JP4177498B2 - Enzyme immunoassay - Google Patents

Description

（上記一般式（１）において、Ｒ¹ 及びＲ² は、それぞれアルキル基、アリール基及びハロゲン化アリール基からなる群より選択され、互いに同一でも又は異なるものでもよく、Ｒ³ 、Ｒ⁴ 、Ｒ⁵ 及びＲ⁶ は、それぞれ水素原子、アルキル基、アリール基、アルコキシ基、アリーロキシ基及びハロゲン原子からなる群より選択され、互いに同一でも又は異なるものでもよく、Ｘ・は前駆体ビスアクリジニウム塩の対アニオンから電子が移動した残基である酸ラジカルを示す。）
で表わされるＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体、
及び下記一般式（２）
【００１０】
【化５】

（上記一般式（２）において、Ｒ₁ は水素原子、炭素数１〜１０のアルキル基、炭素数２〜１０のアルケニル基及び炭素数６〜２０のアリール基からなる群より選択され、該アリール基はアルキル基、ニトロ基、水酸基、アミノ基又はハロゲン原子等で置換されていてもよく、Ｒ₂ はメチル基及びエチル基からなる群より選択され、Ｒ₃ は炭素数１〜１０のアルキル基、炭素数２〜１０のアルケニル基及び炭素数６〜２０のアリール基からなる群より選択され、該アリール基はアルキル基、ニトロ基、水酸基、アミノ基又はハロゲン原子等で置換されていてもよく、Ｒ₁ 及びＲ₃ は、互いに結合して、それぞれが結合しているカルボニル基の炭素原子及びアミド基の窒素原子と共に環を形成していてもよい。）
で表わされるＮ，Ｎ−ジ置換カルボン酸アミド化合物を主成分とする化学発光試薬を用い、水素受容体の存在下において化学発光させ、その発光強度を測定することにより試料中の抗原又は抗体の量を定量することを特徴とする化学発光酵素免疫測定法に関するものである。
【００１１】
また、本発明の第二は、ペルオキシダーゼ酵素標識した抗体若しくは抗原を試料中の測定すべき抗原若しくは抗体、又はそれらの凝集物と混合し、抗原抗体反応によりペルオキシダーゼ酵素標識−抗原抗体錯体からなる免疫複合体を形成させた後、これを分離して、請求項１に記載の化学発光試薬に下記一般式（３）
【００１２】
【化６】

（上記一般式（３）において、Ｒは炭素数１〜５の二価の脂肪族炭化水素を表わし、ｍは１〜３の整数を表わす。）
で表わされるアミノアルコール化合物を添加してなる化学発光試薬を用い、水素受容体の存在下において化学発光させ、その発光強度を測定することにより試料中の抗原又は抗体の量を定量することを特徴とする化学発光酵素免疫測定法に関するものである。
【００１３】
【発明の実施の形態】
以下、本発明につき更に詳しく説明する。
本発明の化学発光酵素免疫測定法は、ペルオキシダーゼ酵素標識した抗体又は抗原を試料中の測定すべき抗原若しくは抗体、又はそれらの凝集物と混合し、抗原抗体反応により測定すべき抗原又は抗体をペルオキシダーゼ酵素標識した免疫複合体として捕捉する免疫反応段階と、生成した該免疫複合体をその分子中に存在する標識酵素を用いる化学発光法により測定する化学発光反応段階とからなる。
【００１４】
免疫反応段階を構成する抗原抗体反応の方法は任意であり、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体及びＮ，Ｎ−ジ置換カルボン酸アミド化合物からなる化学発光試薬、又はＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体、Ｎ，Ｎ−ジ置換カルボン酸アミド化合物及び特定アミノアルコール化合物からなる化学発光試薬を用いることができるものであれば、いずれの方法も採用することができる。
【００１５】
例えば、
▲１▼不溶性担体に結合した抗体に試料中の測定すべき抗原を捕捉させた後にペルオキシダーゼ酵素標識抗体を反応させるサンドイッチ法、
▲２▼サンドイッチ法において、不溶性担体に結合した抗体と異なる動物種に由来する抗体を用い、生成したサンドイッチ錯体に対して、更にこの抗体に対する標識第二抗体を反応させる二抗体法、
▲３▼不溶性担体に結合した抗体に試料中の測定すべき抗原をペルオキシダーゼ酵素標識抗原の存在下で反応させる競合法、
▲４▼測定すべき抗原若しくは抗体を含有する試料にこれらと特異的に反応する標識した抗体若しくは抗原を作用させて凝集沈殿させた後、遠心分離して分離した免疫複合体中の標識物質を検出する凝集沈殿法、
▲５▼不溶性担体に結合した抗原に試料中の測定すべき抗体をペルオキシダーゼ酵素標識抗ヒトガンマグロブリン抗体を作用させる抗体検出法、更に、
▲６▼ビオチン標識抗体にペルオキシダーゼ酵素標識アビジンを反応させるビオチン−アビジン法等
を非限定的に用いることができる。
【００１６】
本発明の化学発光酵素免疫測定法に用いられる不溶性担体としては、例えば、ポリスチレン、ポリエチレン、ポリプロピレン、ポリエステル、ポリアクリロニトリル、フッ素樹脂、架橋デキストラン、ポリサッカライド等の高分子化合物、その他、ガラス、金属、磁性粒子及びこれらの組み合わせ等が挙げられる。また、不溶性担体の形状としては、例えば、トレイ状、球状、繊維状、棒状、盤状、容器状、セル、マイクロプレート、試験管等の種々の形状で用いることができる。更に、これら不溶性担体への抗原若しくは抗体の固定化方法は任意であるが、物理的吸着法、共有結合法、イオン結合法等を用いることができる。
【００１７】
尚、本発明の化学発光酵素免疫測定法において用いられる抗体類は、モノクローナル抗体及びポリクローナル抗体のいずれを使うことも可能であり、その形態としては全抗体或いはＦ（ａｂ’）₂ 、Ｆａｂ等の断片を用いることができる。また、抗体の起源は任意であるが、マウス、ラット、兎、羊、山羊、鶏等に由来する抗体が好適に用いられる。
【００１８】
更に、本発明の化学発光酵素免疫測定法の後段を構成する化学発光反応は、化学発光試薬としてＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体とＮ，Ｎ−ジ置換カルボン酸アミド化合物とからなる化学発光試薬を用い、そして発光増強剤の存在下に水素受容体を作用させて不溶性担体上に捕捉された標識物質であるペルオキシダーゼ酵素の活性を測定するものであり、その測定方法は任意であるが、一般に、化学発光物質及び発光増強剤を含有する測定試薬をペルオキシダーゼ酵素標識抗体又は抗原を免疫学的に捕捉した不溶性担体に添加し、特定塩基性ｐＨ領域において、水素受容体、例えば、過酸化水素水溶液を添加して化学発光反応させ、その化学発光量を発光測定装置で測定する方法等が行なわれている。
【００１９】
次に、本発明の化学発光酵素免疫測定法に用いられる化学発光試薬について具体的に説明する。
化学発光試薬は、必須成分として、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体およびＮ，Ｎ−ジ置換カルボン酸アミド化合物を、任意成分としてさらにアミノアルコール化合物を添加してなるものである。
Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体は、
下記一般式（１）
【００２０】
【化７】

で表わされる。
【００２１】
上記一般式（１）において、Ｒ¹ 及びＲ² は、それぞれアルキル基、アリール基及びハロゲン化アリール基からなる群より選択され、互いに同一でも又は異なるものでもよい。アルキル基、アリール基及びハロゲン化アリール基は、炭素数１〜２０を有するものであり、好ましいアルキル基は炭素数１〜１０のものである。例えば、メチル基、エチル基、プロピル基、ブチル基、ペンチル基、ヘキシル基、ヘプチル基、オクチル基、ノニル基及びデシル基等の直鎖状アルキル基又はこれらの分岐状アルキル基を挙げることができる。また、アリール基は炭素数６〜２０のものが好ましく、例えば、フェニル基、トリール基、キシリル基等を挙げることができ、さらにアルキル基で置換されたものでもよい。アリール基としては、特に、フェニル基が好ましい。ハロゲン化アリール基としてはハロゲン化フェニル基、ハロゲン化トリル基、ハロゲン化キシリル基等を挙げることができ、特に、クロロフェニル基が好ましい。
【００２２】
一般式（１）において、Ｒ³ 、Ｒ⁴ 、Ｒ⁵ 及びＲ⁶ は、それぞれ水素原子、アルキル基、アリール基、アルコキシ基、アリーロキシ基及びハロゲン原子からなる群より選択され、互いに同一でも又は異なるものでよい。これらの炭化水素基としては、炭素数１〜２０、好ましくは１〜１０のものを挙げることができる。例えば、炭素数１〜２０の直鎖状又は分岐状アルキル基、アルコキシ基、炭素数６〜２０のアリール基、アリーロキシ基を挙げることができ、アリール基、アリーロキシ基にはアルキル基が置換されたものでもよい。
また、一般式（１）において、Ｘ・は前駆体ビスアクリジニウム塩の対アニオンから電子が移動した残基である酸ラジカルを示す。
【００２３】
Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体は、紫外吸収スペクトルにおける５５０ｎｍ付近に極大をもつ巾広い吸収帯の出現を分光光度計を用いる方法により測定することができる。
上記Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動体の具体的な例としては、Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジエチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジプロピル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジイソプロピル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジブチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジイソブチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジフェニル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジ−ｍ−クロロフェニル−９，９’−ビスアクリジニウム塩等の電荷移動錯体が挙げられるが、これらのなかで、特に、Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジナイトレート（ルシゲニン）の電荷移動錯体が好適に用いられる。
【００２４】
Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体の前駆体であるＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の対イオンとしては、塩素イオン、臭素イオン、沃素イオン、硝酸イオン、炭酸イオン、硫酸イオン、リン酸イオン及びカルボン酸イオン等が非限定的に挙げられる。
Ｎ，Ｎ’−ジ置換カルボン酸アミド化合物は、
下記一般式（２）
【００２５】
【化８】

で表わされる。
【００２６】
上記一般式（２）において、Ｒ₁ は水素原子、炭素数１〜１０のアルキル基、炭素数２〜１０のアルケニル基及び炭素数６〜２０のアリール基からなる群より選択され、該アリール基はアルキル基、ニトロ基、水酸基、アミノ基又はハロゲン原子等で置換されていてもよい。Ｒ₂ はメチル基及びエチル基からなる群より選択される。Ｒ₃ は炭素数１〜１０のアルキル基、炭素数２〜１０のアルケニル基及び炭素数６〜２０のアリール基からなる群より選択され、該アリール基はアルキル基、ニトロ基、水酸基、アミノ基又はハロゲン原子等で置換されていてもよい。また、Ｒ₁ 及びＲ₃ は、互いに結合して、それぞれが結合しているカルボニル基の炭素原子及びアミド基の窒素原子と共に環を形成していてもよい。
【００２７】
上記一般式（２）のＮ，Ｎ−ジ置換カルボン酸アミド化合物の具体例を例示すると、Ｎ，Ｎ−ジメチルホルムアミド、Ｎ，Ｎ−ジメチルアセトアミド、Ｎ，Ｎ−ジメチルアクリルアミド、Ｎ，Ｎ−ジメチルプロピオンアミド、Ｎ，Ｎ−ジメチルベンズアミド、Ｎ−メチル−２−ピロリドン等が非限定的に挙げられる。
また、アミノアルコール化合物は、
下記一般式（３）
【００２８】
【化９】

で表わされる。
【００２９】
上記一般式（３）において、Ｒは炭素数１〜５の二価の脂肪族炭化水素を表わし、ｍは１〜３の整数を表わす。
上記アミノアルコール化合物の具体例を例示すると、モノエタノールアミン、ジエタノールアミン、トリエタノールアミン、モノイソプロパノールアミン、ジイソプロパノールアミン、トリイソプロパノールアミン等を非限定的に挙げることができる。
【００３０】
本発明の化学発光免疫測定法に用いられる化学発光試薬の一つは、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体及び特定のＮ，Ｎ−ジ置換カルボン酸アミド化合物を必須成分とするものであるが、その成分の一つであるＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体の前駆体であるＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類は、もう一つの成分であるＮ，Ｎ−ジ置換カルボン酸アミド化合物の存在により自然光等により容易に電荷移動錯体に転化する現象が認められる。また、このＮ，Ｎ−ジ置換カルボン酸アミド化合物は、生成した電荷移動錯体のラジカルの安定性にも寄与していると考えられ、求核性の弱い酸からなるＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体の形成及び安定化の必須成分として作用している。また、本発明の化学発光免疫測定法に用いられるもう一つの化学発光試薬は、上記Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体とＮ，Ｎ−ジ置換カルボン酸アミド化合物とからなる化学発光試薬へ特定のアミノアルコール化合物を添加することにより得られるものであるが、このアミノアルコール化合物の添加は、発光反応のブランク値を低下させると共に、より廣いペルオキシダーゼ濃度範囲において安定した化学発光反応を実現することが可能になる。このアミノアルコール化合物の添加効果については必ずしもはっきりはしていないが、対イオンからアクリジン環への電荷移動に関与してその反応を促進すると共に、生成する電荷移動錯体の有するラジカルを一層安定化して発光反応時における溶存酸素との反応を阻止してブランク値を高める要因を排除することにより、ブランク値の低い安定した高感度測定を可能にしているものと考えられる。
【００３１】
上記化学発光試薬の製造方法は任意であるが、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体及びＮ，Ｎ−ジ置換カルボン酸アミド化合物を主成分とする化学発光試薬は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類を等モル以上のＮ，Ｎ−ジ置換カルボン酸アミド化合物と混合して自然光の下に放置することにより得られるが、電荷移動錯体の生成は光により触媒されているので、光照射下に反応を行なうのが好ましい。この光照射に用いる光源としては、波長領域が約２９０〜８００ｎｍの範囲の紫外可視部が用いられ、特に、約４００〜８００ｎｍの範囲の可視光が望ましい。これらの光源としては、高圧水銀灯、低圧水銀灯、殺菌灯、蛍光灯及び白熱電灯等を非限定的に用いることができるが、白熱電灯が好ましく用いられる。
【００３２】
また、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体とＮ，Ｎ−ジ置換カルボン酸アミド化合物との混合比としては、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体に対してＮ，Ｎ−ジ置換カルボン酸アミド化合物を１〜１万倍モル、好ましくは１〜５千倍モルの割合で用いることができる。
【００３３】
本発明に用いられるもう一つの化学発光試薬は、上記Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体とＮ，Ｎ−ジ置換カルボンアミド化合物からなる化学発光試薬に特定のアミノアルコール化合物を添加すことにより得られる。アミノアルコール化合物はＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動反応時及び／又は反応後に添加することができるが、電荷移動反応後に添加するのが好ましい。ここで、電荷移動反応時の添加には電荷移動反応前の添加も含む。上記アミノアルコール化合物の添加量は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の電荷移動錯体に対して、１〜１万倍モル、好ましくは１〜２千倍モルの量でよい。
【００３４】
本発明の化学発光免疫測定法において上記の新規化学発光試薬は、ｐＨ７．５〜１３の塩基性条件下で、過剰の水素受容体の存在下、ペルオキシダーゼの濃度に依存した量で発光する。この発光は、フェノール性化合物等の発光促進剤によって増強することが認められる。このようなフェノール性化合物としては、ｐ−ヒドロキシ桂皮酸、ｐ−フェニルフェノール、ｐ−（４−クロロフェニル）フェノール、ｐ−（４−ブロモフェニル）フェノール、ｐ−（４−ヨードフェニル）フェノール、ｐ−ヨードフェノール、ｐ−ブロモフェノール、ｐ−クロロフェノール、２，４−ジクロロフェノール、ｐ−クマル酸、６−ヒドロキシベンゾチアゾール、２−ナフトール、ホタルルシフェリン等が非限定的に挙げられる。
【００３５】
上記新規化学発光試薬を用いて化学発光分析を行なう場合には、１０^-8〜１Ｍ、好ましくは１０^-6〜１０^-2Ｍの範囲の濃度で用いられ、その使用量は１０〜５００μｌ、好ましくは５０〜３００μｌの範囲でよい。また、発光促進剤の使用量は、化学発光試薬の０．０１〜１００倍モル、好ましくは０．１〜１０倍モルの範囲であり、その濃度としては、１０^-6〜１Ｍ、好ましくは１０^-4〜１０^-2Ｍの範囲が採用される。
【００３６】
また、化学発光反応に用いられる水素受容体としては、ペルオキシダーゼ酵素の基質となり得るものであれば特に限定されるものではなく、無機過酸化物、有機過酸化物等が任意に用いられるが、過酸化水素が特に好ましい。水素受容体は化学発光試薬に対して充分に過剰な量で用いることが必要であり、その使用量は化学発光試薬に対して３〜１万倍モル、好ましくは１０〜１０００倍モルの範囲で採用される。
【００３７】
また、ペルオキシダーゼを標識物質として抗体、核酸等を標識して種々の物質を定量する場合には、特に限定されるものではないが、ペルオキシダーゼとして、西洋ワサビペルオキシダーゼ（ＨＲＰ）が好ましく用いられる。化学発光反応に用いる塩基性緩衝液としては、トリス緩衝液、リン酸緩衝液、ほう酸緩衝液、炭酸緩衝液、グリシン−水酸化ナトリウム緩衝液等を任意に用いることができる。これらの緩衝液の濃度としては１ｍＭ〜１Ｍの範囲が望ましい。また、反応時には界面活性剤、キレート剤等の添加剤を任意に用いることができる。
【００３８】
【実施例】
以下、参考例と共に実施例等を示し、本発明を具体的に説明する。もっとも、本発明は実施例等により限定されるものではない。
尚、参考例及び実施例中の％は重量％を意味する。
【００３９】
［参考例１］
Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジ沃化水素酸塩の電荷移動錯体及びＮ，Ｎ−ジメチルアセトアミドを含む化学発光試薬の調製
ルシゲニンの１×１０^-2モル／ｌの水溶液に、２倍モルの固体の沃化カリウムを添加し、室温で１時間攪拌することにより赤色沈殿が生成した。この赤色沈殿を含む反応液をナス型フラスコに入れ、ロータリーエバポレーターを用いて減圧下に６０℃で溶媒の水を蒸発して乾固させ、赤色沈殿からなる残渣をベンゼンで洗浄して副生物を除去し、ベンゼン不溶分を濾別し乾燥して粗生成物を得た。次に、この粗生成物に少量の水を加え、遮光下に９５℃の湯浴上で加熱して溶解した後、室温に戻してから、更に、４℃に冷却放置し、生成した沈殿を濾別、乾燥して未反応物を除去してＮ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジ沃化水素酸塩を約７０％の収率で得た。次に、この化合物１．５ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルアセトアミド１ｍｌを加えて溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを７時間照射することによりＮ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジ沃化水素酸塩の電荷移動錯体を含有する化学発光試薬を得た。尚、電荷移動錯体の生成は、紫外吸収スペクトルにおける５５０ｎｍ付近に極大をもつ幅広い吸収帯の出現を分光光度計を用いる方法により測定して確認した。
【００４０】
［参考例２］
Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジ沃化水素酸塩の電荷移動錯体、Ｎ，Ｎ−ジメチルアセトアミド及びトリエタノールアミンを含む化学発光試薬の調製
参考例１で得られた化学発光試薬１ｍｌにトリエタノールアミン０．５ｍｌを添加し混合することにより化学発光試薬を得た。
【００４１】
［参考例３］
Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジ硝酸塩の電荷移動錯体及びＮ，Ｎ−ジメチルアセトアミドを含む化学発光試薬の調製
ルシゲニンの１．５ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルアセトアミド１ｍｌを加えて溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを７時間照射することによりＮ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジ硝酸塩の電荷移動錯体を含有する化学発光試薬を得た。
【００４２】
［参考例４］
Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジ硝酸塩の電荷移動錯体、Ｎ，Ｎ−ジメチルアセトアミド及びトリエタノールアミンを含む化学発光試薬の調製
参考例３で得られた化学発光試薬１ｍｌにトリエタノールアミン０．５ｍｌを添加し混合することにより化学発光試薬を得た。
【００４３】
［参考例５］
不溶性担体固定化ポリクローナル抗体の調製
抗原に特異的反応性を有する兎等の動物由来のポリクローナル抗体を１０ｍＭリン酸緩衝生理食塩水（ＰＢＳ）(pH7.4) に１０ｍｇ／ｍｌの濃度で溶解した溶液を、白色マイクロプレート（ラボシステム社）の各ウェルに０．１ｍｌずつ加え、３７℃の温度で１時間放置した後、ＰＢＳで洗浄してから、１％ウシ血清アルブミン（ＢＳＡ）水溶液を０．３ｍｌずつ加えて３７℃の温度で１時間放置してポストコーティング処理を実施してポリクローナル抗体固定化白色マイクロプレートを得た。
【００４４】
［参考例６］
ペルオキシダーゼ酵素標識モノクローナル抗体の調製
抗原に特異的反応性を有するマウス由来等のモノクローナル抗体を１０ｍＭリン酸緩衝生理食塩水（ＰＢＳ）(pH7.4) に１．０ｍｇ／ｍｌの濃度で溶解した溶液１ｍｌに、Ｎ−（ｍ−マレイミド安息香酸）−Ｎ−サクシンイミドエステル（ＭＢＳ）の１０ｍｇ／ｍｌの濃度のジメチルホルムアミド溶液０．１ｍｌを添加し、２５℃の温度で３０分間反応させた。次いで、この反応混合液をセファデックスＧ−２５を充填したカラムを用い、０．１Ｍリン酸緩衝液(pH6.0) でゲル濾過を行ない、マレイミド化モノクローナル抗体と未反応ＭＢＳとを分離した。
一方、ペルオキシダーゼ酵素としてホースラディッシュ・ペルオキシダーゼ（ＨＲＰ）の１．０ｍｇ／ｍｌのＰＢＳ溶液に、Ｎ−サクシンイミジル−３−（２−ピリジルチオ）プロピオネート（ＳＰＤＰ）の１０ｍｇ／ｍｌの濃度のエタノール溶液を添加し、２５℃の温度で３０分間反応させた。
次いで、この反応混合液をセファデックスＧ−２５を充填したカラムを用い、１０ｍＭ酢酸緩衝液(pH4.5) でゲル濾過して精製、ピリジルジスルフィド化ＨＲＰを含有する画分を採取し、これをコロジオンバック中において氷冷下に約１０倍に濃縮した。次に、これに０．１Ｍジチオスレイトールを含有する０．１Ｍ酢酸緩衝生理食塩水(pH4.5) １ｍｌを添加して、２５℃の温度で３０分間攪拌してＨＲＰ分子中に導入したピリジルジスルフィド基を還元した後、この反応混合液をセファデックスＧ−２５を充填したカラムを用いてゲル濾過し、チオール化ＨＲＰを含有する画分を得た。
次に、マレイミド化モノクローナル抗体とチオール化ＨＲＰとを混合し、コロジオンバックを用いて氷冷下に４ｍｇ／ｍｌの蛋白質濃度まで濃縮し、４℃で一昼夜放置した後、ウルトロゲルＡｃＡ４４（ＳＥＰＲＡＣＯＲ社）を充填したカラムを用いてゲル濾過し、ペルオキシダーゼ酵素標識モノクローナル抗体を得た。
【００４５】
［実施例１］
参考例１で調製した化学発光試薬を用いる同時サンドイッチ法ＣＬＥＩＡによるα−フェトプロティン（ＡＦＰ）の測定
参考例５の方法で調製した兎抗ヒトＡＦＰポリクローナル抗体を固定化した白色マイクロプレートに、精製したヒトＡＦＰ（標準物質）を０〜８００ｎｇ／ｍｌの範囲で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) ５０μｌと参考例６の方法で調製したペルオキシダーゼ酵素標識マウス抗ヒトＡＦＰモノクローナル抗体を約３μｇ／ｍｌの濃度で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) １００μｌとを加え、３７℃の温度で１時間インキュベートした。次に、ウェル内の溶液を吸引除去した後、ウェル内を生理食塩水で洗浄してから、各ウェルに７５ｍＭトリス塩酸緩衝液(pH7.8) １００μｌを加え、これに参考例１で調製した化学発光試薬溶液１００μｌ及び０．００１７％過酸化水素を含む７５ｍＭトリス塩酸緩衝液(pH7.8) ５０μｌを順次注入して発光させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で１〜５秒間積算して測定し、この値を標準物質濃度に対してプロットすることにより、図１に示される濃度依存性の良い検量線を得た。この検量線を用いて血清検体中のヒトＡＦＰを０．０１ｎｇ／ｍｌの濃度まで測定することが可能であった。
【００４６】
［実施例２］
参考例２で調製した化学発光試薬を用いる同時サンドイッチ法ＣＬＥＩＡによるヒトプロラクチン（ＰＲＬ）の測定
参考例５の方法で調製した兎抗ヒトＰＲＬポリクローナル抗体を固定化した白色マイクロプレートに、精製したヒトＰＲＬ（標準物質）を０〜１００ｎｇ／ｍｌの範囲で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) ５０μｌと参考例６の方法で調製したペルオキシダーゼ酵素標識マウス抗ヒトＰＲＬモノクローナル抗体を約３μｇ／ｍｌの濃度で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) １００μｌとを加え、３７℃の温度で１時間インキュベートした。次いで、ウェル内の溶液を吸引除去し、ウェル内を生理食塩水で洗浄してから、各ウェルに７５ｍＭトリス塩酸緩衝液(pH7.8) １００μｌを加え、これに参考例２で調製した化学発光試薬溶液１００μｌ及び０．００１７％過酸化水素を含む７５ｍＭトリス塩酸緩衝液(pH7.8) ５０μｌを順次注入して発光させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で１〜５秒間積算して測定し、この値を標準物質濃度に対してプロットすることにより、図２に示される濃度依存性の良い検量線を得た。この検量線を用いて血清検体中のヒトＰＲＬを０．０１ｎｇ／ｍｌの濃度まで測定することが可能であった。
【００４７】
［実施例３］
参考例３で調製した同時サンドイッチ法ＣＬＥＩＡによるヒト絨毛性ゴナドトロピンβ鎖（βｈＣＧ）の測定
参考例５の方法で調製した兎抗ヒトｈＣＧポリクローナル抗体を固定化した白色マイクロプレートに、精製したβｈＣＧ（標準物質）を０〜１００ｍＩＵ／ｍｌの範囲で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) ５０μｌと参考例５の方法で調製したペルオキシダーゼ酵素標識マウス抗βｈＣＧモノクローナル抗体を約２μｇ／ｍｌの濃度で含む２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) １００μｌとを加え、３７℃の温度で１時間インキュベートした。次に、ウェル内の溶液を吸引除去した後、ウェル内を生理食塩水で洗浄してから、各ウェルに７５ｍＭトリス塩酸緩衝液(pH7.8) １００μｌを加え、これに参考例３で調製した化学発光試薬溶液１００μｌ及び０．００１７％過酸化水素を含む７５ｍＭトリス塩酸緩衝液(pH7.8) ５０μｌを順次注入して発光させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で１〜５秒間積算して測定し、この値を標準物質濃度に対してプロットすることにより、図３に示される濃度依存性の良い検量線を得た。この検量線を用いて血清検体中のβｈＣＧを０．０１ｍＩＵ／ｍｌの濃度まで測定することが可能であった。
【００４８】
［実施例４］
参考例４で調製した化学発光試薬を用いる同時サンドイッチ法ＣＬＥＩＡによる α−フェトプロティン（ＡＦＰ）の測定
参考例５の方法で調製した兎抗比とＡＦＰポリクローナル抗体を固定化した白色マイクロプレートに、精製したヒトＡＦＰ（標準物質）を０〜８００ｎｇ／ｍｌの範囲で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) ５０μｌと参考例６の方法で調製したペルオキシダーゼ酵素標識マウス抗ヒトＡＦＰモノクローナル抗体を約３μｇ／ｍｌの濃度で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) １００μｌとを加え、３７℃の温度で１時間インキュベートする。次に、ウェル内の溶液を吸引除去した後、ウェル内を生理食塩水で洗浄してから、各ウェルに７５ｍＭトリス塩酸緩衝液(pH7.8) １００μｌを加え、これに参考例４で調製した化学発光試薬１００μｌ及び０．００１７％過酸化水素を含む７５ｍＭトリス塩酸緩衝液(pH7.8) ５０μｌを順次注入して発光させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で１〜５秒間積算して測定し、この値を標準物質濃度に対してプロットすることにより、図４に示される濃度依存性を有する検量線を得た。この検量線を用いて血清検体中のヒトＡＦＰを０．０１ｎｇ／ｍｌの濃度まで測定することが可能であった。
【００４９】
［比較例１］
ルミノールを用いる同時サンドイッチ法ＣＬＥＩＡによるα−フェトプロティン（ＡＦＰ）の測定
兎抗ヒトＡＦＰポリクローナル抗体を固定化した白色マイクロプレートに、精製したヒトＡＦＰ（標準物質）を０〜８００ｎｇ／ｍｌの範囲で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) ５０μｌとペルオキシダーゼ酵素標識マウス抗ヒトＡＦＰモノクローナル抗体を約３μｇ／ｍｌの濃度で含有する２％ＢＳＡ含有ＰＢＳ溶液(pH7.4) １００μｌとを加え、３７℃の温度で１時間インキュベートした。次に、ウェル内の溶液を吸引除去した後、ウェル内を生理食塩水で洗浄してから、各ウェルにｐ−ヨードフェノールを１０^-3Ｍの濃度で含有する０．１Ｍトリス塩酸緩衝液(pH8.4) １００μｌを加え、これにルミノールを５．６×１０^-5Ｍの濃度で含有する０．１Ｍトリス塩酸緩衝液(pH8.4) １００μｌ、及び０．００３４％過酸化水素の０．１Ｍトリス塩酸緩衝液(pH8.4) ５０μｌを注入して発光させ、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で０〜５秒間積算して測定し、この値を標準物質濃度に対してプロットすることにより、図５に示される濃度依存性を有する検量線を得た。この検量線を用いて血清検体中のヒトＡＦＰを２．０ｎｇ／ｍｌの濃度まで測定することが可能であった。
【００５０】
【発明の効果】
本発明の化学発光酵素免疫測定法は、入手が容易で取り扱いも比較的に容易なペルオキシダーゼ酵素を標識物質として用い、安価で入手が容易であるルシゲニン等を出発原料とし、且つ容易に製造できる新規化学発光試薬を用いる化学発光法により測定対象物質である種々の抗原又は抗体類を免疫学的に高感度に測定することができる。
【図面の簡単な説明】
【図１】 実施例１記載の反応系を用いて化学発光させた化学発光量をヒトαＡＦＰ（標準物質）の濃度の関数としてプロットして作成したヒトαＡＦＰ測定用の検量線である。
【図２】 実施例２記載の反応系を用いて化学発光させた化学発光量をヒトプロラクチン（標準物質）の濃度の関数としてプロットして作成したヒトプロラクチン測定用の検量線である。
【図３】 実施例３記載の反応系を用いて化学発光させた化学発光量をβｈＣＧ（標準物質）の濃度の関数としてプロットして作成したβｈＣＧ測定用の検量線である。
【図４】 実施例４記載の反応系を用いて化学発光させた化学発光量をヒトαＡＦＰ（標準物質）の濃度の関数としてプロットして作成したヒトαＡＦＰ測定用の検量線である。
【図５】 比較例１記載の反応系を用いて化学発光させた化学発光量をヒトαＡＦＰ（標準物質）の濃度の関数としてプロットして作成したヒトαＡＦＰ測定用の検量線である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an enzyme immunoassay method using a chemiluminescence method, and more particularly to an immunoassay method for an antigen or antibody using a peroxidase enzyme as a labeling substance and a specific novel chemiluminescence reagent. It is.
[0002]
[Prior art]
Enzyme immunoassay does not use radioisotopes as a labeling substance, so it can maintain a good measurement environment and has been developed as an immunoassay with little danger to the human body. It is used in the system. In this enzyme immunoassay, various enzymes such as peroxidase, alkaline phosphatase, β-galactosidase and glucose oxidase are used. Examples of methods for detecting enzyme activity of enzyme-labeled antibodies or antigens using these enzymes include hydrogen peroxide / o-phenylenediamine, 4-nitrophenyl-phosphate, 2-nitrophenyl-β-galactoside, etc. A colorimetric method is generally used to measure the amount of color of a chromogenic substance produced by the decomposition reaction of the substrate with the enzyme to quantify the enzyme activity, and to quantify the amount of antibody or antigen having a correlation with the enzyme activity. .
[0003]
However, in clinical chemistry analysis, the measurement target is a biological sample (mainly serum, urine, etc.), and the measured value is often used for diagnosis of a disease state or follow-up, etc. Although high-precision measurement is required, it is difficult to completely satisfy this requirement by the colorimetric method.
[0004]
Therefore, a fluorescence method has been proposed for the purpose of satisfying this requirement. The fluorescence method is a method of decomposing fluorescent substrates such as 4-hydroxyphenylacetic acid, 4-methylumbelliferyl-β-galactoside, 4-methylumbelliferyl-phosphate by the catalytic activity of the enzyme used for labeling. After the generation, the fluorescence intensity is measured to quantify the enzyme activity, and the amount of antibody or antigen having a correlation with the enzyme activity is quantified. However, in the fluorescence method, it is difficult to satisfy the above requirement because of the scattering of excitation light. In addition, the colorimetric method and the fluorescence method require a light source such as a xenon lamp, and the background level increases due to the stray phenomenon derived from the light from the light source and the Raman light derived from the solvent. In principle, it is difficult to increase the sensitivity of the measurement by the colorimetric method and the fluorescence method.
[0005]
In recent years, a chemiluminescent enzyme immunoassay (CLEIA) has been developed and attracts attention as a highly sensitive enzyme immunoassay exceeding the colorimetric method and the fluorescence method. CLEIA determines the enzyme activity by measuring the amount of luminescence emitted when the chemiluminescent substance enters an excited state through an intermediate due to the catalytic activity of the enzyme and returns to the ground state from this state, and correlates with this enzyme activity. This method quantifies the amount of antibodies or antigens that have a high degree of sensitivity, and does not require a light source because it emits a chemiluminescent substance through a chemical reaction. It is. Examples of enzymes used in CLEIA include peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, dehydrogenase, etc., but peroxidase is preferably used in terms of ease of handling, availability, A chemiluminescence system using luminol as a chemiluminescent substance and p-iodophenol as a luminescence enhancer has been developed, and various substances can be quantified immunologically with high sensitivity by a chemiluminescence method.
However, in CLEIA using a peroxidase enzyme as a labeling substance, it is often necessary to further improve the quantitativeness of a measurement target substance in a low concentration region, and the sensitivity of CLEIA using a peroxidase enzyme as a labeling substance is further increased. It was desired.
[0006]
[Problems to be solved by the invention]
Therefore, in view of the above circumstances, an object of the present invention is to provide a chemiluminescent enzyme immunoassay method using a novel immunoassay system based on a chemiluminescence method capable of measuring a measurement target substance with higher sensitivity.
[0007]
[Means for Solving the Problems]
Therefore, as a result of intensive studies to solve the above-mentioned problems, the present inventors have found that N, N'-disubstituted-9,9'-bisacridinium salt charge transfer complexes and N Chemiluminescent reagent comprising N, N-disubstituted carboxylic acid amide compound, or charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts, N, N-disubstituted carboxylic acid amide compound and A chemiluminescent system that uses a chemiluminescent reagent consisting of a specific amino alcohol compound and, if necessary, a specific phenolic compound as a luminescence enhancer measures more sensitively than a chemiluminescent system that uses luminol as the chemiluminescent reagent. It was found that the target substance can be quantified immunologically, and the present invention has been reached based on these findings.
[0008]
That is, the first of the present invention is
The peroxidase enzyme-labeled antibody or antigen is mixed with the antigen or antibody to be measured in the sample, or an aggregate thereof, and an immune complex consisting of a peroxidase enzyme label-antigen-antibody complex is formed by an antigen-antibody reaction. And the following general formula (1)
[0009]
[Formula 4]

(In the above general formula (1), R¹ And R² Are each selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other;^Three , R^Four , R^Five And R⁶ Are each selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other, and X. is from the counter anion of the precursor bisacridinium salt. An acid radical that is a residue from which electrons have been transferred is shown. )
A charge transfer complex of N, N'-disubstituted-9,9'-bisacridinium salts represented by:
And the following general formula (2)
[0010]
[Chemical formula 5]

(In the above general formula (2), R₁ Is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms, and the aryl group is an alkyl group, nitro group, hydroxyl group, amino May be substituted with a group or a halogen atom, and R₂ Is selected from the group consisting of methyl and ethyl;_Three Is selected from the group consisting of an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms, and the aryl group is an alkyl group, nitro group, hydroxyl group, amino group or halogen May be substituted with atoms, etc., R₁ And R_Three May be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which they are bonded. )
A chemiluminescent reagent mainly composed of an N, N-disubstituted carboxylic acid amide compound represented by the above formula is used for chemiluminescence in the presence of a hydrogen acceptor, and the luminescence intensity is measured to measure the antigen or antibody in the sample. The present invention relates to a chemiluminescent enzyme immunoassay characterized by quantifying the amount.
[0011]
In the second aspect of the present invention, a peroxidase enzyme-labeled antibody or antigen is mixed with an antigen or antibody to be measured in a sample or an aggregate thereof, and an immunity comprising a peroxidase enzyme label-antigen-antibody complex by an antigen-antibody reaction. After the complex is formed, it is separated, and the chemiluminescent reagent according to claim 1 is added to the following general formula (3):
[0012]
[Chemical 6]

(In the general formula (3), R represents a divalent aliphatic hydrocarbon having 1 to 5 carbon atoms, and m represents an integer of 1 to 3).
The amount of antigen or antibody in a sample is quantified by chemiluminescence in the presence of a hydrogen acceptor using a chemiluminescence reagent to which an amino alcohol compound represented by the formula is added and measuring the luminescence intensity. To a chemiluminescent enzyme immunoassay.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
In the chemiluminescent enzyme immunoassay method of the present invention, a peroxidase enzyme-labeled antibody or antigen is mixed with an antigen or antibody to be measured in a sample, or an aggregate thereof, and the antigen or antibody to be measured by an antigen-antibody reaction is peroxidized. It comprises an immune reaction stage for capturing as an enzyme-labeled immune complex, and a chemiluminescence reaction stage in which the produced immune complex is measured by a chemiluminescence method using a labeled enzyme present in the molecule.
[0014]
The method of antigen-antibody reaction that constitutes the immune reaction step is arbitrary, and includes a charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts and an N, N-disubstituted carboxylic acid amide compound. Or a chemiluminescent reagent comprising a charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts, an N, N-disubstituted carboxylic acid amide compound and a specific amino alcohol compound Any method can be adopted as long as it can be used.
[0015]
For example,
(1) Sandwich method in which an antibody bound to an insoluble carrier is captured with an antigen to be measured and then reacted with a peroxidase enzyme-labeled antibody.
(2) In the sandwich method, an antibody derived from an animal species different from the antibody bound to the insoluble carrier is used, and the resulting sandwich complex is further reacted with a labeled second antibody against this antibody,
(3) A competitive method in which an antibody to be measured is reacted with an antibody bound to an insoluble carrier in the presence of a peroxidase enzyme-labeled antigen.
(4) The labeled substance in the immune complex separated by centrifugation after the labeled antibody or antigen specifically reacting with the sample to be measured is allowed to act on the sample containing the antigen or antibody to be measured. Coagulation sedimentation method to detect,
(5) An antibody detection method in which a peroxidase enzyme-labeled anti-human gamma globulin antibody is allowed to act on an antibody to be measured in an antigen bound to an insoluble carrier,
(6) Biotin-avidin method in which peroxidase enzyme-labeled avidin is reacted with a biotin-labeled antibody, etc.
Can be used without limitation.
[0016]
Examples of insoluble carriers used in the chemiluminescent enzyme immunoassay method of the present invention include, for example, high molecular compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharides, glass, metals, Examples thereof include magnetic particles and combinations thereof. The insoluble carrier can be used in various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a microplate, and a test tube. Furthermore, the method for immobilizing the antigen or antibody on these insoluble carriers is arbitrary, but a physical adsorption method, a covalent bond method, an ionic bond method, or the like can be used.
[0017]
The antibodies used in the chemiluminescent enzyme immunoassay method of the present invention can be either a monoclonal antibody or a polyclonal antibody, and the form thereof is a whole antibody or F (ab ')₂ Fragments such as Fab can be used. The origin of the antibody is arbitrary, but an antibody derived from mouse, rat, rabbit, sheep, goat, chicken or the like is preferably used.
[0018]
Furthermore, the chemiluminescent reaction that constitutes the latter stage of the chemiluminescent enzyme immunoassay method of the present invention comprises N, N′-disubstituted-9,9′-bisacridinium salts as a chemiluminescent reagent and N, Using a chemiluminescent reagent composed of an N-disubstituted carboxylic acid amide compound, a hydrogen acceptor is allowed to act in the presence of a luminescence enhancer to measure the activity of a peroxidase enzyme that is a labeling substance captured on an insoluble carrier. However, in general, a measurement reagent containing a chemiluminescent substance and a luminescence enhancer is added to a peroxidase enzyme-labeled antibody or an insoluble carrier immunologically captured with an antigen to obtain a specific basicity. In the pH range, a method of adding a hydrogen acceptor, for example, an aqueous hydrogen peroxide solution to cause a chemiluminescence reaction, and measuring the amount of chemiluminescence with a luminescence measuring apparatus is performed.
[0019]
Next, the chemiluminescent reagent used in the chemiluminescent enzyme immunoassay method of the present invention will be specifically described.
The chemiluminescent reagent comprises an N, N′-disubstituted-9,9′-bisacridinium salt charge transfer complex and an N, N-disubstituted carboxylic acid amide compound as essential components, and an amino alcohol as an optional component. A compound is added.
The charge transfer complex of N, N'-disubstituted-9,9'-bisacridinium salts is
The following general formula (1)
[0020]
[Chemical 7]

It is represented by
[0021]
In the general formula (1), R¹ And R² Are each selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same as or different from each other. The alkyl group, aryl group and halogenated aryl group have 1 to 20 carbon atoms, and preferred alkyl groups are those having 1 to 10 carbon atoms. Examples thereof include linear alkyl groups such as methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group and decyl group, or branched alkyl groups thereof. . In addition, the aryl group preferably has 6 to 20 carbon atoms, and examples thereof include a phenyl group, a tolyl group, and a xylyl group, and may further be substituted with an alkyl group. As the aryl group, a phenyl group is particularly preferable. Examples of the halogenated aryl group include a halogenated phenyl group, a halogenated tolyl group, a halogenated xylyl group, and the like, and a chlorophenyl group is particularly preferable.
[0022]
In the general formula (1), R^Three , R^Four , R^Five And R⁶ Are each selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other. Examples of these hydrocarbon groups include those having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms. For example, a linear or branched alkyl group having 1 to 20 carbon atoms, an alkoxy group, an aryl group having 6 to 20 carbon atoms, and an aryloxy group can be exemplified, and the aryl group and the aryloxy group are substituted with an alkyl group. It may be a thing.
In the general formula (1), X. represents an acid radical that is a residue obtained by transferring electrons from the counter anion of the precursor bisacridinium salt.
[0023]
Charge transfer complexes of N, N′-disubstituted-9,9′-bisacridinium salts measure the appearance of a broad absorption band having a maximum near 550 nm in the ultraviolet absorption spectrum by a method using a spectrophotometer. be able to.
Specific examples of the charge transfer body of the N, N′-disubstituted-9,9′-bisacridinium salts include N, N′-dimethyl-9,9′-bisacridinium salts, N , N′-diethyl-9,9′-bisacridinium salt, N, N′-dipropyl-9,9′-bisacridinium salt, N, N′-diisopropyl-9,9′-bisacridin Nium salt, N, N′-dibutyl-9,9′-bisacridinium salt, N, N′-diisobutyl-9,9′-bisacridinium salt, N, N′-diphenyl-9,9 ′ -A charge transfer complex such as bisacridinium salt, N, N'-di-m-chlorophenyl-9,9'-bisacridinium salt, and the like. Among these, in particular, N, N'- Charge of dimethyl-9,9'-bisacridinium dinitrate (lucigenin) Dynamic complex is preferably used.
[0024]
As counter ions of N, N′-disubstituted-9,9′-bisacridinium salts, which are precursors of charge transfer complexes of N, N′-disubstituted-9,9′-bisacridinium salts, Chlorine ions, bromine ions, iodine ions, nitrate ions, carbonate ions, sulfate ions, phosphate ions, carboxylate ions, and the like.
N, N'-disubstituted carboxylic acid amide compounds are:
The following general formula (2)
[0025]
[Chemical 8]

It is represented by
[0026]
In the general formula (2), R₁ Is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms, and the aryl group is an alkyl group, nitro group, hydroxyl group, amino It may be substituted with a group or a halogen atom. R₂ Is selected from the group consisting of methyl and ethyl groups. R_Three Is selected from the group consisting of an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms, and the aryl group is an alkyl group, nitro group, hydroxyl group, amino group or halogen It may be substituted with an atom or the like. R₁ And R_Three May be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which they are bonded.
[0027]
Specific examples of the N, N-disubstituted carboxylic acid amide compound represented by the general formula (2) include N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, and N, N-dimethyl. Nonlimiting examples include propionamide, N, N-dimethylbenzamide, N-methyl-2-pyrrolidone, and the like.
The amino alcohol compound is
The following general formula (3)
[0028]
[Chemical 9]

It is represented by
[0029]
In the general formula (3), R represents a divalent aliphatic hydrocarbon having 1 to 5 carbon atoms, and m represents an integer of 1 to 3.
Specific examples of the amino alcohol compound include monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, diisopropanolamine, triisopropanolamine and the like.
[0030]
One of the chemiluminescent reagents used in the chemiluminescent immunoassay method of the present invention is a charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts and a specific N, N-disubstituted N, which is a precursor of a charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts, which is one of the components, which contains a carboxylic acid amide compound as an essential component. N′-disubstituted-9,9′-bisacridinium salts are easily converted into charge transfer complexes by natural light or the like due to the presence of another component, N, N-disubstituted carboxylic acid amide compound. Is recognized. Further, this N, N-disubstituted carboxylic acid amide compound is considered to contribute to the stability of the radical of the generated charge transfer complex, and N, N′-disubstituted consisting of an acid having a weak nucleophilicity. It acts as an essential component for the formation and stabilization of charge transfer complexes of -9,9'-bisacridinium salts. Another chemiluminescent reagent used in the chemiluminescent immunoassay method of the present invention is a charge transfer complex of the above N, N′-disubstituted-9,9′-bisacridinium salts and N, N-di This is obtained by adding a specific amino alcohol compound to a chemiluminescent reagent comprising a substituted carboxylic acid amide compound, but the addition of this amino alcohol compound reduces the blank value of the luminescence reaction and is more ugly. A stable chemiluminescence reaction can be realized in the peroxidase concentration range. The effect of the addition of this amino alcohol compound is not necessarily clear, but it is involved in charge transfer from the counter ion to the acridine ring to promote the reaction and further stabilize the radicals in the generated charge transfer complex. It is considered that stable high-sensitivity measurement with a low blank value is enabled by eliminating the factor that increases the blank value by preventing the reaction with dissolved oxygen during the luminescence reaction.
[0031]
The method for producing the chemiluminescent reagent is arbitrary, but the charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts and an N, N-disubstituted carboxylic acid amide compound are the main components. The chemiluminescent reagent to be used is to mix N, N′-disubstituted-9,9′-bisacridinium salts with equimolar or more N, N-disubstituted carboxylic acid amide compounds and leave them under natural light. However, since the formation of the charge transfer complex is catalyzed by light, the reaction is preferably performed under light irradiation. As a light source used for this light irradiation, an ultraviolet-visible part having a wavelength region of about 290 to 800 nm is used, and visible light of about 400 to 800 nm is particularly desirable. As these light sources, high-pressure mercury lamps, low-pressure mercury lamps, germicidal lamps, fluorescent lamps, incandescent lamps and the like can be used without limitation, but incandescent lamps are preferably used.
[0032]
The mixing ratio of the charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts to the N, N-disubstituted carboxylic acid amide compound is N, N′-disubstituted- The N, N-disubstituted carboxylic acid amide compound can be used in a ratio of 1 to 10,000 times mol, preferably 1 to 5,000 times mol, of the charge transfer complex of 9,9′-bisacridinium salts. .
[0033]
Another chemiluminescent reagent used in the present invention is a chemiluminescence comprising a charge transfer complex of the N, N′-disubstituted-9,9′-bisacridinium salt and an N, N-disubstituted carbonamide compound. It is obtained by adding a specific amino alcohol compound to the reagent. The amino alcohol compound can be added during and / or after the charge transfer reaction of N, N'-disubstituted-9,9'-bisacridinium salts, but is preferably added after the charge transfer reaction. Here, the addition during the charge transfer reaction includes the addition before the charge transfer reaction. The amino alcohol compound is added in an amount of 1 to 10,000 times mol, preferably 1 to 2000 times mol for the charge transfer complex of N, N'-disubstituted-9,9'-bisacridinium salts. The amount is sufficient.
[0034]
In the chemiluminescent immunoassay method of the present invention, the novel chemiluminescent reagent emits light in an amount depending on the concentration of peroxidase in the presence of an excess of hydrogen acceptor under basic conditions of pH 7.5-13. It is recognized that this light emission is enhanced by a light emission accelerator such as a phenolic compound. Such phenolic compounds include p-hydroxycinnamic acid, p-phenylphenol, p- (4-chlorophenyl) phenol, p- (4-bromophenyl) phenol, p- (4-iodophenyl) phenol, p -Iodophenol, p-bromophenol, p-chlorophenol, 2,4-dichlorophenol, p-coumaric acid, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin, and the like.
[0035]
In the case of performing chemiluminescence analysis using the above novel chemiluminescent reagent, 10^-8~ 1M, preferably 10^-6-10^-2It is used at a concentration in the range of M, and the amount used may be in the range of 10 to 500 μl, preferably 50 to 300 μl. Moreover, the usage-amount of a light emission promoter is 0.01-100 times mole of a chemiluminescent reagent, Preferably it is the range of 0.1-10 times mole, As the density | concentration, it is 10^-6~ 1M, preferably 10^-Four-10^-2A range of M is adopted.
[0036]
The hydrogen acceptor used in the chemiluminescence reaction is not particularly limited as long as it can be a substrate for the peroxidase enzyme, and inorganic peroxides, organic peroxides and the like are arbitrarily used. Hydrogen oxide is particularly preferred. It is necessary to use the hydrogen acceptor in a sufficiently excessive amount with respect to the chemiluminescent reagent, and the amount used thereof is 3 to 10,000 times mol, preferably 10 to 1000 times mol, of the chemiluminescent reagent. Adopted.
[0037]
Further, when various substances are quantified by labeling antibodies, nucleic acids and the like using peroxidase as a labeling substance, horseradish peroxidase (HRP) is preferably used as the peroxidase. As a basic buffer solution used for the chemiluminescence reaction, a Tris buffer solution, a phosphate buffer solution, a borate buffer solution, a carbonate buffer solution, a glycine-sodium hydroxide buffer solution, or the like can be arbitrarily used. The concentration of these buffers is preferably in the range of 1 mM to 1M. Moreover, additives, such as surfactant and a chelating agent, can be arbitrarily used at the time of reaction.
[0038]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples and the like. However, the present invention is not limited to the examples.
In addition,% in a reference example and an example means weight%.
[0039]
[Reference Example 1]
Preparation of chemiluminescent reagent comprising N, N'-dimethyl-9,9'-bisacridinium dihydroiodide charge transfer complex and N, N-dimethylacetamide
1x10 of lucigenin^-2A 2 times mole of solid potassium iodide was added to a mole / l aqueous solution and stirred at room temperature for 1 hour to form a red precipitate. The reaction solution containing this red precipitate is placed in an eggplant-shaped flask, and the solvent water is evaporated to dryness at 60 ° C. under reduced pressure using a rotary evaporator. The residue consisting of the red precipitate is washed with benzene to remove by-products. The benzene insoluble matter was filtered off and dried to obtain a crude product. Next, a small amount of water is added to the crude product, and the mixture is dissolved by heating on a 95 ° C. hot water bath in the dark. After returning to room temperature, the mixture is further left to cool to 4 ° C. to precipitate the produced precipitate. The unreacted material was removed by filtration and drying to obtain N, N′-dimethyl-9,9′-bisacridinium dihydroiodide in a yield of about 70%. Next, 1.5 mg of this compound is taken into a test tube, and 1 ml of N, N-dimethylacetamide is added thereto and dissolved, followed by irradiation with a 250 W copy lamp in a water bath at a temperature of 30 ° C. for 7 hours. A chemiluminescent reagent containing a charge transfer complex of N, N′-dimethyl-9,9′-bisacridinium dihydroiodide was obtained. The formation of the charge transfer complex was confirmed by measuring the appearance of a broad absorption band having a maximum near 550 nm in the ultraviolet absorption spectrum by a method using a spectrophotometer.
[0040]
[Reference Example 2]
Preparation of chemiluminescent reagent comprising N, N'-dimethyl-9,9'-bisacridinium dihydroiodide charge transfer complex, N, N-dimethylacetamide and triethanolamine
A chemiluminescent reagent was obtained by adding and mixing 0.5 ml of triethanolamine to 1 ml of the chemiluminescent reagent obtained in Reference Example 1.
[0041]
[Reference Example 3]
Preparation of chemiluminescent reagent comprising N, N'-dimethyl-9,9'-bisacridinium dinitrate charge transfer complex and N, N-dimethylacetamide
After taking 1.5 mg of lucigenin in a test tube and adding 1 ml of N, N-dimethylacetamide to dissolve it, N, N is irradiated by irradiation with a 250 W copy lamp in a water bath at 30 ° C. for 7 hours. '-Dimethyl-9,9'-bisacridiniumTheA chemiluminescent reagent containing a charge transfer complex of nitrate was obtained.
[0042]
[Reference Example 4]
Preparation of a chemiluminescent reagent comprising a charge transfer complex of N, N'-dimethyl-9,9'-bisacridinium dinitrate, N, N-dimethylacetamide and triethanolamine
A chemiluminescent reagent was obtained by adding 0.5 ml of triethanolamine to 1 ml of the chemiluminescent reagent obtained in Reference Example 3 and mixing them.
[0043]
[Reference Example 5]
Preparation of insoluble carrier-immobilized polyclonal antibody
A solution prepared by dissolving a polyclonal antibody derived from an animal such as a spider having a specific reactivity with an antigen in 10 mM phosphate buffered saline (PBS) (pH 7.4) at a concentration of 10 mg / ml is prepared as a white microplate (lab system 0.1 ml each, and left at 37 ° C. for 1 hour, washed with PBS, then added 0.3 ml of 1% bovine serum albumin (BSA) aqueous solution at a temperature of 37 ° C. Were left standing for 1 hour and post-coating was performed to obtain a polyclonal antibody-immobilized white microplate.
[0044]
[Reference Example 6]
Preparation of peroxidase enzyme-labeled monoclonal antibody
To a 1 ml solution of a monoclonal antibody derived from a mouse having a specific reactivity with an antigen, dissolved in 10 mM phosphate buffered saline (PBS) (pH 7.4) at a concentration of 1.0 mg / ml, N- (m- 0.1 ml of a 10 mg / ml dimethylformamide solution of maleimidobenzoic acid) -N-succinimide ester (MBS) was added and reacted at a temperature of 25 ° C. for 30 minutes. Next, this reaction mixture was subjected to gel filtration with a 0.1M phosphate buffer (pH 6.0) using a column packed with Sephadex G-25 to separate maleimide monoclonal antibody and unreacted MBS.
On the other hand, an ethanol solution of N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) at a concentration of 10 mg / ml was added to a 1.0 mg / ml PBS solution of horseradish peroxidase (HRP) as a peroxidase enzyme. And reacted at a temperature of 25 ° C. for 30 minutes.
Next, this reaction mixture was purified by gel filtration with 10 mM acetate buffer (pH 4.5) using a column packed with Sephadex G-25, and a fraction containing pyridyl disulfide HRP was collected. In the collodion bag, the solution was concentrated about 10 times under ice cooling. Next, 1 ml of 0.1 M acetate buffered saline (pH 4.5) containing 0.1 M dithiothreitol was added thereto, and the mixture was stirred at a temperature of 25 ° C. for 30 minutes to introduce pyridyl introduced into the HRP molecule. After reducing the disulfide group, the reaction mixture was subjected to gel filtration using a column packed with Sephadex G-25 to obtain a fraction containing thiolated HRP.
Next, the maleimidated monoclonal antibody and thiolated HRP were mixed, concentrated to a protein concentration of 4 mg / ml under ice-cooling using a collodion bag, and allowed to stand overnight at 4 ° C., after which Ultrogel AcA44 (SEPRACOR) was used. Gel filtration was performed using a packed column to obtain a peroxidase enzyme-labeled monoclonal antibody.
[0045]
[Example 1]
Measurement of α-fetoprotein (AFP) by simultaneous sandwich method CLEIA using chemiluminescent reagent prepared in Reference Example 1
2% BSA-containing PBS solution (pH 7) containing purified human AFP (standard substance) in a range of 0 to 800 ng / ml on a white microplate on which the anti-human AFP polyclonal antibody prepared by the method of Reference Example 5 is immobilized. .4) Add 50 μl and 100 μl of a 2% BSA-containing PBS solution (pH 7.4) containing a peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody prepared by the method of Reference Example 6 at a concentration of about 3 μg / ml, and add 37 ° C. For 1 hour. Next, after removing the solution in the well by suction, the well was washed with physiological saline, and then 100 μl of 75 mM Tris-HCl buffer (pH 7.8) was added to each well. After sequentially injecting 100 μl of a chemiluminescent reagent solution and 50 μl of 75 mM Tris-HCl buffer (pH 7.8) containing 0.0017% hydrogen peroxide to emit light, this luminescence was measured using a luminometer (Luminous CT-manufactured by Diatron). 9000D) and integrated for 1 to 5 seconds, and by plotting this value against the standard substance concentration, a calibration curve with good concentration dependency shown in FIG. 1 was obtained. Using this calibration curve, it was possible to measure human AFP in serum samples up to a concentration of 0.01 ng / ml.
[0046]
[Example 2]
Measurement of human prolactin (PRL) by simultaneous sandwich method CLEIA using the chemiluminescent reagent prepared in Reference Example 2
2% BSA-containing PBS solution (pH 7) containing purified human PRL (standard substance) in the range of 0 to 100 ng / ml on a white microplate on which the anti-human PRL polyclonal antibody prepared by the method of Reference Example 5 is immobilized. .4) Add 50 μl and 100 μl of a 2% BSA-containing PBS solution (pH 7.4) containing a peroxidase enzyme-labeled mouse anti-human PRL monoclonal antibody prepared by the method of Reference Example 6 at a concentration of about 3 μg / ml, and add 37 ° C. For 1 hour. Next, the solution in the well is removed by aspiration, the inside of the well is washed with physiological saline, and 100 μl of 75 mM Tris-HCl buffer (pH 7.8) is added to each well.2After sequentially injecting 100 μl of the chemiluminescent reagent solution prepared in step 1 and 50 μl of 75 mM Tris-HCl buffer (pH 7.8) containing 0.0017% hydrogen peroxide, the amount of luminescence was measured using a luminometer (manufactured by Diatron). Luminous CT-9000D) was integrated and measured for 1 to 5 seconds, and this value was plotted against the standard substance concentration to obtain a calibration curve with good concentration dependence shown in FIG. Using this calibration curve, it was possible to measure human PRL in serum samples to a concentration of 0.01 ng / ml.
[0047]
[Example 3]
Measurement of human chorionic gonadotropin β chain (βhCG) by simultaneous sandwich method CLEIA prepared in Reference Example 3
A PBS solution containing 2% BSA (pH 7. 7) containing purified βhCG (standard substance) in a range of 0 to 100 mIU / ml on a white microplate on which the anti-human hCG polyclonal antibody prepared by the method of Reference Example 5 is immobilized. 4) Add 50 μl and 100 μl of a 2% BSA-containing PBS solution (pH 7.4) containing a peroxidase enzyme-labeled mouse anti-βhCG monoclonal antibody prepared by the method of Reference Example 5 at a concentration of about 2 μg / ml, at a temperature of 37 ° C. Incubated for 1 hour. Next, after removing the solution in the well by suction, the well was washed with physiological saline, and then 100 μl of 75 mM Tris-HCl buffer (pH 7.8) was added to each well.3After sequentially injecting 100 μl of the chemiluminescent reagent solution prepared in step 1 and 50 μl of 75 mM Tris-HCl buffer (pH 7.8) containing 0.0017% hydrogen peroxide, the amount of luminescence was measured using a luminometer (manufactured by Diatron). Luminous CT-9000D) was integrated and measured for 1 to 5 seconds, and this value was plotted against the standard substance concentration to obtain a calibration curve with good concentration dependency shown in FIG. Using this calibration curve, it was possible to measure βhCG in serum samples up to a concentration of 0.01 mIU / ml.
[0048]
[Example 4]
By simultaneous sandwich method CLEIA using the chemiluminescent reagent prepared in Reference Example 4 Measurement of α-fetoprotein (AFP)
A PBS solution containing 2% BSA containing purified human AFP (standard substance) in a range of 0 to 800 ng / ml on a white microplate on which the resistance ratio prepared by the method of Reference Example 5 and an AFP polyclonal antibody are immobilized. 50 μl and 100 μl of 2% BSA-containing PBS solution (pH 7.4) containing a peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody prepared by the method of Reference Example 6 at a concentration of about 3 μg / ml, Incubate for 1 hour at a temperature of 0C. Next, after removing the solution in the well by suction, the well was washed with physiological saline, and then 100 μl of 75 mM Tris-HCl buffer (pH 7.8) was added to each well. After sequentially injecting 100 μl of a chemiluminescent reagent and 50 μl of 75 mM Tris-HCl buffer (pH 7.8) containing 0.0017% hydrogen peroxide to emit light, this luminescence was measured using a luminometer (Luminous CT-9000D manufactured by Diatron). ) Was integrated for 1 to 5 seconds, and this value was plotted against the standard concentration to obtain a calibration curve having concentration dependency as shown in FIG. Using this calibration curve, it was possible to measure human AFP in serum samples up to a concentration of 0.01 ng / ml.
[0049]
[Comparative Example 1]
Determination of α-fetoprotein (AFP) by simultaneous sandwich method CLEIA using luminol
(2) 50 μl of 2% BSA-containing PBS solution (pH 7.4) containing purified human AFP (standard substance) in the range of 0 to 800 ng / ml on a white microplate immobilized with anti-human AFP polyclonal antibody and peroxidase enzyme labeling 100% of 2% BSA-containing PBS solution (pH 7.4) containing mouse anti-human AFP monoclonal antibody at a concentration of about 3 μg / ml was added and incubated at a temperature of 37 ° C. for 1 hour. Next, the solution in the well is removed by suction, and then the well is washed with physiological saline, and then 10 parts of p-iodophenol is added to each well.^-3100 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing M at a concentration of 9.5 × 10^-FiveInject 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing M at a concentration of 50 and 50 μl of 0.1 M Tris-HCl buffer (pH 8.4) in 0.0034% hydrogen peroxide to emit light. The amount of luminescence is measured by integrating for 0 to 5 seconds with a luminometer (Diatron Luminous CT-9000D), and this value is plotted against the standard concentration, thereby having the concentration dependency shown in FIG. A calibration curve was obtained. Using this calibration curve, it was possible to measure human AFP in serum samples up to a concentration of 2.0 ng / ml.
[0050]
【The invention's effect】
The chemiluminescent enzyme immunoassay method of the present invention uses a peroxidase enzyme that is easily available and relatively easy to handle as a labeling substance, and is a novel material that can be easily manufactured using lucigenin, which is inexpensive and easily available, as a starting material. Various antigens or antibodies that are measurement target substances can be measured immunologically with high sensitivity by a chemiluminescence method using a chemiluminescent reagent.
[Brief description of the drawings]
FIG. 1 is a calibration curve for measuring human αAFP prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 1 as a function of the concentration of human αAFP (standard substance).
FIG. 2 is a calibration curve for measuring human prolactin prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 2 as a function of the concentration of human prolactin (standard substance).
FIG. 3 is a calibration curve for βhCG measurement prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 3 as a function of the concentration of βhCG (standard substance).
FIG. 4 is a calibration curve for measuring human αAFP prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 4 as a function of the concentration of human αAFP (standard substance).
FIG. 5 is a calibration curve for measuring human αAFP prepared by plotting the chemiluminescence amount chemiluminescent using the reaction system described in Comparative Example 1 as a function of the concentration of human αAFP (standard substance).

Claims (8)

The peroxidase enzyme-labeled antibody or antigen is mixed with the antigen or antibody to be measured in the sample, or an aggregate thereof, and an immune complex consisting of a peroxidase enzyme label-antigen-antibody complex is formed by an antigen-antibody reaction. And the following general formula (1)

(In the above general formula (1), R ¹ and R ² are each selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ and R ⁶ are each selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other, and X. is a precursor bisacridinium salt. It shows an acid radical that is a residue obtained by transferring electrons from the counter anion.)
A charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts represented by the formula : wherein the ultraviolet absorption spectrum has an absorption band having a maximum near 550 nm;
And the following general formula (2)

(In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms; The group may be substituted with an alkyl group, a nitro group, a hydroxyl group, an amino group or a halogen atom, R ₂ is selected from the group consisting of a methyl group and an ethyl group, and R ₃ is an alkyl group having 1 to 10 carbon atoms. Selected from the group consisting of an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms, and the aryl group may be substituted with an alkyl group, a nitro group, a hydroxyl group, an amino group, a halogen atom, or the like. R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which they are bonded.
A chemiluminescent reagent mainly composed of an N, N-disubstituted carboxylic acid amide compound represented by the above formula is used for chemiluminescence in the presence of a hydrogen acceptor, and the luminescence intensity is measured to measure the antigen or antibody in the sample. A chemiluminescent enzyme immunoassay characterized by quantifying the amount. The peroxidase enzyme-labeled antibody or antigen is mixed with the antigen or antibody to be measured in the sample, or an aggregate thereof, and an immune complex consisting of a peroxidase enzyme label-antigen-antibody complex is formed by an antigen-antibody reaction. And the following general formula (1)

(In the above general formula (1), R ¹ and R ² are each selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ and R ⁶ are each selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other, and X. is a precursor bisacridinium salt. It shows an acid radical that is a residue obtained by transferring electrons from the counter anion.)
A charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts represented by the formula (1) having an absorption band having a maximum near 550 nm in an ultraviolet absorption spectrum;
And the following general formula (2)

(In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms; The group may be substituted with an alkyl group, a nitro group, a hydroxyl group, an amino group or a halogen atom, R ₂ is selected from the group consisting of a methyl group and an ethyl group, and R ₃ is an alkyl group having 1 to 10 carbon atoms. Selected from the group consisting of an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms, and the aryl group may be substituted with an alkyl group, a nitro group, a hydroxyl group, an amino group, a halogen atom, or the like. R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which they are bonded.
An N, N-disubstituted carboxylic acid amide compound represented by:
And the following general formula (3)

(In the above general formula (3), R represents a divalent aliphatic hydrocarbon having 1 to 5 carbon atoms, and m represents an integer of 1 to 3).
A chemiluminescent reagent comprising an amino alcohol compound represented by the formula: chemiluminescence in the presence of a hydrogen acceptor, and measuring the emission intensity to quantify the amount of antigen or antibody in the sample. Luminescent enzyme immunoassay.   The chemiluminescent enzyme immunoassay method according to claim 1 or 2, wherein the antigen-antibody reaction is carried out in the presence of an antibody immobilized on an insoluble carrier to form a labeled immune complex on the insoluble carrier. The charge transfer complex of N, N′-disubstituted-9,9′-bisacridinium salts is N, N′-dimethyl-9,9′-bisacridinium dinitrate, N, N′-dimethyl. 9,9'-bis acridinium di hydroiodide or N, N, '- dimethyl-9,9' - one of the claims 1 to 3 which is a charge transfer complex of bis acridinium dihydrochloride The chemiluminescent enzyme immunoassay method according to any of the claims.   The chemiluminescent enzyme immunoassay method according to any one of claims 1 to 4, wherein the N, N-disubstituted carboxylic acid amide compound is N, N-dimethylformamide or N, N-dimethylacetamide.   The chemiluminescent enzyme immunoassay method according to any one of claims 1 to 5, wherein the hydrogen acceptor is hydrogen peroxide or a hydrogen peroxide source.   The chemiluminescent enzyme immunoassay method according to any one of claims 1 to 6, further comprising a luminescence enhancer when the chemiluminescent reaction is performed.   The chemiluminescent enzyme immunoassay method according to claim 7, wherein the luminescence enhancer is a phenolic compound.

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