit of anti-human IgG antibody is approximately 10 ng/ml.

[Table] Example 3 The same polymer particles used in Example 1 were dispersed in 2 ml of BSA/PBS solution with a concentration of 5 mg/ml, stirred at 30°C for 7 hours, and then left in a refrigerator at 4°C overnight. did. Next, the microparticles were washed with PBS by centrifugation, redispersed in 4 ml of PBS supplemented with 0.5% human serum albumin, stirred at 30°C for 1 hour, and then left in a refrigerator at 4°C overnight. The polymer particles on which BSA was immobilized in this manner were reacted with anti-BSA antiserum (rabbit) on a slide glass in the same manner as in Example 2. The results are shown in Table 3. anti
It can be seen that the BSA antibody detection limit is 0.1 μg/ml.

[Table] Example 4 Polymerization was carried out in the same manner as in Example 1 except that the mixing ratio of glycidyl methacrylate, 2-oxyethyl methacrylate, and ethylene glycol was changed to 71.4:23.8:4.8 (molar ratio), and the average diameter
10.8 parts of fine particles of 1.0 μm were obtained. Using these polymer particles, BSA was prepared in exactly the same manner as in Example 3.
was fixed. The results of reaction with anti-BSA antiserum on a slide glass in the same manner as in Example 3 were exactly the same as in Example 3, and the anti-BSA antibody detection limit was 0.1 μg/ml. Example 5 Glycidyl methacrylate, 2-oxypropyl methacrylate and triethylene glycol methacrylate were mixed in a ratio of 47.6:47.6:4.8 (molar ratio), and 24 parts of the monomer mixture, methyl n-
A mixture of 76 parts of propylketone and 0.13 parts (4.7 mmol/) of 2,2'-azobis(2,4-dimethyl-4-methoxyvaleronitrile) was polymerized at 40 DEG C. for 3 hours under an argon atmosphere. The cloudy polymer mixture was treated in the same manner as in Example 1 to obtain fine polymer particles.
Got 8.4 copies. The average diameter was 2.5 μm. This polymer fine particle was dispersed in a 0.5% human serum albumin aqueous solution so that the polymer content was 1.25%, and BSA was immobilized in the same manner as in Example 3. Then, in the same manner as in Example 3, it was reacted with anti-BSA antiserum on a slide glass. The results are shown in Table 4.

[Table] Example 6 Using glycidyl acrylate instead of glycidyl methacrylate, glycidyl acrylate,
The mixing ratio of 2-oxyethyl methacrylate and triethylene glycol dimethacrylate is
Example 5 except that the molar ratio was 23.8:714:4.8
Polymerization was carried out in the same manner as above to obtain 7.2 parts of polymer fine particles having an average diameter of about 1 μm. In exactly the same manner as in Example 5
BSA was immobilized on polymer fine particles and reacted with anti-BSA antiserum in the same manner as in Example 5. The results were similar to those in Example 5, and the anti-BSA antibody detection sensitivity was about 10 μg/ml. Example 7 Polymerization was carried out in exactly the same manner as in Example 1, except that 2-oxyethyl acrylate was used in place of 2-oxyethyl methacrylate in Example 1, to obtain 9.5 parts of polymer fine particles having an average diameter of about 3 μm. The TP antigen was immobilized on the polymer particles in the same manner as in Example 1, and the activity was assayed. As a result, the sensitivity for detecting syphilis antibodies in serum was the same as in Example 1.

Claims (1)

[Claims] 1. By polymerizing glycidyl acrylate or glycidyl methacrylate in a medium that dissolves the monomer but precipitates the resulting polymer, fine particulate polymers with an average diameter of 0.03 to 10 μm are precipitated, A method for producing immunoactive fine particles, which comprises then reacting the fine particulate polymer with an immunoactive substance having an amino group.