JP5428166B2 - Aggregation and dispersion method of magnetic particles and separation, detection method and detection kit using the same - Google Patents
Aggregation and dispersion method of magnetic particles and separation, detection method and detection kit using the same Download PDFInfo
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- JP5428166B2 JP5428166B2 JP2008028641A JP2008028641A JP5428166B2 JP 5428166 B2 JP5428166 B2 JP 5428166B2 JP 2008028641 A JP2008028641 A JP 2008028641A JP 2008028641 A JP2008028641 A JP 2008028641A JP 5428166 B2 JP5428166 B2 JP 5428166B2
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- temperature
- sodium
- responsive polymer
- magnetic particles
- salt
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- 239000006249 magnetic particle Substances 0.000 title claims description 116
- 238000000034 method Methods 0.000 title claims description 72
- 238000001514 detection method Methods 0.000 title claims description 48
- 239000006185 dispersion Substances 0.000 title description 17
- 230000002776 aggregation Effects 0.000 title description 15
- 238000004220 aggregation Methods 0.000 title description 15
- 238000000926 separation method Methods 0.000 title description 8
- 229920000208 temperature-responsive polymer Polymers 0.000 claims description 94
- 239000003463 adsorbent Substances 0.000 claims description 62
- 150000003839 salts Chemical class 0.000 claims description 47
- 229920000642 polymer Polymers 0.000 claims description 45
- 239000007864 aqueous solution Substances 0.000 claims description 43
- 239000013076 target substance Substances 0.000 claims description 37
- -1 Nt-butylacrylamide Chemical compound 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 30
- 239000000178 monomer Substances 0.000 claims description 22
- 239000002245 particle Substances 0.000 claims description 20
- 159000000000 sodium salts Chemical class 0.000 claims description 14
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 10
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 10
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 10
- 235000011152 sodium sulphate Nutrition 0.000 claims description 10
- 230000000379 polymerizing effect Effects 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- 239000011734 sodium Substances 0.000 claims description 8
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 7
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 150000002762 monocarboxylic acid derivatives Chemical class 0.000 claims description 6
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims description 6
- 150000003627 tricarboxylic acid derivatives Chemical class 0.000 claims description 6
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 5
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 5
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 5
- 235000019262 disodium citrate Nutrition 0.000 claims description 5
- 239000002526 disodium citrate Substances 0.000 claims description 5
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 5
- MSJMDZAOKORVFC-UAIGNFCESA-L disodium maleate Chemical compound [Na+].[Na+].[O-]C(=O)\C=C/C([O-])=O MSJMDZAOKORVFC-UAIGNFCESA-L 0.000 claims description 5
- CEYULKASIQJZGP-UHFFFAOYSA-L disodium;2-(carboxymethyl)-2-hydroxybutanedioate Chemical compound [Na+].[Na+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O CEYULKASIQJZGP-UHFFFAOYSA-L 0.000 claims description 5
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 5
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 5
- 235000011181 potassium carbonates Nutrition 0.000 claims description 5
- 229940073490 sodium glutamate Drugs 0.000 claims description 5
- PRWXGRGLHYDWPS-UHFFFAOYSA-L sodium malonate Chemical compound [Na+].[Na+].[O-]C(=O)CC([O-])=O PRWXGRGLHYDWPS-UHFFFAOYSA-L 0.000 claims description 5
- ZNCPFRVNHGOPAG-UHFFFAOYSA-L sodium oxalate Chemical compound [Na+].[Na+].[O-]C(=O)C([O-])=O ZNCPFRVNHGOPAG-UHFFFAOYSA-L 0.000 claims description 5
- 229940039790 sodium oxalate Drugs 0.000 claims description 5
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 5
- WTWSHHITWMVLBX-DKWTVANSSA-M sodium;(2s)-2-aminobutanedioate;hydron Chemical compound [Na+].[O-]C(=O)[C@@H](N)CC(O)=O WTWSHHITWMVLBX-DKWTVANSSA-M 0.000 claims description 5
- 150000000000 tetracarboxylic acids Chemical class 0.000 claims description 5
- XLPJNCYCZORXHG-UHFFFAOYSA-N 1-morpholin-4-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCOCC1 XLPJNCYCZORXHG-UHFFFAOYSA-N 0.000 claims description 4
- WLPAQAXAZQUXBG-UHFFFAOYSA-N 1-pyrrolidin-1-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCCC1 WLPAQAXAZQUXBG-UHFFFAOYSA-N 0.000 claims description 4
- LVCMKNCJDCTPIB-UHFFFAOYSA-N 2-methyl-1-pyrrolidin-1-ylprop-2-en-1-one Chemical compound CC(=C)C(=O)N1CCCC1 LVCMKNCJDCTPIB-UHFFFAOYSA-N 0.000 claims description 4
- YQIGLEFUZMIVHU-UHFFFAOYSA-N 2-methyl-n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C(C)=C YQIGLEFUZMIVHU-UHFFFAOYSA-N 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical group [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- HAXVIVNBOQIMTE-UHFFFAOYSA-L disodium;2-(carboxylatomethylamino)acetate Chemical compound [Na+].[Na+].[O-]C(=O)CNCC([O-])=O HAXVIVNBOQIMTE-UHFFFAOYSA-L 0.000 claims description 4
- INHCSSUBVCNVSK-UHFFFAOYSA-L lithium sulfate Inorganic materials [Li+].[Li+].[O-]S([O-])(=O)=O INHCSSUBVCNVSK-UHFFFAOYSA-L 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- QRWZCJXEAOZAAW-UHFFFAOYSA-N n,n,2-trimethylprop-2-enamide Chemical compound CN(C)C(=O)C(C)=C QRWZCJXEAOZAAW-UHFFFAOYSA-N 0.000 claims description 4
- 229940088644 n,n-dimethylacrylamide Drugs 0.000 claims description 4
- YLGYACDQVQQZSW-UHFFFAOYSA-N n,n-dimethylprop-2-enamide Chemical compound CN(C)C(=O)C=C YLGYACDQVQQZSW-UHFFFAOYSA-N 0.000 claims description 4
- ZIWDVJPPVMGJGR-UHFFFAOYSA-N n-ethyl-2-methylprop-2-enamide Chemical compound CCNC(=O)C(C)=C ZIWDVJPPVMGJGR-UHFFFAOYSA-N 0.000 claims description 4
- SWPMNMYLORDLJE-UHFFFAOYSA-N n-ethylprop-2-enamide Chemical compound CCNC(=O)C=C SWPMNMYLORDLJE-UHFFFAOYSA-N 0.000 claims description 4
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 4
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 4
- 235000011151 potassium sulphates Nutrition 0.000 claims description 4
- 239000001632 sodium acetate Substances 0.000 claims description 4
- 235000017281 sodium acetate Nutrition 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- RBTVSNLYYIMMKS-UHFFFAOYSA-N tert-butyl 3-aminoazetidine-1-carboxylate;hydrochloride Chemical compound Cl.CC(C)(C)OC(=O)N1CC(N)C1 RBTVSNLYYIMMKS-UHFFFAOYSA-N 0.000 claims description 4
- RESPXSHDJQUNTN-UHFFFAOYSA-N 1-piperidin-1-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCCCC1 RESPXSHDJQUNTN-UHFFFAOYSA-N 0.000 claims description 3
- AKVUWTYSNLGBJY-UHFFFAOYSA-N 2-methyl-1-morpholin-4-ylprop-2-en-1-one Chemical compound CC(=C)C(=O)N1CCOCC1 AKVUWTYSNLGBJY-UHFFFAOYSA-N 0.000 claims description 3
- RASDUGQQSMMINZ-UHFFFAOYSA-N 2-methyl-1-piperidin-1-ylprop-2-en-1-one Chemical compound CC(=C)C(=O)N1CCCCC1 RASDUGQQSMMINZ-UHFFFAOYSA-N 0.000 claims description 3
- GVGGWUXGMRTNIK-UHFFFAOYSA-N n-(2-amino-2-oxoethyl)prop-2-enamide Chemical compound NC(=O)CNC(=O)C=C GVGGWUXGMRTNIK-UHFFFAOYSA-N 0.000 claims description 3
- 159000000001 potassium salts Chemical class 0.000 claims description 3
- WIEFOJWXOGVOKJ-BYPYZUCNSA-N (2s)-2-amino-n-prop-2-enoylbutanediamide Chemical compound NC(=O)C[C@H](N)C(=O)NC(=O)C=C WIEFOJWXOGVOKJ-BYPYZUCNSA-N 0.000 claims description 2
- IZBQAIGPFVPPJB-UHFFFAOYSA-N n-prop-2-enoylpiperidine-3-carboxamide Chemical compound C=CC(=O)NC(=O)C1CCCNC1 IZBQAIGPFVPPJB-UHFFFAOYSA-N 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims 1
- 235000002906 tartaric acid Nutrition 0.000 claims 1
- 239000011975 tartaric acid Substances 0.000 claims 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 30
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- 238000006243 chemical reaction Methods 0.000 description 13
- 239000003446 ligand Substances 0.000 description 12
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 230000004931 aggregating effect Effects 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 229920002307 Dextran Polymers 0.000 description 9
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- 102000002260 Alkaline Phosphatase Human genes 0.000 description 8
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- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 8
- 238000007885 magnetic separation Methods 0.000 description 7
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 7
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
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Description
本発明は、磁性粒子の凝集及び分散方法並びに該方法を用いた分離または検出方法及び検出用キットに関する。 The present invention relates to a method for aggregating and dispersing magnetic particles, a separation or detection method using the method, and a detection kit.
混合液（検体）中から検出対象物質（目的物質ともいう。）を回収する方法として、検出対象物質を特異的に吸着するリガンドが固定された微粒子を検体に添加し、検出対象物質を吸着した後、微粒子を回収し、検出対象物質を微粒子から分離、回収する方法が知られている。特に磁性粒子は磁石によって容易に回収されるという特徴から、検出対象物質を効率よく回収する手段として用いられている。磁性粒子の粒子径は、５００ｎｍより大きいと磁集しやすくなるが、磁性粒子表面のリガンドと検出対象物質との吸着の反応速度が充分でなく、逆に粒子径を２００ｎｍ以下に小さくすると、吸着の反応速度は大きくなるものの磁集しにくくなり、検出対象物質を回収することができなくなる。 As a method for recovering the detection target substance (also called target substance) from the mixed liquid (specimen), fine particles on which a ligand that specifically adsorbs the detection target substance is immobilized are added to the specimen, and the detection target substance is adsorbed. Thereafter, a method is known in which fine particles are collected and a detection target substance is separated and collected from the fine particles. In particular, magnetic particles are used as a means for efficiently recovering a detection target substance because it is easily recovered by a magnet. If the particle size of the magnetic particle is larger than 500 nm, it is easy to collect the magnetic particles. However, the adsorption reaction rate between the ligand on the surface of the magnetic particle and the detection target substance is not sufficient. However, it is difficult to collect magnetic substances, and the substance to be detected cannot be recovered.
非特許文献１及び非特許文献２には、下限臨界溶液温度（以下「ＬＣＳＴ」と記述することがある。）を有するポリイソプロピルアクリルアミドで、粒子径が１００〜２００ｎｍ程度の磁性粒子を表面修飾した刺激応答性磁性粒子（刺激応答性高分子表面修飾磁性粒子）が開示されている。 In Non-Patent Document 1 and Non-Patent Document 2, magnetic particles having a particle diameter of about 100 to 200 nm are surface-modified with polyisopropylacrylamide having a lower critical solution temperature (hereinafter sometimes referred to as “LCST”). Stimulus-responsive magnetic particles (stimulus-responsive polymer surface-modified magnetic particles) are disclosed.
該刺激応答性高分子表面修飾磁性粒子は、その粒子径が１００〜２００ｎｍ程度と微小であることから水によく分散するが、分散状態では磁集できない。しかしながら、該刺激応答性高分子表面修飾磁性粒子として、温度応答性高分子表面修飾磁性粒子の水溶液を加熱して、その温度をＬＣＳＴ以上とした場合には、該温度応答性高分子表面修飾磁性粒子が析出、凝集する。この凝集物は磁力で容易に回収できることから、該温度応答性高分子表面修飾磁性粒子に抗体や抗原を固定化した温度応答性高分子表面修飾磁性粒子（吸着剤）を用い、検体中の種々の生体分子や微生物の分離を行う試みがなされている。
しかしながら、このような方法で回収を行う場合、温度応答性高分子表面修飾磁性粒子を用いた吸着剤を含む検体を加熱によりＬＣＳＴ以上に昇温して凝集または磁集させるための加熱及び冷却装置が必要である。また、短時間で多種類の分析項目を測定する免疫診断装置等に吸着剤を適用する場合、加熱及び冷却作業が必要なため操作が煩雑である。また、分析及び測定時間をより短縮するため、吸着剤を一度凝集させた後の再分散をより速く行う方法も求められている。 However, in the case of performing the recovery by such a method, a heating and cooling device for heating a sample containing an adsorbent using temperature-responsive polymer surface-modified magnetic particles to a temperature higher than LCST by heating to cause aggregation or magnetic collection is necessary. In addition, when an adsorbent is applied to an immunodiagnosis apparatus or the like that measures many types of analysis items in a short time, the operation is complicated because heating and cooling operations are required. In addition, in order to further shorten the analysis and measurement time, there is also a demand for a method of performing redispersion after aggregating the adsorbent once.
そこで、本発明は、吸着剤と検体とを含む水溶液を加熱または冷却せず、一定温度で簡便に温度応答性高分子表面修飾磁性粒子を凝集させることができる方法、及びこの方法を適用した検体中の検出対象物質の分離方法、検出方法を提供することを課題のひとつとしてなされた。また、吸着剤を短時間で再分散させる方法を提供することを課題のひとつと
してなされた。
Therefore, the present invention provides a method capable of easily aggregating temperature-responsive polymer surface-modified magnetic particles at a constant temperature without heating or cooling an aqueous solution containing an adsorbent and the sample, and a sample to which this method is applied. One of the problems was to provide a method for separating and detecting a substance to be detected. Another object of the present invention is to provide a method for redispersing the adsorbent in a short time.
発明者らは、上記の課題を解決するために鋭意検討した。その結果、温度応答性高分子で表面修飾された磁性粒子を含む水溶液の塩濃度を変化させることにより、凝集及び分散させることができることを見出し、これをもとに本発明を完成させた。 The inventors diligently studied to solve the above problems. As a result, it was found that aggregation and dispersion can be achieved by changing the salt concentration of an aqueous solution containing magnetic particles whose surface is modified with a temperature-responsive polymer, and the present invention has been completed based on this.
すなわち、本発明の要旨は以下の通りである。
［１］検体中の検出対象物質を分離する方法であって、
温度応答性高分子で磁性粒子が表面修飾された平均粒径５０〜１０００ｎｍの温度応答性高分子表面修飾磁性粒子と、該温度応答性高分子表面修飾磁性粒子に結合した、該検出対象物質に対する親和性を有する物質とからなる吸着剤と検体とを混合した水溶液中で、該吸着剤に該検出対象物質を吸着させ、該水溶液中の塩濃度を変化させることより該吸着剤を凝集させる工程、及び、
該吸着剤を磁力により水溶液中から回収する工程、
を含むことを特徴とする方法。
［２］前記温度応答性高分子が、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−イソプロピルアクリルアミド、Ｎ−ｔ−ブチルアクリルアミド、Ｎ−エチルアクリルアミド、Ｎ，Ｎ−ジメチルアクリルアミド、Ｎ−アクリロイルピロリジン、Ｎ−アクリロイルピペリジン、Ｎ−アクリロイルモルホリン、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−エチルメタクリルアミド、Ｎ，Ｎ−ジメチルメタクリルアミド、Ｎ−メタクリロイルピロリジン、Ｎ−メタクリロイルピペリジン、及び、Ｎ−メタクリロイルモルホリンからなる群から選ばれる少なくとも１種のモノマーを重合して得られるポリマーであることを特徴とする［１］記載の方法。
［３］前記温度応答性高分子が、Ｎ−アクリロイルグリシンアミド、Ｎ−アクリロイルニペコタミド、及び、Ｎ−アクリロイルアスパラギンアミドからなる群から選ばれる少なくとも１種のモノマーを重合して得られるポリマーであることを特徴とする［１］記載の方法。
［４］前記温度応答性高分子表面修飾磁性粒子の平均粒径が５０〜２００ｎｍであることを特徴とする「１」〜［３］のいずれか記載の方法。
［５］前記塩が、硫酸リチウム、硫酸ナトリウム、硫酸カリウム、硫酸マグネシウム、硫酸アンモニウム、炭酸ナトリウム、及び、炭酸カリウムからなる群から選ばれる少なくとも１種を含むことを特徴とする［１］〜［４］のいずれか記載の方法。
［６］前記塩が、モノカルボン酸のナトリウム塩、モノカルボン酸のカリウム塩、ジカルボン酸のナトリウム塩、ジカルボン酸のカリウム塩、トリカルボン酸のナトリウム塩、トリカルボン酸のカリウム塩、テトラカルボン酸のナトリウム塩、及び、テトラカルボン酸のカリウム塩からなる群から選ばれる少なくとも１種を含むことを特徴とする［１］〜［４］のいずれか記載の方法。
［７］前記塩が、酢酸ナトリウム、アスパラギン酸ナトリウム、クエン酸二ナトリウム、エチレンジアミン４酢酸二ナトリウム、グルタミン酸ナトリウム、イミノ二酢酸ナトリウム、マレイン酸ナトリウム、マロン酸ナトリウム、シュウ酸ナトリウム、コハク酸二ナトリウム、及び、酒石酸ナトリウムからなる群から選ばれる少なくとも１種を含むことを特徴とする［６］記載の方法。
［８］温度応答性高分子で磁性粒子が表面修飾された温度応答性高分子表面修飾磁性粒子と、該温度応答性高分子表面修飾磁性粒子に結合した、検出対象物質に対する親和性を有する物質とからなる吸着剤と、塩の水溶液を含む、検体中の検出対象物質を分離または検出するためのキット。
［９］温度応答性高分子で磁性粒子が表面修飾された温度応答性高分子表面修飾磁性粒子を、水溶液中の塩濃度を変化させることにより、凝集または分散させることを特徴とする磁性粒子の凝集または分散方法。
［１０］前記温度応答性高分子表面修飾磁性粒子が、検出対象物質に対する親和性を有する物質を表面に有していることを特徴とする［９］記載の方法。
［１１］前記［１］〜［７］のいずれかに記載の方法によって検体中の検出対象物質を吸着剤に吸着させて分離する工程、及び、吸着剤に吸着した該検出対象物質を検出する工程、
を含むことを特徴とする検体中の検出対象物質を検出する方法。
That is, the gist of the present invention is as follows.
[1] A method for separating a detection target substance in a specimen,
A temperature-responsive polymer surface-modified magnetic particle having an average particle size of 50 to 1000 nm, the surface of which is modified with a temperature-responsive polymer, and the detection target substance bound to the temperature-responsive polymer surface-modified magnetic particle A step of causing the adsorbent to adsorb the substance to be detected in an aqueous solution in which an adsorbent comprising a substance having affinity and a sample are mixed, and aggregating the adsorbent by changing a salt concentration in the aqueous solution ,as well as,
Recovering the adsorbent from an aqueous solution by magnetic force;
A method comprising the steps of:
[2] The temperature-responsive polymer is Nn-propylacrylamide, N-isopropylacrylamide, Nt-butylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloyl. Piperidine, N-acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, and N-methacryloyl [1] The method according to [1], which is a polymer obtained by polymerizing at least one monomer selected from the group consisting of morpholine.
[3] The temperature-responsive polymer is a polymer obtained by polymerizing at least one monomer selected from the group consisting of N-acryloylglycinamide, N-acryloylnipecotamide, and N-acryloylasparaginamide. The method according to [1], which is characterized in that it exists.
[4] The method according to any one of [1] to [3], wherein the temperature-responsive polymer surface-modified magnetic particles have an average particle size of 50 to 200 nm.
[5] The salt includes at least one selected from the group consisting of lithium sulfate, sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate, sodium carbonate, and potassium carbonate [1] to [4] ] The method in any one of.
[6] Sodium salt of monocarboxylic acid, potassium salt of monocarboxylic acid, sodium salt of dicarboxylic acid, potassium salt of dicarboxylic acid, sodium salt of tricarboxylic acid, potassium salt of tricarboxylic acid, sodium tetracarboxylic acid The method according to any one of [1] to [4], comprising at least one selected from the group consisting of a salt and a potassium salt of tetracarboxylic acid.
[7] The salt is sodium acetate, sodium aspartate, disodium citrate, disodium ethylenediaminetetraacetate, sodium glutamate, sodium iminodiacetate, sodium maleate, sodium malonate, sodium oxalate, disodium succinate, And the method of [6] characterized by including at least 1 sort (s) chosen from the group which consists of sodium tartrate.
[8] Temperature-responsive polymer surface-modified magnetic particles whose magnetic particles are surface-modified with a temperature-responsive polymer, and a substance having affinity for a detection target substance bound to the temperature-responsive polymer surface-modified magnetic particles A kit for separating or detecting a detection target substance in a specimen, comprising an adsorbent comprising: and an aqueous salt solution.
[9] A magnetic particle characterized in that the temperature-responsive polymer surface-modified magnetic particles whose surface is modified with a temperature-responsive polymer are aggregated or dispersed by changing the salt concentration in the aqueous solution. Aggregation or dispersion method.
[10] The method according to [9], wherein the temperature-responsive polymer surface-modified magnetic particles have a substance having affinity for the substance to be detected on the surface.
[11] The step of adsorbing and separating the detection target substance in the specimen by the method according to any one of [1] to [7], and detecting the detection target substance adsorbed on the adsorbent Process,
A method for detecting a substance to be detected in a specimen, comprising:
本発明の凝集及び分散方法並びにこの方法を適用した検体中の検出対象物質の分離方法または検出方法を用いることで、吸着剤と検体とを含む水溶液を加熱及び冷却せず、一定温度で簡便に温度応答性高分子表面修飾磁性粒子及びこれを用いた吸着剤を凝集でき、この凝集した吸着剤を磁集することで、検出対象物質を分離または検出することが可能になる。特に、短時間で多種類の分析項目を測定する免疫診断装置等に吸着剤を適用する場合、有利である。また、検体中の検出対象物質の迅速な分離または検出が可能になる。 By using the aggregating and dispersing method of the present invention and the method for separating or detecting the detection target substance in the sample to which this method is applied, the aqueous solution containing the adsorbent and the sample is not heated and cooled, and can be easily and at a constant temperature. The temperature-responsive polymer surface-modified magnetic particles and the adsorbent using the same can be aggregated, and by collecting the aggregated adsorbent, it is possible to separate or detect the detection target substance. In particular, it is advantageous when the adsorbent is applied to an immunodiagnostic apparatus or the like that measures many types of analysis items in a short time. In addition, it is possible to quickly separate or detect the detection target substance in the specimen.
以下、本発明について詳細に説明する。
本発明は、温度応答性高分子表面修飾磁性粒子及び吸着剤を、水溶液中で塩濃度を変化させることにより凝集及び分散させる、温度応答性高分子表面修飾磁性粒子及び吸着剤の凝集及び分散方法である。
Hereinafter, the present invention will be described in detail.
The present invention relates to a method for aggregating and dispersing temperature-responsive polymer surface-modified magnetic particles and an adsorbent, wherein the temperature-responsive polymer surface-modified magnetic particles and the adsorbent are aggregated and dispersed by changing the salt concentration in an aqueous solution. It is.
（温度応答性高分子表面修飾磁性粒子）
本発明に用いられる温度応答性高分子表面修飾磁性粒子は、温度応答性高分子で磁性粒子が表面修飾された粒子である。表面修飾とは、温度応答性高分子が、磁性粒子の表面に直接または間接に化学的に固定されている状態や、磁性粒子の表面に直接または間接に絡まった状態をいう。ここで、間接とは、磁性粒子と温度応答性高分子とが、他の物質（例えば、デキストラン等の多価アルコール）を介して表面修飾されていることをいう。
(Temperature-responsive polymer surface-modified magnetic particles)
The temperature-responsive polymer surface-modified magnetic particles used in the present invention are particles whose surface is modified with a temperature-responsive polymer. The surface modification refers to a state in which the temperature-responsive polymer is chemically fixed directly or indirectly to the surface of the magnetic particle, or a state in which the temperature-responsive polymer is directly or indirectly entangled with the surface of the magnetic particle. Here, indirect means that the surface of the magnetic particles and the temperature-responsive polymer is modified through another substance (for example, a polyhydric alcohol such as dextran).
本発明に用いられる磁性粒子は、酸化鉄、またはフェライトからなる粒子でもよく、例えば多価アルコールとマグネタイトから製造した粒子のように、酸化鉄、フェライト、またはマグネタイトとその他の無機物、有機物とからなる粒子でもよい。温度応答性高分子は、酸化鉄、フェライト、またはマグネタイト等に固定（表面修飾）してもよく、また、例えば、磁性粒子の成分である多価アルコールまたは多価アルコール誘導体等に固定してもよい。 The magnetic particles used in the present invention may be particles composed of iron oxide or ferrite, and are composed of iron oxide, ferrite, or magnetite and other inorganic substances and organic substances such as particles produced from polyhydric alcohol and magnetite. Particles may be used. The temperature-responsive polymer may be fixed (surface modified) to iron oxide, ferrite, magnetite, or the like, or may be fixed to, for example, a polyhydric alcohol or a polyhydric alcohol derivative that is a component of magnetic particles. Good.
温度応答性高分子表面修飾磁性粒子の平均粒径は、通常５０〜１０００ｎｍであり、８０〜２００ｎｍであることが好ましい。 The average particle diameter of the temperature-responsive polymer surface-modified magnetic particles is usually 50 to 1000 nm, and preferably 80 to 200 nm.
（磁性粒子）
上記温度応答性高分子表面修飾磁性粒子に用いられる磁性粒子は、例えば、特表２００２−５１７０８５号公報等に開示された方法によって製造することができる。すなわち、鉄（II）化合物、または鉄（II）化合物及び金属（II）化合物を含有する水溶液を、磁性酸化物の形成のために必要な酸化状態下に置き、水溶液のｐＨを７以上に維持して、酸化鉄、またはフェライト磁性体ナノ粒子を形成する方法である。また、金属（II）化合物含有の水溶液と鉄（III）化合物含有の水溶液をアルカリ性条件下で混合することによっても製造することができる。
(Magnetic particles)
The magnetic particles used for the temperature-responsive polymer surface-modified magnetic particles can be produced, for example, by the method disclosed in JP-T-2002-517085. That is, an aqueous solution containing an iron (II) compound or an iron (II) compound and a metal (II) compound is placed in an oxidation state necessary for forming a magnetic oxide, and the pH of the aqueous solution is maintained at 7 or more. Thus, it is a method of forming iron oxide or ferrite magnetic nanoparticles. It can also be produced by mixing an aqueous solution containing a metal (II) compound and an aqueous solution containing an iron (III) compound under alkaline conditions.
あるいは、磁性粒子は、多価アルコールとマグネタイトから製造することもできる。この多価アルコールは、構成単位に水酸基を少なくとも２個有し、鉄イオンと結合可能なアルコール構造体であれば、特に制限なく使用することができる。例えば、デキストラン、
ポリビニルアルコール、マンニトール、ソルビトール、シクロデキストリンなどが挙げられる。例えば、特開２００５−０８２５３８号公報に、デキストランを用いた磁性粒子の製造方法が開示されており、この方法によって製造することもできる。また、グリシジルメタクリレート重合体のように、エポキシ基を有し、開環後多価アルコール構造体を形成する化合物も使用できる。
Alternatively, the magnetic particles can be produced from polyhydric alcohol and magnetite. The polyhydric alcohol can be used without particular limitation as long as it is an alcohol structure having at least two hydroxyl groups in the structural unit and capable of binding to iron ions. For example, dextran,
Polyvinyl alcohol, mannitol, sorbitol, cyclodextrin and the like can be mentioned. For example, JP 2005-082538 A discloses a method for producing magnetic particles using dextran, which can also be produced by this method. Moreover, the compound which has an epoxy group and forms a polyhydric alcohol structure after ring-opening like a glycidyl methacrylate polymer can also be used.
本発明に用いられる磁性粒子は、温度応答性高分子で表面修飾を行った後で良好な分散性を有するように、その平均粒径が、１０００ｎｍ未満であることが好ましい。特に、温度応答性高分子表面修飾磁性粒子表面の検出対象物質と親和性を有する物質と、検出対象物質との吸着の反応速度を高めるためには、平均粒径が２００ｎｍ未満であることが好ましい。温度応答性高分子表面修飾磁性粒子の磁集速度を速めるためには、磁性粒子は３０ｎｍ以上が好ましく、４０ｎｍ以上がより好ましい。 The magnetic particles used in the present invention preferably have an average particle diameter of less than 1000 nm so as to have good dispersibility after surface modification with a temperature-responsive polymer. In particular, the average particle size is preferably less than 200 nm in order to increase the reaction rate of adsorption between the substance having affinity for the surface of the temperature-responsive polymer surface modified magnetic particle and the substance to be detected. . In order to increase the magnetic collection speed of the temperature-responsive polymer surface-modified magnetic particles, the magnetic particles are preferably 30 nm or more, and more preferably 40 nm or more.
（温度応答性高分子）
本発明に用いられる温度応答性高分子は、温度変化に応答して構造変化を起こし、凝集と分散が調整できる高分子である。温度応答性高分子としては、上限臨界溶液温度（以下「ＵＣＳＴ」と記述することがある。）を有する高分子と、下限臨界溶液温度（以下「ＬＣＳＴ」と記述することがある。）を有する高分子が存在するが、操作性などの点からＬＣＳＴを有する高分子がより好ましく利用できる。
(Temperature responsive polymer)
The temperature-responsive polymer used in the present invention is a polymer that undergoes a structural change in response to a temperature change and can adjust aggregation and dispersion. The temperature-responsive polymer has a polymer having an upper critical solution temperature (hereinafter sometimes referred to as “UCST”) and a lower critical solution temperature (hereinafter sometimes referred to as “LCST”). Although a polymer exists, a polymer having LCST is more preferably used from the viewpoint of operability.
ＬＣＳＴを有する高分子としては、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−イソプロピルアクリルアミド、Ｎ−ｔ−ブチルアクリルアミド、Ｎ−エチルアクリルアミド、Ｎ，Ｎ−ジメチルアクリルアミド、Ｎ−アクリロイルピロリジン、Ｎ−アクリロイルピペリジン、Ｎ−アクリロイルモルホリン、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−エチルメタクリルアミド、Ｎ，Ｎ−ジメチルメタクリルアミド、Ｎ−メタクリロイルピロリジン、Ｎ−メタクリロイルピペリジン、Ｎ−メタクリロイルモルホリン等のＮ置換（メタ）アクリルアミド誘導体から選ばれる少なくとも１種のモノマーを重合して得られるポリマー；ヒドロキシプロピルセルロース、ポリビニルアルコール部分酢化物、ポリビニルメチルエーテル、（ポリオキシエチレン−ポリオキシプロピレン）ブロックコポリマー、ポリオキシエチレンラウリルアミン等のポリオキシエチレンアルキルアミン誘導体；ポリオキシエチレンソルビタンラウレート等のポリオキシエチレンソルビタンエステル誘導体；（ポリオキシエチレンノニルフェニルエーテル）アクリレート、（ポリオキシエチレンオクチルフェニルエーテル）メタクリレート等の（ポリオキシエチレンアルキルフェニルエーテル）（メタ）アクリレート類；及び（ポリオキシエチレンラウリルエーテル）アクリレート、（ポリオキシエチレンオレイルエーテル）メタクリレート等の（ポリオキシエチレンアルキルエーテル）（メタ）アクリレート類等のポリオキシエチレン（メタ）アクリル酸エステル誘導体等（以下「ＬＣＳＴ型ポリマー」と記述する。）を挙げることができる。 Examples of polymers having LCST include Nn-propylacrylamide, N-isopropylacrylamide, Nt-butylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N N substitution such as acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Polymer obtained by polymerizing at least one monomer selected from (meth) acrylamide derivatives; hydroxypropylcellulose, polyvinyl alcohol partially acetylated product, polyvinylmethyl Ether, (polyoxyethylene-polyoxypropylene) block copolymer, polyoxyethylene alkylamine derivatives such as polyoxyethylene laurylamine; polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene nonylphenyl ether) ) (Polyoxyethylene alkylphenyl ether) (meth) acrylates such as acrylate, (polyoxyethylene octyl phenyl ether) methacrylate; and (polyoxyethylene lauryl ether) acrylate, (polyoxyethylene oleyl ether) methacrylate (poly) Polyoxyethylene (meth) acrylic acid ester derivatives such as oxyethylene alkyl ether) (meth) acrylates (hereinafter referred to as “L ST-type polymer "and describe.) Can be mentioned.
温度応答性高分子として、上記ＬＣＳＴ型ポリマーを用いることができる。また、上記ＬＣＳＴ型ポリマーを構成するモノマーに、さらにアクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、Ｎ−ビオチニル−Ｎ′−メタクリロイルトリメチレンアミド（ビオチン以外の物質を結合させてモノマーとすることも可能である）、アクリロイルザルコシンアミド、メタクリルザルコシンアミド、アクリロイルメチルウラシル、またはアクリロイルグルタミンアミド等のモノマーを加えて共重合して得られるポリマーも利用できる。通常、該ポリマー中、ＬＣＳＴ型ポリマーを構成するモノマー含有量は、該ポリマーを構成する全モノマー含有量の９０ｍｏｌ％以上である。 The LCST type polymer can be used as the temperature-responsive polymer. Furthermore, acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyltrimethylene amide (a substance other than biotin can also be combined with the monomer constituting the LCST polymer to form a monomer. And a polymer obtained by copolymerization by adding a monomer such as acryloyl sarcosine amide, methacryl sarcosine amide, acryloyl methyl uracil, or acryloyl glutamine amide. Usually, the monomer content constituting the LCST type polymer in the polymer is 90 mol% or more of the total monomer content constituting the polymer.
なかでも、温度応答性高分子として、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−イソプロピルアクリルアミド、Ｎ−エチルアクリルアミド、Ｎ，Ｎ−ジメチルアクリルアミド、Ｎ
−アクリロイルピロリジン、Ｎ−アクリロイルピペリジン、Ｎ−アクリロイルモルホリン、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−エチルメタクリルアミド、Ｎ，Ｎ−ジメチルメタクリルアミド、Ｎ−メタクリロイルピロリジン、Ｎ−メタクリロイルピペリジン、Ｎ−メタクリロイルモルホリンからなる群から選ばれる少なくとも１種のモノマーを重合して得られるポリマーがより好ましく利用できる。
Of these, Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N
-Acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloyl A polymer obtained by polymerizing at least one monomer selected from the group consisting of piperidine and N-methacryloylmorpholine is more preferably used.
本発明では、Ｎ−イソプロピルアクリルアミドを重合して得られるポリマーがさらに好ましく利用できる。 In the present invention, a polymer obtained by polymerizing N-isopropylacrylamide can be used more preferably.
ＵＣＳＴを有する高分子としては、アクリロイルグリシンアミド、アクリロイルニペコタミド、及びアクリロイルアスパラギンアミドからなる群から選ばれる少なくとも１種のモノマーを重合して得られるホモポリマーまたはコポリマー等（以下「ＵＣＳＴ型ポリマー」と記述する。）を挙げることができる。 Examples of the polymer having UCST include a homopolymer or a copolymer obtained by polymerizing at least one monomer selected from the group consisting of acryloylglycinamide, acryloylnipecotamide, and acryloylasparaginamide (hereinafter referred to as “UCST type polymer”). Can be described).
温度応答性高分子として、上記ＵＣＳＴ型ポリマーを用いることができる。また、上記ＵＣＳＴ型ポリマー中に、さらにアクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、Ｎ−ビオチニル−Ｎ′−メタクリロイルトリメチレンアミド（ビオチン以外の物質を結合させてモノマーとすることも可能である）、アクリロイルザルコシンアミド、メタクリルザルコシンアミド、アクリロイルメチルウラシル、及びアクリロイルグルタミンアミド等を重合して得られたポリマーを利用できる。通常、該ポリマー中、ＵＣＳＴ型ポリマーを構成するモノマー含有量は、該ポリマーを構成する全モノマー含有量の９０ｍｏｌ％以上である。 As the temperature-responsive polymer, the UCST polymer can be used. In addition, acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide (a substance other than biotin can be bonded to the UCST type polymer), A polymer obtained by polymerizing acryloyl sarcosine amide, methacryl sarcosine amide, acryloylmethyl uracil, acryloyl glutamine amide and the like can be used. Usually, in the polymer, the monomer content constituting the UCST type polymer is 90 mol% or more of the total monomer content constituting the polymer.
ＬＣＳＴ型ポリマー、ＵＣＳＴ型ポリマーともに、重合または共重合するモノマーの種類、割合を変えることでＬＣＳＴまたはＵＣＳＴを制御できるため、使用する温度に合わせたポリマー設計が可能である。 Since both LCST type polymer and UCST type polymer can control LCST or UCST by changing the kind and ratio of the monomers to be polymerized or copolymerized, it is possible to design a polymer in accordance with the temperature to be used.
本発明に好適に用いることのできる温度応答性高分子の重合度は、通常５０〜１００００である。 The degree of polymerization of the temperature-responsive polymer that can be suitably used in the present invention is usually 50 to 10,000.
温度応答性高分子の製造方法としては、上記モノマーを有機溶媒または水に溶解し、不活性ガスで系中を置換した後、重合温度まで昇温し、有機溶媒中であればアゾビスイソブチロニトリル等のアゾ系開始剤、過酸化ベンゾイル等の過酸化物、水系であれば過硫酸アンモニウム、過硫酸カリウム、２，２’−アゾビス（２−アミジノプロパン）二塩酸塩、４，４’−アゾビス（４−シアノ吉草酸）等の重合開始剤を添加し、攪拌下加熱を続けることにより得ることができる。その後、貧溶媒中で再沈殿を行い、析出したポリマーをろ取したり、ポリマーを凝集させる温度変化刺激を与えて凝集させ、遠心によりポリマーを分離する等の手法で、製造したポリマーを精製することもできる。 As a method for producing a temperature-responsive polymer, the above monomer is dissolved in an organic solvent or water, the inside of the system is replaced with an inert gas, and then the temperature is raised to the polymerization temperature. Azo initiators such as ronitrile, peroxides such as benzoyl peroxide, ammonium persulfate, potassium persulfate, 2,2'-azobis (2-amidinopropane) dihydrochloride, 4,4'- It can be obtained by adding a polymerization initiator such as azobis (4-cyanovaleric acid) and continuing heating with stirring. After that, reprecipitation is performed in a poor solvent, and the produced polymer is purified by techniques such as filtering the precipitated polymer, aggregating by stimulating the temperature change that causes the polymer to aggregate, and separating the polymer by centrifugation. You can also.
磁性粒子と温度応答性高分子との結合は、反応性の官能基を介して結合する方法や、磁性粒子中の多価アルコール上の活性水素または多価アルコールに重合性不飽和結合を導入し、磁性粒子にグラフト重合する方法等の当技術分野で周知の方法で得られる（例えば、ADV.Polym.Sci.,Vol.4、p111、1965やJ.Polymer Sci.,Part-A,3,p1031,1965に記載されている。）。このようにして温度応答性高分子で表面修飾された磁性粒子を得ることができる。温度応答性高分子としてＬＣＳＴ型ポリマーを用い、これにより表面修飾された温度応答性高分子表面修飾磁性粒子を「ＬＣＳＴ型磁性粒子」ということがある。また、温度応答性高分子としてＵＣＳＴ型ポリマーを用い、これにより表面修飾された温度応答性高分子表面修飾磁性粒子を「ＵＣＳＴ型磁性粒子」ということがある。 The bonding between the magnetic particles and the temperature-responsive polymer can be achieved by a method of bonding via a reactive functional group, or by introducing a polymerizable unsaturated bond into the active hydrogen or polyhydric alcohol on the polyhydric alcohol in the magnetic particle. , Obtained by a method known in the art such as a method of graft polymerization to magnetic particles (for example, ADV.Polym.Sci., Vol.4, p111, 1965 and J.Polymer Sci., Part-A, 3, p1031,1965). In this way, magnetic particles whose surface is modified with a temperature-responsive polymer can be obtained. An LCST type polymer is used as the temperature responsive polymer, and the temperature-modified polymer surface-modified magnetic particles surface-modified by this are sometimes referred to as “LCST type magnetic particles”. In addition, a temperature-responsive polymer surface-modified magnetic particle whose surface is modified by using a UCST-type polymer as the temperature-responsive polymer is sometimes referred to as “UCST-type magnetic particle”.
磁性粒子表面に修飾される温度応答性高分子層の厚みは、１〜１００ｎｍであることが
好ましく、５〜５０ｎｍであることがより好ましい。
The thickness of the temperature-responsive polymer layer modified on the surface of the magnetic particles is preferably 1 to 100 nm, and more preferably 5 to 50 nm.
（吸着剤）
本発明に用いられる吸着剤は、温度応答性高分子表面修飾磁性粒子に、検出対象物質に対する親和性を有する物質（リガンド）が結合した粒子である。
(Adsorbent)
The adsorbent used in the present invention is a particle in which a substance (ligand) having affinity for a detection target substance is bound to a temperature-responsive polymer surface-modified magnetic particle.
本発明では、検出対象物質に対する親和性を有する物質を温度応答性高分子表面修飾磁性粒子に固定することで、その物質と相互的に特異的吸着作用を有する検出対象物質を特異的に吸着できる。検出対象物質がタンパク質の場合には、検出対象物質に対する親和性を有する物質として、ビオチン、アビジン、グルタチオン、レクチン及び抗体等を温度応答性高分子表面修飾磁性粒子に固定することで、これらに対する特異的吸着作用を有するタンパク質を特異的に吸着できる。ビオチンの場合は、アビジンとの特異的な結合を介してビオチン化された検出対象タンパク質、またビオチン化された抗体を用いてそれらの抗原である種々のタンパク質を更に吸着することが可能である。本発明では、市販されているアビジン、ビオチン化タンパク質が利用でき、ビオチン化は、当技術分野で周知の方法に従えばよい。グルタチオンの場合は、グルタチオン−Ｓ−トランスフェラーゼ（以下、「ＧＳＴ」という。）を含有するタンパク質を特異的に吸着できる。このようなＧＳＴ含有タンパク質の調製は当技術分野で周知の方法に従えばよい。 In the present invention, by fixing a substance having affinity for a detection target substance to the temperature-responsive polymer surface-modified magnetic particles, it is possible to specifically adsorb the detection target substance having a specific adsorption action mutually with the substance. . When the detection target substance is protein, biotin, avidin, glutathione, lectin, antibody, etc. are immobilized on temperature-responsive polymer surface-modified magnetic particles as substances having affinity for the detection target substance. It is possible to specifically adsorb proteins having a specific adsorption action. In the case of biotin, it is possible to further adsorb various proteins as antigens using biotinylated proteins to be detected through specific binding to avidin and biotinylated antibodies. In the present invention, commercially available avidin and biotinylated protein can be used, and biotinylation may be performed according to a method well known in the art. In the case of glutathione, a protein containing glutathione-S-transferase (hereinafter referred to as “GST”) can be specifically adsorbed. Such a GST-containing protein may be prepared by methods well known in the art.
温度応答性高分子とリガンドとの結合の例として、温度応答性高分子と抗体の結合方法を示す。国際公開第０１／００９１４１号パンフレットに記載されているように、ビオチンをメタクリルやアクリル等の重合性の官能基と結合させて付加重合性モノマーとし、他のモノマーと共重合することにより温度応答性高分子にビオチンを結合させることができる。一方、リガンドとしての抗体にアビジンを結合させ、ビオチン結合温度応答性高分子と混合することにより、アビジンとビオチンの結合を利用して、温度応答性高分子に抗体を吸着させることができる。なお、ビオチンの代わりにグルタチオンを用いた場合は、アビジンではなく、グルタチオンＳトランスフェラーゼを用いればよい。また、ポリマーの製造時にカルボキシル、アミノまたはエポキシ等の官能基を持つモノマーを他のモノマーと共重合させ、当技術分野で周知の方法に従い、この官能基を介して、抗体親和性物質（例えば、メロンゲル、プロテインＡ、プロテインＧなど）をポリマー上に結合させる方法も利用できる。このようにして得られた抗体親和性物質にリガンドとしての抗体を結合させることにより、温度応答性高分子に抗体を結合させることができる。 As an example of the binding between the temperature-responsive polymer and the ligand, a method for binding the temperature-responsive polymer and the antibody will be described. As described in WO 01/009141 pamphlet, biotin is combined with a polymerizable functional group such as methacryl or acryl to form an addition polymerizable monomer, and copolymerized with other monomers to react with temperature. Biotin can be bound to the polymer. On the other hand, by binding avidin to an antibody as a ligand and mixing it with a biotin-binding temperature-responsive polymer, the antibody can be adsorbed to the temperature-responsive polymer using the bond between avidin and biotin. When glutathione is used instead of biotin, glutathione S transferase may be used instead of avidin. In addition, a monomer having a functional group such as carboxyl, amino, or epoxy is copolymerized with another monomer during the production of the polymer, and an antibody affinity substance (for example, Melon gel, protein A, protein G, etc.) can also be used on the polymer. By binding an antibody as a ligand to the antibody affinity substance thus obtained, the antibody can be bound to the temperature-responsive polymer.
このようにして、リガンドと結合した温度応答性高分子を得ることができる。リガンドと結合した温度応答性高分子は、温度応答性高分子が凝集する条件にし、遠心分離等により分離精製することができる。また、温度応答性高分子が磁性粒子表面に固定化されている場合には、磁石で磁性粒子を回収することにより、精製することができる。 In this way, a temperature-responsive polymer bonded with a ligand can be obtained. The temperature-responsive polymer bound to the ligand can be separated and purified by centrifugation or the like under conditions where the temperature-responsive polymer aggregates. In addition, when the temperature-responsive polymer is immobilized on the surface of the magnetic particle, it can be purified by collecting the magnetic particle with a magnet.
（磁集方法）
温度応答性高分子表面修飾磁性粒子及び吸着剤の回収に用いる磁石等の磁力は、用いる磁性粒子の有する磁力の大きさ等によって異なる。磁力は、目的の磁性粒子を磁集可能な程度の磁力を適宜使用できる。磁石の素材には、例えばマグナ社製ネオジ磁石が利用できる。このように本発明では、磁石等の磁力によって、温度応答性高分子表面修飾磁性粒子及び吸着剤等を回収するが、磁性粒子の表面に温度応答性高分子が固定されていることで、分散状態では回収困難なナノサイズの磁性粒子を意図的に凝集させて、回収率を高めることが可能になる。なお、本発明は、このような温度応答性高分子表面修飾磁性粒子及び吸着剤等を、水溶液中の塩濃度を変化させることにより凝集または分散させることが可能である。したがって、本発明では、吸着剤と検体とを含有する水溶液を加熱または冷却せず、一定温度で簡便に吸着剤を凝集及び磁集させ、検体中の検出対象物質を分離または検出することが可能である。
(Magnetic collection method)
The magnetic force of the magnets used for recovering the temperature-responsive polymer surface-modified magnetic particles and the adsorbent varies depending on the magnitude of the magnetic force of the magnetic particles used. As the magnetic force, a magnetic force that can collect magnetic particles of interest can be used as appropriate. For example, a neodymium magnet manufactured by Magna can be used as the magnet material. As described above, in the present invention, the temperature-responsive polymer surface-modified magnetic particles, the adsorbent, and the like are recovered by the magnetic force of a magnet or the like, but the dispersion is achieved by fixing the temperature-responsive polymer on the surface of the magnetic particles. In this state, it is possible to intentionally agglomerate nano-sized magnetic particles that are difficult to recover, thereby increasing the recovery rate. In the present invention, such temperature-responsive polymer surface-modified magnetic particles and adsorbents can be aggregated or dispersed by changing the salt concentration in the aqueous solution. Therefore, in the present invention, it is possible to easily separate and detect the detection target substance in the sample by aggregating and collecting the adsorbent at a constant temperature without heating or cooling the aqueous solution containing the adsorbent and the sample. It is.
（本発明の凝集及び分散方法）
本発明は、温度応答性高分子表面修飾磁性粒子及び吸着剤等を、水溶液中の塩濃度を変化させることにより凝集及び分散させることが可能である。
(Aggregation and dispersion method of the present invention)
In the present invention, temperature-responsive polymer surface-modified magnetic particles, adsorbents, and the like can be aggregated and dispersed by changing the salt concentration in the aqueous solution.
水溶液（塩添加後）中の温度応答性高分子表面修飾磁性粒子及び吸着剤の好ましい濃度は、検出対象物質、温度応答性高分子表面修飾磁性粒子の種類等によって異なるが、操作性などの点から、通常０．１〜１０ｍｇ／ｍＬである。 The preferred concentration of the temperature-responsive polymer surface-modified magnetic particles and adsorbent in the aqueous solution (after the addition of salt) varies depending on the substance to be detected, the temperature-responsive polymer surface-modified magnetic particles, etc. From 0.1 to 10 mg / mL.
本発明で用いられる塩としては、本発明の効果を発揮するものであれば特に限定されないが、硫酸リチウム、硫酸ナトリウム、硫酸カリウム、硫酸マグネシウム、硫酸アンモニウム等の硫酸塩；塩化ナトリウム、塩化カリウム、塩化マグネシウム、塩化カルシウム、塩化バリウム、等のハロゲン化物；硝酸マグネシウム、硝酸カルシウム等の硝酸塩；チオシアン化カリウム等のチオシアン酸塩；炭酸ナトリウム、炭酸カリウム等の炭酸塩；ホウ酸塩；リン酸塩等が挙げられる。これらの塩を単独で、または２種以上を組み合わせて用いることができる。
また、本発明で用いられる塩としては、酢酸ナトリウム等のモノカルボン酸のナトリウム塩、アスパラギン酸ナトリウム、グルタミン酸ナトリウム、イミノ二酢酸ナトリウム、マレイン酸ナトリウム、マロン酸ナトリウム、シュウ酸ナトリウム、コハク酸二ナトリウム、または、酒石酸ナトリウム等のジカルボン酸のナトリウム塩、クエン酸二ナトリウム等のトリカルボン酸のナトリウム塩、エチレンジアミン４酢酸二ナトリウム等のテトラカルボン酸のナトリウム塩等の有機酸塩等が挙げられ、これらのカリウム塩等の有機酸塩等も利用できる。これらの塩を単独で、または２種以上を組み合わせて用いることができる。
The salt used in the present invention is not particularly limited as long as it exhibits the effects of the present invention, but sulfates such as lithium sulfate, sodium sulfate, potassium sulfate, magnesium sulfate, and ammonium sulfate; sodium chloride, potassium chloride, chloride Halides such as magnesium, calcium chloride and barium chloride; nitrates such as magnesium nitrate and calcium nitrate; thiocyanates such as potassium thiocyanide; carbonates such as sodium carbonate and potassium carbonate; borate salts; phosphates and the like It is done. These salts can be used alone or in combination of two or more.
Examples of the salt used in the present invention include sodium salt of monocarboxylic acid such as sodium acetate, sodium aspartate, sodium glutamate, sodium iminodiacetate, sodium maleate, sodium malonate, sodium oxalate, disodium succinate Or organic acid salts such as sodium salt of dicarboxylic acid such as sodium tartrate, sodium salt of tricarboxylic acid such as disodium citrate, sodium salt of tetracarboxylic acid such as disodium ethylenediaminetetraacetate, etc. Organic acid salts such as potassium salts can also be used. These salts can be used alone or in combination of two or more.
なかでも、少量の添加で温度応答性高分子表面修飾磁性粒子または吸着剤を凝集させることができる点で、アスパラギン酸ナトリウム、クエン酸二ナトリウム、エチレンジアミン４酢酸二ナトリウム、グルタミン酸ナトリウム、イミノ二酢酸ナトリウム、マレイン酸ナトリウム、マロン酸ナトリウム、シュウ酸ナトリウム、コハク酸二ナトリウム、酒石酸ナトリウム等の有機酸塩；硫酸リチウム、硫酸ナトリウム、硫酸カリウム、硫酸マグネシウム、硫酸アンモニウム等の硫酸塩；炭酸ナトリウム、炭酸カリウム等の炭酸塩が好ましい。
また、凝集後の吸着剤を再分散しやすいという点で、酢酸ナトリウム等のモノカルボン酸のナトリウム塩、アスパラギン酸ナトリウム、グルタミン酸ナトリウム、イミノ二酢酸ナトリウム、マレイン酸ナトリウム、マロン酸ナトリウム、シュウ酸ナトリウム、コハク酸二ナトリウム、または、酒石酸ナトリウム等のジカルボン酸のナトリウム塩、クエン酸二ナトリウム等のトリカルボン酸のナトリウム塩、エチレンジアミン４酢酸二ナトリウム等のテトラカルボン酸のナトリウム塩等の有機酸塩が好ましく、これらのカリウム塩も好ましい。
Among them, the temperature-responsive polymer surface-modified magnetic particles or the adsorbent can be aggregated by adding a small amount, so that sodium aspartate, disodium citrate, disodium ethylenediaminetetraacetate, sodium glutamate, sodium iminodiacetate Organic acid salts such as sodium maleate, sodium malonate, sodium oxalate, disodium succinate, sodium tartrate; sulfates such as lithium sulfate, sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate; sodium carbonate, potassium carbonate, etc. The carbonate of is preferred.
In addition, it is easy to re-disperse the adsorbent after aggregation. Sodium salt of monocarboxylic acid such as sodium acetate, sodium aspartate, sodium glutamate, sodium iminodiacetate, sodium maleate, sodium malonate, sodium oxalate Organic salts such as disodium succinate or sodium salt of dicarboxylic acid such as sodium tartrate, sodium salt of tricarboxylic acid such as disodium citrate, sodium salt of tetracarboxylic acid such as disodium ethylenediaminetetraacetate These potassium salts are also preferred.
温度応答性高分子表面修飾磁性粒子または吸着剤を凝集させるためには、例えば、所望の塩濃度となるように温度応答性高分子表面修飾磁性粒子または吸着剤の分散液中に塩の水溶液を添加すればよい。 In order to aggregate the temperature-responsive polymer surface-modified magnetic particles or the adsorbent, for example, an aqueous salt solution is added to the dispersion of the temperature-responsive polymer surface-modified magnetic particles or the adsorbent so as to obtain a desired salt concentration. What is necessary is just to add.
温度応答性高分子表面修飾磁性粒子及び吸着剤を凝集させるための塩の必要添加量は、塩の種類、水溶液の温度、温度応答性高分子の種類、温度応答性高分子表面修飾磁性粒子または吸着剤濃度等にもよるが、水溶液中終濃度で概ね５０ｍＭ〜５Ｍ、好ましくは１００〜１０００ｍＭの範囲である。 The required addition amount of the salt for aggregating the temperature-responsive polymer surface-modified magnetic particles and the adsorbent is the type of salt, the temperature of the aqueous solution, the type of temperature-responsive polymer, the temperature-responsive polymer surface-modified magnetic particles or Although depending on the adsorbent concentration and the like, the final concentration in the aqueous solution is generally in the range of 50 mM to 5 M, preferably 100 to 1000 mM.
例えば、４ｍｇ／ｍｌの温度応答性高分子表面修飾磁性粒子または吸着剤を含む水溶液
は、１／３容量の１Ｍ硫酸ナトリウムや炭酸カリウム等の塩の水溶液を添加することで、温度応答性高分子表面修飾磁性粒子または吸着剤の濃度３ｍｇ／ｍｌ、塩濃度２５０ｍＭの条件下で、容易に凝集状態にすることができる。これらの塩の水溶液は酸やアルカリ等により中和して用いてもよく、緩衝液等に溶解して用いてもよい。
For example, an aqueous solution containing 4 mg / ml temperature-responsive polymer surface-modified magnetic particles or adsorbent is added to an aqueous solution of 1/3 volume of a salt such as 1M sodium sulfate or potassium carbonate. It can be easily agglomerated under the conditions of surface modified magnetic particles or adsorbent at a concentration of 3 mg / ml and a salt concentration of 250 mM. An aqueous solution of these salts may be used after being neutralized with an acid, an alkali, or the like, or may be used after being dissolved in a buffer solution or the like.
一方、凝集させた温度応答性高分子表面修飾磁性粒子及び吸着剤を再び分散させるには、分散前の塩濃度になるように所望の濃度の塩の水溶液を添加するか、精製水などで塩濃度を希釈すればよい。 On the other hand, in order to disperse the aggregated temperature-responsive polymer surface-modified magnetic particles and the adsorbent again, an aqueous solution of a salt having a desired concentration is added so that the salt concentration before dispersion is obtained, or the salt is added with purified water or the like. What is necessary is just to dilute a density | concentration.
（本発明の分離方法、検出方法）
本発明では、分離とは、検出対象物質を分けて取り出すことをいう。
本発明の検体中の検出対象物質を分離する方法は、（１）吸着剤に検出対象物質を吸着させた後、検体中の塩濃度を変化させることにより吸着剤を凝集させる工程、及び（２）該吸着剤を磁力により回収する工程、を含むことを特徴とする方法である。
本発明の検体中の検出対象物質を検出する方法は、（１）、（２）の工程の後に、さらに（３）該吸着剤に吸着した該検出対象物質を検出する工程、を含むことを特徴とする方法である。
(Separation method and detection method of the present invention)
In the present invention, the separation means that the detection target substance is separated and taken out.
The method for separating a detection target substance in a specimen of the present invention includes (1) a step of aggregating the adsorbent by changing the salt concentration in the specimen after adsorbing the detection target substance on the adsorbent, and (2 And a step of recovering the adsorbent by magnetic force.
The method for detecting a substance to be detected in a sample of the present invention further comprises (3) a step of detecting the substance to be detected adsorbed on the adsorbent after the steps (1) and (2). It is a characteristic method.
以下に、検出対象物質として抗原を、蛍光色素を用いたサンドイッチ法により検出及び測定する例を示す。
（ａ）検出及び測定しようとする抗原に対する抗体ａを結合させた温度応答性高分子表面修飾磁性粒子（吸着剤）を含む試薬Ａと、検出対象物質である抗原を含む検体を混合し、反応容器中で反応させる。
（ｂ）反応液に吸着剤を凝集させることができる濃度になるように、高濃度の塩の水溶液（試薬Ｂ）を添加、混合し、吸着剤を凝集させる。
（ｃ）該吸着剤を磁石により反応容器壁に磁集し、検体中の不要成分を含む液体部分を除去し、磁石を外して吸着剤が分散する濃度になるようにバッファー（試薬Ｃ）を加え、吸着剤を再分散する。同様な操作を繰り返し、吸着剤を洗浄する。
（ｄ）検出及び測定しようとする抗原に対して、前記抗体ａとは違う部位を認識する抗体ｂを結合させた蛍光色素の水溶液（試薬Ｄ）を混合し、反応容器中で反応させる。
（ｅ）反応液に吸着剤を凝集させることができる濃度になるように、高濃度の塩の水溶液（前記試薬Ｂ）を添加、混合し、吸着剤を凝集させる。
（ｆ）前記吸着剤を磁石により反応容器壁に磁集し、試薬Ｄ中の過剰成分を含む液体部分を除去し、磁石を外して吸着剤が分散する濃度になるようにバッファー（試薬Ｃ）を加え、吸着剤を再分散する。同様な操作を繰り返し、吸着剤を洗浄する。
（ｇ）蛍光色素の蛍光強度を測定する。
An example in which an antigen is detected and measured as a detection target substance by a sandwich method using a fluorescent dye is shown below.
(A) A reagent A containing temperature-responsive polymer surface-modified magnetic particles (adsorbent) bound with an antibody a against an antigen to be detected and measured is mixed with a specimen containing an antigen as a detection target substance, and reacted. React in a container.
(B) A high-concentration salt aqueous solution (reagent B) is added and mixed so that the adsorbent can be aggregated in the reaction solution, and the adsorbent is aggregated.
(C) The adsorbent is magnetically collected on the reaction vessel wall with a magnet, the liquid portion containing unnecessary components in the specimen is removed, and the buffer (reagent C) is removed so that the adsorbent is dispersed by removing the magnet. In addition, the adsorbent is redispersed. The same operation is repeated to wash the adsorbent.
(D) An antigen solution to be detected and measured is mixed with an aqueous solution (reagent D) of a fluorescent dye bound with an antibody b that recognizes a site different from the antibody a, and reacted in a reaction vessel.
(E) A high-concentration salt aqueous solution (the reagent B) is added and mixed so that the adsorbent can be aggregated in the reaction solution, and the adsorbent is aggregated.
(F) The adsorbent is magnetically collected on the reaction vessel wall by a magnet, the liquid portion containing excess components in the reagent D is removed, and the buffer is removed so that the adsorbent is dispersed by removing the magnet. And redisperse the adsorbent. The same operation is repeated to wash the adsorbent.
(G) The fluorescence intensity of the fluorescent dye is measured.
この例では試薬Ｄとして、抗原に対し、前記抗体ａとは違う部位を認識する抗体ｂを結合させた蛍光色素を使用し、蛍光を測定する方法を例として示したが、放射ラベルした抗体ｂを用いて放射能を測定する方法、西洋ワサビペルオキシダーゼやアルカリフォスファターゼ等の酵素で標識した抗体ｂ及び酵素の基質である発光または発色試薬を用いて発光または発色強度を測定する方法等各種方法が適用できる。 In this example, as a reagent D, a method of measuring fluorescence by using a fluorescent dye in which an antibody b that recognizes a site different from the antibody a is bound to an antigen is shown as an example. Various methods, such as a method for measuring radioactivity using an enzyme, a method for measuring luminescence or color intensity using an antibody b labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase, and a luminescence or chromogenic reagent which is a substrate of the enzyme are applied. it can.
本発明の分離方法または検出方法は、環境検査、食品検査、臨床診断等に利用される各種物質の分離、検出及び定量に好適に使用できる。
具体的には、例えば、水や食品中の病原菌、体液、尿、喀痰、糞便中等に含まれるヒトイムノグロブリンＧ、ヒトイムノグロブリンＭ、ヒトイムノグロブリンＡ、ヒトイムノグロブリンＥ、ヒトアルブミン、ヒトフィブリノーゲン（フィブリンおよびそれらの分解産物）、α−フェトプロテイン（ＡＦＰ）、Ｃ反応性タンパク質（ＣＲＰ）、ミオグロビン、ガン胎児性抗原、肝炎ウイルス抗原、ヒト絨毛性ゴナドトロピン（ｈＣＧ）、ヒト胎盤
性ラクトーゲン（ＨＰＬ）、インスリン、ＨＩＶウイルス抗原、アレルゲン、細菌毒素、細菌抗原、酵素、ホルモン、薬剤等を挙げることができる。
The separation method or detection method of the present invention can be suitably used for separation, detection and quantification of various substances used for environmental inspection, food inspection, clinical diagnosis and the like.
Specifically, for example, human immunoglobulin G, human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen contained in pathogens in water and food, body fluids, urine, sputum, feces, etc. (Fibrin and their degradation products), α-fetoprotein (AFP), C-reactive protein (CRP), myoglobin, oncofetal antigen, hepatitis virus antigen, human chorionic gonadotropin (hCG), human placental lactogen (HPL) Insulin, HIV virus antigen, allergen, bacterial toxin, bacterial antigen, enzyme, hormone, drug and the like.
（本発明の分離用キット及び検出用キット）
本発明の検体中の分離対象物質を分離するためのキットは、例えば、下記の試薬Ａ、試薬Ｂ、及び試薬Ｃから構成される。
試薬Ａ：吸着剤（温度応答性高分子表面修飾磁性粒子にリガンドが結合した粒子）の分散液
試薬Ｂ：塩の水溶液
試薬Ｃ：希釈用バッファー（上記試薬Ａ及びＢの希釈、並びに検体の希釈に使用可能な緩衝液である。一例として、トリス塩酸緩衝液、リン酸緩衝液等が挙げられる。）
また、本発明の検体中の検出対象物質を検出するためのキットは、例えば上記検体中の分離対象物質を分離するためのキット（試薬Ａ、試薬Ｂ、及び試薬Ｃ）に加えて、下記試薬Ｄ、試薬Ｅ、及び試薬Ｆから構成される。
試薬Ｄ：検出対象物質に対して、試薬Ａ中のリガンドとは別の部位を認識するリガンドと、検出ユニット（蛍光色素、放射性同位体元素、酵素等）の結合物の溶液。
試薬Ｅ：酵素の基質（検出ユニットとして酵素を用いる場合）
試薬F：検出対象物質の標準品（一例として、精製した抗原が挙げられる。）。
また、検出ユニットの種類により、蛍光、放射能、発光、発色強度等を測定する装置等が必要となる。
(Separation kit and detection kit of the present invention)
The kit for separating the substance to be separated in the specimen of the present invention is composed of, for example, the following reagent A, reagent B, and reagent C.
Reagent A: Dispersion liquid of adsorbent (particles in which ligands are bonded to temperature-responsive polymer surface-modified magnetic particles) Reagent B: Salt aqueous solution Reagent C: Dilution buffer (dilution of reagents A and B and sample dilution) (Examples include Tris-HCl buffer and phosphate buffer.)
The kit for detecting the detection target substance in the specimen of the present invention includes, for example, the following reagents in addition to the kits (reagent A, reagent B, and reagent C) for separating the separation target substance in the specimen. D, reagent E, and reagent F.
Reagent D: A solution of a combination of a ligand that recognizes a site different from the ligand in reagent A and a detection unit (fluorescent dye, radioisotope element, enzyme, etc.) with respect to the detection target substance.
Reagent E: Enzyme substrate (when using enzyme as detection unit)
Reagent F: Standard product of the detection target substance (an example is a purified antigen).
In addition, depending on the type of the detection unit, an apparatus for measuring fluorescence, radioactivity, luminescence, color intensity, etc. is required.
以下、実施例に基いて本発明をより詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
［製造例１］磁性粒子（６０ｎｍ）の調製方法：
１００ｍｌ容量のフラスコに、塩化第二鉄・六水和物（１．０ｍｏｌ）及び塩化第一鉄・四水和物（０．５ｍｏｌ）混合水溶液を３ｍｌ、多価アルコールであるデキストラン（和光純薬社製、分子量３２０００〜４００００）の１０重量％水溶液６０ｍｌを入れ、メカニカルスターラーで攪拌し、この混合水溶液を５０℃に昇温した後、これに２５重量％アンモニア水溶液５．０ｍｌを滴下し、１時間程度攪拌した。この操作で、平均粒径が約６０ｎｍのデキストラン含有磁性粒子が得られた（製造例において、特開２００５−８２５３８を参照した。）。
[Production Example 1] Method for preparing magnetic particles (60 nm):
In a 100 ml flask, 3 ml of a mixed aqueous solution of ferric chloride / hexahydrate (1.0 mol) and ferrous chloride / tetrahydrate (0.5 mol), dextran (polypropylene alcohol) 60 ml of a 10% by weight aqueous solution having a molecular weight of 32,000 to 40,000) was added and stirred with a mechanical stirrer. After the temperature of the mixed aqueous solution was raised to 50 ° C., 5.0 ml of a 25% by weight aqueous ammonia solution was added dropwise thereto. Stir for about an hour. By this operation, dextran-containing magnetic particles having an average particle diameter of about 60 nm were obtained (refer to JP-A-2005-82538 in the production examples).
［製造例２］ビオチンモノマー〔Ｎ−ビオチニル−Ｎ′−メタクリロイルトリメチレンアミド〕の調製方法：
Ｎ−（３−アミノプロピル）メタクリルアミド塩酸塩１８ｇ、ビオチン２４ｇ及びトリエチルアミン３０ｇを３００ｍｌのＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ）に溶解し、０℃に冷却した。ジフェニルホスフォニルアジド２８ｇを５０ｍｌのＤＭＦに溶解させた溶液を１時間かけて、この混合物中に滴下した。滴下終了後、０℃で３時間攪拌し、更に室温で１２時間攪拌した。この後、減圧下で、溶媒を留去し、展開溶媒としてクロロホルム−メタノール混合溶媒を用いて、カラムクロマトグラフィーで精製したところ、白色粉末２２ｇが得られた。これは、目的物であるＮ−ビオチニル−Ｎ′−メタクリロイルトリメチレンアミドであった（収率５９％）。
[Production Example 2] Preparation method of biotin monomer [N-biotinyl-N'-methacryloyl trimethyleneamide]:
18 g of N- (3-aminopropyl) methacrylamide hydrochloride, 24 g of biotin and 30 g of triethylamine were dissolved in 300 ml of N, N-dimethylformamide (DMF) and cooled to 0 ° C. A solution of 28 g of diphenylphosphonlazide dissolved in 50 ml of DMF was dropped into this mixture over 1 hour. After completion of dropping, the mixture was stirred at 0 ° C. for 3 hours, and further stirred at room temperature for 12 hours. Thereafter, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography using a chloroform-methanol mixed solvent as a developing solvent. As a result, 22 g of white powder was obtained. This was N-biotinyl-N′-methacryloyl trimethyleneamide, which was the target product (yield 59%).
［製造例３］リガンドとしてビオチンを固定したＬＣＳＴ型磁性粒子の調製方法：
５０ｍｌの三口フラスコに、Ｎ−イソプロピルアクリルアミド３００ｍｇ、上記方法で調製したビオチンモノマー３ｍｇ、上記方法で調製したデキストラン含有磁性粒子（６０ｎｍ）の２重量％水溶液２ｍｌを入れ、蒸留水で２０ｍｌに調節した。この水溶液を窒素置換した後、更に０．２Ｍ 硝酸二アンモニウムセリウム（IV）硝酸溶液２００μｌを添
加し、２時間攪拌し、反応を進行させることで、ＬＣＳＴ型磁性粒子が得られた。このＬＣＳＴ型磁性粒子の平均粒径は、大塚電子株式会社製レーザーゼータ電位計ＥＬＳ−８０００を用いて測定したところ、約１００ｎｍであることがわかった。またこの粒子はＬＣＳＴを３７℃に有し、ＬＣＳＴ未満の水溶液中では完全に分散し、磁石での回収は困難であったが、水溶液をＬＣＳＴ以上とすると直ちに凝集し、磁石で容易に回収することが可能であった。
[Production Example 3] Preparation method of LCST type magnetic particles having biotin immobilized as a ligand:
A 50 ml three-necked flask was charged with 300 mg of N-isopropylacrylamide, 3 mg of biotin monomer prepared by the above method, and 2 ml of a 2 wt% aqueous solution of dextran-containing magnetic particles (60 nm) prepared by the above method, and adjusted to 20 ml with distilled water. After the aqueous solution was purged with nitrogen, 200 μl of 0.2M diammonium cerium (IV) nitrate solution was added, stirred for 2 hours, and the reaction was allowed to proceed, whereby LCST type magnetic particles were obtained. The average particle size of the LCST type magnetic particles was measured with a laser zeta electrometer ELS-8000 manufactured by Otsuka Electronics Co., Ltd., and was found to be about 100 nm. This particle has LCST at 37 ° C. and is completely dispersed in an aqueous solution lower than LCST, and recovery with a magnet is difficult. However, when the aqueous solution exceeds LCST, it immediately aggregates and is easily recovered with a magnet. It was possible.
［試験例１］磁性粒子への塩添加及び磁気回収実験例：
上記方法で調製したデキストラン含有磁性粒子（濃度0.4重量％、水分散液）とＬＣＳＴ型磁性粒子（濃度0.4重量％、水分散液）を100μlずつ用意し、３０℃恒温で1ＭのNa2SO4水溶液25μlをNa2SO4の最終濃度が200ｍＭになるように加えた。デキストラン含有磁性粒子はNa2SO4水溶液を添加後も完全に分散状態で磁気回収不可能であった。これに対して、ＬＣＳＴ型磁性粒子はNa2SO4を添加後凝集し、１分以内に磁気回収が可能になった。磁気回収したＬＣＳＴ型磁性粒子にH2Oを加えると再び分散した。
[Test Example 1] Example of salt addition to magnetic particles and magnetic recovery experiment:
Prepare 100 μl each of dextran-containing magnetic particles (concentration 0.4 wt%, aqueous dispersion) and LCST type magnetic particles (concentration 0.4 wt%, aqueous dispersion) prepared by the above method, and 1M Na 2 SO 4 at 30 ° C. and constant temperature. 25 μl of aqueous solution was added so that the final concentration of Na 2 SO 4 was 200 mM. The dextran-containing magnetic particles were completely dispersed and could not be magnetically recovered even after the addition of the Na 2 SO 4 aqueous solution. In contrast, the LCST type magnetic particles aggregated after addition of Na 2 SO 4 , and magnetic recovery became possible within 1 minute. When H 2 O was added to the magnetically recovered LCST type magnetic particles, they were dispersed again.
以上のように温度応答性高分子で表面を修飾されたＬＣＳＴ型磁性粒子はNa2SO4の最終濃度が200mMでは磁気分離が可能であるのに対し、温度応答性高分子で表面を修飾されていない、デキストラン含有磁性粒子は、Na2SO4の最終濃度が200mMでは磁気分離が不可能であった。 As described above, the LCST type magnetic particles whose surface is modified with a temperature-responsive polymer can be magnetically separated when the final concentration of Na 2 SO 4 is 200 mM, whereas the surface is modified with a temperature-responsive polymer. In the case of dextran-containing magnetic particles, magnetic separation was impossible at a final concentration of Na 2 SO 4 of 200 mM.
［実施例１］
塩濃度によるＬＣＳＴ型磁性粒子の凝集及び分散効果を検討した例を示す。
なお、温度応答性高分子で表面を修飾されていない磁性粒子として、上記製造例１で調製した磁性粒子を用い、吸着剤として、上記製造例３で調製した、ビオチンを固定したＬＣＳＴ型磁性粒子を用いた。
[Example 1]
The example which examined the aggregation and dispersion | distribution effect of the LCST type magnetic particle by salt concentration is shown.
In addition, the LCST type magnetic particle which fixed the biotin which prepared in the said manufacture example 3 as an adsorption agent using the magnetic particle prepared in the said manufacture example 1 as a magnetic particle by which the surface is not modified with the temperature-responsive polymer | macromolecule Was used.
１．５ｍｌのマイクロチューブ中において、濃度が０．４重量％の前記ＬＣＳＴ型磁性粒子の水分散液100μlに、表１及び表３に記載の各塩の水溶液を所望の最終濃度となるように加え、ピペッティングにより攪拌し、30℃で30秒静置し、目視により凝集を確認した（表１）。
凝集が確認されたサンプルは、マイクロチューブごと、磁石付きのスタンド（マグナビート株式会社製Magna-Stand6）にセットし、30℃で1分、磁気分離し、上澄を除去した。精製水（MILLIPORE社製 Direct-Q（商品名）により精製した水）100μlを加え、再分散を確認した。
In an aqueous dispersion of the LCST type magnetic particles having a concentration of 0.4% by weight in a 1.5 ml microtube, an aqueous solution of each salt described in Tables 1 and 3 is adjusted to a desired final concentration. In addition, the mixture was stirred by pipetting, allowed to stand at 30 ° C. for 30 seconds, and aggregation was visually confirmed (Table 1).
The sample in which aggregation was confirmed was set in a micro-tube and a magnet-equipped stand (Magna-Stand 6 manufactured by Magnabeat Co., Ltd.), magnetically separated at 30 ° C. for 1 minute, and the supernatant was removed. 100 μl of purified water (water purified by Direct-Q (trade name) manufactured by MILLIPORE) was added, and redispersion was confirmed.
［比較例１］
ＬＣＳＴ型磁性粒子の代わりに、製造例１で調製した温度応答性高分子で表面を修飾されていない磁性粒子を用いた以外は、実施例１と同様にして、目視により凝集を確認したところ、凝集が確認できなかった（表２及び表４）。
[Comparative Example 1]
Instead of the LCST type magnetic particles, the aggregation was visually confirmed in the same manner as in Example 1 except that the magnetic particles whose surface was not modified with the temperature-responsive polymer prepared in Production Example 1 were used. Aggregation could not be confirmed (Tables 2 and 4).
結果を表１〜表４に示す。前記ＬＣＳＴ型磁性粒子は、各種塩の水溶液を用いて塩濃度を調整することにより、吸着剤を凝集・分離することが可能であることがわかった。これに対して、温度応答性高分子で表面を修飾されていない磁性粒子は、各種塩の水溶液により塩濃度を調整しても、吸着剤を凝集・分離できないことがわかった。 The results are shown in Tables 1 to 4. It was found that the LCST type magnetic particles can aggregate and separate the adsorbent by adjusting the salt concentration using an aqueous solution of various salts. In contrast, it has been found that magnetic particles whose surface is not modified with a temperature-responsive polymer cannot aggregate and separate the adsorbent even when the salt concentration is adjusted with an aqueous solution of various salts.
［実施例２］
温度応答性高分子表面修飾磁性粒子に結合した抗体及びアルカリフォスファターゼの結合した抗体を用い、サンドイッチ法によりTSH（甲状腺刺激ホルモン）を定量した例を示す。
ビオチン化抗TSHα抗体（Leinco technologies社製マウス抗TSHα抗体をビオチン化(ビオチン化は旭テクノグラス社に依頼)したもの。濃度0.75mg/ml）2μl、アルカリフォスファターゼ結合抗TSHβ抗体（Leinco technologies社製マウス抗TSHβ抗体にアルカリフォ
スファターゼを付加(アルカリフォスファターゼ付加は旭テクノグラス社に依頼)したもの。濃度1.09mg/ml）0.5μl、TBS バッファー（20mM Tris-HCl(pH7.5)、150mM NaCl）12.5μl、温度応答性高分子表面修飾磁性粒子（マグナビート株式会社製Therma-Max（登録商標） LA Avidin（濃度4mg/ml））20μlの量で混合し、必要量を用意し、35μlずつマイクロチューブに分注した（２系列）。ここに、アーキテクト（登録商標）・TSHキャリブレーター（アボットジャパン株式会社製）をTSH濃度0μIU/ml、4.0μIU/ml、及び40.0μIU/mlの濃度で65μl加えてピペッティングにて攪拌し、1分間室温で反応させた。反応終了後、反応液に1M硫酸ナトリウム液を30μl加えて（塩濃度合計268mM）、ピペッティングにて攪拌し、30℃で30秒反応後、磁石付きのスタンド（マグナビート株式会社製Magna-Stand6（商品名））にセットし、30℃で1分、磁気分離し、上澄を除去した。TBS-T バッファー（20mM Tris-HCl(pH7.5)、150mM NaCl、0.05（w/v）%Tween20）100μlにて再分散を行い、1M硫酸ナトリウム液を30μl加えて（塩濃度合計380mM）、ピペッティングにて攪拌し、30℃で30秒反応させた後、30℃で1分、磁気分離し、洗浄を行なった。洗浄は同様の手順で２回繰り返した。最後にTBS バッファー100μlにてペレットを再分散し、100μlの発光基質（Lumigen社製LUMIGEN（登録商標） APS-5）を添加し、5秒間攪拌した後、10秒反応させ、マルチラベルプレートリーダー（ベルトールドジャパン株式会社製マルチラベルプレートリーダー Mithras LB940）にて0.1秒間発光強度を測定した。
[Example 2]
An example in which TSH (thyroid stimulating hormone) was quantified by the sandwich method using an antibody bound to temperature-responsive polymer surface-modified magnetic particles and an antibody bound to alkaline phosphatase is shown.
Biotinylated anti-TSHα antibody (Leinco technologies mouse anti-TSHα antibody biotinylated (biotinylated requested by Asahi Techno Glass). Concentration 0.75 mg / ml) 2 μl, alkaline phosphatase-conjugated anti-TSHβ antibody (Leinco technologies) Alkaline phosphatase added to mouse anti-TSHβ antibody (alkaline phosphatase added to Asahi Techno Glass Co., Ltd.) 0.5 μl in concentration 1.09 mg / ml), TBS buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl) 12.5 μl, temperature-responsive polymer surface-modified magnetic particles (Therma-Max (registered trademark) LA Avidin (concentration: 4 mg / ml) manufactured by Magnabeat Co., Ltd.) 20 μl are mixed, and the required amount is prepared. (2 series). Add Architect (registered trademark) and TSH calibrator (Abbott Japan Co., Ltd.) at a TSH concentration of 0 μIU / ml, 4.0 μIU / ml, and 40.0 μIU / ml and stir by pipetting. The reaction was allowed to proceed at room temperature for minutes. After completion of the reaction, add 30 μl of 1M sodium sulfate solution to the reaction solution (salt concentration: 268 mM), stir by pipetting, react at 30 ° C. for 30 seconds, and then stand with magnet (Magna-Stand6 from Magnabeat Co., Ltd.) (Trade name)), magnetically separated at 30 ° C. for 1 minute, and the supernatant was removed. Redispersion with 100 μl of TBS-T buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05 (w / v)% Tween20), and 30 μl of 1M sodium sulfate solution (salt concentration 380 mM in total) After stirring by pipetting and reacting at 30 ° C. for 30 seconds, magnetic separation was performed at 30 ° C. for 1 minute, and washing was performed. Washing was repeated twice with the same procedure. Finally, the pellet was redispersed with 100 μl of TBS buffer, 100 μl of luminescent substrate (Lumigen (registered trademark) APS-5 manufactured by Lumigen) was added, stirred for 5 seconds, reacted for 10 seconds, and a multi-label plate reader ( The luminescence intensity was measured for 0.1 seconds using a multi-label plate reader (Mithras LB940) manufactured by Berthold Japan Co., Ltd.
発光強度の測定結果を表５及び図１に示した。表５及び図１によると、TSHの濃度に比例して発光強度が変化していることがわかる。すなわち、本手法を用いることで、温度30℃の一定の条件で良好にTSHを検出及び濃度を定量できることがわかった。 The measurement results of the emission intensity are shown in Table 5 and FIG. According to Table 5 and FIG. 1, it can be seen that the emission intensity varies in proportion to the concentration of TSH. That is, by using this method, it was found that TSH can be detected and the concentration quantified satisfactorily at a constant temperature of 30 ° C.
［実施例３］
温度応答性高分子表面修飾磁性粒子に結合した抗体及びアルカリフォスファターゼの結合した抗体を用い、サンドイッチ法によりTSH（甲状腺刺激ホルモン）を定量した例を示す。
ビオチン化抗TSHα抗体（Leinco technologies社製マウス抗TSHα抗体をビオチン化(ビオチン化は旭テクノグラス社に依頼)したもの。濃度0.75mg/ml）0.2μl、アルカリフォスファターゼ結合抗TSHβ抗体（Leinco technologies社製マウス抗TSHβ抗体にアルカリフォスファターゼを付加(アルカリフォスファターゼ付加は旭テクノグラス社に依頼)したもの。濃度1.09mg/ml）0.2μl、TBS バッファー（20mM Tris-HCl(pH7.5)、150mM NaCl）12.5μl、温度応答性高分子表面修飾磁性粒子（マグナビート株式会社製Therma-Max（登録商標） LA Avidin濃度4mg/ml）20μlの量で混合し、必要量を用意し、35μlずつマイクロチューブに分注した（２系列）。ここに、TSH溶液（富士レビオ株式会社製ルミパルス（登録商標）・TSH-N標準TSH溶液（WHO STANDARD 2nd基準））をTSH濃度0μIU/ml、5.0μIU/ml、60.0μIU/ml及び200.0μIU/mlの濃度で65μl加えてピペッティングにて攪拌し、5分間室温で反応させた。反応終了後、反応液に1M酒石酸ナトリウム液を30μl加えて（塩濃度合計268mM）、ピペッティングにて攪拌し、30℃で30秒反応後、磁石付きのスタンド（マグナビート株式会社製Magna-Stand6（商品名））にセットし、30℃で1分、磁気分離し、上澄を除去した。TBS-T バッファー（20mM Tris-HCl(pH7.5)、150mM NaCl、0.05(w/v)%Tween20）100μlにて再分散を行い、1M酒石酸ナトリウム液を30μl加えて（塩濃度合計380mM）、ピペッティングにて攪拌し、30℃で30秒反応後、30℃で1分、磁気分離し、洗浄を行
なった。洗浄は同様の手順で２回繰り返した。最後にTBS バッファー100μlにてペレットを再分散し、100μlの発光基質（Lumigen社製LUMIGEN（登録商標） APS-5）を添加し、5秒間攪拌した後、10秒反応させ、マルチラベルプレートリーダー（ベルトールドジャパン株式会社製マルチラベルプレートリーダー Mithras LB940）にて0.1秒間、発光強度を測定した。
[Example 3]
An example in which TSH (thyroid stimulating hormone) was quantified by the sandwich method using an antibody bound to temperature-responsive polymer surface-modified magnetic particles and an antibody bound to alkaline phosphatase is shown.
Biotinylated anti-TSHα antibody (Leinco technologies mouse anti-TSHα antibody biotinylated (biotinylation requested by Asahi Techno Glass Co., Ltd., concentration 0.75 mg / ml) 0.2 μl, alkaline phosphatase-conjugated anti-TSHβ antibody (Leinco technologies) Alkaline phosphatase added to mouse anti-TSHβ antibody (concentration 1.09 mg / ml) 0.2 μl, TBS buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl) 12.5 μl, temperature-responsive polymer surface modified magnetic particles (Therma-Max (registered trademark) LA Avidin concentration 4 mg / ml, manufactured by Magnabeat Co., Ltd.) 20 μl are mixed in the required amount, and 35 μl each is prepared in a microtube. Dispensing (2 series). Here, TSH solutions (Lumipulse (registered trademark), TSH-N standard TSH solution (WHO STANDARD 2nd standard) manufactured by Fujirebio Inc.) were added with TSH concentrations of 0μIU / ml, 5.0μIU / ml, 60.0μIU / ml and 200.0μIU / 65 μl was added at a concentration of ml, and the mixture was stirred by pipetting and reacted at room temperature for 5 minutes. After completion of the reaction, add 30 μl of 1M sodium tartrate solution to the reaction solution (salt concentration: 268 mM), stir by pipetting, react at 30 ° C. for 30 seconds, and then stand with a magnet (Magna-Stand6, manufactured by Magna Beat Co., Ltd.) (Trade name)), magnetically separated at 30 ° C. for 1 minute, and the supernatant was removed. Redispersion with 100 μl of TBS-T buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05 (w / v)% Tween20), 30 μl of 1M sodium tartrate solution (salt concentration 380 mM in total), The mixture was stirred by pipetting, reacted at 30 ° C. for 30 seconds, magnetically separated at 30 ° C. for 1 minute, and washed. Washing was repeated twice with the same procedure. Finally, the pellet was redispersed with 100 μl of TBS buffer, 100 μl of luminescent substrate (Lumigen (registered trademark) APS-5 manufactured by Lumigen) was added, stirred for 5 seconds, reacted for 10 seconds, and a multi-label plate reader ( The luminescence intensity was measured for 0.1 second using a multi-label plate reader (Mithras LB940) manufactured by Berthold Japan Co., Ltd.
発光強度の測定結果を表６及び図２に示した。表６及び図２によると、TSHの濃度に比例して発光強度が変化していることがわかる。すなわち、本手法を用いることで、温度30℃の一定の条件で良好にTSHを検出及び濃度を定量できることがわかった。 The measurement results of the emission intensity are shown in Table 6 and FIG. According to Table 6 and FIG. 2, it can be seen that the emission intensity changes in proportion to the concentration of TSH. That is, by using this method, it was found that TSH can be detected and the concentration quantified satisfactorily at a constant temperature of 30 ° C.
［実施例４］
実施例２および実施例３と同様の方法で、体液、尿、喀痰、糞便中などに含まれるヒトイムノグロブリンＧ、ヒトイムノグロブリンＭ、ヒトイムノグロブリンＡ、ヒトイムノグロブリンＥ、ヒトアルブミン、ヒトフィブリノーゲン（フィブリン及びそれらの分解産物）、α−フェトプロテイン（ＡＦＰ）、Ｃ反応性タンパク質（ＣＲＰ）、ミオグロビン、ガン胎児性抗原、肝炎ウイルス抗原、ヒト絨毛性ゴナドトロピン（ｈＣＧ）、ヒト胎盤性ラクトーゲン（ＨＰＬ）、インスリン、ＨＩＶウイルス抗原、アレルゲン、細菌毒素、細菌抗原、酵素、ホルモン、薬剤等を測定することができる。
[Example 4]
Human immunoglobulin G, human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen contained in body fluid, urine, sputum, feces etc. in the same manner as in Example 2 and Example 3. (Fibrin and their degradation products), α-fetoprotein (AFP), C-reactive protein (CRP), myoglobin, oncofetal antigen, hepatitis virus antigen, human chorionic gonadotropin (hCG), human placental lactogen (HPL) Insulin, HIV virus antigens, allergens, bacterial toxins, bacterial antigens, enzymes, hormones, drugs and the like can be measured.
［実施例５］
カルボン酸塩を用いると、短時間で再分散が可能であることを検討した例を示す。
マイクロチューブ中において、上記製造例３で調製したＬＣＳＴ型磁性粒子の水分散液（０．４重量％）１００μlを４２℃で３０秒静置し、凝集させた。つづいてマイクロチューブごと、磁石付きのスタンド（マグナビート株式会社製Magna-Stand6（商品名））にセットし、４２℃で上澄が透明になるまで磁気分離を行った。このとき磁気分離に要した時間は６０秒であった。次に上澄を除去し、精製水（MILLIPORE社製 Direct-Q（商品名）により精製した水）１００μlを加え、ピペッティングにより攪拌し、再分散させた。このとき再分散に要した時間は６０秒であった。
同様に、上記製造例３で調製した、ビオチンを固定したＬＣＳＴ型磁性粒子の水分散液（０．４重量％）１００μl中に、硫酸ナトリウム、亜硫酸ナトリウム、及びクエン酸ナトリウムを、表７に記載の各最終濃度となるように加え、ピペッティングにより攪拌し、３０℃で３０秒静置し、凝集させた。つづいてMagna-Stand6にセットし、３０℃で上澄が透明になるまで磁気分離を行った。このとき磁気分離に要した時間を表７に記した。次に上澄を除去し、精製水１００μlを加え、ピペッティングにより攪拌し再分散に要した時間を測定した。
結果を表７に示した。塩を添加せず４２℃に昇温させて凝集及び磁気分離した場合、再分散には６０秒を要した。硫酸ナトリウム、及び亜硫酸ナトリウムを用いて、３０℃で同様の操作を行った場合、再分散には６０秒を要した。一方、カルボン酸塩であるクエン酸ナトリウムを用いて、３０℃で同様の操作を行った場合、再分散に要した時間は３０秒であった。この結果より、カルボン酸塩を用いると、再分散が短時間で可能であることが示された。
[Example 5]
An example in which redispersion is possible in a short time when a carboxylate is used will be described.
In a microtube, 100 μl of an aqueous dispersion (0.4 wt%) of the LCST type magnetic particles prepared in Production Example 3 was allowed to stand at 42 ° C. for 30 seconds for aggregation. Subsequently, each microtube was set on a stand with a magnet (Magna-Stand 6 (trade name) manufactured by Magna Beat Co., Ltd.), and magnetic separation was performed at 42 ° C. until the supernatant became transparent. At this time, the time required for magnetic separation was 60 seconds. Next, the supernatant was removed, 100 μl of purified water (water purified by Direct-Q (trade name) manufactured by MILLIPORE) was added, stirred by pipetting, and redispersed. At this time, the time required for redispersion was 60 seconds.
Similarly, Table 100 shows sodium sulfate, sodium sulfite, and sodium citrate in 100 μl of an aqueous dispersion (0.4 wt%) of LCST type magnetic particles immobilized with biotin prepared in Production Example 3 above. The final concentrations of each were added, stirred by pipetting, and allowed to stand at 30 ° C. for 30 seconds for aggregation. Subsequently, it was set on Magna-Stand 6 and magnetic separation was performed at 30 ° C. until the supernatant became transparent. Table 7 shows the time required for magnetic separation. Next, the supernatant was removed, 100 μl of purified water was added, the mixture was stirred by pipetting, and the time required for redispersion was measured.
The results are shown in Table 7. In the case of aggregation and magnetic separation by heating to 42 ° C. without adding salt, re-dispersion took 60 seconds. When the same operation was performed at 30 ° C. using sodium sulfate and sodium sulfite, re-dispersion took 60 seconds. On the other hand, when the same operation was performed at 30 ° C. using sodium citrate which is a carboxylate, the time required for redispersion was 30 seconds. From this result, it was shown that redispersion is possible in a short time when a carboxylate is used.
Claims (10)
温度応答性高分子で磁性粒子が表面修飾された平均粒径５０〜１０００ｎｍの温度応答性高分子表面修飾磁性粒子と、該温度応答性高分子表面修飾磁性粒子に結合した、該検出対象物質に対する親和性を有する物質とからなる吸着剤と検体とを混合した水溶液中で、該吸着剤に該検出対象物質を吸着させ、加熱または冷却せず、一定温度で該水溶液中の塩濃度を変化させることより該吸着剤を凝集させる工程、及び、
該吸着剤を磁力により水溶液中から回収する工程、
を含むことを特徴とする方法。 A method for separating a detection target substance in a specimen,
A temperature-responsive polymer surface-modified magnetic particle having an average particle size of 50 to 1000 nm, the surface of which is modified with a temperature-responsive polymer, and the detection target substance bound to the temperature-responsive polymer surface-modified magnetic particle In an aqueous solution in which an adsorbent composed of a substance having affinity and a sample are mixed, the detection target substance is adsorbed on the adsorbent, and the salt concentration in the aqueous solution is changed at a constant temperature without heating or cooling. And agglomerating the adsorbent, and
Recovering the adsorbent from an aqueous solution by magnetic force;
A method comprising the steps of:
を含むことを特徴とする検体中の検出対象物質を検出する方法。 A step of adsorbing and separating a detection target substance in a specimen by an adsorbent by the method according to any one of claims 1 to 7, and a step of detecting the detection target substance adsorbed on the adsorbent,
A method for detecting a substance to be detected in a specimen, comprising:
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