JP3717582B2 - Fine particles - Google Patents

Description

しかしながら、本発明の微粒子を得る場合は、上記の計算式より導き出される量よりも多い量のシランカップリング剤を用いるのが好ましく、特に、上記の計算式で導き出される量の３〜７倍量のシランカップリング剤を用いるのが好ましい。これにより、無機微粒子を多量のポリアクロレインで被覆することが可能となり、染料にて染色した場合、効率良く濃染色が可能になる。
【００１１】
シランカップリング剤としては、例えばアミノ基を有するシランカップリング剤（γーアミノプロピルトリエトキシシラン、Ｎ−β( アミノエチル) γーアミノプロピルメチルジメトキシシラン、Ｎ−β( アミノエチル) γーアミノプロピルトリメトキシシラン等）、ビニル基を有するシランカップリング剤（ビニルエトキシシラン、ビニルトリス( βーメトキシエトキシ) シラン等）、メタアクリル基を有するシランカップリング剤（γーメタアクリロキシプロピルトリメトキシシラン等）が挙げられる。但し、ビニル基を有するシランカップリング剤やメタアクリル基を有するシランカップリング剤による処理はトルエン等の有機溶媒中で行うため、カップリング処理後の溶剤洗浄と乾燥に難点があり、適正な設備、機器等が必要となる。
【００１２】
シランカップリング剤による処理等により表面に官能基が導入された無機微粒子（以下
、「処理済無機微粒子」という）の存在下に溶媒中でアクロレインを重合する際、アクロレインは処理済無機微粒子に対して５０〜２００重量％用いるのが好ましく、特に７５〜１５０重量％用いるのが好ましい。又、アクロレインは溶媒に対しては１０〜３０重量％の範囲になるようにするのが好ましい。更に、アクロレインの重合を促進し、得られる微粒子のアクロレインの刺激臭を減少させ、未反応アクロレイン及び副性する可溶性重合体の除去を容易にするために重合開始剤を使用するのが好ましい。重合開始剤はアクロレインに対して０. ０１〜２重量％用いるのが好ましく、重合開始剤としては、過硫酸塩、アゾ化合物、過酸化ベンゾイル等、公知の重合開始剤が使用できる。
【００１３】
このようにして得られる本発明の微粒子は、更に他の共重合可能なモノマーを共重合させることもできる。他の共重合可能なモノマーとしては、メチル（メタ）アクリレート、エチル（メタ）アクリレート、ブチル（メタ）アクリレート、２ーエチルヘキシル（メタ）アクリレート、スチレン、（メタ）アクリレート等が挙げられる。微粒子に他の共重合可能なモノマーを共重合する場合、アクロレインの重合途中又は重合がほとんど終了した後に反応系に該モノマーを加え重合開始剤で重合させればよい。この際に用いる重合開始剤は、過硫酸塩、アゾ化合物、過酸化ベンゾイル等が適当である。
【００１４】
本発明の微粒子を染料にて染色したものを用いて得た診断試薬（特にマイクロタイター法用の）は凝集像が鮮明で判定が容易になる。微粒子の染色に使用される染料は染色可能な染料なら何でも良く酸性染料、直接染料、分散染料、反応染料等の大部分の染料は使用できる。染料について、具体的に染料名を挙げると、
Ｋａｙａｒｕｓ Ｓｕｐｒａ Ｂｌｕｅ ＢＲＬ
Ｋａｙａｒｕｓ Ｓｕｐｒａ Ｒｅｄ ６ＢＬ
Ｋａｙａｃｒｙｌ Ｂｌｕｅ ＧＲＬ
Ｋａｙａｎｏｌ Ｍｉｌｌｉｎｇ Ｒｅｄ ６ＢＷ
Ｋａｙａｌｏｎ Ｐｏｌｙｅｓｔｅｒ Ｂｌｕｅ ２Ｒ−ＳＦ
Ｋａｙａｌｏｎ Ｐｏｌｙｅｓｔｅｒ Ｒｕｂｉｎｅ ＧＬ−ＳＥ
Ｋａｙａｃｉｏｎ Ｒｅｄ Ａ−３Ｂ
Ｋａｙａｃｉｏｎ Ｂｌｕｅ Ａ−５Ｒ
（以上 日本化薬（株）製）
等が挙げられる。しかし、染料はブルー系、赤系に限られるものではなく、例えば、Ｋａｙｌｏｎ Ｐｏｙｅｓｔｅｒ Ｌｉｇｈｔ Ｙｅｌｌｏｗ ５ＧＳ（黄色）、Ｆｕｃｈｓｉｎ（赤系）、Ｍａｌａｃｈｉｔｅ Ｇｒｅｅｎ（緑系）、（以上 和光純薬（株）製）
等でも当然非常に良く染色できる。また染料の混合によって中間色に染色することも可能である。
【００１５】
微粒子を染色する方法は、織布を染色するときと同様にして行なうことができる。染料は０. ００１〜５％重量％の濃度となるように水に溶かすか又は分散させて用いるのが好ましく、被染色物である微粒子の濃度は０. １〜２０重量％の範囲とするのが好ましい。染色温度は常温〜１００℃が好ましく、３０分〜５時間で染色される。
【００１６】
本発明の微粒子に、例えば抗原又は抗体を感作することにより診断試薬が得られる。抗原又は抗体による感作は、一般的な感作方法によって行なうことができる。即ち、リン酸等の緩衝生理食塩水中で微粒子と抗原又は抗体を接触させることにより感作を行なうことができる。
【００１７】
本発明の特に染料で染色した微粒子に抗原又は抗体を感作して得たものを診断試薬としてマイクロタイター法で検査した場合、判定の凝集像が鮮明で判定が容易になる。また、染料 で染色した微粒子を長期保存した場合、染色前のものより分散性は良好であり、診断試薬にした場合も染料で染色したものを用いた場合の方が長期安定性が良好である。即ち、染色することで保存安定性が向上する。これは、微粒子表面が染料で覆われることにより安定化したためと思われる。これは、アクロレインモノマーの重合に際してビニル重合のみならず共役するカルボニル二重結合も重合に関与した結果として、構造が複雑になり不安定な結合を持ち、これが染料によって表面処理され、染料分子と結合したり、染料による強い親和力等によって安定化したためと考えられる。
【００１８】
本発明の染料で染色された微粒子は比重が高く（例えば、実施例３で得られる微粒子の比重は１. ８）、そのため、これを用いて得た診断試薬をマイクロタイター法で使用した場合、判定時間は３０分〜１時間程度で短時間判定が可能になる。従来の着色したポリアクロレイン微粒子は比重１. ３２のため判定時間は１時間〜２. ５時間必要であった。
【００１９】
本発明の染料で染色された微粒子は、マイクロタイター法のみならず、粒径の小さい微粒子を用いることでラテックス凝集比濁法、スライドテスト法にも応用することが可能である。
【００２０】
【実施例】
以下、実施例を挙げて本発明を具体的に説明する。但し、本発明はこれら実施例に限定されるものではない。
【００２１】
製造例１
シリカ微粒子（シーホスターＫＥ−Ｐ１００，平均粒径1.０μｍ，日本触媒（株）製）１０ｇを含有する５０％エタノール水溶液５０ｇにシランカップリング剤としてγーアミノプロピルトリエトキシシランを２ｇ添加し、酢酸でｐＨ４. ６に調整後、４０℃で２時間攪拌する。その後、エタノール水溶液を除去するため、遠心分離機（１５００ｒｐｍ×５分）によりカップリング処理したシリカ微粒子のみ沈殿させ蒸留水で２回洗浄後、１００℃で乾燥してカップリング処理したシリカ微粒子を得た。
【００２２】
製造例２
シリカ微粒子（シーホスターＫＥ−Ｐ１００，平均粒径1.０μｍ，日本触媒（株）製）１０ｇを含有するトルエン５０ｇにシランカップリング剤としてγーメタアクリロキシプロピルトリメトキシシランを１ｇ添加し、８０℃で２時間攪拌する。冷却後、遠心分離機（１５００ｒｐｍ×５分）により上清のトルエンを除去する。沈渣のカップリング処理したシリカ微粒子を５０％エタノール５０ｇ中に添加し攪拌し、遠心分離機（１５００ｒｐｍ×５分）により５０％エタノールによる上清の交換を２回行いトルエンの洗浄を行う。次に沈殿物に蒸留水５０ｇ添加し、同様の操作でエタノール洗浄を行った後、１００℃で乾燥してカップリング処理したシリカ微粒子を得た。
【００２３】
実施例１
製造例１で得たカップリング処理したシリカ微粒子１０ｇを含有する水１００ｇにアクロレインモノマー１５ｍｌを添加し、窒素ガスを導入して酸素を追い出し、２％過硫酸アンモニウム水溶液１ｍｌ添加し、５０℃で５時間反応させ、更に２％過硫酸アンモニウム水溶液１ｍｌ添加後、７０℃で２時間、後反応を行う。得られた微粒子中の少量の未反応アクロレイン及び可溶性重合体を除去するため、遠心分離機（１５００ｒｐｍ×５分）により微粒子のみ沈殿させ分散媒を水と交換してポリアクロレインで被覆された微粒子（固形分濃度１０ｗｔ％に調製しておく）が得られた。得られた微粒子の平均粒径は１. ０８μｍであり、ポリアクロレイン量はシリカ微粒子に対して１４. ２重量％であった。
【００２４】
実施例２
製造例２で得たカップリング処理したシリカ微粒子１０ｇを用い、実施例１と同様にして反応を行いポリアクロレインで被覆された微粒子が得られた。得られた微粒子の平均粒径は１. ０７μｍであり、ポリアクロレイン量はシリカ微粒子に対して１３. ３重量％であった。
【００２５】
実施例３
実施例１で得たポリアクロレインで被覆された微粒子（固形分濃度１０ｗｔ％）１００ｇに対して、Ｋａｙａｌｏｎ Ｐｏｌｙｅｓｔｅｒ Ｒｕｂｉｎｅ ＧＬ−ＳＥ（日本化薬（株）製）０. ４ｇを１００ｍｌの蒸留水に溶解したものを混合して酢酸でｐＨ５に調整後、９５℃で２時間攪拌した。冷却後、遠心分離機（１５００ｒｐｍ×５分）により蒸留水による上清の交換を３回行い、その後沈殿物を酢酸１ｍｌを含む蒸留水２００ｍｌ中に分散し、９５℃で３０分攪拌した。冷却後、遠心分離機（１５００ｒｐｍ×５分）により蒸留水による上清の交換を５回行い洗浄して、濃赤色に着色した微粒子（固形分濃度１０ｗｔ％に調製しておく）が得られた。
【００２６】
実施例４
実施例２で得たポリアクロレインで被覆された微粒子（固形分濃度１０ｗｔ％）１００ｇを実施例３と同様に染色して、濃赤色に着色した微粒子（固形分濃度１０ｗｔ％に調製しておく）が得られた。
【００２７】
実施例５
実施例１で得たポリアクロレインで被覆された微粒子（固形分濃度１０ｗｔ％）１００ｇに対して、Ｋａｙａｎｏｌ Ｍｉｌｌｉｎｇ Ｒｅｄ ６ＢＷ（日本化薬（株）製）０. ２ｇを用い、実施例３と同様に染色して、濃赤色に着色した微粒子（固形分濃度１０ｗｔ％に調製しておく）が得られた。
【００２８】
試験例
着色微粒子の診断試薬への応用として梅毒抗体検査法を例として説明する。但し、これらの検査項目に限定されるものではない。
【００２９】
１．梅毒抗原の調製
ＴＰ菌（Ｎｉｃｈｏｌｓ株）をウサギ睾丸内で増殖させ、摘出した睾丸からクエン酸緩衝液で抽出、円心分画して集菌し、菌数１〜２×１０⁹Cells／ｍｌとなるようにＰＢＳに再浮遊した。このＴＰ菌浮遊液５ｍｌをＰＢＳで１０倍に希釈し、超音波破砕器（久保田商事（株）２０１型）を用い、９ｋＨｚ、１８０Ｗで２０分処理してＴＰ菌体成分を含む抗原液を得た。
【００３０】
２．抗原感作、試薬の調製
着色微粒子（固形分１０ｗｔ％）１ｍｌをＰＢＳ １０ｍｌに分散させ、ＴＰ抗原液４ｍｌ添加し３７℃で３０分間ゆっくり攪拌した。その後、２ｗｔ％牛血清アルブミン（ＢＳＡと略す）ーＰＢＳ １０ｍｌをさらに添加し、３７℃で１時間ゆっくり攪拌した。
これを、遠心分離機（１５００ｒｐｍ×５分）により分離し上清を捨て、０. ５ｗｔ％ＢＳＡーＰＢＳで２回同様に遠心して沈渣を洗浄する。その後、この沈渣に０. ５ｗｔ％ＢＳＡーＰＢＳ ４０ｍｌ添加し、よく攪拌して固形分０. ２５ｗｔ％のＴＰ抗原感作微粒子分散液（試薬）を得た。
【００３１】
なお、着色微粒子としては、前記実施例３〜５で得た着色微粒子及び特開昭６４ー１４８号公報の実施例４と同様にして作製した従来の着色ポリアクロレイン微粒子を使用した。得られた試薬を以下のとおり、それぞれＡ，Ｂ，Ｃ，Ｄとした。
試薬Ａ：実施例３の着色微粒子（固形分１０ｗｔ％）を用いたもの 試薬Ｂ：実施例４の着色微粒子（固形分１０ｗｔ％）を用いたもの 試薬Ｃ：実施例５の着色微粒子（固形分１０ｗｔ％）を用いたもの 試薬Ｄ：従来の着色ポリアクロレイン微粒子（固形分１０ｗｔ％）を用いたもの
【００３２】
３．感作微粒子の評価
ＴＰ抗原感作微粒子を用いてマイクロタイター法にて検査を行った。Ｕ型マイクロプレートの第１管目に０. ５ｗｔ％ＢＳＡーＰＢＳを１００μｌ、２管目以降に２５μｌずつ滴下し、第１管目に血清２５μｌを加えた。これをダイリューターにて１管目より連続２倍希釈した。ＴＰ抗原感作微粒子（試薬Ａ〜Ｄ）を第２管目以降に２５μｌずつ滴下し、プレートミキサーで１分間振とう後、フタをして静置した。管底の凝集像で陰性か陽性を判定した。この場合、最終希釈倍数は、２管目が２０倍、第３管目以降は倍々の希釈倍数となり、ＴＰＨＡと同様に４０倍をカットオフ値とした。８０倍以上で凝集が認められたものは陽性となる。結果は表１に示した。
【００３３】
【表１】

【００３４】
４．結論
上記結果から明らかなように、本発明の着色微粒子を用いたＴＰ抗原感作微粒子は、赤血球を担体としたＴＰＨＡと同等の感度に判定され大幅に判定時間を短縮した。
【００３５】
【発明の効果】
本発明の微粒子を用いることにより、従来品に比べ保存安定性が良好で、大幅に判定時間の短縮した診断試薬を提供することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fine particle useful as a fine particle for a diagnostic reagent utilizing an antigen-antibody reaction or the like used for diagnosis of a disease.
[0002]
[Prior art]
Conventionally, fine particles of synthetic polymers such as polystyrene fine particles have been used as fine particles for immunodiagnostic reagents. However, the polymers are essentially colorless to light-colored, and in recent years, the aggregation image is clearly determined by coloring the fine particles. Attempts have been made to make it easier. However, since the specific gravity of polystyrene microparticles is small, a diagnostic reagent for microtiter method using this has the disadvantage that it takes time to make a determination.
JP-B-5-48245 and JP-A-64-148 have proposed polyacrolein microparticles or colored microparticles obtained by dyeing polyacrolein microparticles with a dye, which can be used as diagnostic reagents for microtiter methods. When used, the determination time is shorter because the specific gravity is larger than that of polystyrene fine particles, but the diagnostic reagent for the microtiter method has a higher specific gravity because the sedimentation time becomes the rate-determining test, and the sedimentation time can be further shortened. Fine particles are desired.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide a microparticle that can be used particularly as a diagnostic reagent for a microtiter method and that can shorten the determination time when the reagent is used.
[0004]
[Means for Solving the Problems]
The present inventors have intensively studied to solve the above problems and completed the present invention.
[0005]
That is, the present invention
(1) Fine particles comprising inorganic fine particles coated with polyacrolein,
(2) The fine particles according to the above (1), wherein the inorganic fine particles are silica fine particles,
(3) Fine particles according to (2) above, obtained by polymerizing acrolein in the presence of silica fine particles treated with a silane coupling agent,
(4) The fine particles according to (1), (2) or (3), which are dyed with a dye,
(5) Fine particles according to the above (1), (2), (3) or (4) for diagnostic reagents,
(6) The fine particles according to (5) above for use in a microtiter method.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the inorganic fine particles include fine particles of silica (silicon dioxide), alumina, calcium carbonate, magnesium carbonate, barium sulfate and the like, and silica fine particles are particularly preferable. The average particle size of the inorganic fine particles is preferably from 0.05 to 10 μm, particularly preferably from 0.2 to 5 μm.
[0007]
The amount of polyacrolein coating the inorganic fine particles is preferably in the range of 5 to 30 parts by weight, particularly preferably in the range of 8 to 20 parts by weight with respect to 100 parts by weight of the inorganic fine particles serving as the nucleus. .
The average particle size of the fine particles of the present invention (inorganic fine particles coated with polyacrolein) is preferably from 0.05 to 12 μm, particularly preferably from 0.2 to 7 μm.
[0008]
In the fine particles of the present invention, functional groups such as amino groups, vinyl groups, and methacryl groups are introduced on the surface of the inorganic fine particles, and acrolein is added in the presence of the inorganic fine particles introduced with functional groups on the surface, such as water and alcohol aqueous solutions It can be obtained by polymerizing in a solvent.
Examples of a method for introducing a functional group onto the surface of the inorganic fine particles include a method of treating the inorganic fine particles with a silane coupling agent and a method of halogenation.
[0009]
Here, as an example, a method of treating inorganic fine particles with a silane coupling agent will be described. This treatment can be performed by mixing inorganic fine particles and a silane coupling agent in a medium such as water, an aqueous alcohol solution, an aqueous acetone solution, or toluene. This treatment is preferably performed at pH 3-7, particularly preferably at pH 3-5.
[0010]
The silane coupling agent is preferably used in an amount of 1 to 50% by weight, particularly 10 to 40% by weight, based on the inorganic fine particles.
In general, when an inorganic material is treated with a silane coupling agent, the best effect is exhibited when the surface is coated with a monomolecular film, and it is appropriate to use the amount derived from the following formula for the inorganic material. It is said that.

However, when obtaining the fine particles of the present invention, it is preferable to use a larger amount of the silane coupling agent than the amount derived from the above formula, and in particular, 3 to 7 times the amount derived from the above formula. It is preferable to use a silane coupling agent. As a result, it is possible to coat the inorganic fine particles with a large amount of polyacrolein, and when dyeing with a dye, it is possible to efficiently perform deep dyeing.
[0011]
Examples of the silane coupling agent include a silane coupling agent having an amino group (γ-aminopropyltriethoxysilane, N-β (aminoethyl) γ-aminopropylmethyldimethoxysilane, N-β (aminoethyl) γ-amino. Propyltrimethoxysilane, etc.), silane coupling agents having a vinyl group (vinylethoxysilane, vinyltris (β-methoxyethoxy) silane, etc.), silane coupling agents having a methacrylic group (γ-methacryloxypropyltrimethoxysilane) Etc.). However, since the treatment with a silane coupling agent having a vinyl group or a silane coupling agent having a methacryl group is performed in an organic solvent such as toluene, there are difficulties in washing and drying the solvent after the coupling treatment, and appropriate equipment. , Equipment is required.
[0012]
When acrolein is polymerized in a solvent in the presence of inorganic fine particles having functional groups introduced on the surface by treatment with a silane coupling agent (hereinafter referred to as “treated inorganic fine particles”), It is preferable to use 50 to 200% by weight, particularly 75 to 150% by weight. Acrolein is preferably in the range of 10 to 30% by weight with respect to the solvent. Furthermore, it is preferable to use a polymerization initiator in order to accelerate the polymerization of acrolein, reduce the irritating odor of acrolein in the resulting fine particles, and facilitate the removal of unreacted acrolein and by-product soluble polymers. The polymerization initiator is preferably used in an amount of 0.01 to 2% by weight based on acrolein. As the polymerization initiator, a known polymerization initiator such as a persulfate, an azo compound, or benzoyl peroxide can be used.
[0013]
The fine particles of the present invention thus obtained can be further copolymerized with other copolymerizable monomers. Examples of other copolymerizable monomers include methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, styrene, and (meth) acrylate. In the case of copolymerizing other copolymerizable monomers with the fine particles, the monomers may be added to the reaction system during polymerization of acrolein or after the polymerization is almost completed, and polymerization is performed with a polymerization initiator. As the polymerization initiator used in this case, persulfates, azo compounds, benzoyl peroxide and the like are suitable.
[0014]
A diagnostic reagent (particularly for the microtiter method) obtained by using the fine particles of the present invention dyed with a dye has a clear agglomerated image and is easy to judge. The dye used for dyeing fine particles may be any dye that can be dyed, and most dyes such as acid dyes, direct dyes, disperse dyes, and reactive dyes can be used. Regarding dyes, if you give specific dye names,
Kayarus Supra Blue BRL
Kayarusu Supra Red 6BL
Kayacryl Blue GRL
Kayanol Milling Red 6BW
Kayalon Polyester Blue 2R-SF
Kayalon Polyester Rubin GL-SE
Kayacion Red A-3B
Kayacion Blue A-5R
(Nippon Kayaku Co., Ltd.)
Etc. However, the dye is not limited to blue and red. For example, Kaylon Poester Light Yellow 5GS (yellow), Fuchsin (red), Malachite Green (green), (Wako Pure Chemical Industries, Ltd.)
Of course, it can be dyed very well. It is also possible to dye in a neutral color by mixing dyes.
[0015]
The method for dyeing the fine particles can be performed in the same manner as for dyeing the woven fabric. The dye is preferably dissolved or dispersed in water so as to have a concentration of 0.001 to 5% by weight, and the concentration of fine particles that are to be dyed is in the range of 0.1 to 20% by weight. Is preferred. The dyeing temperature is preferably from room temperature to 100 ° C., and the dyeing is performed in 30 minutes to 5 hours.
[0016]
A diagnostic reagent can be obtained by sensitizing the microparticles of the present invention with, for example, an antigen or an antibody. Sensitization with an antigen or antibody can be performed by a general sensitization method. That is, sensitization can be performed by bringing the microparticles into contact with the antigen or antibody in a buffered physiological saline such as phosphate.
[0017]
When the microparticle method of the present invention, which is obtained by sensitizing a fine particle stained with a dye with an antigen or antibody, is examined as a diagnostic reagent by the microtiter method, the determination aggregate image is clear and the determination is easy. In addition, when the fine particles dyed are stored for a long time, the dispersibility is better than that before dyeing, and the long-term stability is better when using a dye dyed with a diagnostic reagent. . That is, the storage stability is improved by dyeing. This seems to be because the surface of the fine particles was stabilized by being covered with the dye. This is because, as a result of the polymerization of acrolein monomer, not only vinyl polymerization but also conjugated carbonyl double bonds are involved in the polymerization, the structure becomes complex and has unstable bonds, which are surface-treated by dyes and bonded to dye molecules. It is thought that it was stabilized by strong affinity with dyes.
[0018]
The microparticles dyed with the dye of the present invention have a high specific gravity (for example, the specific gravity of the microparticles obtained in Example 3 is 1.8). Therefore, when a diagnostic reagent obtained using this is used in the microtiter method, The determination time is about 30 minutes to 1 hour, and a short time determination is possible. Since the conventional colored polyacrolein fine particles have a specific gravity of 1.32, the determination time required 1 hour to 2.5 hours.
[0019]
The fine particles dyed with the dye of the present invention can be applied not only to the microtiter method but also to the latex agglutination turbidimetry method and the slide test method by using fine particles having a small particle diameter.
[0020]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.
[0021]
Production Example 1
2 g of γ-aminopropyltriethoxysilane as a silane coupling agent was added to 50 g of 50% ethanol aqueous solution containing 10 g of silica fine particles (Seahoster KE-P100, average particle size 1.0 μm, manufactured by Nippon Shokubai Co., Ltd.) After adjusting to pH 4.6, stir at 40 ° C. for 2 hours. Thereafter, in order to remove the ethanol aqueous solution, only the silica fine particles that have been subjected to the coupling treatment are precipitated by a centrifuge (1500 rpm × 5 minutes), washed twice with distilled water, and then dried at 100 ° C. to obtain the silica fine particles that have undergone the coupling treatment. It was.
[0022]
Production Example 2
1 g of γ-methacryloxypropyltrimethoxysilane as a silane coupling agent was added to 50 g of toluene containing 10 g of silica fine particles (Seahoster KE-P100, average particle size 1.0 μm, manufactured by Nippon Shokubai Co., Ltd.) at 80 ° C. For 2 hours. After cooling, the toluene in the supernatant is removed by a centrifuge (1500 rpm × 5 minutes). Silica fine particles subjected to the coupling treatment of the sediment are added to 50 g of 50% ethanol and stirred, and the supernatant is washed twice with 50% ethanol by a centrifuge (1500 rpm × 5 minutes) to wash toluene. Next, 50 g of distilled water was added to the precipitate, and ethanol washing was performed in the same manner, followed by drying at 100 ° C. to obtain a coupling-treated silica fine particle.
[0023]
Example 1
15 ml of acrolein monomer is added to 100 g of water containing 10 g of the silica fine particles subjected to the coupling treatment obtained in Production Example 1, nitrogen gas is introduced to expel oxygen, 1 ml of 2% aqueous ammonium persulfate solution is added, and 50 hours at 50 ° C. After the reaction, 1 ml of a 2% ammonium persulfate aqueous solution was further added, followed by post-reaction at 70 ° C. for 2 hours. In order to remove a small amount of unreacted acrolein and soluble polymer in the obtained fine particles, only fine particles were precipitated by a centrifuge (1500 rpm × 5 minutes), and the dispersion medium was replaced with water, and the fine particles coated with polyacrolein ( Prepared to a solid content concentration of 10 wt%). The average particle diameter of the obtained fine particles was 1.08 μm, and the amount of polyacrolein was 14.2% by weight based on the silica fine particles.
[0024]
Example 2
Using 10 g of the coupling-treated silica fine particles obtained in Production Example 2, the reaction was carried out in the same manner as in Example 1 to obtain fine particles coated with polyacrolein. The average particle diameter of the obtained fine particles was 1.07 μm, and the amount of polyacrolein was 13.3 wt% with respect to the silica fine particles.
[0025]
Example 3
For 100 g of the fine particles (solid content concentration: 10 wt%) coated with polyacrolein obtained in Example 1, 0.4 g of Kayalon Polyester Rubin GL-SE (manufactured by Nippon Kayaku Co., Ltd.) was dissolved in 100 ml of distilled water. The mixture was mixed, adjusted to pH 5 with acetic acid, and stirred at 95 ° C. for 2 hours. After cooling, the supernatant was replaced with distilled water three times using a centrifuge (1500 rpm × 5 minutes), and then the precipitate was dispersed in 200 ml of distilled water containing 1 ml of acetic acid and stirred at 95 ° C. for 30 minutes. After cooling, the supernatant was replaced with distilled water five times with a centrifuge (1500 rpm × 5 minutes) and washed to obtain fine red-colored fine particles (prepared to a solid content concentration of 10 wt%). .
[0026]
Example 4
100 g of the fine particles coated with polyacrolein obtained in Example 2 (solid content concentration: 10 wt%) were dyed in the same manner as in Example 3 and colored in deep red (prepared to a solid content concentration of 10 wt%) was gotten.
[0027]
Example 5
In the same manner as in Example 3, 0.2 g of Kayanol Milling Red 6BW (manufactured by Nippon Kayaku Co., Ltd.) was used for 100 g of the fine particles (solid content concentration: 10 wt%) coated with polyacrolein obtained in Example 1. Fine particles colored with a deep red color (prepared to a solid content concentration of 10 wt%) were obtained.
[0028]
Test Example A syphilis antibody test method will be described as an example as an application of colored fine particles to a diagnostic reagent. However, it is not limited to these inspection items.
[0029]
1. Preparation of syphilis antigen TP bacteria (Nichols strain) were grown in rabbit testicles, extracted from extracted testicles with citrate buffer, and collected by centrifugal fractionation, and the number of bacteria was 1-2 × 10 ⁹ cells / ml. Resuspended in PBS so that Dilute 5 ml of this suspension of TP bacteria 10 times with PBS and use an ultrasonic crusher (Kubota Shoji Co., Ltd. Model 201) for 20 minutes at 9 kHz and 180 W to obtain an antigen solution containing TP cell components. It was.
[0030]
2. Antigen sensitization, reagent preparation 1 ml of colored fine particles (solid content 10 wt%) was dispersed in 10 ml of PBS, 4 ml of TP antigen solution was added, and the mixture was slowly stirred at 37 ° C. for 30 minutes. Thereafter, 2 wt% bovine serum albumin (abbreviated as BSA) -PBS (10 ml) was further added, and the mixture was slowly stirred at 37 ° C. for 1 hour.
This is separated with a centrifuge (1500 rpm × 5 minutes), the supernatant is discarded, and the precipitate is washed by centrifugation twice with 0.5 wt% BSA-PBS. Thereafter, 40 ml of 0.5 wt% BSA-PBS was added to this sediment and stirred well to obtain a TP antigen-sensitized fine particle dispersion (reagent) having a solid content of 0.25 wt%.
[0031]
As the colored fine particles, the colored fine particles obtained in Examples 3 to 5 and the conventional colored polyacrolein fine particles prepared in the same manner as in Example 4 of JP-A No. 64-148 were used. The obtained reagents were designated as A, B, C, and D, respectively, as follows.
Reagent A: Using colored fine particles of Example 3 (solid content 10 wt%) Reagent B: Using colored fine particles of Example 4 (solid content 10 wt%) Reagent C: Colored fine particles of Example 5 (solid content) Reagent D: Using conventional colored polyacrolein fine particles (solid content: 10 wt%) [0032]
3. Evaluation of sensitized microparticles TP antigen-sensitized microparticles were used for examination by the microtiter method. 100 μl of 0.5 wt% BSA-PBS was added dropwise to the first tube of the U-shaped microplate, and 25 μl of serum was added to the second tube and thereafter, and 25 μl of serum was added to the first tube. This was diluted twice continuously from the first tube with a diluter. TP antigen-sensitized microparticles (reagents A to D) were added dropwise by 25 μl to the second and subsequent tubes, shaken with a plate mixer for 1 minute, and then left to stand. Negative or positive was determined from the aggregate image of the tube bottom. In this case, the final dilution factor was 20 times for the second tube and doubled for the third tube and thereafter, and 40 times was set as the cut-off value in the same manner as TPHA. Those in which aggregation is observed at 80 times or more are positive. The results are shown in Table 1.
[0033]
[Table 1]

[0034]
4). Conclusion As is clear from the above results, the TP antigen-sensitized microparticles using the colored microparticles of the present invention were judged to have the same sensitivity as TPHA using erythrocytes as a carrier, and the judgment time was greatly shortened.
[0035]
【The invention's effect】
By using the microparticles of the present invention, it is possible to provide a diagnostic reagent that has better storage stability than conventional products and has a significantly reduced determination time.

Claims (2)

Silica fine particles treated with a silane coupling agent are coated with polyacrolein and stained with a dye for diagnostic reagents . Microparticles according to claim 1 for use in the microtiter method.