JP3543001B2 - Method for labeling or detecting substances using luminescent aryl sulfonate cyanine dye - Google Patents

Description

メロシアニン
【００５６】
【化２】

スチリル
【００５７】
【化３】

下記のものはポリメチンタイプ染料の更に特定した例である：
シアニン
【００５８】
【化４】

メロシアニン
【００５９】
【化５】

スチリル
【００６０】
【化６】

シアニン
【００６１】
【化７】

メロシアニン
【００６２】
【化８】

スチリル
【００６３】
【化９】

上記構造において、
ＸおよびＹはＯ，Ｓおよび
【００６４】
【化１０】

よりなる群から選ばれ、
ＺはＯおよびＳよりなる群から選ばれ、そして、
ｍは１，２，３および４よりなる群から選ばれる整数である。
【００６５】
上記式において、メチン基の数は部分的に励起カラーを決定する。環式アジン構造も亦、部分的に励起構造を決定しうる。しばしば、ｍのより高い値は増加ルミネッセンスないし吸光度に寄与する。４より高いｍの値では、化合物は不安定になる。そこでは、環構造での変性により更にルミネッセンスが付与されうる。ｍ＝２のとき、励起波長は約６５０ｎｍで、該化合物は正しく蛍光を発生する。最大発光波長は最大励起波長より概ね１５〜１００ｎｍ大きい。
【００６６】
各分子中Ｒ_１ ，Ｒ_２ ，Ｒ_３ ，Ｒ_４ ，Ｒ_５ ，Ｒ_６ およびＲ_７ 基の少なくとも一つ（好ましくは一つのみ場合により二つないし三つ以上）は、染料を標識成分に結合させるための反応基である。或る試薬に関しては、各分子上Ｒ_１ ，Ｒ_２ ，Ｒ_３，Ｒ_４ ，Ｒ_５ ，Ｒ_６ およびＲ_７ 基の少なくとも一つは、発色団の溶解性を高め或は標識成分の標識の選択性ないし染料による標識成分の標識位置に影響する基でありうる。
【００６７】
上記式において、Ｒ_８，Ｒ_９（存在時）およびＲ_１０（存在時）の少なくとも一つはスルホネート基の少なくとも一つを含む。用語スルホネート基はスルホン酸を含むものとする。なぜなら、スルホネート基はイオン化スルホン酸に過ぎないからである。
【００６８】
発色団に直接若しくは間接に結合してＲ_１，Ｒ_２，Ｒ_３，Ｒ_４，Ｒ_５，Ｒ_６およびＲ_７基を形成しうる反応基として、イソチオシアネート、イソシアネート、モノクロロトリアジン、ジクロロトリアジン、モノ−ないしジ−ハロゲン置換ピリジン、モノ−ないしジ−ハロゲン置換ジアジン、マレイミド、アジリジン、スルホニルハリド、酸ハリド、ヒドロキシスクシンイミドエステル、ヒドロキシスルホスクシンイミドエステル、イミドエステル、ヒドラジン、アジドニトロフェニル、アジド、３−（２−ピリジルジチオ）プロピオンアミド、グリオキサルおよびアルデヒドを含有する基の如き反応部分が含まれうる。
【００６９】
有効アミノ、ヒドロキシおよびスルフヒドリル基を有する成分の標識に特に有用なＲ_１，Ｒ_２，Ｒ_３，Ｒ_４，Ｒ_５，Ｒ_６およびＲ_７基の特定例として次のものが含まれる。
【００７０】
【化１１】

（ここで、Ｑ又はＷの少なくとも一つはＩ、Ｂｒ又はＣｌの如き残基である）、
【００７１】
【化１２】

２段階法で抗体を標識するのに用いることのできる有効スルフヒドリルを有する成分の標識に特に有用なＲ_１，Ｒ_２，Ｒ_３，Ｒ_４，Ｒ_５，Ｒ_６およびＲ_７基の特定例は次のものである：
【００７２】
【化１３】

（ここで、ＱはＩ又はＢｒの如き残基である）、
【００７３】
【化１４】

および
【００７４】
【化１５】

（ここで、ｎは０又は整数である）。
【００７５】
光賦活架橋による成分の標識に特に有用なＲ_１，Ｒ_２，Ｒ_３，Ｒ_４，Ｒ_５，Ｒ_６およびＲ_７基の特定例として
【００７６】
【化１６】

が含まれる。
【００７７】
水溶性を高め、或は試料中不適当な成分への標識成分の望ましくない非特異性結合を減じ、或いはまた蛍光のケンチングをもたらしうる標識成分上の反応性発色団２種以上の相互作用を低下させるために、Ｒ_１，Ｒ_２，Ｒ_３，Ｒ_４，Ｒ_５，Ｒ_６およびＲ_７基を周知の極性ないし帯電化学基から選定することができる。例は−Ｅ−Ｆであり、ここでＦはヒドロキシ、スルホネート、スルフェート、カルボキシレート、置換アミノ又は第四アミノを示し、Ｅは−（ＣＨ_２）_ｎ（ｎ＝０，１，２，３又は４）の如きスペーサー基を示す。有用な例にはアルキルスルホネート−（ＣＨ_２）_３ＳＯ^３−および−（ＣＨ_２）_４−ＳＯ^３−が含まれる。
【００７８】
本発明の発光染料のポリメチン鎖はまた、ポリメチン鎖の炭素原子２個以上の間のブリッジを形成する環式化学基１個以上を含みうる。これらのブリッジは染料の化学的安定性若しくは光安定性を高めるのに役立ち得、また染料の吸光ないし発光波長を変えたり量子収量の吸光係数を変化させるのに用いられうる。改良された溶解特性はこの変性によって達成することができる。
【００７９】
本発明に従えば、標識成分は抗体、蛋白、ペプチド、酵素基質、ホルモン、リンフォカイン、代謝産物、レセプタ、抗原、ハプテン、レクチン、アビジン、ストレプタビジン、トキシン、炭水化物、寡糖類、多糖類、核酸、デオキシ核酸、誘導核酸、誘導デオキシ核酸、ＤＮＡフラグメント、ＲＮＡフラグメント、誘導ＤＮＡフラグメント、誘導ＲＮＡフラグメント、天然薬物、ウイルス粒子、バクテリア粒子、ウイルス成分、イースト成分、血液細胞、血液細胞成分、生物細胞、非細胞血液成分、バクテリア、バクテリア成分、天然ないし合成脂質嚢、合成薬物、毒薬、環境汚染物質、重合体、重合体粒子、ガラス粒子、ガラス表面材、プラスチック粒子、プラスチック表面材、重合体膜、導体および半導体でありうる。
【００８０】
或る成分との反応時６３３ｎｍで光を吸収しうるシアニン又は関連発色団を調製することができ、而して検出工程は、このスペクトル波長で発光するヘリウムネオンレーザーを用いることができる。また、或る成分との反応時７００ｎｍと９００ｎｍとの間で最大限に光を吸収しうるシアニン又は関連染料を調製することができ、而して検出工程は、スペクトルのこの領域で光を放出するレーザーダイオードを用いることができる。
【００８１】
選択性
上に列挙した反応性基は、適当なｐＨ条件を含む適当な反応条件が用いられる限り蛋白その他生物学的若しくは非生物学的分子、巨大分子、表面材又は粒子上の特定官能基を標識するのに比較的特異性である。
【００８２】
反応性シアニン、メロシアニンおよびスチリル染料並びにそれらの生成物の特性
本発明の染料のスペクトル特性は、本明細書に記載の官能化によっては認めうるほど変化しない。標識蛋白その他の化合物のスペクトル特性も亦、蛋白その他の物質に結合してないベース染料分子とさほど異ならない。単独若しくは標識物質に結合した本発明に記載の染料は概ね、大きな吸光係数（ε＝１００，０００〜２５０，０００）と、或る場合には０．４程度の高い量子収量を有し、そして４００〜９００ｎｍのスペクトル範囲で光を発生する。斯くして、それらは発光検出用試薬を標識するものとして特に価値がある。
【００８３】
光学的検出方法
標識ないし染色成分を検出するのに任意の方法を用いることができる。検出方法は、初めに定義された波長の光で標識物質を含む混合物を照明する光源を用いることができる。該混合物により伝達され或は該混合物により蛍光ないし発光される第二波長で光を検出する既知装置が用いられる。斯かる検出装置に蛍光分光計、吸収分光測光、蛍光顕微鏡、透過光顕微鏡ないしフロー血球計算器、ファイバーオプチックセンサーおよび免疫検定装置が含まれる。
【００８４】
本発明の方法は、単数ないし複数の標識成分への染料結合を検出するのに化学的分析方法を用いることもできる。化学的分析方法には赤外スペクトロメトリー、ＮＭＲスペクトロメトリー、吸収スペクトロメトリー、蛍光スペクトロメトリー、質量スペクトロメトリーおよびクロマトグラフィー方法が含まれうる。
【００８５】
【実施例】
例 １
スルホインドジカルボシアニンと蛋白との結合に対するｐＨの影響
０．１Ｍ炭酸塩緩衝液中の羊γ−グロブリン（４ｍｇ／ｍｌ）試料を室温で１０倍モル過剰の下記スルホインドジカルボシアニン活性エステル（ｍ＝２）と室温で混合した：
【００８６】
【化１７】

５秒〜３０分範囲の適当な時間、セファデックスＧ−５０上でのゲル透過クロマトグラフィーにより蛋白試料を非共有結合染料から分離した。蛋白の最大標識は１０分後に生じて、ｐＨ８．５、８．９および９．４でインキュベートした試料に関し夫々５．８、６．４および８．２の最終的染料／蛋白モル比をもたらした。染料／蛋白比を５とするのに必要な時間および異なるｐＨでの生成物の量子収量を次表に示す：
ｐＨ 時間（ｓｅｃ） 量子収量
８．５ １１５ ０．０９
８．９ ５３ ０．０９
９．４ ６．５ ０．１７
上記データーは、この染料で標識した蛋白がｐＨ９未満よりもｐＨ９．４において良好であることを示している。緩衝液のｐＨが高いほど、結合反応は非常に迅速であるが、標識効率が優れ、生成物の蛍光度が高い。量子収率は標識蛋白上の染料分子当りの平均量子収量を表わす。
【００８７】
例 ２
スルホインドカルボシアニン活性エステルと蛋白との結合
ｐＨ７．４の燐酸塩緩衝剤入り塩水（ＰＢＳ）に溶解せる羊γ−グロブリン（１ｍｇ／ｍｌ）を、０．１Ｍ炭酸ナトリウムを用いてｐＨ９．４に調節した。種々の染料／蛋白モル比を得るためにこの蛋白溶液のアリコートにシアニン染料標識剤（例１の構造、ｍ＝１）を加えた。室温で３０分のインキュベーション後、ＰＢＳを溶離剤とするセファデックスＧ−５０ゲル透過クロマトグラフィーにより分離した。生成物中蛋白に共有結合した染料のモル比は、初期染料／蛋白比３、６、１２、２４および３０に関し夫々１．２、３．５、５．４、６．７および１１．２であった。
【００８８】
例 ３
スルホインドジカルボシアニンによるＡＥＣＭデキストランの標識
デキストラン分子当り平均１６個のアミノ基を含有するＮ−アミノエチル−カルボキシアミドメチル（ＡＥＣＭ）デキストランを平均分子量７００００のデキストランから合成した〔Ｉｎｍａｎ、Ｊ．Ｋ．，Ｊ．Ｉｍｍｕｎｏｌ．１１４：７０４−７０９（１９７５）〕。ｐＨ９．４の、０．１Ｍ炭酸塩緩衝液に溶解せるＡＥＣＭ−デキストラン（１ｍｇ／２５０μｌ）の一部分をスルホインドジカルボシアニン活性エステル（例１の構造、ｍ＝２）０．２ｍｇに加えて染料／蛋白モル比１０を得た。混合物を室温で３０分間攪拌した。次いで、溶離緩衝液として酢酸アンモニウム（５０ｍＭ）を用いたセファデックスＧ−５０ゲル透過クロマトグラフィーによりデキストランを非結合染料から分離した。各デキストラン分子に平均２．２個の染料分子が共有結合した。
【００８９】
例 ４
特異抗体のスルホインドジカルボシアニン活性エステル標識
１．１Ｍ炭酸塩緩衝液（ｐＨ９．４）中のネズミＩｇＧ（１ｍｇ／ｍｌ）に対し特異な羊γ−グロブリンとスルホインドジカルボシアニン活性エステル（例１の構造、ｍ＝２）とを染料分子／蛋白分子比８で混合した。室温で３分間のンキュベーション後、標識混合物を、燐酸塩緩衝剤入り塩水（ｐＨ７．４）で平衡させたセファデックスＧ−５０上でのゲル濾過により分離した。回収された蛋白には、各蛋白に共有結合した平均４．４の染料分子が含まれた。
【００９０】
例 ５
羊抗マウスＩｇＧ抗体に結合したスルホインドジカルボシアニン染料によるヒトリンパ球の染色ないし顕微鏡可視化
新たに単離した抹消血リンパ球をマウス抗−β２−ミクログロブリン（０．２５μｇ１０^６細胞）により０℃で３０分間処理した。細胞をＤＭＥＭ緩衝液で二回洗浄し、次いでスルホインドジカルボシアニン標識羊抗−マウスＩｇＧ抗体（１μｇ／１０^６細胞）で処理した。０℃で３０分間のインキュベーション後、細胞を遠心処理して過剰の抗体を除去し、細胞を再度ＤＭＥＭ緩衝液で洗浄した。蛍光顕微鏡で分析すべく細胞のアリコートをスライド上に固定させた。顕微鏡下、スライド上のリンパ球を６１０〜６３０ｎｍの光で照射し、イメージデジタル化装置およびテレビモニターに取付けたＣＯＴＵ赤色感知性増強テレビカメラによって６５０〜７００ｎｍの蛍光を検出した。この方法で染色した細胞は顕微鏡下蛍光を示した。対照実験で、一次マウス抗−β２−ミクログロブリン抗体の使用を省いたほかは上記の如く染色および分析を行なった。対照試料は顕微鏡下で蛍光を示さず、而してスルホインドシアニン標識羊抗−マウス抗体がリンパ球への有意な非特異性結合をもたらさないことを示した。
【図面の簡単な説明】
【図１】第１図は、Ｎ，Ｎ’−ジスルホブチルインドジカルボシアニン染料の単量体吸収スペクトルおよび二量体スペクトルを示す。
【図２】第２図は、Ｎ，Ｎ’−ジエチルインドジカルボシアニン−５，５’−ジスルホン酸染料のスペクトルを示す。
【図３】第３図は、Ｎ，Ｎ’−ジカルボキシペンチルインドジカルボシアニン−５，５’−ジスルホン酸のビス−Ｎ−ヒドロキシスクシンイミド染料による羊免疫グロブリンの標識反応における染料／蛋白モル比を示す。
【図４】第４図は、Ｎ，Ｎ’−ジカルボブチルインドジカルボシアニン−５，５’−酢酸のビス−Ｎ−ヒドロキシスクシンイミドエステルで標識された抗体の吸収スペクトルを示す。
【図５】第５図は、新規なスルホインドジカルボシアニン染料と結合した蛋白の吸収スペクトルを示す。
【図６】第６図は、染料／蛋白比の上昇に伴うアリールスルホシアニン染料の量子収量（菱形印）変化と非アリールスルホシアニン染料のそれ（丸印）との比較を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to labeling and detection of substances using luminescent aryl sulfonate cyanine dyes.
[0002]
[Prior art]
Cyanines and related dyes with light absorbing properties have been used in photographic films. Such dyes require light absorbing properties but do not require luminescent (fluorescent or phosphorescent) properties. Heretofore, cyanine dyes having luminescent properties have had very limited uses. Such uses include, among other things, labeling the sulfhydryl groups of the protein. "Sulfhydryl reagent dye is a rapid Ca from sarcoplasmic reticulum (SR)²⁺Initiate release (Sulfhydryl Reagent Dies Trigger the Rapid Release of Ca)²  from Sacoplasmic Reticulum Vehicles (SR)). Wagoner A. S. Abramson J. Indicates that the cyanine chromophore having an iodoacetyl group is Ca²⁺It has been reported that it was used to form a covalent bond with a sulfhydryl group on myoplasmic reticulum protein at pH 6.7 to initiate release (Biophysical Journal, 47, 456a (1985)). The article also states that fluorescent dyes have been used for labeling or isolating proteins.
[0003]
"Kinetics of Conformal Changes in a Region of the Rhodopsin Molecule Away from the Retiredene's Journal of Aging from the Retinylide," A Kinetics of Conformal Changes in a Region of the Rhodopsin Molecule Away from the Retinylidene. S. Jenkins P .; L. Carpenter J. et al. P. And Gupta R.A. Report that a sulfhydryl group on the F1 region of bovine rhodopsin was covalently labeled with a cyanine dye having an absorbance at 660 nm [Biophysical Journal, 33, 292a (1985)]. Again, it is reported that a cyanine dye was used to label the sulfhydryl group of the protein, but the use of a fluorescent dye was not disclosed.
[0004]
"International Works on the Application of Fluorescence photobleaching Technologies to Problems in Cell biology in the International Seminar on the Application of Fluorescent Photobleaching Techniques to Problems in Cell Biology" Jacobson K. for cyanine-type fluorescent probes that can be excited by Elson E. et al. Koppel D .; And Webb W. (Fed. Proc. 42: 72-79 (1983)).
[0005]
The only cyanine probes described in any of the above three articles are those which covalently bind, especially to the sulfhydryl groups of proteins. The only specific cyanine compounds described have an iodoacetyl group, which causes the cyanine dye to covalently react with the sulfhydryl group. None of the above articles disclose the covalent reaction of a cyanine dye with a substance other than a protein or any group on a protein other than a sulfhydryl group.
[0006]
[Problems to be solved by the invention]
However, many non-proteinaceous materials do not have sulfhydryl groups, and many proteins do not have enough of these groups to be useful for fluorescence probing. Moreover, the sulfhydryl groups (-SHSH-) readily oxidize to disulfides (-SS-) in the presence of air, thereby rendering them unacceptable for covalent bonding to the fluorescent probe.
[0007]
[Means for Solving the Problems]
The present invention provides a method for labeling components in an aqueous liquid. The method comprises:
(A) To the liquid containing the component, a luminescent dye selected from the group consisting of cyanine, merocyanine, and styryl dyes having at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus is added. Is reactive with the components, and
(B) reacting the dye with the component such that the dye labels the component
Including.
[0008]
In another aspect of the invention, a method is provided for labeling a component in an aqueous liquid. The method comprises:
(A) a sulfoindolenine base and a naphthosulfoindolenine containing at least one sulfonic acid group or sulfonate group bonded to one or more rings of a heterocyclic nucleus in the liquid containing the above components; Adding a luminescent dye selected from the group consisting of base polymethine dyes, said dye being reactive with said components;
(B) reacting the dye with the component such that the dye labels the component;
(C) detecting the label component by an optical method;
Including.
[0009]
In another aspect of the invention, there is provided a method of labeling a first component and using the labeled first component to bind to a second component to form a labeled second component. The method comprises:
(A) adding, to the aqueous liquid containing the first component, a luminescent dye selected from the group consisting of cyanine, merocyanine, and styryl dyes containing at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus; A dye is reactive with the first component;
(B) reacting the dye with the first component such that the dye luminescently labels the first component to provide a labeled first component;
(C) binding the labeled first component to the second component to form a labeled second component;
(D) detecting the labeled second component by an optical method;
Including.
[0010]
In yet another embodiment of the present invention, a sulfocyanine dye-labeled material is combined with one or more different fluorescent materials, all of which significantly absorb light at a single excitation wavelength but fluoresce at different wavelengths. A method comprising mixing, then exciting all of the fluorescent materials with a single wavelength of light, and weighing each of the fluorescent materials by detecting their respective fluorescent signals at their different fluorescent wavelengths. Provided.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
According to the present invention, under appropriate reaction conditions for the fluorescence or phosphorescence detection of a substance, not only sulfhydryl groups on proteins and other substances but also amines (-NH₂  ) And related polymethine dyes having substituents that covalently react with groups such as hydroxy (OH) groups and other aldehyde (-CHO) groups have been developed. The present invention provides significant benefits over the use of conventional iodoacetyl cyanine dyes and their specific reactivity with sulfhydryl groups. Amine and hydroxy groups predominate and are more stable in proteins or other substances than sulfhydryl groups. Accordingly, when a fluorescent cyanine dye is used for the search for the presence or absence of a specific protein, a large amount of dye molecules can bind to the protein to be searched, so that a stronger fluorescent or phosphorescent light intensity signal is output. Moreover, the amine or hydroxy groups are naturally more easily added to the desired components of the label, such as sulfhydryl, amine or hydroxy group free polymer particles.
[0012]
The present invention also provides a luminescent cyanine containing an amine or hydroxy group or a group that covalently reacts with another reactive group so that the presence or absence or amount of a labeled protein or other substance can be detected after separating the labeled component by chromatography. The present invention relates to a method for labeling a protein or other raw material having an amine or a hydroxy group or another group capable of reacting with a dye in a mixture using the dye. According to the references cited earlier, apparently sulfhydryl groups are chosen for covalent reactions, especially because the sulfhydryl groups on protein molecules are very few and in some cases they play a significant role in the function of the protein. Was. Therefore, it was possible for the author to attempt to identify the specific position of the sulfhydryl group on the protein structure. Further, in the above-mentioned literature, the sulfhydryl group was used as a probe to probe a structural change of a specific protein or to cause the change. Thus, it was necessary to know the location of the probe to determine the change in absorbance due to the dye or the change in free calcium ion due to dye binding.
[0013]
Because the number of sulfhydryl groups on most proteins is very small, the number of sulfhydryl groups may not be sufficient to provide sufficient total luminescence for probing. In contrast, the number of amine or hydroxy groups is significantly higher in number and widely dispersed on the protein molecule, allowing the fluorescent probe to bind to multiple locations on the molecule, thereby facilitating protein detection. Therefore, determination of light absorption or change in fluorescence is eliminated.
[0014]
The present invention relates to the use of luminescent polymethinecyanine or related polymethine dyes for proteins, other nucleic acids, DNAs, drugs, toxins, blood cells, microbial substances, particles, plastic or glass surface materials, polymer films, etc. For labeling at the hydroxy site. Advantageously, the dye is soluble in aqueous or other media containing the label. The present invention relates to the two-stage labeling method in addition to the one-stage labeling method. In the two-step labeling method, a first component, such as an antibody, can be labeled at a location on the component that includes an amine, hydroxy, aldehyde, or sulfhydryl moiety. The labeled component is used as a probe for a second component such as an antigen specific for the antibody.
[0015]
In the prior art described above, specificity of a binding site by a cyanine probe was achieved by using a probe that covalently reacts with a sulfhydryl group. According to the two-step method of the present invention, in a first step, a cyanine or related probe can be reacted with an amine, aldehyde, sulfhydryl, hydroxy or other group on a first component, such as an antibody, and thus a second step. At the stage of staining, the antibody can achieve the desired specificity with a second component, such as an antigen. This specificity is determined by the site of antigen binding to the antibody.
[0016]
The present invention also relates to luminescent polymethinecyanines and related compounds containing groups that allow covalent attachment to amine, hydroxy, aldehyde or sulfhydryl groups on the target molecule. The present invention relates to monoclonal antibodies and other components labeled with these luminescent cyanine compounds that can serve as antigen probes. When the target is a single cell, the present invention can be used to determine the amount of labeled antibody that binds to the cell. This measurement can be performed by examining the relative brightness of the luminescence of the cells.
[0017]
The present invention can be used to determine the concentration of specific proteins or other components of the system. If the number of reactive groups on the protein that can react with the probe is known, the fluorescence per molecule can be known, and the molecular concentration of the system can be determined by the total luminescence intensity of the system.
[0018]
The method can be used to quantitate various proteins or other substances in the system by labeling the entire mixture of proteins in the system and then separating the labeled proteins by any means such as chromatography. Next, the amount of the luminescent protein to be separated can be examined, and the position of the dye on the labeling substance can be confirmed by a chromatography detection system.
[0019]
The present invention can also be used to determine the number of various cells labeled with an antibody. A survey of this number can be performed by labeling multiple cells of the system and then separating the labeled cells out of the system. Labeled cells can also be separated from unlabeled cells outside the system.
[0020]
Another embodiment of the present invention provides a plurality of different luminescent cyanines or related dyes, each bound to a different first component, such as an antibody, each of which is bound to a first component specific to a different second component, such as an antigen. Include multiparameter methods used to identify each of a plurality of antigens in an antigen mixture. According to this embodiment, each antibody is separately labeled with a dye having a different absorbance and luminescence wavelength characteristic than the dye used to label the other probe. All labeled antibodies are then added to the biological preparation which has been analyzed to contain a second component, such as an antigen, each of which can be stained by the particular labeled antibody. Unreacted dye material can be entirely removed from the preparation by washing if it interferes with the analysis. The biological preparation is then subjected to various excitation wavelengths. Each excitation wavelength used is that of the particular conjugate dye. A luminescence microscope for examining the luminous intensity of the emission wavelength corresponding to the excitation wavelength, or other luminescence such as a flow cytometer or a fluorescence spectrophotometer having a filter or monochromator that selects the light of the excitation wavelength and selects the wavelength of the luminescence. A detection system is used. The luminescence intensity at a wavelength corresponding to the emission wavelength of a particular conjugate dye indicates the amount of antigen bound to the antibody to which the dye binds. In some cases, a single excitation wavelength can be used to excite luminescence from two or more substances in a mixture, each emitting fluorescence at a different wavelength, and detecting the individual fluorescence intensity at each of the fluorescence wavelengths By doing so, the amount of each labeled species can be measured. If desired, absorbance detection can be used. The two-stage method of the present invention can be applied to any system that uses a first substance bound to a dye to probe for the presence of another substance to which the first substance-dye complex is directed in a luminescence or absorbance detection system. For example, a dye can bind to a DNA or RNA fragment to form a dye-bound DNA or RNA fragment, which is then directed to a DNA or RNA backbone where the fragment is complementary. The same test method can be used to probe for the complementary backbone of DNA.
[0021]
The cyanine and related dyes of the present invention are particularly well-suited for component mixture analysis that requires dyes of various excitation and emission wavelengths because specific cyanine and related dyes having a wide range of excitation and emission wavelengths can be synthesized. Certain cyanines and related dyes having particular excitation and emission wavelengths can be synthesized by changing the number of methine groups or the cyanine ring structure. In this way, a dye having a specific excitation wavelength corresponding to a specific excitation light source, such as a laser, for example HeNe or a diode laser, can be synthesized.
[0022]
The present invention provides a method for converting cyanine and related dye molecules that are highly luminescent and have high absorbance under reaction conditions into proteins, peptides, carbohydrates, nucleic acids, derived nucleic acids, lipids, other specific biological molecules, amines on biological cells. Co-reacting with non-biological substances such as hydroxy, aldehyde, sulfhydryl or other groups and soluble polymers, polymer particles, polymer facings, polymer membranes, glass facings and other particles or facings About. Since luminescence involves sensitive optical techniques, the presence or absence of a dye `` label '' can be probed and quantified even when the label is present in very low amounts, and thus labeled using a dye labeling reagent. The amount of the substance can be measured. The most useful dyes have high absorbance (ε = 70,000-250,000 lb / mol cm or more), are very luminescent, and have quantum yields of at least 5-80% or more. The amount applies to the dye itself and also to the dye bound to the label.
[0023]
An important application of such color labeling reagents is in the production of luminescent monoclonal antibodies. Monoclonal antibodies are protein molecules that bind very tightly and specifically to specific chemical sites or "markers" on or in the cell surface. Therefore, these antibodies have enormous research and clinical applications in identifying specific cell types (eg, HLA, T cells, bacteria and viruses, etc.) and abnormal cells. Conventionally, antibody bound to cells has been quantified by labeling the antibody by various methods, and the label can be a radioactive label (radioimmunoassay), an enzyme (ELISA technique) or a fluorescent dye (usually fluorescein, rhodamine, Texas Red®). ) Or red algae). Most manufacturers or users of clinical antibody reagents want to eliminate the problems associated with the use of radiotracers, so luminescence is considered one of the most promising alternatives. In fact, many vendors today market fluorescein, Texas Red®, rhodamine, and red algain labeled with monoclonal antibodies.
[0024]
In recent years, optical / electronic devices for detecting fluorescent antibodies on cells have become increasingly complex. For example, flow cytometry can be used to measure the amount of fluorescent antibody on individual cells at rates up to 5,000 cells / second, and microscopy and solution fluorescence techniques have advanced. These devices can excite fluorescence at many wavelengths in the UV, visible and near IR regions of the spectrum. Most of the useful fluorescent labeling reagents accepted today can be excited in the 400-580 nm spectral region. The exception is that they can be covalently attached to proteins and can be excited at slightly longer wavelengths. Some phycobiliprotein-type pigments isolated from marine organisms. Therefore, there is a large spectral window in the 580 to about 900 nm range where new labeling reagents need to be effective at labeling biological or non-biological substances that are analyzed on instruments commercially available today. New reagents that can be excited in this region of the spectrum allow the performance of multicolor luminescence analysis of markers on cells since antibodies with different specificities can each be labeled with different colored fluorescent dyes. In this way, the presence or absence of several markers can be checked simultaneously for each cell analyzed.
[0025]
The invention also relates to the luminescent (fluorescent or phosphorescent) cyanine, merocyanine and styryl dyes themselves which can be covalently linked to biological or non-biological substances. Merocyanine and styryl dyes are recognized as being related to the cyanine dyes for the present invention. The new labeling reagent itself can be excited with light of a wavelength that is initially defined, particularly when it is bound to a labeling component, for example, light having a wavelength in the 450-900 nm spectral range. Background fluorescence of the cells generally occurs at lower wavelengths. Therefore, the labeling reagent is distinguished from the background fluorescence. Particularly advantageous are derivatives that absorb light at 633 nm. Because the derivatives can be excited by a cheap, strong, stable and long-lived HeNe laser source. The light of the second defined wavelength, which is fluorescent or phosphorescent emitted by the labeling component, can then be detected. Fluorescence or phosphorescence generally has a longer wavelength than the excitation light. The detection step can use a luminescence microscope having a filter to absorb scattered light at the excitation wavelength and to pass the corresponding wavelength of luminescence corresponding to the particular dye label used with the analyte. Such an optical microscope is described in U.S. Pat. No. 4,621,911.
[0026]
Not all cyanines and related dyes are luminescent. However, the dyes of the present invention include cyanine and related dyes that are luminescent. They are relatively stable and are often soluble in the reaction solution, preferably an aqueous solution. The binding dye itself has a molecular extinction coefficient (ε) of at least 50,000, preferably 100,000 l / mol cm, especially when it is bound to a labeling component. The extinction coefficient is a measure of the ability of a molecule to absorb light. The binding dyes of the present invention have a quantum yield of at least 2%, preferably at least 10%. In addition, the binding dyes of the present invention absorb and generate light in the 400-900 nm, preferably 600-900 nm, spectral range.
[0027]
Aryl sulfonated dye
The arylsulfonate or arylsulfonic acid-substituted dyes as described herein have been found to be inherently more fluorescent and have improved photostability and water solubility as compared to analogous dyes without arylsulfonate or arylsulfonic acid groups. Was. As used herein, the term arylsulfonate or arylsulfonate group refers to a compatible arylsulfonate or arylsulfonate group attached to a single ring aromatic structure or to an aromatic ring structure including a fused ring structure such as a naphthalene structure. Means a group. Single ring or fused ring aromatic structures can be present in polymethine, cyanine, merocyanine or styryl type dyes.
[0028]
Many dyes having a coplanar molecular structure, usually including cyanine dyes, are liable to form dimers or more conjugates in aqueous solution, especially when inorganic salts are also present, such as in buffers or saline. . These conjugates usually have an absorption band shifted to the shorter wavelength side of the monomer absorption, and are generally very weak fluorescent species. The tendency of cyanine dyes to form conjugates in aqueous solutions is particularly well known in the photographic industry [West W. et al. & Pierce S. ,J. Phys. Chem. Sturmer D., 69; 1894 (1965); M. ,Spec. Top in Heterocyclic Chemistry, 30 (1974)].
[0029]
Many dye molecules, especially cyanine dye molecules, tend to form conjugates in aqueous solution. Thus, aryl sulfonate dyes have been found to have a minimal tendency to form these conjugates. When an arylsulfonate dye is used in forming a fluorescent labeling reagent, it has a reduced tendency to form conjugates when it binds to other molecules, such as proteins or antibodies, at a high surface density. The tendency of a particular dye molecule to form a conjugate in a salt solution (eg, 150 mM sodium chloride) can be taken as a measure of the tendency of the same dye molecule to form a conjugate on the surface of a protein. It is therefore desirable that the dye molecules have a minimal tendency to form conjugates in aqueous salt solutions. The data shown in FIG. 2 can be used to illustrate that certain arylsulfonated dyes have a low tendency to form conjugates even at high concentrations in aqueous salt solutions.
[0030]
FIG. 1 shows a monomer absorption spectrum and a dimer absorption spectrum of a typical cyanine dye dissolved in an aqueous buffer. The dye N, N'-di-sulfobutylindodicarbocyanine used to generate these spectra has no arylsulfonate groups and readily forms dimers even at concentrations below the millimolar range. The dimer spectrum was calculated from the dye spectra at different concentrations (see West & Peerce, 1965). At a concentration of 3 mM in phosphate buffered saline solution, the absorption of the monomer and dimer bands was approximately equal.
[0031]
The spectrum of the improved sulfoindodicarbocyanine N, N'-diethylindodicarbocyanine-5,5'-disulfonic acid is shown in FIG. This dye showed no sign of conjugate in saline solution up to a concentration of 10 mM.
[0032]
It is common practice to measure the efficiency with which a particular reactive dye binds to a protein, such as an antibody, under defined reaction conditions. The test dye was N, N'-dicarboxypentylindodicarbocyanine-5,5'-disulfonic acid bis-N-hydroxysuccinimide. FIG. 3 shows that the sulfocyanine dye active ester efficiently reacts with sheep immunoglobulin in a pH 9.2 carbonate buffer, and ranges from less than 1 to more than 20 depending on the relative dye or antibody concentration in the reaction solution. Illustrating the formation of covalently labeled antibody molecules having a dye: antibody molar ratio over a range. The slope obtained by applying the least-squares method to the data indicates that the labeling efficiency of the dye under such conditions is about 80%. In a similar study, fluorescein isothiocyanate (FITC) reacted with an efficiency of about 20%.
[0033]
The reactivity of the active ester of the novel sulfoindodicarbocyanine dye was determined by labeling sheep immunoglobulin (IgG). The protein (4 mg / ml) was dissolved in a 0.1 molar carbonate buffer (pH 9.2). An aliquot of the reactive dye dissolved in anhydrous dimethylformamide was added to the protein sample to obtain an initial dye: protein molar ratio. After 30 minutes, proteins were separated from sheep-bound dye by gel permeation chromatography (Sephadex G-50). The resulting dye: protein molar ratio was determined with a spectrophotometer and is shown in FIG.
[0034]
At low dye: protein ratios, the absorption spectrum of the labeled protein shows a band that closely corresponds to that of the free monomeric dye. Antibody molecules that are highly labeled (high dye: protein ratio) or labeled with a dye that has a high tendency to aggregate in aqueous solutions often have new absorption peaks that appear at shorter wavelengths than the monomer dye absorption bands in aqueous solutions. Has been found to have. The wavelength of the new absorption peak often falls in the region exhibiting the dimer absorption spectral properties of the dye (see FIG. 1).
[0035]
The higher the degree of labeling of the antibody, the higher the short wavelength: long wavelength absorption peak ratio. This shorter wavelength absorption peak can be seen at about 590 nm in FIG. In FIG. 4, the longer wavelength (645 nm) peak is due to monomer dye molecules bound to the antibody. The labeling reagent N, N'-dicarbobutylindodicarbocyanine-5,5'-acetic acid bis-N-hydroxysuccinimide ester used to produce the antibody absorption spectrum of FIG. 4 has no arylsulfonate group. In aqueous salt solutions, it also readily forms dimers on the antibody with which it has reacted. Importantly, the fluorescence excitation spectra of these antibodies indicate that excitation of the labeled antibody at the short wavelength peak does not produce fluorescence proportionally as much as it does at the long wavelength peak. This consideration is consistent with the notion that the short wavelength absorption peak is due to the formation of a non-fluorescent dimer or conjugate on the antibody molecule.
[0036]
We have found that the arylsulfonate cyanine dye used to obtain the data of FIG. 3 shows that the antibody molecule has a much smaller absorption peak at the characteristic absorption wavelength of the dimer (see FIG. 5). It has been found above that it does not bind easily. This is important because antibodies and other "non-binding" labeling reagents should produce more fluorescently labeled proteins. In fact, aryl sulfocyanines produce sharp fluorescent antibodies even when the average dye / antibody ratio is relatively high (see FIG. 6).
[0037]
FIG. 4 shows sheep immunoglobulin labeled with a carboxyindodicarbocyanine dye. FIG. 5 shows a protein conjugated with a novel sulfoindodicarbocyanine dye. The presence of increased dimer in the former sample (see FIG. 1) is evident. Although the dye: protein molar ratio was approximately the same in both preparations, the protein shown in FIG. 5 was more fluorescent than the sample shown in FIG.
[0038]
To have a clear fluorescent antibody or other protein labeled with a fluorescent dye, it is important that the average quantum yield per dye molecule on the protein be as high as possible. In general, as the surface density of dye molecules on protein increases (ie, the dye / protein ratio increases), the average quantum yield of the dye decreases. This effect has sometimes been attributed to quenching as a result of dye interactions on the surface of highly labeled proteins. Indeed, the formation of non-fluorescent dimers on the protein surface can contribute to this quenching. FIG. 6 shows that the average quantum yield of the arylsulfocyanine dye decreases slowly with increasing dye / protein ratio (curve connecting diamonds). In contrast, the curves connecting the circles relate to the binding of the non-arylsulfocyanine dye (N, N'-di-sulfobutylindodicarbocyanine-5-isothiocyanate) as the dye / protein ratio increases. This shows that there is a very rapid drop in the average quantum yield. Therefore, the sulfocyanine dyes illustrated in FIG. 6 produce sharper fluorescent antibodies than other dyes, especially in the labeling range of 1-10 dye molecules per antibody molecule.
[0039]
The average fluorescence quantum yield of individual dye molecules on a labeled protein is a measure of the fluorescence signal obtained from these biomolecules. Data from sheep immunoglobulin (IgG) labeled with the novel sulfoindodicarbocyanine dye in phosphate buffered saline solution is shown in FIG. 6 by the curve connecting the diamonds. The curve connecting the circles in FIG. 6 shows the protein labeled with the indodicarbocyanine-isothiocyanate reactive dye for comparison. In FIG. 6, the quantum yield at a dye / protein ratio of 0 shows a value for a methylamine adduct of a reactive dye (free dye) in a buffer solution.
[0040]
Background method
Luminescent probes are useful reagents for analyzing and separating molecules and cells and detecting and quantifying other substances. A very small number of luminescent molecules can be detected under optimal circumstances. Barak and Webb used a SIT camera to visualize less than 50 fluorescent lipid analogs associated with LDL reception in cells (J. Cell Biol. 90: 595-604 (1981)). Flow cytometry can be used to detect fewer than 10,000 fluorescein molecules [Mirhead, Horan and Poste, Bio / Technology 3: 337-356 (1985)] Some specific examples of fluorescent probe applications. Can be used to identify and separate subpopulations of cells in a cell mixture by techniques such as (1) fluorescence flow cytometry, fluorescence-activated cell sorting and fluorescence microscopy, and (2) separate species by fluorescence immunoassay techniques. Technique for measuring the concentration of a substance to be bound (for example, an antigen-antibody reactant) and (3) fluorescent staining The localization of substances in gels and other insoluble carriers according to Herzenberg et al., "Cellular Immunology", 3rd edition, Chapter 22; Blackwell Scientific Publications, 1978. Fluorescence Activated Medicine and Fluorescence Inspection and Fluorescence Inspection and Fluorescence Inspection and Fluorescence Inspection and Fluorescence Inspection and Fluorescence Inspection and Fluorescence Inspection and Fluorescence Inspection encodes, edited by Taior et al., Alan Lis Inc., 1986.
[0041]
When using fluorescence for the above purposes, there are many restrictions on the choice of fluorescent material. One limitation is the absorption and emission characteristics of the phosphor. This is because many ligands, receptors and substances in analytes such as blood, urea, and cerebrospinal fluid fluoresce and interfere with the accurate measurement of the fluorescence of the fluorescent label. This phenomenon is called autofluorescence or background fluorescence. Another consideration is the ability of the fluorescent substance to bind ligands and receptors, as well as other biological or non-biological substances, and the effect of such binding to the fluorescent substance. In many situations, binding to another molecule can result in a substantial change in the fluorescent properties of the fluorescent material, in some cases substantially impairing or reducing the quantum efficiency of the fluorescent material. It is also possible that the binding to the fluorescent substance makes the function of the label molecule inactive. A third consideration is the quantum efficiency of the fluorescent material, which should be high for sensitive detection. A fourth consideration is the absorbance or extinction of the fluorescent material, which should be as large as possible. Further, it is related whether or not the self-quenching occurs when the fluorescent molecules interfere with each other when in the proximity state. It also concerns whether the fluorescent substance non-specifically binds to the container wall in relation to other compounds or to the fluorescent substance itself or to the compound to which the substance is bound.
[0042]
The applicability and value of the above method is closely tied to the availability of suitable fluorescent compounds. Particularly, a fluorescent substance which emits light in a long wavelength visible region (yellow to near infrared) is necessary. This is because the excitation of these chromophores produces less autofluorescence and multiple chromophores emitting at different wavelengths can be analyzed simultaneously when the full visible and near infrared region of the spectrum can be used. Although the widely used fluorescent compound fluorescein is a useful emitter in the green region, background autofluorescence caused by excitation at the fluorescein absorption wavelength limits detection sensitivity in certain immunoassays and cell analysis systems. However, conventional red fluorescently labeled rhodamine was found to be less effective than fluorescein. Texas Red® is a useful labeling reagent that can be excited at 578 nm and emits maximal fluorescence at 610 nm.
[0043]
Phycobiliprotein has made an important contribution due to its high extinction coefficient and high quantum yield. Such chromophore-containing proteins can be covalently linked to many proteins, and are thus used in microscopy and fluorescent antibody assays in flow cytometry. Phycobiliproteins are: (1) protein labeling methods are relatively complex, (2) protein labeling efficiency is usually not high (typically an average of 0.5 phycobiliprotein molecules / protein), and (3) phycobiliprotein The protein is a natural product, its preparation and purification are complicated, (4) phycobiliprotein is expensive, and (5) as a labeling reagent which emits fluorescence in the red region spectrum more than allophycocyanin which emits maximum fluorescence at 680 nm. No effective phycobiliprotein exists, (6) phycobiliprotein is relatively chemically unstable, (7) it is relatively easily photobleached, and (8) phycobiliprotein is 33,000. Large proteins with molecular weights of up to 240,000, and labels such as metabolites, drugs, hormones, derived nucleotides, and many proteins, including antibodies, are desirable. It has the disadvantage that have greater than a lot of substance. The latter disadvantage is particularly significant. Because antibodies, avidin, DNA hybridization probes, hormones and small molecules, labeled with large phycobiliproteins, cannot bind to the target due to steric limitations imposed by the size of the complex complex, and will not bind to the target. This is because the binding rate is slower for the low molecular weight binder.
[0044]
Other techniques, including histology, cytology, and immunoassays, also exhibit high quantum efficiency at long wavelengths, absorption and emission properties, have simple binding means, and are virtually free of non-specific interference. Benefit from use.
[0045]
Invention outline
The present invention uses fluorescent arylsulfonated cyanines and related dyes with relatively high extinction and high quantum yields for the purpose of assaying and quantifying labeled components. Fluorescent cyanines and related dyes include antibodies, antigens, avidin, streptavidin, proteins, peptides, derived nucleotides, carbohydrates, lipids, biological cells, bacteria, viruses, blood cells, tissue cells, hormones, lymphokines, trace biomolecules, It can be used to label biological substances such as toxins and drugs. Fluorescent dyes can also be used to label non-biological substances such as soluble polymers, polymer or glass particles, drugs, conductors, semiconductors, glass or polymer facings, polymer films and other solid particles. Can be used. The component to be labeled can be in a mixed form with other substances. The mixture in which the labeling reaction takes place can be a liquid mixture, especially a water mixture. The detection step can occur with a liquid or a dry mixture, such as a microscope slide.
[0046]
The present invention requires a cyanine dye to be modified by incorporation into the cyanine molecule of a reactive group that is covalently bonded to the target molecule, preferably at an amine or hydroxy site and in some cases a sulfhydryl or aldehyde site. The present invention also enhances the solubility of cyanine and related dye structures in test liquids, (1) facilitates handling in labeling reactions, and (2) helps prevent dye binding on the surface of the labeled protein. And (3) the use or modification of cyanine and related dye structures to help prevent non-specific binding of the label to biological materials and surface materials or assay devices.
[0047]
Cyanines and related dyes offer important advantages over existing fluorescent labeling reagents. First, cyanines and related dyes that absorb or emit in the spectral range of 400 to about 1100 nm are synthesized. Thus, the reactive derivatives of these dyes can be prepared for assays that require simultaneous measurement of multiple labeling substances. This type of multi-color (or multi-parameter) analysis can be simplified, cost-effective, or the ratio of different labeled species on each particle in a complex mixture of particles (e.g., multi-parameter flow cytometry or complexities with fluorescence microscopy). It is desirable to determine the ratio of antigen markers on individual blood cells in the mixture. Second, many cyanines and related dyes absorb light strongly and fluoresce. Third, many cyanines and related dyes are relatively photostable and do not bleach quickly under a fluorescence microscope. Fourth, it is possible to produce cyanine and related dye derivatives, which are simple and effective coupling reagents. Fifth, many structures and synthetic methods are available, and this type of dye is versatile. Therefore, many structural modifications can be made to make the reagent somewhat water soluble. The charge can vary so as not to disturb the molecule to which it binds and to reduce non-specific binding. Sixth, unlike phycobiliproteins, cyanine dyes are relatively small (molecular weight = 1,000) and therefore do not sterically hinder the labeled molecule's ability to reach its binding zone quickly or perform its function. .
[0048]
Thus, cyanine type dye labels offer many potential advantages. Such dyes can be used for the selective labeling of one or more components in liquids, especially aqueous liquids. The label component can then be detected by an optical or luminescence method. Alternatively, the labeled component is used to stain another component for which it has a strong affinity, and the latter component is detected by optical or luminescent methods. In this case, the dye reacts with the amine, hydroxy, aldehyde or sulfhydryl group of the labeling component. For example, the labeling component can be an antibody, and the stained component that has strong affinity can be a biological cell, an antigen or hapten, or a biological cell or particle containing an antigen or hapten. In another example, the labeling component can be avidin and the component to be stained can be a biotinylated substance. In addition, a specific carbohydrate group can be detected and quantified using a lectin bound to a polymethine cyanine type dye. In addition, luminescent cyanines and related dyes can bind to DNA or RNA fragments. The DNA or RNA-labeled fragment can be used as a fluorescent hybridization probe for examining the presence and amount of a specific complementary nucleotide sequence in a sample containing DNA or RNA. Also, the dye binds to a hormone or ligand (eg, a hormone, protein, peptide, lymphokine, metabolite), which can then bind to a receptor.
[0049]
Patent description for other uses of reactive cyanine
(U.S. Pat. No. 4,337,063) and Masuda et al. (U.S. Pat. Nos. 4,404,289, 4,405,711 and 4,414,325) disclose N-hydroxy Various cyanine dyes having succinimide active ester groups have been synthesized. These patents show that the above reagents can be used as photographic photosensitizers. The potential fluorescent properties of the reagents are not described in the above patents, and in fact the process does not require fluorescence. Most of the dyes listed in the above patents emit only weak fluorescence, are not particularly photostable and their solubility properties are not optimal for many applications involving fluorescence detection of labeled substances.
[0050]
Exekiel et al. (GB 1,529,202) present a number of cyanine dye derivatives that can be used as covalently reactive molecules. The reactive group used in this reagent is an azine group to which a mono- or dichlorotriazine group belongs. The UK patent relates to the development and use of the above reagents as photographic film sensitizers. Fluorescence is not required for the process and most of the reagents described do not fluoresce. This UK patent does not relate to the development and use of reactive cyanine dyes for the purpose of detecting or quantifying labeling substances.
[0051]
Description of incarnation
The present invention relates to the use of luminescent cyanines and cyanine-type dyes for making biological substances, non-biological molecules and luminescent cyanines and cyanine-type dyes to render the substance being labeled luminescent so that it can be detected and / or quantified by luminescence detection methods. The present invention relates to a method for covalently bonding to macromolecules and particles.
[0052]
The present invention relates to a cyanine, merocyanine containing a substituent which is soluble in the liquid when the component is detected in the liquid and which covalently bonds the dye to an amine or a hydroxy group on the component and optionally an aldehyde or a sulfhydryl group. And a method of adding a dye selected from the group consisting of styryl dyes so that it labels the above components. The label component is detected and / or quantified by luminescence or light absorption methods. When the labeling component is an antibody, a DNA fragment, a hormone, a lymphokine or a drug, the labeling component can be used to determine the presence or absence of another component to which it binds, thus detecting the other component. And / or can be quantified.
[0053]
Any effective luminescence or absorbance detection step can be used. For example, the detecting step may be an optical detecting step in which the liquid is illuminated with light of the initially defined wavelength. Next, light at another defined wavelength that is fluorescent or phosphorescent by the labeling component is detected. Detection may also be by optical absorption. For example, the detecting step includes passing light of an initially defined wavelength through the liquid and then identifying the light wavelength transmitted by the liquid.
[0054]
If desired, the detecting step can include a chemical analysis that chemically detects binding of the cyanine or related chromophore to the component.
The basic structures of cyanine, merocyanine and styryl dyes that can be modified to create covalent labeling reagents are shown below:
Cyanine
[0055]
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Merocyanine
[0056]
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Styryl
[0057]
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The following are more specific examples of polymethine type dyes:
Cyanine
[0058]
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Merocyanine
[0059]
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Styryl
[0060]
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Cyanine
[0061]
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Merocyanine
[0062]
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Styryl
[0063]
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In the above structure,
X and Y are O, S and
[0064]
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Selected from the group consisting of
Z is selected from the group consisting of O and S;
m is an integer selected from the group consisting of 1, 2, 3, and 4.
[0065]
In the above formula, the number of methine groups partially determines the excitation color. Cyclic azine structures can also partially determine the excited structure. Often, higher values of m contribute to increased luminescence or absorbance. At values of m higher than 4, the compound becomes unstable. There, further luminescence can be imparted by modification in the ring structure. When m = 2, the excitation wavelength is about 650 nm and the compound correctly emits fluorescence. The maximum emission wavelength is approximately 15 to 100 nm larger than the maximum excitation wavelength.
[0066]
R in each molecule₁  , R₂  , R₃  , R₄  , R₅  , R₆  And R₇  At least one of the groups (preferably only one and possibly two or more) is a reactive group for attaching the dye to the labeling component. For some reagents, R on each molecule₁  , R₂  , R₃, R₄  , R₅  , R₆  And R₇  At least one of the groups can be a group that enhances the solubility of the chromophore or affects the selectivity of the label of the label component or the location of the label on the label component.
[0067]
In the above formula, R₈, R₉(If present) and R₁₀At least one of the (when present) contains at least one of the sulfonate groups. The term sulfonate group shall include sulfonic acid. This is because the sulfonate group is only an ionized sulfonic acid.
[0068]
Linked directly or indirectly to the chromophore₁, R₂, R₃, R₄, R₅, R₆And R₇As a reactive group capable of forming a group, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide Reactive moieties such as esters, hydroxysulfosuccinimide esters, imide esters, hydrazine, azidonitrophenyl, azido, 3- (2-pyridyldithio) propionamide, glyoxal and groups containing aldehydes can be included.
[0069]
R which is particularly useful for labeling components having effective amino, hydroxy and sulfhydryl groups₁, R₂, R₃, R₄, R₅, R₆And R₇Specific examples of groups include:
[0070]
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(Where at least one of Q or W is a residue such as I, Br or Cl),
[0071]
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R that is particularly useful for labeling components having effective sulfhydryls that can be used to label antibodies in a two-step method₁, R₂, R₃, R₄, R₅, R₆And R₇Particular examples of groups are:
[0072]
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(Where Q is a residue such as I or Br),
[0073]
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and
[0074]
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(Where n is 0 or an integer).
[0075]
Particularly useful for labeling components by photoactivated crosslinking₁, R₂, R₃, R₄, R₅, R₆And R₇As a specific example of a group
[0076]
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Is included.
[0077]
Increase the solubility in water or reduce the undesired non-specific binding of the labeled component to unsuitable components in the sample, or otherwise inhibit the interaction of two or more reactive chromophores on the labeled component that can result in quenching of fluorescence. To reduce, R₁, R₂, R₃, R₄, R₅, R₆And R₇The groups can be selected from known polar or charged chemical groups. An example is -EF, where F represents hydroxy, sulfonate, sulfate, carboxylate, substituted amino or quaternary amino, and E is-(CH₂)_nAnd a spacer group such as (n = 0, 1, 2, 3, or 4). Useful examples include alkylsulfonate- (CH₂)₃SO^3-And-(CH₂)₄-SO^3-Is included.
[0078]
The polymethine chain of the luminescent dyes of the present invention may also include one or more cyclic chemical groups that form a bridge between two or more carbon atoms of the polymethine chain. These bridges can help to increase the chemical or photostability of the dye and can be used to change the absorption or emission wavelength of the dye or to change the extinction coefficient of the quantum yield. Improved dissolution properties can be achieved by this modification.
[0079]
According to the present invention, the labeling component is an antibody, protein, peptide, enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin, carbohydrate, oligosaccharide, polysaccharide, nucleic acid, deoxy. Nucleic acid, derived nucleic acid, derived deoxynucleic acid, DNA fragment, RNA fragment, derived DNA fragment, derived RNA fragment, natural drug, virus particle, bacterial particle, virus component, yeast component, blood cell, blood cell component, biological cell, non-cell Blood components, bacteria, bacterial components, natural or synthetic lipid sac, synthetic drugs, poisons, environmental pollutants, polymers, polymer particles, glass particles, glass surface materials, plastic particles, plastic surface materials, polymer films, conductors and It can be a semiconductor.
[0080]
Cyanines or related chromophores that can absorb light at 633 nm upon reaction with certain components can be prepared, and the detection step can use a helium-neon laser emitting at this spectral wavelength. Also, it is possible to prepare cyanines or related dyes that are capable of maximally absorbing light between 700 nm and 900 nm upon reaction with certain components, so that the detection step emits light in this region of the spectrum. A laser diode can be used.
[0081]
Selectivity
The reactive groups listed above label proteins or other specific functional groups on biological or non-biological molecules, macromolecules, surface materials or particles as long as appropriate reaction conditions are used, including appropriate pH conditions. Relatively specific.
[0082]
Characteristics of reactive cyanine, merocyanine and styryl dyes and their products
The spectral properties of the dyes of the present invention are not appreciably changed by the functionalization described herein. The spectral properties of the labeled protein or other compound also do not differ significantly from the base dye molecule which is not bound to the protein or other substance. The dyes according to the invention, alone or bound to a label, generally have a large extinction coefficient (ε = 100,000-250,000), and in some cases as high a quantum yield as 0.4, and It emits light in the 400-900 nm spectral range. Thus, they are particularly valuable as labels for luminescence detection reagents.
[0083]
Optical detection method
Any method can be used to detect the label or the staining component. The detection method can use a light source that illuminates the mixture containing the labeling substance with light of the wavelength defined initially. Known devices are used that detect light at a second wavelength transmitted by or fluoresced or emitted by the mixture. Such detection devices include fluorescence spectrometers, absorption spectroscopy, fluorescence microscopes, transmission light microscopes or flow cytometers, fiber optic sensors, and immunoassay devices.
[0084]
The methods of the present invention may also employ chemical analysis methods to detect dye binding to one or more labeling components. Chemical analysis methods can include infrared spectrometry, NMR spectrometry, absorption spectrometry, fluorescence spectrometry, mass spectrometry and chromatographic methods.
[0085]
【Example】
Example 1
Effect of pH on binding of sulfoindodicarbocyanine to protein
A sample of sheep γ-globulin (4 mg / ml) in 0.1 M carbonate buffer was mixed at room temperature with a 10-fold molar excess of the following sulfoindodicarbocyanine active ester (m = 2) at room temperature:
[0086]
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The protein sample was separated from the non-covalent dye by gel permeation chromatography on Sephadex G-50 for an appropriate time ranging from 5 seconds to 30 minutes. Maximum labeling of the protein occurred after 10 minutes, resulting in final dye / protein molar ratios of 5.8, 6.4 and 8.2 for samples incubated at pH 8.5, 8.9 and 9.4, respectively. . The time required for a dye / protein ratio of 5 and the quantum yield of the product at different pHs are shown in the following table:
pH  Time (sec)  Quantum yield
8.5 115 0.09
8.9 53 0.09
9.4 6.5 0.17
The above data show that the protein labeled with this dye is better at pH 9.4 than below pH 9. The higher the pH of the buffer, the faster the binding reaction, but the better the labeling efficiency and the higher the fluorescence of the product. The quantum yield indicates the average quantum yield per dye molecule on the labeled protein.
[0087]
Example 2
Binding of sulfoindocarbocyanine active ester to protein
Sheep γ-globulin (1 mg / ml) dissolved in phosphate buffered saline (PBS) at pH 7.4 was adjusted to pH 9.4 using 0.1 M sodium carbonate. An aliquot of this protein solution was added with a cyanine dye label (the structure of Example 1, m = 1) to obtain various dye / protein molar ratios. After incubation at room temperature for 30 minutes, the cells were separated by Sephadex G-50 gel permeation chromatography using PBS as an eluent. The molar ratio of dye covalently bound to protein in the product was 1.2, 3.5, 5.4, 6.7 and 11.2 for the initial dye / protein ratios of 3, 6, 12, 24 and 30, respectively. there were.
[0088]
Example 3
Labeling of AECM dextran with sulfoindodicarbocyanine
N-aminoethyl-carboxamidomethyl (AECM) dextran containing an average of 16 amino groups per dextran molecule was synthesized from dextran with an average molecular weight of 70000 [Inman, J. et al. K. , J. et al. Immunol.114: 704-709 (1975)]. A portion of AECM-dextran (1 mg / 250 μl) dissolved in 0.1 M carbonate buffer, pH 9.4, is added to 0.2 mg of sulfoindodicarbocyanine active ester (structure of Example 1, m = 2) and dye / Protein molar ratio of 10. The mixture was stirred at room temperature for 30 minutes. Dextran was then separated from unbound dye by Sephadex G-50 gel permeation chromatography using ammonium acetate (50 mM) as elution buffer. An average of 2.2 dye molecules were covalently attached to each dextran molecule.
[0089]
Example 4
Sulfoindodicarbocyanine active ester labeling of specific antibodies
Dyeing of a sheep γ-globulin specific for murine IgG (1 mg / ml) in a 1.1 M carbonate buffer (pH 9.4) and a sulfoindodicarbocyanine active ester (structure of Example 1, m = 2) The mixture was mixed at a molecular / protein molecular ratio of 8. After incubation for 3 minutes at room temperature, the labeling mixture was separated by gel filtration on Sephadex G-50 equilibrated with phosphate buffered saline (pH 7.4). The recovered proteins contained an average of 4.4 dye molecules covalently linked to each protein.
[0090]
Example 5
Staining or microscopic visualization of human lymphocytes with sulfoindodicarbocyanine dye conjugated to sheep anti-mouse IgG antibody
Freshly isolated peripheral blood lymphocytes were purified from mouse anti-β2-microglobulin (0.25 μg10⁶Cells) at 0 ° C. for 30 minutes. The cells were washed twice with DMEM buffer and then sulfonedodicarbocyanine-labeled sheep anti-mouse IgG antibody (1 μg / 10⁶Cells). After a 30 minute incubation at 0 ° C., the cells were centrifuged to remove excess antibody and the cells were washed again with DMEM buffer. Aliquots of cells were fixed on slides for analysis by fluorescence microscopy. Under a microscope, lymphocytes on slides were illuminated with light at 610-630 nm, and fluorescence at 650-700 nm was detected by a COTU red-sensitive enhanced television camera attached to an image digitizer and television monitor. Cells stained by this method showed fluorescence under the microscope. In control experiments, staining and analysis were performed as described above, except that the use of the primary mouse anti-β2-microglobulin antibody was omitted. Control samples did not show fluorescence under the microscope, indicating that sulfoindocyanine-labeled sheep anti-mouse antibody did not result in significant non-specific binding to lymphocytes.
[Brief description of the drawings]
FIG. 1 shows a monomer absorption spectrum and a dimer spectrum of an N, N′-disulfobutylindodicarbocyanine dye.
FIG. 2 shows the spectrum of N, N′-diethylindodicarbocyanine-5,5′-disulfonic acid dye.
FIG. 3 shows a dye / protein molar ratio in a labeling reaction of sheep immunoglobulin with a bis-N-hydroxysuccinimide dye of N, N′-dicarboxypentylindodicarbocyanine-5,5′-disulfonic acid. Is shown.
FIG. 4 shows the absorption spectrum of an antibody labeled with bis-N-hydroxysuccinimide ester of N, N′-dicarbobutylindodicarbocyanine-5,5′-acetic acid.
FIG. 5 shows an absorption spectrum of a protein bound to a novel sulfoindodicarbocyanine dye.
FIG. 6 shows a comparison of the change in quantum yield (diamonds) of arylsulfocyanine dyes with that of non-arylsulfocyanine dyes (circles) with increasing dye / protein ratio.

Claims (4)

A method for labeling a component in an aqueous liquid,
(A) To the liquid containing the component, a luminescent dye selected from the group consisting of cyanine, merocyanine, and styryl dyes having at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus is added. Reactive with the components,
(B) reacting the dye with the component such that the dye labels the component; and (c) detecting the labeled component by an optical method. The component is an antibody, protein, peptide, enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, toxin, carbohydrate, saccharide, oligosaccharide, polysaccharide, nucleotide, derived nucleotide, nucleic acid, deoxy nucleic acid, The method according to claim 1, wherein the method is selected from an induced nucleic acid, an induced deoxynucleic acid, a DNA fragment, an RNA fragment, an induced DNA fragment, an induced RNA fragment, and a drug . A luminescent label component in an aqueous liquid, wherein the luminescent dye is selected from the group consisting of cyanine, merocyanine, and styryl dyes having at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus; and A luminescent labeling component that is covalently reactive with the component and comprises the luminescent dye attached to the component . The component is an antibody, protein, peptide, enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, toxin, carbohydrate, saccharide, oligosaccharide, polysaccharide, nucleotide, derived nucleotide, nucleic acid, deoxy nucleic acid, The luminescent label component according to claim 3, which is selected from the group consisting of a derivative nucleic acid, a derivative deoxynucleic acid, a DNA fragment, an RNA fragment, a derivative DNA fragment, a derivative RNA fragment and a drug.

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