JP3911200B2 - Sensitivity enhancer for immunochromatography measurement method, measurement method and instrument for measurement method - Google Patents
Sensitivity enhancer for immunochromatography measurement method, measurement method and instrument for measurement method Download PDFInfo
- Publication number
- JP3911200B2 JP3911200B2 JP2002151415A JP2002151415A JP3911200B2 JP 3911200 B2 JP3911200 B2 JP 3911200B2 JP 2002151415 A JP2002151415 A JP 2002151415A JP 2002151415 A JP2002151415 A JP 2002151415A JP 3911200 B2 JP3911200 B2 JP 3911200B2
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- Prior art keywords
- group
- sensitivity enhancer
- general formula
- methacrylate
- monomer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- 239000003623 enhancer Substances 0.000 title claims description 35
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- 238000000034 method Methods 0.000 claims description 16
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- CQEYYJKEWSMYFG-UHFFFAOYSA-N butyl acrylate Chemical compound CCCCOC(=O)C=C CQEYYJKEWSMYFG-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N cinnamic acid Chemical compound OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
- SUPCQIBBMFXVTL-UHFFFAOYSA-N ethyl 2-methylprop-2-enoate Chemical compound CCOC(=O)C(C)=C SUPCQIBBMFXVTL-UHFFFAOYSA-N 0.000 description 1
- ZAFFWOKULJCCSA-UHFFFAOYSA-N ethyl 2-methylprop-2-enoate;trimethylazanium;chloride Chemical compound [Cl-].C[NH+](C)C.CCOC(=O)C(C)=C ZAFFWOKULJCCSA-UHFFFAOYSA-N 0.000 description 1
- ZJXZSIYSNXKHEA-UHFFFAOYSA-N ethyl dihydrogen phosphate Chemical compound CCOP(O)(O)=O ZJXZSIYSNXKHEA-UHFFFAOYSA-N 0.000 description 1
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- YMRDXPCIIDUQFY-UHFFFAOYSA-N n-(2-phenylethyl)prop-2-enamide Chemical compound C=CC(=O)NCCC1=CC=CC=C1 YMRDXPCIIDUQFY-UHFFFAOYSA-N 0.000 description 1
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- YPHQUSNPXDGUHL-UHFFFAOYSA-N n-methylprop-2-enamide Chemical compound CNC(=O)C=C YPHQUSNPXDGUHL-UHFFFAOYSA-N 0.000 description 1
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- 125000003935 n-pentoxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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Description
【０００１】
【発明の属する技術分野】
本発明は、梅毒関連抗体を測定対象物質とするイムノクロマト測定法に於ける感度増強剤、当該感度増強剤の共存下で行う、梅毒関連抗体を測定対象物質とするイムノクロマト測定法並びに当該感度増強剤を含有してなる、梅毒関連抗体を測定対象物質とするイムノクロマト測定法用器具に関する。
【０００２】
【従来の技術】
イムノクロマト法は、多孔質膜の毛細管現象を利用したクロマトグラフィーの手法と免疫学的手法を組み合わせた方法であり、迅速且つ簡便に、そして特別な器具を用いることなく免疫測定を行える方法であり、各種抗原や抗体の検出等に用いられている。
【０００３】
しかしながら、この方法は、簡易に測定できる代わりに感度の精度が低く、陽性検体を用いても偽陰性と判定される場合もあり、より高い感度のイムノクロマト測定法が望まれていた。
【０００４】
【発明が解決しようとする課題】
本発明の第１の目的は、従来のものと比較し、より精度が優れたイムノクロマト用感度増強剤を提供することにある。また、本発明の第２の目的は、前記イムノクロマト用感度増強剤を用いた測定法を提供することにある。また更に、本発明の第３の目的は、前記感度増強剤を含んでなるイムノクロマト測定法用器具を提供するものである。
【０００５】
【課題を解決するための手段】
本発明者らは、このような現状に鑑み、鋭意検討した結果、本発明がなされたものである。
即ち、本発明者等は、梅毒関連抗体を測定対象物質とする免疫学的測定法に於ける、公知の感度増強剤に比較して、より強い感度増強効果を有する化合物を見出すべく鋭意研究の結果、上記一般式［１］で示される、ホスホベタイン構造を有する基を側鎖に有するポリマーが目的の性能を有していることを見出し、本発明を完成するに至った。
その手段として以下のものを提供するものである。
【０００６】
（１）下記一般式［１］
【化７】
（式中、Ｒ１〜Ｒ３は夫々独立して水素原子又は水酸基を有していてもよいアルキル基を示し、Ｒ４はアルキレン基を示す。）で表される基を側鎖に有するポリマーを含んでなる、梅毒関連抗体を測定対象物質とするイムノクロマト測定法用感度増強剤。
【０００８】
（２）一般式［１］で表される基を側鎖に有するポリマーの存在下に抗原抗体反応を行わせることを特徴とする、梅毒関連抗体を測定対象物質とするイムノクロマト測定法。
【０００９】
（３）（１）の感度増強剤を含んでなる、梅毒関連抗体を測定対象物質とするイムノクロマト測定法用器具。
【００１０】
【発明の実施の形態】
本発明に於いて、感度増強剤として用いられるポリマーは、上記一般式［１］で表される基を側鎖に有するものであればよく、ホモポリマーでもコポリマーでも特に限定されないが、通常分子量が10,000〜1,000,000、好ましくは10,000〜500,000、より好ましくは50,000〜500,000である。
【００１１】
より具体的には、下記一般式［２］
【化８】
（式中、Ｒ５は、置換基を有していてもよく且つ鎖中に酸素原子を有していてもよいアルキレン基を示し、Ｒ６は水素原子又はメチル基を示し、Ｘは酸素原子又は−ＮＨ−基を示し、Ｒ１〜Ｒ４は前記に同じ。）
で表されるモノマーに基づく構成単位に基づく構成単位を有するものが好ましく挙げられる。
【００１３】
上記一般式［１］又は［２］に於いて、Ｒ１〜Ｒ３で示される水酸基を有していてもよいアルキル基のアルキル基としては、直鎖状、分枝状、環状の何れでもよく、通常炭素数１〜６、好ましくは１〜４、より好ましくは１〜２、更に好ましくは１のものが挙げられ、具体的には、例えばメチル基、エチル基、n-プロピル基、イソプロピル基、n-ブチル基、イソブチル基、sec-ブチル基、tert-ブチル基、n-ペンチル基、イソペンチル基、sec-ペンチル基、tert-ペンチル基、n-ヘキシル基、イソヘキシル基、sec-ヘキシル基、tert-ヘキシル基、シクロプロピル基、シクロヘキシル基、シクロペンチル基等が挙げられ、好ましくはメチル基、エチル基等であり、より好ましくはメチル基等である。
【００１４】
また、水酸基を有するアルキル基としては、上記した如きアルキル基の水素原子の１〜２個、好ましくは１個が水酸基に置換したものが挙げられ、具体的には、例えばヒドロキシメチル基、ヒドロキシエチル基、ヒドロキシ-n-プロピル基、ヒドロキシイソプロピル基、ヒドロキシ-n-ブチル基、ヒドロキシ-イソブチル基、ヒドロキシ-sec-ブチル基、ヒドロキシ-tert-ブチル基、ヒドロキシ-n-ペンチル基、ヒドロキシ-イソペンチル基、ヒドロキシ-sec-ペンチル基、ヒドロキシ-tert-ペンチル基、ヒドロキシ-n-ヘキシル基、ヒドロキシ-イソヘキシル基、ヒドロキシ-sec-ヘキシル基、ヒドロキシ-tert-ヘキシル基、ヒドロキシ-シクロプロピル基、ヒドロキシ-シクロヘキシル基、ヒドロキシ-シクロペンチル基等が挙げられ、好ましくはヒドロキシメチル基、ヒドロキシエチル基等である。
【００１５】
Ｒ４で示されるアルキレン基としては、例えば炭素数１〜６、好ましくは２〜３のものが挙げられ、これらは直鎖状、分枝状、環状の何れでもよい。具体的には、例えばメチレン基、エチレン基、プロピレン基、トリメチレン基、ブチレン基、１−エチルエチレン基、２−メチルトリメチレン基、２−エチルトリメチレン基、へキシレン基、シクロプロピレン基、シクロブチレン基、シクロペンチレン基、シクロへキシレン基等が挙げられ、好ましくはエチレン基、プロピレン基、トリメチレン基等である。
【００１６】
一般式［２］に於いてＲ５で表される、置換基を有していてもよく且つ鎖中に酸素原子を有していてもよいアルキレン基において、酸素を有さない場合のアルキレン基としては、例えば炭素数１〜１０、好ましくは１〜６、より好ましくは２〜６のものが挙げられ、これらは直鎖状、分枝状、環状の何れでもよい。具体的には、例えばメチレン基、エチレン基、プロピレン基、トリメチレン基、ブチレン基、１−エチルエチレン基、２−メチルトリメチレン基、２−エチルトリメチレン基、へキシレン基、シクロプロピレン基、シクロブチレン基、シクロペンチレン基、シクロへキシレン基等が挙げられる。また、その置換基としては、例えば炭素数１〜６、好ましくは１〜３のアルコキシル基〔直鎖状、分枝状、環状の何れにてもよい。〕、より具体的には例えばメトキシ基、エトキシ基、n-プロポキシ基、イソプロポキシ基、n-ブトキシ基、イソブトキシ基、sec-ブトキシ基、tert-ブトキシ基、n-ペンチルオキシ基、イソペンチルオキシ基、sec-ペンチルオキシ基、tert-ペンチルオキシ基、n-ヘキシルオキシ基、イソヘキシルオキシ基、sec-ヘキシルオキシ基、tert-ヘキシルオキシ基、シクロプロポキシ基、シクロヘキシルオキシ基、シクロペンチルオキシ基等、例えばハロゲン原子、より具体的にはフッ素原子、塩素原子、臭素原子、ヨウ素原子等が挙げられ、好ましくはエチレン基、プロピレン基、トリメチレン基、ブチレン基等である。また、鎖中に酸素原子を有する場合、酸素原子としては１〜５個、好ましくは１〜３個であり、より具体的には−（Ｃ2Ｈ4Ｏ）n−Ｃ2Ｈ4−（式中、ｎは１〜５の整数を表す。）等が挙げられる。上記したＲ５で表される、置換基を有していてもよく且つ鎖中に酸素原子を有していてもよいアルキレン基の中でも、エチレン基、プロピレン基が好ましく、エチレン基がより好ましい。
【００１７】
本発明に係る一般式［１］で示される基を側鎖に有するモノマーに基づく構成単位を有するポリマーは、市販されているものを用いてもよいし、例えば特開平１０−４５７９４号公報、特開２０００−２３９６９６号公報等に記載された方法に準じて合成されたものを用いてもよい。
【００１８】
上記一般式［２］で表されるモノマーに基づく構成単位としては、上記した如きＲ1〜Ｒ6を有するものであればよいが、具体的には例えば下記一般式［５］
【００１９】
【化９】
で表されるモノマーに基づく構成単位等が挙げられる。
【００２１】
本発明に係る一般式［２］で表されるモノマーに基づく構成単位を有するポリマーがコポリマーである場合、上記一般式［２］で表されるモノマーに基づく構成単位以外のモノマー単位としては、アクリル酸又はアクリル酸エステル、メタクリル酸又はメタクリル酸エステル、アクリルアミド又はそのＮ置換体、メタクリルアミド又はそのＮ置換体、或いはスチレン又はその誘導体から選ばれるモノマー由来のものが挙げられる。尚、これらモノマー単位は、コポリマー中に２種類以上含まれていてもよい。また、コポリマーにおける、一般式［２］で表されるモノマーにに基づく構成単位の比率は、通常２０％以上１００％未満であり、好ましくは３０〜９５％であり、より好ましくは３０〜９０％である。
【００２２】
上記一般式［２］で表されるモノマーに基づく構成単位以外のモノマー単位としてのアクリル酸エステルとしては、アルキルアクリレート、アラルキルアクリレート等が、メタクリル酸エステルとしては、アルキルメタクリレート、アラルキルメタクリレート等が挙げられ、アクリルアミドのＮ置換体は、Ｎ−アルキルアクリルアミド又はＮ−アラルキルアクリルアミドであり、メタクリルアミドのＮ置換体は、Ｎ−アルキルメタクリルアミド又はＮ−アラルキルメタクリルアミドであり、スチレン誘導体としては、α−メチルスチレン、置換基を有するスチレン又はα−メチルスチレン等が挙げられる。
【００２３】
上記のアルキルアクリレート、アルキルメタクリレート、Ｎ−アルキルアクリルアミド及びＮ−アルキルメタクリルアミドに於けるアルキル基としては、直鎖状、分枝状、環状の何れでもよく、通常炭素数１〜６、より好ましくは、１〜４のものが挙げられ、具体的には、例えばメチル基、エチル基、n-プロピル基、イソプロピル基、n-ブチル基、イソブチル基、sec-ブチル基、tert-ブチル基、n-ペンチル基、イソペンチル基、sec-ペンチル基、tert-ペンチル基、n-ヘキシル基、イソヘキシル基、sec-ヘキシル基、tert-ヘキシル基、シクロプロピル基、シクロヘキシル基、シクロペンチル基等が挙げられる。このアルキル基は置換基を有していてもよく、その置換基としては、例えばヒドロキシル基、炭素数１〜３の低級アルコキシル基、トリアルキルアンモニオ基（アルキル基としては、例えばメチル基、エチル基、プロピル基、イソプロピル基等の炭素数１〜３のものが挙げられる。尚、置換基としてトリアルキルアンモニオ基を有する場合、本置換基はプラスに荷電しているため、通常カウンターアニオンが結合しているが、このようなカウンターアニオンとしては、フッ素イオン、塩素イオン、臭素イオン、ヨウ素イオン等のハロゲン化物イオン等が挙げられる。）等が挙げられる。また、置換基を有するアルキル基としては、例えば以下のような基
【化１０】
（尚、Ｒ７は炭素数1〜３のアルキル基を表し、ｍは１〜１００を表す。）で表されるものも含まれる。
【００２５】
また、アラルキルアクリレート、アラルキルメタクリレート、Ｎ−アラルキルアクリルアミド及びＮ−アラルキルメタクリルアミドに於けるアラルキル基としては、炭素数７〜１０のものが挙げられ、具体的には、例えばベンジル基、フェニルエチル基、フェニルプロピル基、フェニルブチル基等が挙げられる。
【００２６】
スチレン若しくはα−メチルスチレンが有していてもよい置換基としては、例えば直鎖状、分枝状、環状の、通常炭素数１〜６、より好ましくは１〜４のアルキル基（具体的には、例えばメチル基、エチル基、n-プロピル基、イソプロピル基、n-ブチル基、イソブチル基、sec-ブチル基、tert-ブチル基、n-ペンチル基、イソペンチル基、sec-ペンチル基、tert-ペンチル基、n-ヘキシル基、イソヘキシル基、sec-ヘキシル基、tert-ヘキシル基、シクロプロピル基、シクロヘキシル基、シクロペンチル基等）、例えば直鎖状、分枝状、環状の、通常炭素数１〜６、より好ましくは１〜４のアラルキル基（具体的には、例えばメトキシ基、エトキシ基、n-プロポキシ基、イソプロポキシ基、n-ブトキシ基、イソブトキシ基、sec-ブトキシ基、tert-ブトキシ基、n-ペンチルオキシ基、イソペンチルオキシ基、sec-ペンチルオキシ基、tert-ペンチルオキシ基、n-ヘキシルオキシ基、イソヘキシルオキシ基、sec-ヘキシルオキシ基、tert-ヘキシルオキシ基、シクロプロポキシ基、シクロヘキシルオキシ基、シクロペンチルオキシ基等）、例えばフッ素原子、塩素原子、臭素原子、ヨウ素原子等のハロゲン原子、カルボキシル基、ヒドロキシ基、アミノ基等が挙げられる。
【００２７】
上記一般式［２］で表されるモノマーに基づく構成単位以外のモノマー単位の具体例としては、例えばメタクリル酸、メタクリル酸メチル、メタクリル酸エチル、メタクリル酸プロピル、メタクリル酸ブチル、メタクリル酸ドデシル、メタクリル酸オクタデシル、メタクリル酸2-エチルヘキシル、メタクリル酸ラウリル、メタクリル酸ステアリル、メタクリル酸2-トリメチルアンモニオエチル、メタクリル酸ベンジル、メタクリル酸フェニルエチル、アクリル酸、アクリル酸メチル、アクリル酸エチル、アクリル酸ブチル、アクリル酸2-エチルヘキシル、アクリル酸ラウリル、アクリル酸ステアリル、アクリル酸2-トリメチルアンモニオエチル、アクリル酸ベンジル、アクリル酸フェニルエチル、アクリルアミド、Ｎ−メチルアクリルアミド、Ｎ−エチルアクリルアミド、Ｎ−ブチルアクリルアミド、Ｎ−2-エチルヘキシルアクリルアミド、Ｎ−ラウリルアクリルアミド、Ｎ−ステアリルアクリルアミド、Ｎ−2-トリメチルアンモニオエチルアクリルアミド、Ｎ−ベンジルアクリルアミド、Ｎ−フェニルエチルアクリルアミド、メタクリルアミド、Ｎ−メチルメタクリルアミド、Ｎ−エチルメタクリルアミド、Ｎ−ブチルメタクリルアミド、Ｎ−2-エチルヘキシルメタクリルアミド、Ｎ−ラウリルメタクリルアミド、Ｎ−ステアリルメタクリルアミド、Ｎ−2-トリメチルアンモニオエチルメタクリルアミド、Ｎ−ベンジルメタクリルアミド、Ｎ−フェニルエチルメタクリルアミド、スチレン、カルボキシスチレン、ヒドロキシスチレン、アミノスチレン、メチルスチレン、エチルスチレン、メトキシスチレン、エトキシスチレン、クロロスチレン、ブロモスチレン、α−メチルスチレン、α−メチル−カルボキシスチレン、α−メチル−ヒドロキシスチレン、α−メチル−アミノスチレン、α−メチル−メチルスチレン、α−メチル−エチルスチレン、α−メチル−メトキシスチレン、α−メチル−エトキシスチレン、α−メチル−クロロスチレン、α−メチル−ブロモスチレン、N,N,N-トリエチルアンモニウムエチルメタクリレートブロミド、N,N,N-トリメチルアンモニウムエチルメタクリレートクロリド、N,N,-ジエチル−Ｎ−プロピルアンモニウムエチルメタクリレートブロミド、N,N,N-トリメチルアンモニウム−２−ヒドロキシプロピルメタクリレートクロリド（ＱＭ）、N,N,N-トリメチルアンモニウムメチルスチレンブロミド等由来のものが挙げられ、また、下記一般式［４］
【００２８】
【化１１】
（式中、ｍは１〜１００を表す。）で表されるもの等も具体例として挙げられる。
【００３０】
上記した中でも、メタクリル酸、メタクリル酸ステアリル、メタクリル酸ベンジル、メタクリル酸ブチル、メタクリル酸ドデシル、メタクリル酸オクタデシルN,N,N-トリメチルアンモニウム−２−ヒドロキシプロピルメタクリレートクロリド（ＱＭ）等由来のもの、一般式［４］で表されるもの等が好ましい。
【００３１】
本発明の感度増強剤は、上記した如き一般式［１］で表される基を側鎖に有するポリマーを含んでなるものであり、好ましくは一般式［２］で表されるモノマーに基づく構成単位を有するポリマーである。該感度増強剤は、イムノクロマト測定法に於いて用いられる各種試薬や試料に溶解させて用いてもよく、また、イムノクロマト測定法用器具に担持させて用いてもよいが、操作の簡便さから後者の方がより好ましい。
【００３２】
本発明のイムノクロマト測定法で用いられるイムノクロマト測定法用器具としては、例えば（１）展開膜からなるもの、（２）検体標識部並びに展開膜からなり、且つ該検体標識部と該展開膜とが毛管現象により移動可能なように形成されたもの、（３）検体標識部、展開膜並びに液体吸収部からなり、且つ該検体標識部と該展開膜と該液体吸収部とが、この順に毛管現象により移動可能なように形成されたもの、（４）検体滴下部、検体標識部、展開膜並びに液体吸収部からなり、且つ該検体滴下部と該検体標識部と該展開膜と該液体吸収部とが、この順に毛管現象により移動可能なように形成されたもの等が挙げられる。
【００３３】
上記展開膜には測定部が設けられており、当該測定部は、測定対象物質と結合能を有する物質（以下、測定対象結合物質と略記する場合がある。）が固定化されて担持された部分であり、数種の測定対象物質を同時に測定する場合には、互いに重なりを持たないように数種の測定対象結合物質が、展開膜上に、例えば線状、或いはスポット状に固定化されて担持されてなる。また、上記検体標識部は、標識された測定対象結合物質を、毛管現象により移動可能な状態で保持してなるものであり、数種の測定対象物質を同時に測定する場合には、数種の標識された測定対象結合物質を、毛管現象により移動可能な状態でに保持してなる。さらに、検体滴下部は、検体滴下用に展開膜又は検体標識部と重なるように形成される部位であり、液体吸収部は、検体の毛管現象による移動をし易くするために設けられるものであり、展開膜に重なるように形成される部位を表す。
【００３４】
上記した如き器具は、市販品を用いても、公知の方法により作製したものを用いてもよい。公知の方法により作製する場合、上記展開膜で用いられる基材としては、通常この分野で用いられるものを用いればよく、例えばセルロース、ニトロセルロース、ナイロン等が好ましいものとして挙げられる。尚、上記検体標識部、検体滴下部及び液体吸収部で用いられる基材も、これらに準じたものを用いればよい。また、上記展開膜上には、非特異的な吸着による測定への影響を防止するために、所謂ブロッキング処理を施してもよい。このようなブロッキング処理は、通常この分野で用いられているブロッキング剤を通常この分野で行われる方法により行えばよい。検体標識部に保持される標識された測定対象結合物質としては、測定対象物質である抗体に対する抗原に標識物質を担持させたもの等が挙げられる。その標識物質としては、通常この分野で用いられているものであれ何れでもよいが、金コロイド、セレニウムコロイド、着色ラテックス等の視覚的に検知し得るシグナルが得られる標識物質が好ましく、特に取扱いのし易さや感度等の点から金コロイドがより好ましい。上記した如き標識物質は、常法により調製されたものを用いても、市販品を用いてもよく、その使用濃度も、この分野で通常用いられる濃度範囲で用いればよい。
【００３５】
本発明の感度増強剤を、イムノクロマト測定法用器具に担持させて用いる場合、その量は、担持される場所、使用する感度増強剤の種類、使用する測定対象物質の種類、使用する標識物質等により変動するが、例えばイムノクロマト測定法用器具の展開膜、検体標識部、検体滴下部等に担持させる場合、単位面積（cm2）当たりに含まれる感度増強剤の量として、通常0.01μg〜10ｍｇ、好ましくは0.1μg〜４ｍｇ、より好ましくは1〜800μgである。また、担持される面積は、用いられるイムノクロマト測定法用器具の種類及び大きさ、測定用試料の量により変動するが、イムノクロマト測定法用器具の展開膜の場合には、通常総面積の10〜60％、好ましくは20〜30％、検体標識部の場合には、通常総面積の1〜30％、好ましくは5〜15％、検体滴下部の場合には、通常総面積の5〜40％、好ましくは10〜20％である。また、イムノクロマト測定法用器具に於いて担持される部位としては、測定用試料と接触し得る部位、即ち測定用試料が通液される部位であればよく、具体的には展開膜、展開膜上の測定部位等が挙げられ、好ましくは測定部位である。尚、本発明の感度増強剤を担持させる方法としては、通常この分野で用いられる方法であればよく、例えば展開膜に上記した如き感度増強剤を含有する溶液を、例えば塗布、滴下或いは噴霧等した後、これを乾燥して物理的吸着により担持させる方法、例えば展開膜等作製時に用いられる緩衝液や洗浄液等に本発明の感度増強剤を溶解させ、これらを用いて展開膜を作製することにより担持させる方法等が挙げられる。
【００３６】
本発明に係るイムノクロマト測定法に用いられるイムノクロマト測定法用試薬は、本発明の感度増強剤以外に、例えば、測定対象物質である抗体に対する抗原若しくは標識物質を担持してなる抗原等を含有していてもよい。尚、この際用いられる標識物質としては、上記のイムノクロマト測定法用器具で用いられるものと同じものが挙げられ、その濃度、調製方法等も同様に行えばよい。更に、該反応試薬中には、緩衝剤（例えばトリス緩衝剤、リン酸緩衝剤、ベロナール緩衝剤、ホウ酸緩衝剤、グッド緩衝剤等）、安定化剤（例えばアルブミン、グロブリン、水溶性ゼラチン、界面活性剤、糖類等）、防腐剤（例えばサリチル酸、安息香酸、アジ化ナトリウム等）、その他この分野で用いられているものであって、共存する試薬等の安定性を阻害したり、抗原抗体反応を阻害しないものを含有していてもよい。またその使用濃度も、通常この分野で通常用いられる濃度範囲で用いればよい。
【００３７】
本発明の感度増強剤を、各種試薬や試料中に溶解させて用いる場合、その濃度は、抗原抗体反応がなされる際の濃度として、通常0.1〜20w/v％、好ましくは0.1〜10w/v％、より好ましくは0.1〜５w/v％となるように用いられる。
【００３８】
また、本発明のイムノクロマト測定法を実施するには、本発明に係る一般式［１］で示される基を側鎖に有するポリマーを上記した如き濃度試薬中に共存させて行うか、又は本発明に係る一般式［１］で示される基を側鎖に有するポリマーを上記した濃度イムノクロマト法用器具に担持させる以外は、自体公知のイムノクロマト測定法に於いて用いられる各種試薬を用い、自体公知の操作法に準じて行えばよい。
【００３９】
本発明のイムノクロマト測定法の測定対象物質としては、例えば抗リン脂質抗体、抗トレポネーマ抗体等の梅毒関連抗体である。
【００４０】
本発明の測定方法で用いられる本発明の感度増強剤は、何れの測定対象物質であっても感度増強は見られるが、測定対象物質によりその好ましいものが異なる。例えば、測定対象物質が抗リン脂質抗体の場合、例えば上記一般式［２］で表されるモノマーに基づく構成単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸ブチル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸ベンジル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位と上記一般式［４］で表されるモノマーに基づく構成単位からなるポリマー等が好ましく挙げられ、中でも例えば上記一般式［２］で表されるモノマーに基づく構成単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸ブチル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位と上記一般式［４］で表されるモノマーに基づく構成単位からなるポリマー等がより好ましいものとして挙げられる。また、例えば測定対象物質が抗TP（Treponema Pallidum）抗体の場合、例えば上記一般式［２］で表されるモノマーに基づく構成単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸ブチル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸オクタデシル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸ベンジル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位と上記一般式［４］で表されるモノマーに基づく構成単位からなるポリマー等が好ましく挙げられ、中でも例えば上記一般式［２］で表されるモノマーに基づく構成単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸ブチル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位とメタクリル酸ベンジル由来のモノマー単位からなるポリマー、上記一般式［２］で表されるモノマーに基づく構成単位と上記一般式［４］で表されるモノマーに基づく構成単位からなるポリマー等がより好ましいものとして挙げられる。
【００４１】
また、本発明のイムノクロマト測定法用器具は、上記一般式［１］で示される基を側鎖に有するポリマーを、試料滴下部、試料標識部、展開膜（測定部）等の試料検体が滴下、展開等通液する場所に担持させたものであり、好ましくは上記一般式［１］で示される基を側鎖に有するポリマーを展開膜に添加したもの、上記一般式［１］で示される基を側鎖に有するポリマーを試料標識部に添加したもの、上記一般式［１］で示される基を側鎖に有するポリマーを測定部に添加したもの等が挙げられる。器具の構成要素の好ましい態様、具体例、使用濃度等は、イムノクロマト測定法で用いられるもので述べた通りである。
【００４２】
以下実施例及び比較例によって本発明を説明するが、本発明はこれによって限定されるものでない。
【００４３】
また、実施例及び比較例に於けるＭＰＣポリマーは、例えば特開2001-228149号公報記載の方法に準じて以下の１種又は２種のモノマーから合成したポリマーを表す。
ＰＭＰＣ：２−(メタクリロイルオキシ)エチル−２’−(トリメチルアンモニオ)エチルホスフェート（以下、ＭＰＣモノマーと略記する。）からなるポリマー、分子量；1030×103
ＰＭＢ８２：ＭＰＣモノマーとメタクリル酸ブチルとからなるコポリマー、分子量；600×103、ＭＰＣモノマーとメタクリル酸ブチルの構成比；８：２
ＰＭＢ８２−１Ｍ：ＭＰＣモノマーとメタクリル酸ブチルとからなるコポリマー、分子量；1210×103、ＭＰＣモノマーとメタクリル酸ブチルの構成比；８：２
ＰＭＢ３７：ＭＰＣモノマーとメタクリル酸ブチルとからなるコポリマー、、分子量；93×103、ＭＰＣモノマーとメタクリル酸ブチルの構成比；３：７
ＰＭＥ４９１：ＭＰＣモノマーとモノメチルエーテルポリエチレングリコールメタクリレート（一般式［４］に於いてｍ＝４のモノマー）とからなるコポリマー、分子量；501×103、ＭＰＣモノマーとモノメチルエーテルポリエチレングリコールメタクリレートの構成比；９：１
ＰＭＥ４７３：ＭＰＣモノマーとモノメチルエーテルポリエチレングリコールメタクリレート（一般式［４］に於いてｍ＝４のモノマー）とからなるコポリマー、分子量；138×103、ＭＰＣモノマーとモノメチルエーテルポリエチレングリコールメタクリレートの構成比；７：３
ＰＭＣ18９１：ＭＰＣモノマーとメタクリル酸オクタデシルとからなるポリマー、分子量；130×103、ＭＰＣモノマーとメタクリル酸オクタデシルの構成比；９：１
ＰＭＣ18８２：ＭＰＣモノマーとメタクリル酸オクタデシルとからなるポリマー、分子量；43×103、ＭＰＣモノマーとメタクリル酸オクタデシルの構成比；８：２
ＰＭＣ12８２：ＭＰＣモノマーとメタクリル酸ドデシルとからなるポリマー、分子量；184×103、ＭＰＣモノマーとメタクリル酸オクタデシルの構成比；８：２
ＰＭＢｚ８２：ＭＰＣモノマーとメタクリル酸ベンジルとからなるポリマー、分子量；240×103、ＭＰＣモノマーとメタクリル酸オクタデシルの構成比；８：２
【００４４】
実施例１
（1）TP(Treponema Pallidum、トレポネーマパリダム)抗原及びリン脂質抗原の標識
先ず、TP抗原（1.0mg／ml）8μlを遠心管に分注し、次いでそこに平均粒径35nm、Amax＝1.2の金コロイド懸濁液5mlを注ぎ室温で30分間反応させた後、遠心分離（8000G、15分間）して金コロイド標識TP抗原を回収した。尚、TP抗原は、公知のリコンビナント抗原作製法（Infection and Immunity, volume 54, p500-506, Michael V. Norgard）に従って作製したものを用いた。
【００４５】
リン脂質抗原液は、5mg/mlカルジオライピン（牛心臓由来、シグマ社製）エタノール溶液、10mg/ml フォスファチジルコリン（卵黄由来、キューピー社製）エタノール溶液をそれぞれ2ml、0.5ml（重量比２:１）ずつ混合したものを調製して用いた。
【００４６】
調製したリン脂質抗原液0.25mlに前処理済み金コロイド懸濁液（平均粒径35nm、Amax＝20.0）0.8mlを添加し、室温で2時間転倒混和した。その後、ブロッキング液（１％BSA、２％スクロースを含む50mM モルホリノエタンスルホン酸(MES）緩衝液）30mlを加え15分間放置し、遠心分離（8000G、15分間）して金コロイド標識リン脂質抗原を回収した。
【００４７】
（２）検体滴下部の作製
先ず、ガラス繊維シートを、0.5％BSA及び1％ スクロースを含む25mMリン酸緩衝液にて15分間マスキングし、さらに1％ ポリ（１−ビニルピロリドンコビニルアセテート）［poly(1-vinylpyrrolidone-co-vinyl acetate)、以下、poly-PAと略記する。(アルドリッチ社製)]及び1％ スクロースを含む25ｍＭリン酸緩衝液で洗浄し乾燥させ、検体滴下部とした。
【００４８】
（３）検体標識部の作製
（１）で作製した金コロイド標識TP抗原をAmax＝1.3となるように、金コロイド標識リン脂質抗原をAmax＝1.4となるように添加した、1％ poly-PA及び１％ スクロースを含む50mM MES緩衝液を、検体滴下部に5mm×200mm当り500μl含浸させた後、50℃で乾燥し、検体標識部とした。
【００４９】
（４）展開膜の作製
リコンビナントTP抗原（1.0mg／ml）40μl及び10％ スクロースを含む250mMリン酸緩衝液20μlを混合したものをTP抗原液とした。
【００５０】
また、5mg/mlカルジオライピン（牛心臓由来、シグマ社製）エタノール溶液125μl、10mg/mlフォスファチジルコリン（卵黄由来、日本油脂（株）製）エタノール溶液250μl及び20mg/mlコレステロール（和光純薬工業（株）製）エタノール溶液125μlからなるリン脂質抗原溶液をエバポレーターで溶媒留去し脂質薄膜を形成させた。2時間真空乾燥した後、該脂質薄膜に100mMホウ酸緩衝液 500μlを加えて懸濁し、該懸濁液を更に超音波処理したものをリポソーム懸濁液とした。
次いで、TP抗原液とリポソーム懸濁液を夫々ナイロン膜(20mm×200mm)の別の部分へ0.25μl/cmで線塗布し(200mm)、50℃で5分間乾燥した。次いで、１％BSA及び２%スクロースを含む50mM MES緩衝液中に15分間浸した。さらに、１％Poly-PA、２％スクロースを含む50mM MES緩衝液にて2回洗浄した後、50℃で15分間乾燥したものを、抗TP抗体・抗リン脂質抗体同時検出用の測定部を有する展開膜とした。
【００５１】
（５）測定用器具の組立て
両面テープをラミネートしたポリエチレンテレフタレート（ＰＥＴ）シート（8cm×20cm）の下端より1.2cmの部位に（４）で作製した展開膜を貼り付け、該展開膜上端に1mm重なるように吸収部としてガラス繊維シート（４cm×20cm）を貼り合わせた。次いで、展開膜下端に1mm重なるように検体標識部を貼り、最後に検体滴下部をＰＥＴシート下端に検体標識部と重ねて張り合わせたシートを4mm幅に切断したものをTP抗体・リン脂質抗体検出用器具とした。得られた器具は除湿条件下にて室温で保存した。模式図を図１に示す。尚、図１に於いて各数字は夫々以下のものを示す。
【００５２】
１：支持体
２：検体滴下部
３：検体標識部（標識リン脂質抗原及び標識梅毒抗原を保持した部分）
４：展開膜
５：リン脂質抗原が固定化された担持部（第１測定部）
６：TP抗原担持部（第２測定部）
７：液体吸収部
【００５３】
（６）測定
（５）で作製した試験用具の検体滴下部に、表１に記載の各種濃度の各種MPCポリマーを0.5〜3%添加した検体50〜100μlを滴下して、15分放置した。その後、測定ラインの有無で抗TP抗体・抗リン脂質抗体の測定を行った。その結果を表１に示す。尚、表中、＋は着色をしているもの、＋ｗは薄い着色をしているもの、±は着色をしていると思われるもの、×は着色をしていないものを表す。
【００５４】
比較例１
検体にMPCポリマーを添加する代わりに、ポリビニルピロリドン（PVP）、ポリビニルアルコール（PVA）又はポリエチレングリコール（PEG）を添加した以外は実施例１と同様に実験を行った。その結果を表１に併せて示す。
【００５５】
【表１】
この結果から、ＭＰＣポリマーの種類によりその効果は多少異なるものの、試料に各種ＭＰＣポリマーを添加することにより、抗TP抗体又は／及び抗リン脂質抗体の判定部における判定ラインの増強が認められ、ＭＰＣポリマー無添加の試料では検出できなかった弱陽性検体が検出できるようになることが分かった。
【００５７】
一方、PVP、PVA、PEGを添加しても、弱陽性検体を検出することはできなかった。
【００５８】
実施例２
さらに、MPCポリマーを添加する場所を変えて以下の実験を行った。
（１）MPCポリマーを検体滴下部作製時の洗浄液に添加した場合
実施例１（２）で検体滴下部を作製する際に、1％ poly-PA(アルドリッチ社製)及び1％ スクロースを含む25ｍＭリン酸緩衝液中に、表１に記載の各種MPCポリマーを0.5〜3%添加させた以外は、実施例１と同様に試験用具を作製した。また、測定は、検体にMPCポリマーを添加しなかった以外は実施例１と同様にして、実験を行った。
【００５９】
（２）MPCポリマーを検体標識部作製時の緩衝液に添加した場合
実施例１（３）で検体標識部を作製する際に、金コロイド標識TP抗原及び金コロイド標識リン脂質抗原を所定濃度含有する1％ poly-PA(アルドリッチ社製)及び１％ スクロースを含む50mM MES緩衝液に、表１に記載の各種MPCポリマーを0.5〜3%添加させた以外は、実施例１と同様に試験用具を作製した。また、測定は、検体にMPCポリマーを添加しなかった以外は実施例１と同様に行った。
（３）MPCポリマーを展開膜作製時の洗浄溶液に添加した場合
実施例１（４）で展開膜を作製する際に、1％ poly-PA(アルドリッチ社製)及び２％ スクロースを含む50mM MES緩衝液に、表１に記載の各種濃度の各種MPCポリマーを0.5〜3%添加させた以外は、実施例１と同様に試験用具を作製した。また、測定は、検体にMPCポリマーを添加しなかった以外は実施例１と同様に行った。
【００６０】
上記した如きMPCポリマー添加場所を変えた実験を行った結果、表１と同じ結果が得られた。
【００６１】
よって、MPCポリマーは、試料中にのみではなく、イムノクロマト測定用器具の試料が通液する場所の何れかに添加されることでも、その感度増強効果を発揮することが分かった。
【００６２】
【発明の効果】
以上述べたことから明らかな如く、本発明は、従来公知の梅毒関連抗体を測定対象物質とするイムノクロマト測定法で用いられていた感度増強剤に比較して、著しく効果が優れているものを提供するものであり、該感度増強剤を用いるイムノクロマト測定法により、更に精度の高い測定を可能にするものである。
【図面の簡単な説明】
【図１】本発明のイムノクロマト測定法用器具の模式図を表す。
【符号の説明】
１：支持体
２：検体滴下部
３：検体標識部（標識リン脂質抗原及び標識梅毒抗原を保持した部分）
４：展開膜
５：リン脂質抗原が固定化された担持部（第１測定部）
６：TP抗原担持部（第２測定部）
７：液体吸収部[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sensitivity enhancer in an immunochromatography measurement method using a syphilis-related antibody as a measurement target substance, an immunochromatography measurement method using a syphilis-related antibody as a measurement target substance in the presence of the sensitivity enhancement agent, and the sensitivity enhancement agent. The present invention relates to an instrument for an immunochromatographic assay using a syphilis-related antibody as a measurement target substance.
[0002]
[Prior art]
The immunochromatography method is a method that combines a chromatographic method using capillary action of a porous membrane and an immunological method, and is a method that can perform immunoassay quickly and easily without using a special instrument. It is used for detection of various antigens and antibodies.
[0003]
However, this method can be easily measured, but has low sensitivity accuracy and may be determined to be false negative even if a positive sample is used. Thus, an immunochromatographic measurement method with higher sensitivity has been desired.
[0004]
[Problems to be solved by the invention]
The first object of the present invention is to provide an immunochromatographic sensitivity enhancer that is more accurate than conventional ones. A second object of the present invention is to provide a measurement method using the immunochromatographic sensitivity enhancer. Furthermore, a third object of the present invention is to provide an instrument for immunochromatographic measurement comprising the sensitivity enhancer.
[0005]
[Means for Solving the Problems]
The present inventors have made the present invention as a result of intensive studies in view of such a current situation.
That is, the present inventors have intensively studied to find a compound having a stronger effect of enhancing the sensitivity compared to known sensitivity enhancers in an immunological assay using a syphilis-related antibody as a measurement target substance. As a result, the present inventors have found that the polymer represented by the above general formula [1] having a group having a phosphobetaine structure in the side chain has the target performance, and has completed the present invention.
The following are provided as the means.
[0006]
(1) The following general formula [1]
[Chemical 7]
(Wherein R1~ R3Each independently represents a hydrogen atom or an alkyl group optionally having a hydroxyl group, R4Represents an alkylene group. A sensitivity enhancer for an immunochromatographic assay using a syphilis-related antibody as a substance to be measured, comprising a polymer having a group represented by) in the side chain.
[0008]
(2) An immunochromatographic measurement method using a syphilis-related antibody as a measurement target substance, wherein an antigen-antibody reaction is performed in the presence of a polymer having a group represented by the general formula [1] in the side chain.
[0009]
(3) An instrument for an immunochromatographic assay using a syphilis-related antibody, which comprises the sensitivity enhancer of (1).
[0010]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the polymer used as the sensitivity enhancer is not particularly limited as long as it has a group represented by the general formula [1] in its side chain, and is not particularly limited as a homopolymer or a copolymer. It is 10,000 to 1,000,000, preferably 10,000 to 500,000, more preferably 50,000 to 500,000.
[0011]
More specifically, the following general formula [2]
[Chemical 8]
(Wherein R5Represents an alkylene group which may have a substituent and may have an oxygen atom in the chain;6Represents a hydrogen atom or a methyl group, X represents an oxygen atom or a —NH— group, R1~ R4Is the same as above. )
Those having a structural unit based on a structural unit based on a monomer represented by
[0013]
In the general formula [1] or [2], R1~ R3The alkyl group of the alkyl group optionally having a hydroxyl group may be linear, branched or cyclic, and usually has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 To 2, more preferably 1, specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group N-pentyl group, isopentyl group, sec-pentyl group, tert-pentyl group, n-hexyl group, isohexyl group, sec-hexyl group, tert-hexyl group, cyclopropyl group, cyclohexyl group, cyclopentyl group, etc. Preferably a methyl group, an ethyl group, and the like, and more preferably a methyl group and the like.
[0014]
Examples of the alkyl group having a hydroxyl group include those in which 1 to 2, preferably 1 of the hydrogen atoms of the alkyl group as described above are substituted with a hydroxyl group. Specific examples thereof include a hydroxymethyl group and a hydroxyethyl group. Group, hydroxy-n-propyl group, hydroxyisopropyl group, hydroxy-n-butyl group, hydroxy-isobutyl group, hydroxy-sec-butyl group, hydroxy-tert-butyl group, hydroxy-n-pentyl group, hydroxy-isopentyl group , Hydroxy-sec-pentyl group, hydroxy-tert-pentyl group, hydroxy-n-hexyl group, hydroxy-isohexyl group, hydroxy-sec-hexyl group, hydroxy-tert-hexyl group, hydroxy-cyclopropyl group, hydroxy-cyclohexyl Group, hydroxy-cyclopentyl group, etc., preferably hydroxymethyl group, Dorokishiechiru is a group, and the like.
[0015]
R4Examples of the alkylene group represented by are those having 1 to 6 carbon atoms, preferably 2 to 3 carbon atoms, which may be linear, branched or cyclic. Specifically, for example, methylene group, ethylene group, propylene group, trimethylene group, butylene group, 1-ethylethylene group, 2-methyltrimethylene group, 2-ethyltrimethylene group, hexylene group, cyclopropylene group, cyclohexane A butylene group, a cyclopentylene group, a cyclohexylene group and the like can be mentioned, and an ethylene group, a propylene group, a trimethylene group and the like are preferable.
[0016]
In the general formula [2], R5In the alkylene group which may have a substituent and may have an oxygen atom in the chain, the alkylene group in the case of having no oxygen, for example, has 1 to 10 carbon atoms, Preferably 1-6, More preferably, 2-6 is mentioned, These may be any of linear, branched, and cyclic. Specifically, for example, methylene group, ethylene group, propylene group, trimethylene group, butylene group, 1-ethylethylene group, 2-methyltrimethylene group, 2-ethyltrimethylene group, hexylene group, cyclopropylene group, cyclohexane Examples include butylene, cyclopentylene, cyclohexylene and the like. The substituent may be, for example, an alkoxyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms [straight, branched, or cyclic. More specifically, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy Group, sec-pentyloxy group, tert-pentyloxy group, n-hexyloxy group, isohexyloxy group, sec-hexyloxy group, tert-hexyloxy group, cyclopropoxy group, cyclohexyloxy group, cyclopentyloxy group, etc. For example, a halogen atom, more specifically a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like can be mentioned, and an ethylene group, a propylene group, a trimethylene group, a butylene group and the like are preferable. Moreover, when it has an oxygen atom in a chain | strand, as an oxygen atom, it is 1-5 pieces, Preferably it is 1-3 pieces, More specifically,-(C2HFourO)n-C2HFour-(Wherein n represents an integer of 1 to 5). R mentioned above5Among the alkylene groups which may have a substituent and may have an oxygen atom in the chain, an ethylene group and a propylene group are preferable, and an ethylene group is more preferable.
[0017]
As the polymer having a structural unit based on a monomer having a group represented by the general formula [1] in the side chain according to the present invention, a commercially available polymer may be used, for example, JP-A-10-45794, You may use what was synthesize | combined according to the method described in the open 2000-239696 gazette.
[0018]
Examples of the structural unit based on the monomer represented by the general formula [2] include R as described above.1~ R6Specifically, for example, the following general formula [5]
[0019]
[Chemical 9]
The structural unit etc. based on the monomer represented by these are mentioned.
[0021]
When the polymer having the structural unit based on the monomer represented by the general formula [2] according to the present invention is a copolymer, the monomer unit other than the structural unit based on the monomer represented by the general formula [2] may be acrylic. Examples include those derived from monomers selected from acids or acrylic esters, methacrylic acid or methacrylic esters, acrylamide or N-substituted products thereof, methacrylamide or N-substituted products thereof, or styrene or derivatives thereof. Two or more kinds of these monomer units may be contained in the copolymer. The proportion of the structural units based on the monomer represented by the general formula [2] in the copolymer is usually 20% or more and less than 100%, preferably 30 to 95%, more preferably 30 to 90%. It is.
[0022]
Examples of the acrylic acid ester as a monomer unit other than the structural unit based on the monomer represented by the general formula [2] include alkyl acrylate and aralkyl acrylate, and examples of the methacrylic acid ester include alkyl methacrylate and aralkyl methacrylate. The N-substituted product of acrylamide is N-alkylacrylamide or N-aralkylacrylamide, the N-substituted product of methacrylamide is N-alkylmethacrylamide or N-aralkylmethacrylamide, and styrene derivatives include α-methyl Examples include styrene, styrene having a substituent, and α-methylstyrene.
[0023]
The alkyl group in the above alkyl acrylate, alkyl methacrylate, N-alkyl acrylamide and N-alkyl methacrylamide may be linear, branched or cyclic, and usually has 1 to 6 carbon atoms, more preferably 1-4, specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n- Examples thereof include a pentyl group, an isopentyl group, a sec-pentyl group, a tert-pentyl group, an n-hexyl group, an isohexyl group, a sec-hexyl group, a tert-hexyl group, a cyclopropyl group, a cyclohexyl group, and a cyclopentyl group. The alkyl group may have a substituent, and examples of the substituent include a hydroxyl group, a lower alkoxyl group having 1 to 3 carbon atoms, and a trialkylammonio group (the alkyl group includes, for example, a methyl group, an ethyl group, Group having 1 to 3 carbon atoms such as a propyl group, an isopropyl group, etc. In addition, when the substituent has a trialkylammonio group, since this substituent is positively charged, Such counter anions include halide ions such as fluorine ions, chlorine ions, bromine ions, iodine ions, etc.). Examples of the alkyl group having a substituent include the following groups:
Embedded image
(In addition, R7Represents an alkyl group having 1 to 3 carbon atoms, and m represents 1 to 100. ) Are also included.
[0025]
Examples of the aralkyl group in aralkyl acrylate, aralkyl methacrylate, N-aralkyl acrylamide and N-aralkyl methacrylamide include those having 7 to 10 carbon atoms, specifically, for example, benzyl group, phenylethyl group, A phenylpropyl group, a phenylbutyl group, etc. are mentioned.
[0026]
Examples of the substituent that styrene or α-methylstyrene may have include, for example, linear, branched, and cyclic alkyl groups having 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms (specifically, Are, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert- Pentyl group, n-hexyl group, isohexyl group, sec-hexyl group, tert-hexyl group, cyclopropyl group, cyclohexyl group, cyclopentyl group, etc.), for example, linear, branched, cyclic, usually having 1 to 1 carbon atoms 6, more preferably 1 to 4 aralkyl groups (specifically, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group) , N-pe Nthyloxy group, isopentyloxy group, sec-pentyloxy group, tert-pentyloxy group, n-hexyloxy group, isohexyloxy group, sec-hexyloxy group, tert-hexyloxy group, cyclopropoxy group, cyclohexyloxy group And cyclopentyloxy group), for example, halogen atoms such as fluorine atom, chlorine atom, bromine atom and iodine atom, carboxyl group, hydroxy group and amino group.
[0027]
Specific examples of the monomer unit other than the structural unit based on the monomer represented by the general formula [2] include, for example, methacrylic acid, methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, dodecyl methacrylate, methacrylic acid. Octadecyl acid, 2-ethylhexyl methacrylate, lauryl methacrylate, stearyl methacrylate, 2-trimethylammonioethyl methacrylate, benzyl methacrylate, phenylethyl methacrylate, acrylic acid, methyl acrylate, ethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, lauryl acrylate, stearyl acrylate, 2-trimethylammonioethyl acrylate, benzyl acrylate, phenylethyl acrylate, acrylamide, N-methylacrylamide, -Ethylacrylamide, N-butylacrylamide, N-2-ethylhexylacrylamide, N-laurylacrylamide, N-stearylacrylamide, N-2-trimethylammonioethylacrylamide, N-benzylacrylamide, N-phenylethylacrylamide, methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, N-butylmethacrylamide, N-2-ethylhexylmethacrylamide, N-laurylmethacrylamide, N-stearylmethacrylamide, N-2-trimethylammonioethylmethacrylamide, N -Benzylmethacrylamide, N-phenylethylmethacrylamide, styrene, carboxystyrene, hydroxystyrene, aminostyrene, methylstyrene, ethylstyrene Methoxystyrene, ethoxystyrene, chlorostyrene, bromostyrene, α-methylstyrene, α-methyl-carboxystyrene, α-methyl-hydroxystyrene, α-methyl-aminostyrene, α-methyl-methylstyrene, α-methyl-ethyl Styrene, α-methyl-methoxystyrene, α-methyl-ethoxystyrene, α-methyl-chlorostyrene, α-methyl-bromostyrene, N, N, N-triethylammonium ethyl methacrylate bromide, N, N, N-trimethylammonium Ethyl methacrylate chloride, N, N, -diethyl-N-propylammonium ethyl methacrylate bromide, N, N, N-trimethylammonium-2-hydroxypropyl methacrylate chloride (QM), N, N, N-trimethylammonium methylstyrene bromide, etc. Derived from And the like, also, the following general formula [4]
[0028]
Embedded image
Specific examples include those represented by the formula (wherein m represents 1 to 100).
[0030]
Among those mentioned above, those derived from methacrylic acid, stearyl methacrylate, benzyl methacrylate, butyl methacrylate, dodecyl methacrylate, octadecyl methacrylate N, N, N-trimethylammonium-2-hydroxypropyl methacrylate chloride (QM), etc. Those represented by the formula [4] are preferred.
[0031]
The sensitivity enhancer of the present invention comprises a polymer having a group represented by the general formula [1] in the side chain as described above, and is preferably based on a monomer represented by the general formula [2]. A polymer having units. The sensitivity enhancer may be used by dissolving it in various reagents and samples used in immunochromatography, or may be used by being supported on an instrument for immunochromatography. Is more preferable.
[0032]
Examples of the instrument for immunochromatographic measurement used in the immunochromatographic measurement method of the present invention include (1) one comprising a developing membrane, (2) comprising a specimen labeling part and a developing film, and the specimen labeling part and the developing film comprise (3) a specimen labeling part, a development film, and a liquid absorption part, and the specimen labeling part, the development film, and the liquid absorption part in this order. (4) a specimen dropping section, a specimen labeling section, a developing film, and a liquid absorbing section, and the specimen dropping section, the specimen labeling section, the developing film, and the liquid absorbing section. And the like formed so as to be movable by capillary action in this order.
[0033]
The spread membrane is provided with a measurement unit, and the measurement unit is fixedly loaded with a substance capable of binding to the measurement target substance (hereinafter sometimes abbreviated as the measurement target binding substance). When measuring several types of substances to be measured at the same time, several kinds of substances to be measured are immobilized on the development film, for example, in the form of a line or spot so as not to overlap each other. To be carried. In addition, the sample labeling unit holds the labeled measurement target binding substance in a movable state by capillary action, and when measuring several types of measurement target substances at the same time, The labeled binding substance to be measured is held in a movable state by capillary action. Furthermore, the specimen dropping part is a part formed so as to overlap with the development film or the specimen labeling part for dropping the specimen, and the liquid absorption part is provided to facilitate the movement of the specimen due to capillary action. , Represents a site formed so as to overlap the spreading membrane.
[0034]
The appliances as described above may be commercially available products or those produced by known methods. In the case of producing by a known method, as a base material used in the spread membrane, those usually used in this field may be used. For example, cellulose, nitrocellulose, nylon and the like are preferable. In addition, what is necessary is just to use the base material used by the said sample label | marker part, a sample dripping part, and a liquid absorption part according to these. In addition, so-called blocking treatment may be performed on the spread film in order to prevent the influence of nonspecific adsorption on the measurement. Such blocking treatment may be performed by a method usually used in this field with a blocking agent usually used in this field. Examples of the labeled measurement target binding substance held in the sample labeling part include those in which a labeling substance is supported on an antigen against the antibody that is the measurement target substance. As the labeling substance, any labeling substance usually used in this field may be used. However, a labeling substance capable of obtaining a visually detectable signal such as gold colloid, selenium colloid, and colored latex is preferable. Gold colloid is more preferable from the viewpoints of easiness and sensitivity. The labeling substance as described above may be prepared by a conventional method or may be a commercially available product, and the concentration used may be within the concentration range usually used in this field.
[0035]
When the sensitivity enhancer of the present invention is used by being supported on an instrument for immunochromatography, the amount thereof depends on the place where it is supported, the type of sensitivity enhancer used, the type of substance to be measured, the labeling substance used, etc. For example, when it is carried on the development membrane, specimen labeling part, specimen dropping part, etc. of an instrument for immunochromatography, the unit area (cm2) The amount of the sensitivity enhancer contained per unit is usually 0.01 μg to 10 mg, preferably 0.1 μg to 4 mg, more preferably 1 to 800 μg. In addition, the supported area varies depending on the type and size of the instrument for immunochromatography used and the amount of the sample for measurement. 60%, preferably 20-30%, in the case of a specimen labeling part, usually 1-30% of the total area, preferably 5-15%, in the case of a specimen dropping part, usually 5-40% of the total area , Preferably 10 to 20%. Further, the part carried in the immunochromatographic measurement instrument may be a part that can come into contact with the measurement sample, that is, a part through which the measurement sample is passed. The above measurement site and the like can be mentioned, and the measurement site is preferable. The method for supporting the sensitivity enhancer of the present invention may be any method that is usually used in this field. For example, a solution containing the sensitivity enhancer as described above may be applied to, for example, spread, sprayed, sprayed, etc. After that, the method of drying and supporting by physical adsorption, for example, dissolving the sensitivity enhancer of the present invention in a buffer solution or a washing solution used at the time of production of the development film, etc., and producing a development film using them. And the like.
[0036]
In addition to the sensitivity enhancer of the present invention, the reagent for immunochromatography used in the immunochromatographic measurement method according to the present invention contains, for example, an antigen against an antibody that is a measurement target substance or an antigen carrying a labeling substance. May be. The labeling substance used at this time may be the same as that used in the above-mentioned instrument for immunochromatography, and its concentration, preparation method, etc. may be carried out in the same manner. Further, in the reaction reagent, a buffer (for example, Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, etc.), stabilizer (for example, albumin, globulin, water-soluble gelatin, Surfactants, saccharides, etc.), preservatives (eg salicylic acid, benzoic acid, sodium azide, etc.), and others used in this field, which may inhibit the stability of coexisting reagents or antigen antibodies You may contain what does not inhibit reaction. Also, the concentration used may be used in the concentration range normally used in this field.
[0037]
When the sensitivity enhancer of the present invention is used by dissolving in various reagents or samples, the concentration is usually 0.1 to 20 w / v%, preferably 0.1 to 10 w / v, as the concentration at the time of antigen-antibody reaction. %, More preferably 0.1 to 5 w / v%.
[0038]
In order to carry out the immunochromatographic measurement method of the present invention, the polymer having the side chain of the group represented by the general formula [1] according to the present invention is allowed to coexist in the concentration reagent as described above, or the present invention is performed. In addition to the above-described concentration immunochromatography apparatus having the group having the group represented by the general formula [1] in the side chain supported thereon, various reagents used in known immunochromatography methods are used. What is necessary is just to follow according to the operation method.
[0039]
Examples of the measurement target substance of the immunochromatography measurement method of the present invention include syphilis-related antibodies such as antiphospholipid antibodies and anti-treponema antibodies.
[0040]
The sensitivity enhancer of the present invention used in the measurement method of the present invention is enhanced in sensitivity regardless of the substance to be measured, but the preferred one varies depending on the substance to be measured. For example, when the measurement target substance is an antiphospholipid antibody, for example, a polymer composed of a structural unit based on the monomer represented by the general formula [2], a structural unit based on the monomer represented by the general formula [2], and methacryl Polymers composed of monomer units derived from butyl acid, polymers composed of structural units based on monomers represented by the above general formula [2] and monomer units derived from benzyl methacrylate, based on monomers represented by the above general formula [2] Preferred examples include a polymer composed of a structural unit based on the monomer represented by the general formula [4] and a structural unit based on the monomer represented by the general formula [2]. A polymer comprising a structural unit based on the monomer represented by the formula [2] and a monomer unit derived from butyl methacrylate, [2] consisting of structural units based on a monomer represented by structural units based on a monomer represented by the above general formula [4] polymer, and the like as preferred. For example, when the substance to be measured is an anti-TP (Treponema Pallidum) antibody, for example, a polymer composed of a structural unit based on the monomer represented by the general formula [2], based on the monomer represented by the general formula [2] A polymer composed of a structural unit and a monomer unit derived from butyl methacrylate, a polymer composed of a structural unit based on the monomer represented by the general formula [2] and a monomer unit derived from octadecyl methacrylate, and represented by the general formula [2]. A polymer comprising a structural unit based on a monomer and a monomer unit derived from methacrylic acid, a polymer comprising a structural unit based on the monomer represented by the general formula [2] and a monomer unit derived from benzyl methacrylate, the general formula [2] And a structural unit based on the monomer represented by the general formula [4]. Preferred examples include polymers, among which, for example, a polymer composed of a structural unit based on the monomer represented by the general formula [2], a structural unit based on the monomer represented by the general formula [2], and a monomer derived from butyl methacrylate Polymer composed of units, a structural unit based on the monomer represented by the general formula [2] and a polymer composed of a monomer unit derived from benzyl methacrylate, a structural unit based on the monomer represented by the general formula [2] and the above general More preferred are polymers composed of structural units based on the monomer represented by the formula [4].
[0041]
In addition, the immunochromatographic measurement instrument of the present invention is prepared by dropping a sample having a group represented by the general formula [1] in a side chain into a sample specimen such as a sample dropping part, a sample labeling part, and a developing film (measuring part). , Which is supported in a place where liquid is passed, such as a development, and preferably a polymer having a side chain having a group represented by the general formula [1] added to the development membrane, represented by the general formula [1] Examples include those obtained by adding a polymer having a group in the side chain to the sample labeling part, and those obtained by adding a polymer having a group represented by the general formula [1] in the side chain to the measurement part. Preferred embodiments, specific examples, use concentrations, and the like of the components of the instrument are as described for those used in the immunochromatography measurement method.
[0042]
Hereinafter, the present invention will be described with reference to Examples and Comparative Examples, but the present invention is not limited thereto.
[0043]
Further, the MPC polymer in Examples and Comparative Examples represents a polymer synthesized from the following one or two monomers according to the method described in, for example, JP-A-2001-228149.
PMPC: polymer composed of 2- (methacryloyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate (hereinafter abbreviated as MPC monomer), molecular weight: 1030 × 10Three
PMB82: copolymer of MPC monomer and butyl methacrylate, molecular weight; 600 × 10Three, Composition ratio of MPC monomer and butyl methacrylate; 8: 2
PMB82-1M: copolymer of MPC monomer and butyl methacrylate, molecular weight: 1210 × 10Three, Composition ratio of MPC monomer and butyl methacrylate; 8: 2
PMB37: Copolymer composed of MPC monomer and butyl methacrylate, molecular weight: 93 × 10Three, Composition ratio of MPC monomer and butyl methacrylate; 3: 7
PME491: Copolymer comprising MPC monomer and monomethyl ether polyethylene glycol methacrylate (monomer of general formula [4], m = 4), molecular weight: 501 × 10Three, Composition ratio of MPC monomer to monomethyl ether polyethylene glycol methacrylate; 9: 1
PME473: Copolymer comprising MPC monomer and monomethyl ether polyethylene glycol methacrylate (monomer of general formula [4], m = 4), molecular weight: 138 × 10Three, Composition ratio of MPC monomer and monomethyl ether polyethylene glycol methacrylate; 7: 3
PMC1891: Polymer composed of MPC monomer and octadecyl methacrylate, molecular weight: 130 × 10Three, Composition ratio of MPC monomer and octadecyl methacrylate; 9: 1
PMC1882: Polymer composed of MPC monomer and octadecyl methacrylate, molecular weight: 43 × 10Three, Composition ratio of MPC monomer and octadecyl methacrylate; 8: 2
PMC1282: Polymer composed of MPC monomer and dodecyl methacrylate, molecular weight; 184 × 10Three, Composition ratio of MPC monomer and octadecyl methacrylate; 8: 2
PMBz82: polymer composed of MPC monomer and benzyl methacrylate, molecular weight; 240 × 10Three, Composition ratio of MPC monomer and octadecyl methacrylate; 8: 2
[0044]
Example 1
(1) Labeling of TP (Treponema Pallidum) antigen and phospholipid antigen
First, 8 μl of TP antigen (1.0 mg / ml) was dispensed into a centrifuge tube, and then there was an average particle size of 35 nm, Amax= 5 ml of a colloidal gold suspension of 1.2 was poured and allowed to react at room temperature for 30 minutes, followed by centrifugation (8000 G, 15 minutes) to recover the colloidal gold-labeled TP antigen. The TP antigen was prepared according to a known recombinant antigen preparation method (Infection and Immunity, volume 54, p500-506, Michael V. Norgard).
[0045]
Phospholipid antigen solutions were 5 mg / ml cardiolipin (derived from bovine heart, manufactured by Sigma) ethanol solution and 10 mg / ml phosphatidylcholine (derived from egg yolk, manufactured by Kewpie) ethanol solution, 2 ml and 0.5 ml, respectively (weight ratio 2: 1) A mixture was prepared and used.
[0046]
Pretreated gold colloid suspension (average particle size 35nm, A in 0.25ml of prepared phospholipid antigen solutionmax= 20.0) 0.8 ml was added and mixed by inverting at room temperature for 2 hours. Then, add 30 ml of blocking solution (50 mM morpholinoethanesulfonic acid (MES) buffer containing 1% BSA, 2% sucrose) and leave for 15 minutes, then centrifuge (8000G, 15 minutes) to collect the colloidal gold-labeled phospholipid antigen. It was collected.
[0047]
(2) Preparation of specimen dropping part
First, the glass fiber sheet was masked with 25 mM phosphate buffer containing 0.5% BSA and 1% sucrose for 15 minutes, and then 1% poly (1-vinylpyrrolidone-co-vinyl acetate) [poly (1-vinylpyrrolidone-co- vinyl acetate), hereinafter abbreviated as poly-PA. (Manufactured by Aldrich)] and 25 mM phosphate buffer containing 1% sucrose and dried to form a specimen dropping part.
[0048]
(3) Preparation of specimen labeling part
The gold colloid-labeled TP antigen prepared in (1) is Amax= Colloidal gold-labeled phospholipid antigen A so that = 1.3max= 50 μM MES buffer containing 1% poly-PA and 1% sucrose added so as to be 1.4 was impregnated in the specimen dropping part with 500 μl per 5 mm × 200 mm, dried at 50 ° C., and the specimen labeling part did.
[0049]
(4) Production of spread membrane
A mixture of 40 μl of recombinant TP antigen (1.0 mg / ml) and 20 μl of 250 mM phosphate buffer containing 10% sucrose was used as a TP antigen solution.
[0050]
In addition, 5mg / ml cardiolipin (from bovine heart, Sigma) ethanol solution 125μl, 10mg / ml phosphatidylcholine (from egg yolk, Nippon Oil & Fats Co., Ltd.) ethanol solution 250μl and 20mg / ml cholesterol (Wako Pure Chemical Industries) A phospholipid antigen solution consisting of 125 μl of ethanol solution was distilled off with an evaporator to form a lipid thin film. After vacuum drying for 2 hours, 500 μl of 100 mM borate buffer was added to the lipid thin film to suspend it, and the suspension was further sonicated to obtain a liposome suspension.
Next, the TP antigen solution and the liposome suspension were respectively applied to different portions of a nylon membrane (20 mm × 200 mm) at a rate of 0.25 μl / cm (200 mm) and dried at 50 ° C. for 5 minutes. It was then immersed for 15 minutes in 50 mM MES buffer containing 1% BSA and 2% sucrose. Furthermore, after washing twice with 50 mM MES buffer containing 1% Poly-PA and 2% sucrose, and drying for 15 minutes at 50 ° C, a measurement part for simultaneous detection of anti-TP antibody and antiphospholipid antibody was prepared. It was set as the deployment film | membrane which has.
[0051]
(5) Assembly of measuring instruments
A spread film prepared in (4) is attached to a 1.2 cm portion from the lower end of a polyethylene terephthalate (PET) sheet (8 cm x 20 cm) laminated with double-sided tape, and glass fiber is used as an absorbent part so as to overlap 1 mm on the upper end of the spread film. A sheet (4 cm × 20 cm) was bonded. Next, the specimen labeling part is pasted so that it overlaps the lower end of the developed film by 1 mm, and finally the specimen dropping part is overlapped with the specimen labeling part at the lower end of the PET sheet and the sheet is cut to 4 mm width to detect TP antibody / phospholipid antibody A tool was used. The resulting instrument was stored at room temperature under dehumidified conditions. A schematic diagram is shown in FIG. In FIG. 1, each numeral indicates the following.
[0052]
1: Support
2: Specimen dropping part
3: Specimen labeling part (part holding labeled phospholipid antigen and labeled syphilis antigen)
4: Deployment membrane
5: Supporting part on which phospholipid antigen is immobilized (first measuring part)
6: TP antigen carrying part (second measuring part)
7: Liquid absorption part
[0053]
(6) Measurement
50 to 100 μl of a sample to which various MPC polymers having various concentrations shown in Table 1 were added in an amount of 0.5 to 3% was dropped onto the sample dropping portion of the test tool prepared in (5) and left for 15 minutes. Thereafter, anti-TP antibody and anti-phospholipid antibody were measured with or without a measurement line. The results are shown in Table 1. In the table, + represents colored, + w represents lightly colored, ± represents colored, and x represents uncolored.
[0054]
Comparative Example 1
The experiment was performed in the same manner as in Example 1 except that polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), or polyethylene glycol (PEG) was added instead of adding the MPC polymer to the specimen. The results are also shown in Table 1.
[0055]
[Table 1]
From this result, although the effect is somewhat different depending on the type of MPC polymer, by adding various MPC polymers to the sample, the determination line in the determination part of anti-TP antibody or / and anti-phospholipid antibody was enhanced, and MPC It was found that weak positive samples that could not be detected with the sample without addition of polymer could be detected.
[0057]
On the other hand, even when PVP, PVA, and PEG were added, weakly positive specimens could not be detected.
[0058]
Example 2
Further, the following experiment was conducted by changing the place where the MPC polymer was added.
(1) When MPC polymer is added to the cleaning solution when preparing the sample dropping part
When preparing the specimen dropping part in Example 1 (2), 0.5% of various MPC polymers described in Table 1 were added to 25 mM phosphate buffer containing 1% poly-PA (Aldrich) and 1% sucrose. A test tool was prepared in the same manner as in Example 1 except that ˜3% was added. The measurement was performed in the same manner as in Example 1 except that no MPC polymer was added to the specimen.
[0059]
(2) When MPC polymer is added to the buffer used when preparing the sample labeling part
When preparing the specimen labeling part in Example 1 (3), 50 mM containing 1% poly-PA (manufactured by Aldrich) and 1% sucrose containing colloidal gold-labeled TP antigen and gold colloid-labeled phospholipid antigen at predetermined concentrations. Test tools were prepared in the same manner as in Example 1 except that 0.5 to 3% of various MPC polymers described in Table 1 were added to the MES buffer. The measurement was performed in the same manner as in Example 1 except that no MPC polymer was added to the specimen.
(3) When MPC polymer is added to the cleaning solution during the development of the spread membrane
When producing a development membrane in Example 1 (4), various MPC polymers having various concentrations shown in Table 1 were mixed with 0.5 mM in 50 mM MES buffer containing 1% poly-PA (Aldrich) and 2% sucrose. A test tool was prepared in the same manner as in Example 1 except that ˜3% was added. The measurement was performed in the same manner as in Example 1 except that no MPC polymer was added to the specimen.
[0060]
As a result of conducting an experiment in which the MPC polymer addition place was changed as described above, the same results as in Table 1 were obtained.
[0061]
Therefore, it was found that the MPC polymer exerts its sensitivity enhancement effect not only in the sample but also when added to any place where the sample of the instrument for immunochromatography passes.
[0062]
【The invention's effect】
As is apparent from the above description, the present invention provides a significantly superior effect compared to a sensitivity enhancer used in an immunochromatographic assay using a conventionally known syphilis-related antibody as a measurement target substance. Therefore, it is possible to perform measurement with higher accuracy by an immunochromatographic measurement method using the sensitivity enhancer.
[Brief description of the drawings]
FIG. 1 shows a schematic diagram of an instrument for immunochromatography measurement of the present invention.
[Explanation of symbols]
1: Support
2: Specimen dropping part
3: Specimen labeling part (part holding labeled phospholipid antigen and labeled syphilis antigen)
4: Deployment membrane
5: Supporting part on which phospholipid antigen is immobilized (first measuring part)
6: TP antigen carrying part (second measuring part)
7: Liquid absorption part
Claims (14)
で表されるモノマーに基づく構成単位を有するものである、請求項１に記載の感度増強剤。The polymer has the following general formula [2]
The sensitivity enhancer of Claim 1 which has a structural unit based on the monomer represented by these.
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Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4628110B2 (en) * | 2005-01-06 | 2011-02-09 | シスメックス株式会社 | Immunochromatographic test equipment |
| WO2007007392A1 (en) | 2005-07-11 | 2007-01-18 | Wako Pure Chemical Industries, Ltd. | Novel polymer and method of measuring cholesterol therewith |
| EP1949108B1 (en) | 2005-11-18 | 2013-09-25 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES | Modified cardiolipin and uses therefor |
| US8778619B2 (en) | 2005-11-18 | 2014-07-15 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Oxidized cardiolipin and uses to detect cardiolipin antibodies |
| JP4629563B2 (en) * | 2005-12-07 | 2011-02-09 | 積水メディカル株式会社 | Antiphospholipid antibody measurement reagent |
| JP4629564B2 (en) * | 2005-12-07 | 2011-02-09 | 積水メディカル株式会社 | Anti-Treponema / Paridum antibody measurement reagent |
| CN102565397B (en) * | 2005-12-07 | 2015-07-08 | 积水医疗株式会社 | Method for preventing serum interference and improving storage stability of reagent of anti-phospholipid antibody |
| JP4984080B2 (en) * | 2008-03-19 | 2012-07-25 | Ｊｓｒ株式会社 | Signal enhancer for immunochemical reaction and immunological measurement method |
| JP4559510B2 (en) | 2008-07-14 | 2010-10-06 | 田中貴金属工業株式会社 | Developing solution for immunochromatography and measurement method using the same |
| WO2016068225A1 (en) * | 2014-10-31 | 2016-05-06 | 日油株式会社 | Sensitizer for immunochromatographic measuring method with dengue virus as substance to be measured, and measuring method using sensitizer |
| WO2018181263A1 (en) * | 2017-03-27 | 2018-10-04 | 日本ハム株式会社 | Substance that prevents antigen-antibody reaction inhibition by body fluid |
| WO2019124532A1 (en) | 2017-12-22 | 2019-06-27 | 株式会社 三和化学研究所 | Immunochromatography device |
| JPWO2022091923A1 (en) * | 2020-10-29 | 2022-05-05 | ||
| WO2022124270A1 (en) * | 2020-12-11 | 2022-06-16 | 日油株式会社 | Sensitizer for immunochromatographic assays, and assay |
| US20250197922A1 (en) * | 2021-09-30 | 2025-06-19 | Nof Corporation | Nucleic acid amplification promoter and test method using same |
| CN120693521A (en) * | 2023-02-09 | 2025-09-23 | 学校法人川崎学园 | Immunochromatographic assay sensitizer for hepatitis virus assay, immunochromatographic assay sample diluent, immunochromatographic assay method, immunochromatographic assay device, and hepatitis virus testing kit |
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2002
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