JPH0376426B2 - - Google Patents
Info
- Publication number
- JPH0376426B2 JPH0376426B2 JP57129691A JP12969182A JPH0376426B2 JP H0376426 B2 JPH0376426 B2 JP H0376426B2 JP 57129691 A JP57129691 A JP 57129691A JP 12969182 A JP12969182 A JP 12969182A JP H0376426 B2 JPH0376426 B2 JP H0376426B2
- Authority
- JP
- Japan
- Prior art keywords
- particles
- polymer
- polymerization
- fine particles
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
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- RPQRDASANLAFCM-UHFFFAOYSA-N oxiran-2-ylmethyl prop-2-enoate Chemical group C=CC(=O)OCC1CO1 RPQRDASANLAFCM-UHFFFAOYSA-N 0.000 claims description 9
- 230000003100 immobilizing effect Effects 0.000 claims description 6
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- 238000006243 chemical reaction Methods 0.000 description 21
- 239000000126 substance Substances 0.000 description 20
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- 238000006116 polymerization reaction Methods 0.000 description 19
- 238000000034 method Methods 0.000 description 16
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- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
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- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 2
- WYGWHHGCAGTUCH-UHFFFAOYSA-N 2-[(2-cyano-4-methylpentan-2-yl)diazenyl]-2,4-dimethylpentanenitrile Chemical compound CC(C)CC(C)(C#N)N=NC(C)(C#N)CC(C)C WYGWHHGCAGTUCH-UHFFFAOYSA-N 0.000 description 2
- HWSSEYVMGDIFMH-UHFFFAOYSA-N 2-[2-[2-(2-methylprop-2-enoyloxy)ethoxy]ethoxy]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOCCOCCOC(=O)C(C)=C HWSSEYVMGDIFMH-UHFFFAOYSA-N 0.000 description 2
- SYBYTAAJFKOIEJ-UHFFFAOYSA-N 3-Methylbutan-2-one Chemical compound CC(C)C(C)=O SYBYTAAJFKOIEJ-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010074051 C-Reactive Protein Proteins 0.000 description 2
- 102100032752 C-reactive protein Human genes 0.000 description 2
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 2
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- 239000004471 Glycine Substances 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108010058683 Immobilized Proteins Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical group CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
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- 239000000969 carrier Substances 0.000 description 2
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- 238000006482 condensation reaction Methods 0.000 description 2
- 229920006037 cross link polymer Polymers 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- FKRCODPIKNYEAC-UHFFFAOYSA-N ethyl propionate Chemical compound CCOC(=O)CC FKRCODPIKNYEAC-UHFFFAOYSA-N 0.000 description 2
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- XNLICIUVMPYHGG-UHFFFAOYSA-N pentan-2-one Chemical compound CCCC(C)=O XNLICIUVMPYHGG-UHFFFAOYSA-N 0.000 description 2
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- YKYONYBAUNKHLG-UHFFFAOYSA-N propyl acetate Chemical compound CCCOC(C)=O YKYONYBAUNKHLG-UHFFFAOYSA-N 0.000 description 2
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- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- QCUFYOBGGZSFHY-UHFFFAOYSA-N depsidomycin Chemical compound O=C1C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(NC=O)C(C)CC)C(C)OC(=O)C2CCCNN2C(=O)C(CC(C)C)NC(=O)C2CCCNN21 QCUFYOBGGZSFHY-UHFFFAOYSA-N 0.000 description 1
- LSXWFXONGKSEMY-UHFFFAOYSA-N di-tert-butyl peroxide Chemical compound CC(C)(C)OOC(C)(C)C LSXWFXONGKSEMY-UHFFFAOYSA-N 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000328 estrogen antagonist Substances 0.000 description 1
- FFYWKOUKJFCBAM-UHFFFAOYSA-N ethenyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OC=C FFYWKOUKJFCBAM-UHFFFAOYSA-N 0.000 description 1
- YVKSGVDJQXLXDV-BYPYZUCNSA-N ethyl (2r)-2-amino-3-sulfanylpropanoate Chemical compound CCOC(=O)[C@@H](N)CS YVKSGVDJQXLXDV-BYPYZUCNSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229940106780 human fibrinogen Drugs 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000010954 inorganic particle Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- JMMWKPVZQRWMSS-UHFFFAOYSA-N isopropanol acetate Natural products CC(C)OC(C)=O JMMWKPVZQRWMSS-UHFFFAOYSA-N 0.000 description 1
- 229940011051 isopropyl acetate Drugs 0.000 description 1
- GWYFCOCPABKNJV-UHFFFAOYSA-M isovalerate Chemical compound CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229960001913 mecysteine Drugs 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 1
- 229920003145 methacrylic acid copolymer Polymers 0.000 description 1
- 229940117841 methacrylic acid copolymer Drugs 0.000 description 1
- 125000005397 methacrylic acid ester group Chemical group 0.000 description 1
- MCYHPZGUONZRGO-VKHMYHEASA-N methyl L-cysteinate Chemical compound COC(=O)[C@@H](N)CS MCYHPZGUONZRGO-VKHMYHEASA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- IHYNKGRWCDKNEG-UHFFFAOYSA-N n-(4-bromophenyl)-2,6-dihydroxybenzamide Chemical compound OC1=CC=CC(O)=C1C(=O)NC1=CC=C(Br)C=C1 IHYNKGRWCDKNEG-UHFFFAOYSA-N 0.000 description 1
- QYZFTMMPKCOTAN-UHFFFAOYSA-N n-[2-(2-hydroxyethylamino)ethyl]-2-[[1-[2-(2-hydroxyethylamino)ethylamino]-2-methyl-1-oxopropan-2-yl]diazenyl]-2-methylpropanamide Chemical compound OCCNCCNC(=O)C(C)(C)N=NC(C)(C)C(=O)NCCNCCO QYZFTMMPKCOTAN-UHFFFAOYSA-N 0.000 description 1
- 229920006173 natural rubber latex Polymers 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229940078552 o-xylene Drugs 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical class [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002689 polyvinyl acetate Polymers 0.000 description 1
- 239000011118 polyvinyl acetate Substances 0.000 description 1
- 229920002717 polyvinylpyridine Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- FBCQUCJYYPMKRO-UHFFFAOYSA-N prop-2-enyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC=C FBCQUCJYYPMKRO-UHFFFAOYSA-N 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000007870 radical polymerization initiator Substances 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 108010075210 streptolysin O Proteins 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Compositions Of Macromolecular Compounds (AREA)
Description
【発明の詳細な説明】
本発明は免疫学的検査用試薬として有効な免疫
活性微粒子に関し、特に粒子状担体に免疫活性物
質を固定してなる免疫活性粒子を用いてヒトまた
は動物の体液中の成分を検出若しくは測定または
細胞を識別する免疫学的検査用試薬として有効な
免疫活性微粒子の改良に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunoactive microparticles that are effective as reagents for immunological testing, and in particular to immunoactive particles in which an immunoactive substance is immobilized on a particulate carrier. The present invention relates to improvements in immunoactive microparticles that are effective as immunological test reagents for detecting or measuring components or identifying cells.
抗原と抗体との反応を利用してその一方を免疫
学的に検出または定量する場合に、測定したい物
質に結合する側の物質を粒子状担体に固定化させ
ておき、その粒子が被測定物質の存在下で凝集を
起こす現象を利用して高感度の測定を行なう方法
は免疫学的臨床検査の重要な手段となつている。
また逆に測定したい物質を粒子状担体に固定化さ
せておき、その被測定物質と特異的に反応する抗
原または抗体の存在による被測定物質固定化粒子
の凝集が、被検液中の被測定物質の存在により阻
止されることにより被測定物質を検出または定量
する方法も免疫学的臨床検査において広く用いら
れている。また特定の細胞と選択的に結合する物
質を粒子状担体に固定化させておき、その粒子が
細胞に結合するか否かによつて細胞の識別を行な
い方法も免疫学的検査の手段としてしばしば採用
されている。 When using the reaction between an antigen and an antibody to immunologically detect or quantify one of them, the substance that binds to the substance to be measured is immobilized on a particulate carrier, and the particles become the substance to be measured. A highly sensitive measurement method that takes advantage of the phenomenon of agglutination in the presence of ions has become an important means for immunological clinical testing.
Conversely, when the substance to be measured is immobilized on a particulate carrier, aggregation of the particles immobilized with the analyte due to the presence of antigens or antibodies that specifically react with the analyte can cause the analyte to be measured in the sample solution. Methods for detecting or quantifying a substance to be measured by inhibiting the presence of the substance are also widely used in immunological clinical tests. In addition, a method in which a substance that selectively binds to specific cells is immobilized on a particulate carrier and cells are identified depending on whether or not the particles bind to the cells is often used as a means of immunological testing. It has been adopted.
このような免疫活性粒子を用いた免疫学的検査
用試薬における粒子状担体としては、従来、ヒト
を含む哺乳動物や鳥類の赤血球、カオリンや炭素
など無機物の粒子、天然ゴムラテツクスやポリス
チレンなどの有機高分子化合物のラテツクスが凝
集反応用として広く用いられている。 Particulate carriers in immunological test reagents using such immunoactive particles have conventionally been red blood cells of mammals including humans and birds, inorganic particles such as kaolin and carbon, and organic polymers such as natural rubber latex and polystyrene. Latexes of molecular compounds are widely used for agglutination reactions.
これらのうち赤血球は多種類の抗原・抗体を固
定化することが可能で応用範囲が最も広い。しか
し採取する動物個体によつて品質等に差があるこ
と、安定性に難があり保存が難しいこと、またヒ
ト血清により非特異的に凝集する場合があること
などの問題点がある。 Among these, red blood cells can immobilize many types of antigens and antibodies and have the widest range of applications. However, there are problems such as differences in quality depending on the individual animal collected, poor stability and difficulty in storage, and non-specific agglutination by human serum.
非生物由来の粒子として最も広く用いられてい
るのはポリエチレン粒子であり、これは合成高分
子化合物であるところから品質を一定にすること
が可能でまたそれ自体では安定である。ポリスチ
レンは疎水性で種々の蛋白質を吸着する性質があ
るため、通常ポリスチレンへの抗原または抗体の
固定化は物理吸着によつて行なわれる。しかし物
理吸着によつて抗原または抗体を固定化した場合
には固定化した抗原（または抗体）と遊離の抗原
（または抗体）との間に平衡が存在し、そのため
測定の目的物質である対応する抗体（または抗
原）に対し粒子に固定化した抗原（または抗体）
と遊離の抗原（または抗体）との間に競争反応が
起こり、その競争反応は凝集に対して抑制的に作
用する。その結果、多くの例において感度と安定
性の不足が指摘されている。また当然のことなが
らポリステレンに対して物理的に吸着されにくい
物質は固定化することができない。これらの問題
点のためにポリスチレン粒子は赤血球を担体とす
る場合に比較して限られた範囲でしか実用に供さ
れていない。 Polyethylene particles are the most widely used particles of non-biological origin, and since they are synthetic polymer compounds, they can have a 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 that the corresponding target substance of measurement Antigen (or antibody) immobilized on particles against antibody (or antigen)
A competitive reaction occurs between the free antigen (or antibody) and the free antigen (or antibody), and the competitive reaction has an inhibitory effect on agglutination. 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 polysterene cannot be immobilized. Due to these problems, polystyrene particles have been put to practical use only to a limited extent compared to when red blood cells are used as a carrier.
これらの問題点の解決を図る目的で最近、スチ
レン−メタクリル酸コポリマーラテツクスにヒト
絨毛性ゴナドトロピンをカルボジイミドを使用し
て結合させた試薬（DT2649218）、カルボキシル
化スチレン−ブタジエンコポリマー、カルボキシ
ル化ポリスチレン、アミノ基をもつカルボキシル
化ポリスチレン、アクリル酸ポリマー、アクリロ
ニトリルポリマー、メタクリル酸ポリマー、アク
リロニトリル−ブタジエン−スチレンコポリマ
ー、ポリ酢酸ビニルアクリレート、ポリビニルピ
リジン、塩化ビニル−アクリレートコポリマーな
ど種々のラテツクスポリマーにカルボイミドを縮
合剤としてヒト絨毛性ゴナドトロピン、ヒト血清
アルブミンまたは変性ガンマグロブリンをアミド
結合を介して縮合させた粒径0.01〜0.9ミクロン
の粒子からなる試薬（特公昭63−12966）、メタク
リル酸、２−ヒドロキシエチルメタクリレートお
よびメチルメタクリレートを共重合して製造した
ヒドロキシル基とカルボキシル基を含有するメチ
ルメタクリレート系ラテツクスにトレボネーマ抗
原を臭化シアノゲンまたはカルボジイミド法で結
合させた試薬（臨床病理27、補冊、522頁
（1978））、ポリスチレン粒子を芯として、それを
スチレン−グリシジルメタクリレートコポリマー
の外皮で被覆したラテツクスのエポキシ基とヒト
絨毛ゴナドトロピンまたはインシユリンを反応さ
せて、それらをラテツクスに結合させた試薬（特
開昭55−110118）など、共有結合により抗原また
は抗体や担体に結合させた試薬が提案されてい
る。 In order to solve these problems, we have recently developed a reagent (DT2649218) in which human chorionic gonadotropin is bonded to a styrene-methacrylic acid copolymer latex using carbodiimide, carboxylated styrene-butadiene copolymer, carboxylated polystyrene, amino carboxylated polystyrene, acrylic acid polymer, acrylonitrile polymer, methacrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate acrylate, polyvinylpyridine, vinyl chloride-acrylate copolymer, etc. with carboimide as a condensing agent. A reagent consisting of particles with a particle size of 0.01 to 0.9 microns condensed with human chorionic gonadotropin, human serum albumin or modified gamma globulin via an amide bond (Japanese Patent Publication No. 63-12966), methacrylic acid, 2-hydroxyethyl methacrylate and methyl A reagent in which Trebonema antigen is bound to a methyl methacrylate latex containing hydroxyl and carboxyl groups produced by copolymerizing methacrylate using the cyanogen bromide or carbodiimide method (Clinical Pathology 27, Supplement, p. 522 (1978)); A reagent that binds human chorionic gonadotropin or insulin to a latex by reacting the epoxy groups of a latex with polystyrene particles as a core and covering it with a styrene-glycidyl methacrylate copolymer shell (Japanese Patent Application Laid-open No. 110118-1982), etc. , reagents that are covalently bound to antigens, antibodies, or carriers have been proposed.
これら先行技術においてはカルボジイミドによ
り免疫活性物質を粒子に結合させる方法が多用さ
れているが、カルボジイミドを使用すると免疫活
性物質分子間および分子内の縮合反応を惹起す
る。これはのぞましくなく副反応であつて免疫活
性物質の活性を損なうものである。臭化シアノゲ
ンを用いれば免疫活性物質分子間および分子内の
縮合反応を回避することはできるが、この場合に
はヒドロキシル基を有するポリマーと臭化シアノ
ーゲンとの反応の再現性を得ることが難かしく、
その結果免疫活性物質を固定化した粒子の免疫活
性が変動しやすい。これらの免疫活性物質固定化
法に比較して重合体に導入されたエポキシ基と蛋
白質またはポリペプチドを反応させる方法は免疫
活性の失活も少なく再現性も良好である。しかし
エポキシ基を利用する上記先行技術において重合
体粒子表面にスチレンに由来する部分が存在する
ため蛋白質を非特異的に吸着する傾向を有してい
る。 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 unwanted side reaction and impairs the activity of the immunoactive substance. If cyanogen bromide is used, 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 an 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, there is a styrene-derived portion on the surface of the polymer particles, which tends to non-specifically adsorb proteins.
一般にヒトまたは動物の体液中には多種類の蛋
白質が含まれ、とくに血漿または血清中にはこれ
のは高濃度で含有されている。検体体液から蛋白
質が担体粒子に吸着されると、それが目的とする
抗原−抗体反応など免疫学的反応に干渉し、凝集
反応の選択性や感度の低下をもたらすおそれがあ
る。 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 may interfere with the target immunological reaction such as an antigen-antibody reaction, leading to a decrease in the selectivity and sensitivity of the agglutination reaction.
本発明者らはこのような問題点を解消すること
を目的に検討を行なつた結果、担体として２，３
−ジオキシプロピレンメタクリレートを主成分と
する架橋重合体よりなる平均直径0.03μｍ〜10μｍ
の親水性微粒子がすぐれていることを見出した
（特開昭56−30405、特開昭56−171559）。上記２，
３−ジオキシプロピルメタクリレートを主成分と
する架橋重合体にはアミノ基またはカルボキシル
基を容易に導入できるので、これらの官能基を用
いて免疫学的活性物質を固定化することができ
る。しかしアミノ基を導入した微粒子は陽電荷
を、カルボキシル基を導入した微粒子は陰電荷を
帯びる。微粒子が帯電するのを避けたい場合には
２，３−ジオキシプロピルメタクリレート単位の
ヒドロキシル基を官能基として利用し、微粒子を
臭化シアノゲンで処理することにより活性化した
のち、免疫学的活性物質と反応させる方法で、免
疫学的活性物質を微粒子とに固定化することがで
きる。しかし臭化シアノゲンは毒性が強くて取扱
いに危険が伴なう上に、臭化シアノゲン活性化法
による免疫学的活性物質固定化は既述のように再
現性に難がある。 The present inventors conducted studies with the aim of solving these problems, and as a result, they found that 2,3
- Average diameter of 0.03 μm to 10 μm made of a crosslinked polymer mainly composed of dioxypropylene methacrylate
It was discovered that the hydrophilic fine particles of 2000 were superior (Japanese Patent Application Laid-Open No. 56-30405, 1982-171559). Above 2,
Since amino groups or carboxyl groups can be easily introduced into crosslinked polymers containing 3-dioxypropyl methacrylate as a main component, immunologically active substances can be immobilized using these functional groups. However, fine particles introduced with amino groups are positively charged, and fine particles introduced with carboxyl groups are negatively charged. If you want to avoid charging the fine particles, use the hydroxyl group of the 2,3-dioxypropyl methacrylate unit as a functional group, activate the fine particles by treating them with cyanogen bromide, and then use the immunologically active substance. An immunologically active substance can be immobilized on microparticles by a method of reacting with microparticles. However, cyanogen bromide is highly toxic and dangerous to handle, and as mentioned above, immobilization of immunologically active substances by the cyanogen bromide activation method has difficulty in reproducibility.
本発明者らはできるだけ中性の微粒子を担体と
して、免疫学的活性物質を、簡便にかつ再現性よ
く固定化する方法を検討した結果、本発明に達し
た。すなわち本発明は、グリシジルアクリレート
および（または）グリシジルメタクリレートのく
り返し単位に有する重合体からなり、かつグリシ
ジルアクリレートまたはグリシジルメタクリレー
ト単位以外に疎水性成分を表面に持たない平均直
径が0.03〜10μｍの微粒子に、メルカプト基の導
入させてなる免疫学的活性物質固定化用担体微粒
子を提供するものである。 The present inventors have studied a method of immobilizing an immunologically active substance simply and with good reproducibility using microparticles as neutral as possible as a carrier, and as a result, the present invention has been achieved. That is, the present invention provides fine particles having an average diameter of 0.03 to 10 μm, which are made of a polymer having repeating units of glycidyl acrylate and/or glycidyl methacrylate, and which have no hydrophobic component on the surface other than the glycidyl acrylate or glycidyl methacrylate units. The present invention provides fine carrier particles for immobilizing an immunologically active substance into which a mercapto group is introduced.
グリシジルアクリレートおよび／またはグリシ
ジルメタクリレートのくり返し単位を有する重合
体からなり、かつグリシジルアクリレートまたは
グリシジルメタクリレート単位以外に疎水性成分
を表面に持たない、平均粒径0.03〜10μｍの微粒
子は、たとえば特願昭57−54245に記載した方法
により製造できる。 Fine particles with an average particle size of 0.03 to 10 μm, which are made of a polymer having repeating units of glycidyl acrylate and/or glycidyl methacrylate and have no hydrophobic component on the surface other than the glycidyl acrylate or glycidyl methacrylate units, are disclosed in, for example, Japanese Patent Application No. 57 It can be produced by the method described in -54245.
重合体微粒子製造時のグリシジルアクリレート
とグリシジルメタクリレートとの混合割合は任意
であり、いずれか一方のみを単独で使用してもよ
い。 The mixing ratio of glycidyl acrylate and glycidyl methacrylate during production of polymer fine particles is arbitrary, and either one may be used alone.
本発明の重合体微粒子は、他の共重合成分を加
えることがしばしば好ましい結果をもたらす。共
重合成分添加の効用は粒径の調節である。共重合
成分としてのぞましいのは親水性単量体であり、
とくに水溶性単量体がのぞましい。共重合に用い
る水溶性単量体として適当なものは、例えば２−
オキシエチルアクリレート、２−オキシエチルメ
タクリレート、２−オキシプロピルアクリレー
ト、２−オキシプロピルメタクリレート、重合度
２ないし25のポリエチレングリコールモノアルキ
ルエーテルのアクリリル酸エステルまたはメタク
リル酸エステル、アクリルアミド、メタクリルア
ミド、Ｎ−ビニルピロリドン、グリセロールメタ
クリレートなどである。これらの水溶性単量体は
２種以上併用してもよい。これら水溶性単量体を
共重合した場合には、免疫活性物質を固定化した
後に、重合体微粒子表面で何らの結合物によつて
覆われることなく露出しているのは、水溶性単量
体に由来する親水性部分である。蛋白質は水性媒
体中では親水性重合体には吸着しにくいので、本
発明による免疫活性物質固定化微粒子は、検体体
液に対して安定で非特異的凝集を起こしにくく、
また細胞に対する非特異的付着がない。グリシジ
ルアクリレートとグリシジルメタクリレートの和
に対する共重合成分の和の比率はモル比で100：
０ないし５：95の範囲で変えることができる。 Addition of other copolymerization components to the polymer fine particles of the present invention often yields favorable results. The effect of adding a copolymer component is to adjust the particle size. Hydrophilic monomers are preferred as copolymerization components,
Particularly desirable are water-soluble monomers. Suitable water-soluble monomers for copolymerization include, for example, 2-
Oxyethyl acrylate, 2-oxyethyl methacrylate, 2-oxypropyl acrylate, 2-oxypropyl methacrylate, acrylic acid ester or methacrylic acid ester of polyethylene glycol monoalkyl ether with a degree of polymerization of 2 to 25, acrylamide, methacrylamide, N-vinyl These include pyrrolidone 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 the body. Since proteins are difficult to adsorb to hydrophilic polymers in aqueous media, the immunoactive substance-immobilized microparticles according to the present invention are stable with respect to sample body fluids and do not easily cause non-specific aggregation.
Also, there is no non-specific attachment to cells. The molar ratio of the sum of copolymer components to the sum of glycidyl acrylate and glycidyl methacrylate is 100:
It can be changed from 0 to 5:95.
本発明の重合体微粒子は例えば次の方法によつ
て製造することができる。 The polymer fine particles of the present invention can be produced, for example, by the following method.
重合反応は通常乳化重合、沈澱重合または懸濁
重合によつて好ましく行なわれる。これらいずれ
の方法も重合と同時に重合体が粒子状になつて析
出するので本発明の目的に適している。 The polymerization reaction is usually preferably carried out by emulsion polymerization, precipitation polymerization or suspension polymerization. All of these methods are suitable for the purpose of the present invention because the polymer is precipitated in the form of particles at the same time as the polymerization.
沈澱重合は、単量体は溶解するが重合によつて
生成する重合体は溶解しない媒体中で重合を行な
う方法であつて、単量体と重合媒体との組合せを
選択することによつて生成する重合体粒子の平均
直径を0.03ないし10μｍの範囲に入るよう調節す
ることが比較的容易であり、粒径の分布も比較的
狭い。また沈澱重合は、乳化重合や懸濁重合の場
合と異なつて、乳化剤や懸濁安定剤を使用しない
ので、重合反応後これらの添加剤を除去する必要
がないのも利点の一つである。 Precipitation polymerization is a method in which polymerization is carried out in a medium that dissolves the monomer but does not dissolve the polymer produced by polymerization. It is relatively easy to adjust the average diameter of the polymer particles to fall within the range of 0.03 to 10 μm, and the particle size distribution is also relatively narrow. Another advantage of precipitation polymerization, unlike emulsion polymerization or suspension polymerization, is that it does not use emulsifiers or suspension stabilizers, so there is no need to remove these additives after the polymerization reaction.
沈澱重合に用いられる媒体としては、例えば酢
酸エチル、酢酸ｎ−プロピル、酢酸イソプロピ
ル、酢酸ブチル各異性体およびプロピオン酸の上
記相当エステルなどのエステル類、メチルエチル
ケトン、メチルｎ−プロピルケトン、メチルイソ
プロピルケトン、メチルブチルケトン各異性体な
どのケトン類、ベンゼン、トルエン、ｏ−キシレ
ン、ｍ−キシレン、ｐ−キシレン、四塩化炭素な
どである。 Examples of the medium used in precipitation polymerization include esters such as ethyl acetate, n-propyl acetate, isopropyl acetate, butyl acetate isomers and the above-mentioned corresponding esters of propionic acid, methyl ethyl ketone, methyl n-propyl ketone, methyl isopropyl ketone, These include ketones such as methyl butyl ketone isomers, benzene, toluene, o-xylene, m-xylene, p-xylene, carbon tetrachloride, and the like.
架橋剤を重合系に添加することは必須ではない
が、通常重合に当つて重合性炭素炭素二重結合を
分子内に２基以上含む多官能性単量体を添加して
積極的に重合体を架橋させることがのぞましい。
そのような目的で重合系に添加するに適した多官
能性単量体は多数存在するが、若干例をあげれ
ば、ジビニルベンゼン、エチレングリコールメタ
クリレート、Ｎ，N′−メチレンビスアクリルア
ミド、コハク酸ジビニル、コハク酸ジアリル、メ
タクリル酸ビニル、メタクリル酸アリル、トリア
リルシアヌレート、トリアリルイソシアヌレート
などである。架橋剤の添加量は通常全単量体中の
30モル％以下である。また架橋剤結合は重合反応
後生成重合体の反応性を利用してこれを多官能化
合物と反応させることによつて導入することもで
きる。例えば生成重合体に含まれるエポキシ基と
エチレンジアミンなどのジアミンとを反応させる
ことにより重合体を架橋させることができる。 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 methacrylate, N,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
It is 30 mol% or less. Further, the crosslinking agent bond 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.
重合開始剤としては通常のラジカル重合開始
剤、例えば２，2′−アゾビスイソブチロニトリ
ル、２，2′−アゾビス（２，４−ジメチルバレロ
ニトリル）、２，2′−アゾビス（２，４−ジメチ
ル−４−メトキシバレロニトニル）、などのアゾ
化合物、過酸化ベンゾイル、ジラウロイルパーオ
キサイド、ジ−tertブチルパーオキサイドなどの
過酸化物を用いることができる。重合温度も通常
のラジカル重合の温度範囲でよく、20℃ないし80
℃がとくに好ましい。重合開始剤の重合混合液中
の濃度は、通常0.001ないし0.03モル／程度で
ある。単量体の重合混合液中の濃度は通常５ない
し50重量％の範囲が好ましい。単量体濃度が50重
量％を超えると生成する重合体粒子が凝集する傾
向がある。また単量体濃度が５重量％未満でも本
発明は実施可能であるが、得られる重合体微粒子
が少なくなるので生産性が低くなる。なお重合は
窒素またはアルゴンなどの不活性ガスで置換して
行なうのがのぞましい。 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-methoxyvaleronitonyl), 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 the monomer in the polymerization mixture is usually preferably in the range of 5 to 50% by weight. 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.
乳化重合は直径0.5μｍ以下の微粒子を製造する
のに適している。乳化剤としては、アニオン性、
カチオン性およびノニオン性いずれのタイプの界
面活性剤も使用できる。重合開始剤としては、た
とば過硫酸のナトリウム、カリウムまたはアンモ
ニウム塩のような水溶性無機過酸化物、２，2′−
アゾビス（２−アミノプロパン）塩酸塩、４，
4′−アゾビス−４−シアンペンタン酸のような水
溶性有機アゾ化合物が好ましく用いられる。これ
らの重合開始剤から遊離基を発生させるために
は、通常温度を上げて熱反応により分解させる
が、紫外線を照射して光反応により分解させても
よい。重合温度は、グリシジルアクリレートまた
はグリシジルメタクリレートのエポキシ基の加水
分解を避けるために70℃以下で行なうのがのぞま
しい。 Emulsion polymerization is suitable for producing fine particles with a diameter of 0.5 μm or less. As emulsifiers, anionic,
Both cationic and nonionic types of surfactants can be used. As polymerization initiators, water-soluble inorganic peroxides such as sodium, potassium or ammonium salts of persulfate, 2,2'-
Azobis(2-aminopropane) hydrochloride, 4,
Water-soluble organic azo compounds such as 4'-azobis-4-cyanpentanoic acid are preferably used. In order to generate free radicals from these polymerization initiators, they are usually decomposed by a thermal reaction by raising the temperature, but they may also be decomposed by a photoreaction by irradiation with ultraviolet rays. The polymerization temperature is preferably 70° C. or lower to avoid hydrolysis of the epoxy groups of glycidyl acrylate or glycidyl methacrylate.
粒子の形状は多くの場合球形であるが球形であ
ることは必要条件ではなく不規則な形状であつて
も差し支えない。不規則な形状の粒子の直径は最
大径と最小径の和の1/2とする。平均直径は式(1)
によつて定義されるｄによつて表わされる。ただ
しdiはｉ番目の粒子の直径、Ｎは粒子の総数であ
る。 The shape of the particles is often spherical, but sphericity is not a necessary condition, and irregular shapes are also acceptable. The diameter of irregularly shaped particles is 1/2 of the sum of the maximum and minimum diameters. The average diameter is given by formula (1)
d defined by . However, di is the diameter of the i-th particle, and N is the total number of particles.
ｄ＝N
〓i=1
di／Ｎ ……(1)
凝集反応が判定しやすいのは経験的に平均直径
が0.1μｍ以上10μｍ以下の場合である。また細胞
標識の目的には平均直径は0.03μｍ以上5μｍ以下
の範囲が好ましい。また染料ないし顔料により適
当に着色した粒子は凝集反応、細胞標識いずれの
目的に対しても好都合である。また細胞標識に対
しては螢光を付与した粒子も好ましい。 d= N 〓 i=1 di/N...(1) Empirically, it is easy to determine the agglutination reaction when the average diameter is 0.1 μm or more and 10 μm or less. Further, for the purpose of cell labeling, the average diameter is preferably in the range of 0.03 μm or more and 5 μm or less. 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.
重合体 微粒子にメルカプト基を導入するため
には、重合体微粒子を水または不活性な液状有機
化合物に分散させて、メルカプト基を導入するた
めの試薬と反応させる。メルカプト基を導入する
ための試薬としては、硫化水素、分子内にメルカ
プト基を２基以上有するポリチオールまたは分子
内にアミノ基とメルカプト基の両方を有する化合
物などが好ましい。硫化水素はガス状で反応系に
吹き込んでもよく、またナトリウム塩すなわち水
硫化ナトリウムなどのアルカリ金属塩として使用
してもよい。分子内にメルカプト基を２基以上有
するポリチオールの例を若干あげれば、ジチオグ
リコール、ジチオエリスリトール、ジチオスレイ
トール、トルエン−３，４−ジチオールなどであ
る。また、分子内にアミノ基とメルカプト基の両
方を有する化合物としては、たとえば２−アミノ
エタンチオール、２−アミノチオフエノール、４
−アミノチオフエノール、システイン、システイ
ンエチルエステル、システインメチルエステルな
どがあげられる。メルカプト基導入反応の温度は
０℃ないし100℃の範囲が適当である。また反応
は撹拌しつつ行なうのがよい。 In order to introduce mercapto groups into polymer particles, the polymer particles are dispersed in water or an inert liquid organic compound and reacted with a reagent for introducing mercapto groups. As a reagent for introducing a mercapto group, hydrogen sulfide, a polythiol having two or more mercapto groups in the molecule, a compound having both an amino group and a mercapto group in the molecule, etc. are preferable. Hydrogen sulfide may be blown into the reaction system in gaseous form or may be used as an alkali metal salt such as the sodium salt, ie, sodium bisulfide. Some examples of polythiols having two or more mercapto groups in the molecule include dithioglycol, dithioerythritol, dithiothreitol, and toluene-3,4-dithiol. In addition, examples of compounds having both an amino group and a mercapto group in the molecule include 2-aminoethanethiol, 2-aminothiophenol,
-Aminothiophenol, cysteine, cysteine ethyl ester, cysteine methyl ester and the like. The temperature for the mercapto group introduction reaction is suitably in the range of 0°C to 100°C. Further, the reaction is preferably carried out with stirring.
メルカプト基を導入された重合体微粒子に免疫
学的活性物質を固定化するためは結合剤を使用す
る。結合剤としては分子内にメルカプト基と反応
して結合する官能基を有し、この他にアミノ基ま
たは、カルボキシル基と反応して結合する官能基
を有する化合物が好適である。そのような化合物
として容易に入手できるものに、Ｎ−ヒドロキシ
スクシンイミドと４−（マレイミドメチル）シク
ロヘキサン−１−カルボン酸とのエステル（略
称：CHM）があるが、勿論これに限定されるも
のではない。メルカプト基含有重合体微粒子と結
合剤とを反応させると、結合剤のメルカプト基反
応性官能基が重合体微粒子のメルカプト基と反応
して結合し、重合体微粒子にアミノ基またはカル
ボキシル基と反応する官能基が導入される。免疫
学的活性物質は蛋白質であるかまたはポリペプチ
ド部分を含んでいるので、アミノ基およびカルボ
キシル基を含有しており、上記結合剤によつて処
理された重合体微粒子と接触させると、重合体微
粒子上に固定化される。免疫学的活性物質を固定
化したのち、過剰の結合剤官能基が残存する場合
には血清アルブミン、ゼラチンなど目的とする免
疫学的検査に干渉しない親水性蛋白質を過剰の官
能基と反応させることによつて、その反応性を失
わせることができる。その際官能基消去用の親水
性蛋白質は固定化の目的である免疫活性物質と混
合して同時に反応させてもよく、また免疫活性物
質を先に単独で反応させてその後で反応させても
よい。また上記アルブミンやゼラチンなどの親水
性蛋白質の代りにグリシン、アラニンなどのアミ
ノ酸を用いることも可能である。 A binding agent is used to immobilize the immunologically active substance on the polymer particles into which mercapto groups have been introduced. As the binder, a compound having a functional group in its molecule that reacts with a mercapto group and bonds therewith, and also has a functional group that reacts with and bonds with an amino group or a carboxyl group is suitable. One of the easily available such compounds is ester of N-hydroxysuccinimide and 4-(maleimidomethyl)cyclohexane-1-carboxylic acid (abbreviation: CHM), but it is of course not limited to this. . When the mercapto group-containing polymer fine particles and the binder are reacted, the mercapto group-reactive functional group of the binder reacts and bonds with the mercapto group of the polymer fine particles, and reacts with the amino group or carboxyl group on the polymer fine particles. A functional group is introduced. Since the immunologically active substance is a protein or contains a polypeptide moiety, it contains amino and carboxyl groups, and when brought into contact with the polymer microparticles treated with the above binding agent, the polymer Immobilized on microparticles. If an excess of binding agent functional groups remains after immobilizing an immunologically active substance, a hydrophilic protein such as serum albumin or gelatin that does not interfere with the intended immunological test may be reacted with the excess functional groups. The reactivity can be lost by In this case, the hydrophilic protein for functional group elimination may be mixed with the immunoactive substance to be immobilized and reacted at the same time, 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.
ここで免疫活性物質とは抗原および抗体のみで
なく、補体、Feレセプター、C3レセプターなど
液性免疫反応ないし細胞性免疫反応に関与してあ
る物質に特異的に結合する物質を意味するものと
する。具体例を若干あげれば、梅毒トレポネーマ
抗原、Ｂ型肝炎表面抗原（HBs抗原）、Ｂ型肝炎
表面抗原に対する抗体（抗HBs抗体）、風疹抗
原、トキソプラズマ抗原、ストレプトリジンＯ、
抗ストレプトリジンＯ抗体、マイコプラズマ抗
原、ヒト絨毛性ゴナドトロピン（HCG）、抗
HCG抗体、熱凝集ヒトIgG、リウマチ因子、該
蛋白、DNA、抗DNA抗体、Ｃ反応性蛋白
（CRP）、抗CRP抗体、抗エストロゲン抗体、α
−フエトプロテイン（α−FP）、抗α−FP抗体、
癌胎児性抗原（CEA）、抗CEA抗体、C1q、抗C1q
抗体、C3、抗C3抗体、抗3b抗体、抗C3bi抗体、
C4、抗C4抗体、プロテイン−Ａ、コングルチニ
ン、イムノコングルチニンなどである。 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, Fe receptors, and C3 receptors. do. Some specific examples include Treponema pallidum antigen, hepatitis B 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, this protein, DNA, anti-DNA antibody, C-reactive protein (CRP), anti-CRP antibody, anti-estrogen antibody, α
-fetoprotein (α-FP), anti-α-FP antibody,
carcinoembryonic antigen (CEA), anti-CEA antibody, C1 q , anti-C1 q
antibody, C3, anti-C3 antibody, anti-3 b antibody, anti-C3 bi antibody,
These include C4, anti-C4 antibody, protein-A, conglutinin, and immunoconglutinin.
次に実施例に基づいて本発明の説明する。 Next, the present invention will be explained based on examples.
実施例 １
グリシジルメタクリレート14.50部（重量基準、
以下同じ）、２−ヒドロキシエチルメタクリレー
ト1.50部、トリエチレングリコールジメタクリレ
ート1.65部、プロピオン酸エチル25.00部および
四塩化炭素25.00部を混合し、２，2′−アゾビス
（２，４−ジメチルバレロニトリル）を0.050部添
加溶解したのち、アルゴン雰囲気下45℃で16時間
重合させた。0.41部の凝集塊を除いて16.00部の
よく分散した重合体微粒子を得た。重合体微粒子
を走査電子顕微鏡で観察すると、直径は2.7μｍで
ほとんど均一の球形であつた。この重合体微粒子
を50倍（重量）の水に分散し、60℃に昇温して硫
化水素ガスを吹き込みつつ、３時間撹拌した。次
に重合体微粒子を遠心沈降させて、Ｎ／200水酸
化ナトリウム水溶液で洗浄し、さらに水洗してか
ら0.16％硫酸水溶液に２％の含量で分散させ、残
存エポキシ基を加水分解する目的で50℃に24時間
保つた。次に重合体微粒子を再び水洗し、水をジ
メチルスルホキシド（以下DMSOと略記）で置
換して、重合体微粒子をDMSO中に２％の割合
で分散させた。この分散液５mlを等容の0.5％
CHM／DMSO溶液と混合し、窒素雰囲気下に30
℃で１時間撹拌した。反応後重合体微粒子を
DMSOで洗浄して遠沈した。沈降した重合体微
粒子を１mg／mlのヒトガンマグロブリン／PBS
溶液（PBS：リン酸塩緩衝食塩水、リン酸塩濃
度0.01モル／、食塩0.14モル／、PH7.0）３ml
に分散し、窒素雰囲気下４℃に16時間保つた。次
いでこの分散液２mlを水洗乾燥したのち、6N塩
酸中で110℃に20時間加熱することにより固定化
された蛋白質を加水分解してアミノ酸分析により
生成アミノ酸を測定した。その結果、ガンマグロ
ブリン固定化量は重合体微粒子１mg当り10.5μｇ
であつた。前記４℃で16時間保つたのち乾燥しな
かつたヒトガンマグロブリン固体化重合体微粒子
分散液１mlを、２モル／グリシン水溶液（PH
7.5）１mlと混合し、窒素雰囲気下に25℃で１時
間撹拌した。そしてPBSで洗浄したのち、１％
ウシ血清アルブミン（以下BSAと略記）添加
PBSに、重合体微粒子含量が１％になるように
分散させた。このヒトガンマグロブリン固定化重
合体微粒子分散液10μと抗ヒトIgG抗血清（ヤ
ギ）希釈液（希釈は１％BSA添加PBSによる）
10μとを、スライドグラス上で混合し、肉眼に
より凝集の有無を判定した。その結果、無希釈、
10倍希釈および100倍希釈の抗血清では明らかに
凝集が認められたが、1000倍希釈抗血清では凝集
が認められなかつた。この凝集が抗原抗体反応に
よる特異的にものであることは、次のようにして
阻止試験により確認した。ヒトIgGの１mg／ml、
0.1mg／ml、0.01mg／mlおよび０mg／mlの溶液
（溶媒は１％BSA添加PBS）を各々等容の５倍希
釈抗ヒトIgG抗血清と混合し、37℃で30分インキ
ユベートした。しかる後、その混合分散液10μ
とヒトガンマグロブリン固定化重合体微粒子分散
液10μとをスライドグラス上で混合して凝集を
観察した。その結果、ヒトIgG濃度0.01および０
mg／mlの場合は強い凝集が、0.1mg／mlの場合は
弱め凝集が認められたが、１mg／mlの場合は凝集
が完全に阻止された。Example 1 14.50 parts of glycidyl methacrylate (by weight,
The same applies hereinafter), 1.50 parts of 2-hydroxyethyl methacrylate, 1.65 parts of triethylene glycol dimethacrylate, 25.00 parts of ethyl propionate, and 25.00 parts of carbon tetrachloride are mixed to form 2,2'-azobis(2,4-dimethylvaleronitrile). After adding and dissolving 0.050 part of the solution, polymerization was carried out at 45° C. for 16 hours under an argon atmosphere. After removing 0.41 parts of agglomerates, 16.00 parts of well-dispersed polymer particles were obtained. When the polymer fine particles were observed using a scanning electron microscope, they were found to be almost uniformly spherical with a diameter of 2.7 μm. The polymer particles were dispersed in 50 times (by weight) water, heated to 60° C., and stirred for 3 hours while blowing hydrogen sulfide gas. Next, the polymer particles were centrifuged, washed with an N/200 aqueous sodium hydroxide solution, further washed with water, and then dispersed in a 0.16% sulfuric acid aqueous solution at a concentration of 2%. Keep at ℃ for 24 hours. Next, the polymer fine particles were washed again with water, and the water was replaced with dimethyl sulfoxide (hereinafter abbreviated as DMSO) to disperse the polymer fine particles in DMSO at a ratio of 2%. Add 5 ml of this dispersion to an equal volume of 0.5%
Mix with CHM/DMSO solution and store under nitrogen atmosphere for 30 min.
Stirred at ℃ for 1 hour. After reaction, polymer fine particles are
It was washed with DMSO and centrifuged. The precipitated polymer particles were dissolved in 1 mg/ml human gamma globulin/PBS.
Solution (PBS: phosphate buffered saline, phosphate concentration 0.01 mol/, salt 0.14 mol/, PH 7.0) 3 ml
The mixture was dispersed in water and kept at 4°C for 16 hours under a nitrogen atmosphere. Next, 2 ml of this dispersion was washed with water and dried, and then heated in 6N hydrochloric acid at 110°C for 20 hours to hydrolyze the immobilized protein, and the produced amino acids were measured by amino acid analysis. As a result, the amount of gamma globulin immobilized was 10.5 μg per 1 mg of polymer fine particles.
It was hot. 1 ml of the human gamma globulin solidified polymer fine particle dispersion that did not dry after being kept at 4°C for 16 hours was mixed with a 2 mol/glycine aqueous solution (PH
7.5) and stirred for 1 hour at 25°C under nitrogen atmosphere. After washing with PBS, 1%
Added bovine serum albumin (hereinafter abbreviated as BSA)
It was dispersed in PBS so that the polymer fine particle content was 1%. 10μ of this human gamma globulin-immobilized polymer particle dispersion and anti-human IgG antiserum (goat) diluted solution (diluted with PBS supplemented with 1% BSA)
10μ were mixed on a slide glass, and the presence or absence of aggregation was determined visually. As a result, undiluted,
Agglutination was clearly observed with the 10-fold diluted and 100-fold diluted antisera, but no agglutination was observed with the 1000-fold diluted antiserum. It was confirmed by the following inhibition test that this agglutination was caused specifically by an antigen-antibody reaction. 1 mg/ml of human IgG,
Solutions of 0.1 mg/ml, 0.01 mg/ml and 0 mg/ml (solvent: PBS supplemented with 1% BSA) were each mixed with an equal volume of 5-fold diluted anti-human IgG antiserum and incubated at 37°C for 30 minutes. After that, 10μ of the mixed dispersion
and 10μ of a human gamma globulin-immobilized polymer particle dispersion were mixed on a slide glass, and aggregation was observed. As a result, the human IgG concentration was 0.01 and 0.
Strong aggregation was observed at mg/ml, weak aggregation was observed at 0.1 mg/ml, but aggregation was completely inhibited at 1 mg/ml.
実施例 ２
実施例１と同様に重合して得た重合体微粒子２
部を100部の水に分散し、ジオスレイトール１部
を添加溶解して窒素雰囲気下60℃で３時間撹拌し
た。遠沈水洗をくり返したのち、重合体微粒子を
0.16％硫酸水溶液中に２％の含量で分散させ、50
℃で24時間加水分解処理を行なつた。このように
してメルカプト基を導入した重合体微粒子の元素
分析値はC48.67％、H6.86％、S8.75％であつた。
このメルカプト基導入重合体微粒子を実施例１と
同様にしてCHMで処理し、ヒトガンマグロブリ
ンを固定化した。実施例１と同様にして固定化蛋
白質量を定量した結果、重合体微粒子１mg当り
7.8μｇであつた。また実施例１と同様にしてスラ
イドグラス上で抗ヒトIgG抗血清（ヤギ）と反応
させた結果、無希釈、10倍希釈、20倍希釈、およ
び40倍希釈抗血清では凝集が認められたが、100
倍希釈抗血清では凝集が認められたかつた。なお
抗ヒトIgG抗血清（ヤギ）の代りに抗ヒトフイブ
リノーゲン抗血清（ヤギ）を用いた場合には上記
どの希釈倍率でも凝集は認められなかつた。Example 2 Polymer fine particles 2 obtained by polymerization in the same manner as Example 1
part was dispersed in 100 parts of water, 1 part of diothreitol was added and dissolved, and the mixture was stirred at 60°C for 3 hours under a nitrogen atmosphere. After repeated centrifugation and water washing, the polymer particles were
Dispersed at 2% content in 0.16% sulfuric acid aqueous solution, 50
Hydrolysis treatment was carried out at ℃ for 24 hours. The elemental analysis values of the polymer fine particles into which mercapto groups were introduced were C48.67%, H6.86%, and S8.75%.
The mercapto group-introduced polymer fine particles were treated with CHM in the same manner as in Example 1 to immobilize human gamma globulin. As a result of quantifying the amount of immobilized protein in the same manner as in Example 1, it was found that per 1 mg of polymer fine particles.
It was 7.8 μg. In addition, as a result of reacting with anti-human IgG antiserum (goat) on a slide glass in the same manner as in Example 1, agglutination was observed with undiluted, 10-fold diluted, 20-fold diluted, and 40-fold diluted antiserum. , 100
Agglutination was observed with the double diluted antiserum. Note that when anti-human fibrinogen antiserum (goat) was used instead of anti-human IgG antiserum (goat), no agglutination was observed at any of the above dilution ratios.
実施例 ３
（コロイド微粒子の調製）
グリシジルメタクリレート、スルホプロピルメ
タクリレート、トリエチレングリコールジメタク
リレートをモル比で85：10：５となるように混合
した。0.01％ドデシル硫酸ナトリウムと0.01モ
ル／の４，4′−アゾビス−４−シアンペンタン
酸の存在下で全モノマー濃度が10％となるように
して反応をおこなつた。反応液のPHは7.2となる
ようにNaOHによりあわせておいた。激しく撹
拌し乳化状態を保ちながら、100Wの高圧水銀灯
の光を照射して20℃で約15分間反応をおこなつ
た。Example 3 (Preparation of colloidal fine particles) Glycidyl methacrylate, sulfopropyl methacrylate, and triethylene glycol dimethacrylate were mixed in a molar ratio of 85:10:5. The reaction was carried out in the presence of 0.01% sodium dodecyl sulfate and 0.01 mol/l of 4,4'-azobis-4-cyanpentanoic acid at a total monomer concentration of 10%. The pH of the reaction solution was adjusted to 7.2 using NaOH. While stirring vigorously to maintain an emulsified state, the reaction was carried out at 20°C for about 15 minutes by irradiating light from a 100W high-pressure mercury lamp.
反応後、CCl4により未反応のモノマーを抽出
し、除去した。生成ポリマーの収率は62％であつ
た。 After the reaction, unreacted monomers were extracted and removed with CCl 4 . The yield of the produced polymer was 62%.
30mg／mlの微粒子分散液と30mg／mlのジチオエ
リスリトール溶液とを等容混合し、50℃で一晩反
応させた。微粒子を25400G、30分の遠心分離、
再分散を繰り返すことで、0.5モル／水酸化ナ
トリウム水溶液で洗浄し、さらに水で洗浄した。
得られたメルカプト基含有重合体微粒子を10mg／
mlとなるように水に分散させ、N2雰囲気下に保
存した。この微粒子の直径を走査電子顕微鏡によ
り測定したところ0.17μｍであつた。 Equal volumes of a 30 mg/ml fine particle dispersion and a 30 mg/ml dithioerythritol solution were mixed and reacted overnight at 50°C. Centrifuge the particles at 25400G for 30 minutes,
By repeating redispersion, the mixture was washed with a 0.5 mol/aqueous sodium hydroxide solution and further washed with water.
10 mg of the obtained mercapto group-containing polymer fine particles
ml of water and stored under N2 atmosphere. The diameter of the fine particles was measured using a scanning electron microscope and was found to be 0.17 μm.
（コロイド微粒子へのBSAの固定化）
10mgのメルカプト基含有コロイド微粒子を１ml
のDMSOに分散させた。分散後に10mgのCHMを
加えて30℃で１時間反応させた。DMSOでコロ
イド微粒子を洗浄した後、10mgのCHM処理コロ
イド微粒子を１mgのBSAを含むPBS1mlに分散さ
せ、30℃で１時間反応させ、さらに10mgのヒト血
清アルブミン（HSAと略す）を加えて４℃で一
晩反応させた。PH8.0の0.1モル／トリス塩酸塩
緩衝液（Trisと略す）で粒子を洗浄し、１％の
HSAや含むTris緩衝液に分散させ、４℃で保存
した。 (Immobilization of BSA on colloidal particles) 1ml of 10mg of mercapto group-containing colloidal particles
of DMSO. After dispersion, 10 mg of CHM was added and reacted at 30°C for 1 hour. After washing the colloidal microparticles with DMSO, 10mg of CHM-treated colloidal microparticles were dispersed in 1ml of PBS containing 1mg of BSA, reacted for 1 hour at 30℃, and further added with 10mg of human serum albumin (abbreviated as HSA) and incubated at 4℃. The mixture was allowed to react overnight. The particles were washed with 0.1 M/Tris-HCl buffer (abbreviated as Tris) at pH 8.0 and 1%
It was dispersed in Tris buffer containing HSA and stored at 4°C.
（BSAの測定）
特願昭56−158193に記した方法でBSAの測定
をおこなつた。すなわち、実施例１に記した微粒
子の重合においてCCl4を溶媒に混合しないで、
同様の条件で重合をおこない、直径4μｍの粒子
を調製した。この粒子をアミノ化し、加水分解し
た。特願昭55−43618に記載の方法に準じてグル
タルアルデヒドで活性化し、PBSに濃度が１％
になるように分散させた。この分散液と抗BSA
抗血清（ウサギ）を等容混合し、30℃で３時間反
応させた後、HSAを粒子分散液中で１％となる
ように加え、さらに１時間反応を続けた。1500G
の遠心沈降と再分散を繰り返すことによる洗浄の
後、１％HSAを含むTrisに分散させ、抗BSA抗
血清固定化固相を調製した。BSAを1μｇ／ml、
100nｇ／ml、10nｇ／ml、1nｇ／ml含むTris100μ
の各々抗BSA抗血清固定化固相分散液100μ
を加え、２時間30℃で反応させた後に0.025％の
BSA結合コロイド微粒子分散液10μを加え、２
時間反応させた。反応後Tris6mlを加え1500G5分
の遠心で固相粒子とコロイド微粒子を分離し、上
清のコロイド微粒子をポイツク積分球式濁度計
（日本精密光学）により測定した。第１図に、
100nｇ／mlでの濁度を100％とした各BSA濃度で
の散乱光強度を示した。第１図からわかるよう
に、BSAは1nｇ／ml〜100nｇ／mlの範囲で測定
できた。 (Measurement of BSA) BSA was measured by the method described in Japanese Patent Application No. 56-158193. That is, in the polymerization of fine particles described in Example 1, CCl 4 was not mixed with the solvent,
Polymerization was carried out under similar conditions to prepare particles with a diameter of 4 μm. The particles were aminated and hydrolyzed. Activated with glutaraldehyde according to the method described in Japanese Patent Application No. 55-43618, and added to PBS at a concentration of 1%.
It was distributed so that This dispersion and anti-BSA
After equal volumes of antiserum (rabbit) were mixed and reacted at 30°C for 3 hours, HSA was added to the particle dispersion at a concentration of 1%, and the reaction was continued for an additional hour. 1500G
After washing by repeating centrifugal sedimentation and redispersion, the solid phase was dispersed in Tris containing 1% HSA to prepare an anti-BSA antiserum-immobilized solid phase. BSA 1 μg/ml,
Tris 100μ containing 100ng/ml, 10ng/ml, 1ng/ml
100μ of each anti-BSA antiserum immobilized solid phase dispersion
was added and reacted at 30℃ for 2 hours, then 0.025%
Add 10μ of BSA-bound colloidal fine particle dispersion and
Allowed time to react. After the reaction, 6 ml of Tris was added and centrifuged at 1500G for 5 minutes to separate solid phase particles and colloidal particles, and the colloidal particles in the supernatant were measured using a Poitzk integrating sphere turbidity meter (Nippon Seimitsu Optics). In Figure 1,
The scattered light intensity at each BSA concentration is shown with the turbidity at 100 ng/ml as 100%. As can be seen from FIG. 1, BSA could be measured in the range of 1 ng/ml to 100 ng/ml.
第１図は実施例３のBSA測定結果を示す線図
である。
FIG. 1 is a diagram showing the BSA measurement results of Example 3.
Claims (1)
シジルメタクリレートのくり返し単位を有する重
合体からなり、かつグリシジルアクリレートまた
はグリシジルメタクリレート単位以外に疎水性成
分を表面に持たない平均粒径0.03〜10μｍの微粒
子に、メルカプト基を導入させてなる免疫学的活
性物質固定化用担体微粒子。1. A mercapto group is introduced into fine particles having an average particle size of 0.03 to 10 μm, which are made of a polymer having repeating units of glycidyl acrylate and/or glycidyl methacrylate, and which have no hydrophobic component on the surface other than glycidyl acrylate or glycidyl methacrylate units. Fine carrier particles for immobilizing immunologically active substances.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12969182A JPS5919856A (en) | 1982-07-27 | 1982-07-27 | Fine carrier particle for immobilizing amynologically active material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12969182A JPS5919856A (en) | 1982-07-27 | 1982-07-27 | Fine carrier particle for immobilizing amynologically active material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5919856A JPS5919856A (en) | 1984-02-01 |
| JPH0376426B2 true JPH0376426B2 (en) | 1991-12-05 |
Family
ID=15015800
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12969182A Granted JPS5919856A (en) | 1982-07-27 | 1982-07-27 | Fine carrier particle for immobilizing amynologically active material |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5919856A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59204601A (en) * | 1983-05-09 | 1984-11-20 | Unitika Ltd | Manufacture of molded article having physiological activity |
| CN111213632B (en) * | 2019-11-19 | 2022-03-01 | 长春中医药大学 | Method for preparing animal medicine epoxy resin specimen |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55136261A (en) * | 1979-04-12 | 1980-10-23 | Toyo Jozo Co Ltd | Novel compound and kit comprising it |
| JPS55156865A (en) * | 1978-10-06 | 1980-12-06 | Toyo Jozo Co Ltd | Organism component measuring compound and its manufacture plus organism component measuring composition, organism component measuring method and organism component measuring kit |
| JPS5796261A (en) * | 1980-12-09 | 1982-06-15 | Toray Ind Inc | Preparation of immunoactive particle |
| JPS5796260A (en) * | 1980-12-09 | 1982-06-15 | Toray Ind Inc | Immunoactive particle |
-
1982
- 1982-07-27 JP JP12969182A patent/JPS5919856A/en active Granted
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55156865A (en) * | 1978-10-06 | 1980-12-06 | Toyo Jozo Co Ltd | Organism component measuring compound and its manufacture plus organism component measuring composition, organism component measuring method and organism component measuring kit |
| JPS55136261A (en) * | 1979-04-12 | 1980-10-23 | Toyo Jozo Co Ltd | Novel compound and kit comprising it |
| JPS5796261A (en) * | 1980-12-09 | 1982-06-15 | Toray Ind Inc | Preparation of immunoactive particle |
| JPS5796260A (en) * | 1980-12-09 | 1982-06-15 | Toray Ind Inc | Immunoactive particle |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5919856A (en) | 1984-02-01 |
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