JPH055742A - Fluorometric analysis - Google Patents
Fluorometric analysisInfo
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- JPH055742A JPH055742A JP28785891A JP28785891A JPH055742A JP H055742 A JPH055742 A JP H055742A JP 28785891 A JP28785891 A JP 28785891A JP 28785891 A JP28785891 A JP 28785891A JP H055742 A JPH055742 A JP H055742A
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、蛍光分析法において、
蛍光物質に塩基性条件下にて最大励起波長の約２倍の波
長の光を照射すると、二光子吸収によって短波長の蛍光
を放射することを利用して、小型の装置を用いて高感度
に蛍光分析を行う方法に関する。BACKGROUND OF THE INVENTION The present invention relates to a fluorescence analysis method,
When a fluorescent substance is irradiated with light having a wavelength about twice the maximum excitation wavelength under basic conditions, it emits fluorescence of a short wavelength due to two-photon absorption, making it highly sensitive using a small device. It relates to a method for performing a fluorescence analysis.
【０００２】[0002]
【従来の技術及び発明が解決しようとする課題】従来、
蛍光分析法により生体試料を分析するには、蛍光物質、
とくにフルオレセイン、ウンベリフェロンなどの蛍光物
質に３００〜６００ｎｍの励起光を照射して、発する蛍
光を測定する方法が知られているが、励起光と蛍光との
波長の差は２０〜９０ｎｍと殆んど差がないため、励起
光を吸収除去するために用いるフィルターに蛍光も吸収
され、蛍光の損失が大きかった。また、励起光の発生装
置の小型化が望まれているが、３００〜６００ｎｍの領
域では励起光源として使用できる半導体レーザーがなか
った。2. Description of the Related Art Conventionally, the problems to be solved by the invention
To analyze a biological sample by fluorescence analysis, a fluorescent substance,
In particular, a method is known in which a fluorescent substance such as fluorescein and umbelliferone is irradiated with excitation light of 300 to 600 nm and the emitted fluorescence is measured. However, the wavelength difference between the excitation light and the fluorescence is 20 to 90 nm, which is almost the same. Since there is almost no difference, fluorescence was also absorbed by the filter used to absorb and remove the excitation light, and the loss of fluorescence was large. Further, there is a demand for miniaturization of a pumping light generator, but there is no semiconductor laser that can be used as a pumping light source in the region of 300 to 600 nm.
【０００３】[0003]
【課題を解決するための手段】本発明者は、塩基性条件
下では蛍光物質が、最大励起波長の１／２のエネルギー
の光子を２個吸収して１個の光子を放出する二光子吸収
により蛍光放射することを見い出した。つまり、従来の
３００〜６００ｎｍの波長の励起光で蛍光を発する物質
は、塩基性条件下では約２倍の６００〜１２００ｎｍの
波長でも励起し、その蛍光は３００〜６００ｎｍで励起
される蛍光と同波長で、同強度又はそれ以上の強度の蛍
光を発する。このため市販の半導体レーザーなどで、高
効率で蛍光物質を励起できるため、装置を小型化するこ
とが可能であり、またフィルターによる蛍光の吸収損失
が少なく、また長波長領域の励起光を使用するので量子
収率がよく、高感度化が実現できることを見い出した。DISCLOSURE OF THE INVENTION The present inventors have found that under basic conditions, a fluorescent substance absorbs two photons having an energy half the maximum excitation wavelength and emits one photon. Have been found to emit fluorescent light. That is, a conventional substance that fluoresces with excitation light having a wavelength of 300 to 600 nm excites even at a wavelength of 600 to 1200 nm, which is about twice as much under basic conditions, and its fluorescence is the same as that of fluorescence excited at 300 to 600 nm. It emits fluorescence of the same intensity or higher at the wavelength. For this reason, a commercially available semiconductor laser or the like can excite the fluorescent substance with high efficiency, so that the device can be downsized, the absorption loss of the fluorescence by the filter is small, and the excitation light in the long wavelength region is used. Therefore, they have found that the quantum yield is good and high sensitivity can be realized.
【０００４】本発明で述べる最大励起波長（λｎｍ）と
は、塩基性条件下にて、一光子励起により発する蛍光の
強度が、最大となる励起波長である。最大励起波長は、
主に塩基性条件下における蛍光物質の極大吸収波長に相
当する。The maximum excitation wavelength (λnm) described in the present invention is an excitation wavelength at which the intensity of fluorescence emitted by one-photon excitation becomes maximum under basic conditions. The maximum excitation wavelength is
It mainly corresponds to the maximum absorption wavelength of the fluorescent substance under basic conditions.
【０００５】また、最大励起波長（λｎｍ）の約２倍の
波長とは、２λ±１００（ｎｍ）以下の波長を意味し、
より好ましくは２λ±５０（ｎｍ）の波長である。本発
明においては、蛍光物質の最大励起波長（λｎｍ）の約
２倍の波長領域のレーザー光ならば、１種類の波長に限
定されず、波長の異なる複数のレーザー光を励起光源と
して使用できる。The wavelength approximately twice the maximum excitation wavelength (λ nm) means a wavelength of 2λ ± 100 (nm) or less,
More preferably, the wavelength is 2λ ± 50 (nm). In the present invention, the laser light having a wavelength region approximately twice the maximum excitation wavelength (λ nm) of the fluorescent substance is not limited to one wavelength, and a plurality of laser lights having different wavelengths can be used as the excitation light source.
【０００６】本発明の方法は、次のような蛍光分析に利
用することができる。 （１）塩基に可溶性の蛍光性被測定物質を、塩基性条件
下で該蛍光物質の最大励起波長の約２倍の波長のレーザ
ー光で励起し、蛍光を測定することを特徴とする蛍光分
析法。The method of the present invention can be used for the following fluorescence analysis. (1) Fluorescence analysis characterized in that a fluorescent substance to be measured soluble in a base is excited under a basic condition with a laser beam having a wavelength which is about twice the maximum excitation wavelength of the fluorescent substance, and fluorescence is measured. Law.
【０００７】（２）光ファイバーのコア表面に免疫物質
を固定化し、（ａ）該コア表面の免疫物質に対して、被
測定物質及び塩基に可溶性の蛍光物質で標識された被測
定物質と同一の免疫反応を示す物質を競合的に反応させ
るか、或いは、（ｂ）該コア表面の免疫物質と被測定物
質を反応させ、次いで塩基に可溶性の蛍光色素で標識さ
れた被測定物質と免疫反応する物質を反応させた後、塩
基性条件下で該蛍光物質の最大励起波長の約２倍の波長
のレーザー光で励起し、蛍光を測定することを特徴とす
る蛍光免疫分析法。(2) An immunological substance is immobilized on the core surface of the optical fiber, and (a) the immunological substance on the core surface is the same as the measured substance and the substance to be measured labeled with a base-soluble fluorescent substance. A substance exhibiting an immune reaction is allowed to react competitively, or (b) an immune substance on the surface of the core is reacted with a substance to be measured, and then, a substance to be measured labeled with a base-soluble fluorescent dye is immunoreacted. A fluorescent immunoassay method, which comprises reacting a substance and then exciting it with a laser beam having a wavelength about twice the maximum excitation wavelength of the fluorescent substance under basic conditions to measure fluorescence.
【０００８】（３）光ファイバーのコア表面に免疫物質
を固定化し、（ａ）該コア表面の免疫物質に対して、被
測定物質及びビオチンが結合した被測定物質と同一の免
疫反応を示す物質を競合的に反応させるか、或いは、
（ｂ）該コア表面の免疫物質と被測定物質を反応させ、
次いで、ビオチンが結合した被測定物質と免疫反応する
物質を反応させた後、塩基に可溶性の蛍光物質で標識し
たアビジンを反応させ、塩基性条件下で該蛍光物質の最
大励起波長の約２倍の波長のレーザー光で励起し、蛍光
を測定することを特徴とする蛍光免疫分析法。(3) An immunizing substance is immobilized on the core surface of the optical fiber, and (a) a substance exhibiting the same immune reaction as the to-be-measured substance and the to-be-measured substance to which biotin is bound to the immunity substance on the core surface. React competitively, or
(B) reacting the immunological substance on the core surface with the substance to be measured,
Next, after reacting the substance to be measured which is bound with biotin with the substance to be immunoreacted, avidin labeled with a fluorescent substance soluble in a base is allowed to react, and about 2 times the maximum excitation wavelength of the fluorescent substance under basic conditions. Fluorescence immunoassay method, which comprises exciting with a laser beam having a wavelength of and measuring fluorescence.
【０００９】蛍光物質としては、塩基に可溶性の４−メ
チルウンベリフェロン、フルオレセイン、ジクロロフル
オレセイン、ビス（ｐ−ヒドロキシフェニルプロピオン
酸）などがあげられる。励起波長と蛍光波長の関係は表
１のとおりである。Examples of the fluorescent substance include base-soluble 4-methylumbelliferone, fluorescein, dichlorofluorescein and bis (p-hydroxyphenylpropionic acid). Table 1 shows the relationship between the excitation wavelength and the fluorescence wavelength.
【００１０】[0010]
【表１】 [Table 1]
【００１１】塩基性条件はpH８〜１３が望ましく、pHが
これより高すぎると蛍光物質が加水分解されるおそれが
あり好ましくない。方法（１）は、塩基に可溶性の蛍光
性被測定物質を、塩基性条件下に該蛍光物質の最大励起
波長の約２倍の波長のレーザー光で励起し、蛍光を測定
するものである（実施例１参照）。蛍光性被測定物質は
蛍光標識された被測定物質であってもよい。被測定物質
を蛍光標識する方法としては、被測定物質に蛍光物質を
直接結合させてもよく、被測定物質と特異的に結合する
物質や、アビジン−ビオチンなどを介して結合させても
よい。The basic condition is preferably pH 8 to 13. If the pH is too high, the fluorescent substance may be hydrolyzed, which is not preferable. Method (1) is a method in which a fluorescent substance to be measured which is soluble in a base is excited under a basic condition with a laser beam having a wavelength about twice the maximum excitation wavelength of the fluorescent substance, and fluorescence is measured ( See Example 1). The fluorescent substance to be measured may be a fluorescent substance to be measured. As a method of fluorescently labeling the substance to be measured, a fluorescent substance may be directly bound to the substance to be measured, or a substance that specifically binds to the substance to be measured or avidin-biotin may be used for binding.
【００１２】方法（２）は、競合法（ａ）とサンドイッ
チ法（ｂ）に大別される。競合法（ａ）では、濃度既知
である塩基に可溶性の蛍光性物質で標識された被測定物
質と同一の免疫反応を示す物質（例えば抗原）と被測定
物質を混合し、次いで、この溶液に免疫物質（例えば抗
体）を固定化した光ファイバーを浸漬し、競合的に反応
させる（抗原−抗体反応）。競合法では、被測定試料の
濃度が高ければ、蛍光性物質で標識された被測定物質の
光ファイバーへの結合量が少ないので、蛍光強度が低下
する（実施例５参照）。The method (2) is roughly classified into a competitive method (a) and a sandwich method (b). In the competitive method (a), a substance (for example, an antigen) that shows the same immune reaction as the substance to be measured, which is labeled with a fluorescent substance soluble in a base whose concentration is known, is mixed with the substance to be measured, and then this solution is added to this solution. An optical fiber on which an immunological substance (eg, an antibody) is immobilized is dipped and reacted competitively (antigen-antibody reaction). In the competitive method, when the concentration of the sample to be measured is high, the binding amount of the substance to be measured labeled with the fluorescent substance to the optical fiber is small, so that the fluorescence intensity is lowered (see Example 5).
【００１３】サンドイッチ法（ｂ）では、被測定物質
（例えば抗原）の溶液に、免疫物質（例えば抗体）を固
定化した光ファイバーを浸漬して反応させ（抗原−抗体
反応）、次いでこの光ファイバーを、塩基に可溶性の蛍
光性物質で標識され、被測定物質と特異的に反応する被
測定物質と免疫反応する物質（例えば抗体）の溶液に浸
漬して反応させる。サンドイッチ法では、光ファイバー
上の免疫物質と蛍光物質で標識された被測定物質と免疫
反応する物質で被測定物質がサンドイッチされた状態と
なる。サンドイッチ法では、被測定物質の濃度が高けれ
ば、蛍光性物質で標識された被測定物質と免疫反応する
物質の光ファイバーへの結合量も多いので、蛍光強度が
高くなる（実施例４参照）。In the sandwich method (b), an optical fiber having an immunizing substance (eg, antibody) immobilized thereon is immersed in a solution of a substance to be measured (eg, antigen) to react (antigen-antibody reaction), and then this optical fiber is It is soaked in a solution of a substance (for example, an antibody) which is labeled with a fluorescent substance soluble in a base and which specifically reacts with the substance to be measured and which immunoreacts with the substance to be measured. In the sandwich method, the substance to be measured is sandwiched by a substance that immunoreacts with the substance to be measured labeled with the immunological substance and the fluorescent substance on the optical fiber. In the sandwich method, when the concentration of the substance to be measured is high, the amount of the substance that immunoreacts with the substance to be measured, which is labeled with the fluorescent substance, is large in the amount bound to the optical fiber, and therefore the fluorescence intensity is high (see Example 4).
【００１４】方法（２）により、被測定物質の濃度を測
定する方法において、測定感度を向上させるためには、
免疫分子（抗原又は抗体分子）１個あたりの蛍光物質の
結合量を増やす必要があり、このために、免疫分子すな
わち、被測定物質と同一の免疫反応を示す物質又は被測
定物質と免疫反応する物質が、ビオチンと結合し、該ビ
オチンは塩基に可溶性の蛍光物質で標識されたアビジン
が結合しているか、あるいは複数の反応活性基を有する
物質と結合し、該複数の反応活性基にはビオチンを介し
て塩基に可溶性の蛍光物質で標識されたアビジンが結合
していることが好ましい。このような方法においては、
被測定物質と同一の免疫反応を示す物質又は被測定物質
と免疫反応する物質に、塩基に可溶性の蛍光物質で標識
されたアビジンが多数結合していることにより、免疫分
子すなわち、被測定物質と同一の免疫反応を示す物質分
子又は被測定物質と免疫反応する物質の１分子当りの蛍
光物質の結合量を増やすことができ、検出感度を飛躍的
に向上させるのに役立つ。In the method of measuring the concentration of the substance to be measured by the method (2), in order to improve the measurement sensitivity,
It is necessary to increase the binding amount of the fluorescent substance per immune molecule (antigen or antibody molecule), and for this reason, immunoreact with the immune molecule, that is, the substance that shows the same immune reaction as the substance to be measured or the substance to be measured. A substance binds to biotin, the biotin is bound to a base-soluble fluorescent substance-labeled avidin, or binds to a substance having a plurality of reactive groups, and the plurality of reactive groups include biotin. It is preferable that avidin labeled with a base-soluble fluorescent substance is bound via a. In this way,
A substance that exhibits the same immune reaction as the substance to be measured or a substance that immunoreacts with the substance to be measured is bound to a large number of avidin labeled with a base-soluble fluorescent substance, so that an immune molecule, that is, a substance to be measured, It is possible to increase the binding amount of the fluorescent substance per molecule of the substance molecule that shows the same immune reaction or the substance that immunoreacts with the substance to be measured, which is useful for dramatically improving the detection sensitivity.
【００１５】アビジンとビオチンは、これらと同等の作
用を有する化合物で置き換えることができる。例えば抗
体−プロテインＡなどの組合わせなどが使用できる。複
数の反応活性基を有する物質としては、ポリリジン、キ
トサン、ポリガラクトサミン、ポリノイラミン酸のよう
なポリペプチド又はアミノグリカンが用いられ、とくに
キトサンが好適である。反応活性基は１分子当り２０〜
１０万個、好ましくは４０００〜５０００個が存在して
いることが望ましい。Avidin and biotin can be replaced with a compound having an action equivalent to these. For example, a combination of antibody-protein A and the like can be used. As the substance having a plurality of reactive groups, a polypeptide such as polylysine, chitosan, polygalactosamine, polyneuraminic acid or aminoglycan is used, and chitosan is particularly preferable. 20 to 20 reactive groups per molecule
It is desirable that 100,000, preferably 4000-5000, exist.
【００１６】さらに、方法（２）において、不安定な蛍
光物質を標識として使用する場合には、方法（３）が望
ましい。方法（３）は、競合法（ａ）とサンドイッチ法
（ｂ）による蛍光免疫分析法であるが、アビジンとビオ
チンが特異的に結合することを利用して、光ファイバー
上に免疫物質を固定化した後に、前記被測定物質と同一
の免疫反応を示す物質又は被測定物質と免疫反応する物
質に、ビオチンが結合した物質、あるいは複数の反応活
性基を有する物質が結合し、該複数の反応活性基にはビ
オチンが結合した物質を前記光ファイバー上の免疫物質
と反応させた後に、塩基に可溶性の蛍光物質で標識され
たアビジンを結合させるものである。この理由は、水溶
液中ではある種の蛍光物質は加水分解や酸化を受けやす
いので、蛍光物質による標識は測定直前がよいからであ
る。Furthermore, in the method (2), the method (3) is preferable when an unstable fluorescent substance is used as a label. The method (3) is a fluorescence immunoassay method based on the competitive method (a) and the sandwich method (b). The immunological substance was immobilized on the optical fiber by utilizing the specific binding of avidin and biotin. After that, a substance to which biotin is bound or a substance having a plurality of reactive groups is bound to a substance that shows the same immune reaction as the substance to be measured or a substance that immunoreacts with the substance to be measured, Is to react a substance bound with biotin with the immunological substance on the optical fiber, and then bind avidin labeled with a base-soluble fluorescent substance. The reason for this is that certain fluorescent substances are susceptible to hydrolysis or oxidation in an aqueous solution, and thus labeling with fluorescent substances should be performed immediately before measurement.
【００１７】競合法（ａ）では、被測定物質とビオチン
が結合した被測定物質と同一の免疫反応を示す物質を混
合し、この溶液に免疫物質を固定化した光ファイバーを
浸漬して競合的に反応させる（抗原−抗体反応）。次い
で、塩基に可溶性の蛍光物質で標識されたアビジンを反
応させると、ビオチンにアビジンが結合し、被測定物質
と同一の免疫反応を示す物質がビオチン−アビジンを介
して蛍光物質で標識される。競合法では、被測定物質の
濃度が高ければ、蛍光物質で標識された被測定物質と同
一の免疫反応を示す物質の光ファイバーへの結合量が少
ないので、蛍光強度が低下する（実施例３参照）。In the competitive method (a), a substance to be measured is mixed with a substance to which biotin is bound and which shows the same immune reaction as that of the substance to be measured, and the optical fiber on which the immune substance is immobilized is immersed in this solution to competitively compete. React (antigen-antibody reaction). Then, when avidin labeled with a base-soluble fluorescent substance is reacted, avidin is bound to biotin, and a substance showing the same immunological reaction as the substance to be measured is labeled with the fluorescent substance via biotin-avidin. In the competitive method, when the concentration of the substance to be measured is high, the amount of the substance that shows the same immune reaction as the substance to be measured, which is labeled with the fluorescent substance, bound to the optical fiber is small, so that the fluorescence intensity decreases (see Example 3). ).
【００１８】サンドイッチ法（ｂ）では、被測定物質の
溶液に免疫物質を固定化した光ファイバーを浸漬して反
応させ、光ファイバー上の免疫物質と被測定物質を結合
させる。次いで、この光ファイバーをビオチンが結合し
た被測定物質と免疫反応する物質の溶液に浸漬する。こ
れにより、光ファイバー上の免疫物質とビオチンが結合
した被測定物質と免疫反応する物質が、被測定物質をサ
ンドイッチした状態で結合される。その後、この光ファ
イバーを塩基に可溶性の蛍光物質で標識されたアビジン
の溶液に浸漬すると、ビオチンにアビジンが結合し、被
測定物質と免疫反応する物質がビオチン−アビジンを介
して蛍光物質で標識される。サンドイッチ法では、被測
定物質の濃度が高ければ、蛍光物質で標識された被測定
物質と免疫反応する物質の光ファイバーへの結合量も多
いので、蛍光強度が高くなる（実施例２参照）。In the sandwich method (b), an optical fiber on which an immunological substance is immobilized is immersed in a solution of the substance to be measured and reacted to bond the immunological substance on the optical fiber with the substance to be measured. Next, this optical fiber is immersed in a solution of a substance that immunoreacts with the substance to be measured to which biotin is bound. As a result, the substance that immunoreacts with the substance to be measured in which the immunological substance on the optical fiber and biotin are bound is bound in the state where the substance to be measured is sandwiched. Then, when this optical fiber is immersed in a solution of avidin labeled with a base-soluble fluorescent substance, avidin is bound to biotin, and a substance that immunoreacts with the substance to be measured is labeled with the fluorescent substance via biotin-avidin. .. In the sandwich method, when the concentration of the substance to be measured is high, the amount of the substance that immunoreacts with the substance to be measured, which is labeled with the fluorescent substance, is also large in the amount bound to the optical fiber, so that the fluorescence intensity becomes high (see Example 2).
【００１９】方法（２）で述べたように、被測定物質と
同一の免疫反応を示す物質又は被測定物質と免疫反応す
る物質が複数の反応活性基を有する物質に結合し、その
複数の反応活性基にはビオチンが結合していることが望
ましい。アビジンとビオチンはこれらと同等の作用を有
する化合物の組み合わせで置き換えることができる。As described in the method (2), a substance which shows the same immune reaction as the substance to be measured or a substance which immunoreacts with the substance to be measured is bound to a substance having a plurality of reactive groups, and the plurality of reactions It is desirable that biotin is bound to the active group. Avidin and biotin can be replaced by a combination of compounds having equivalent effects.
【００２０】方法（２）及び方法（３）における、被測
定物質と免疫反応する物質又は被測定物質と同一の免疫
反応を示す物質が、複数の反応活性基を有する物質と結
合し、該反応活性基にビオチンを介して蛍光物質で標識
されたアビジンと結合したものは、次の方法で製造する
ことができる。すなわち、ビオチンを複数の反応活性基
を有する物質の大部分の反応活性基に反応させた後、つ
いで被測定物質と免疫反応する物質または被測定物質と
同一の免疫反応を示す物質と反応させ、さらにビオチン
を蛍光物質で標識されたアビジンで修飾して製造する。
また、複数の反応活性基を有する物質の大部分の反応活
性基に直接蛍光色素を結合させてもよい。この場合に
は、蛍光物質を複数の反応活性基を有する物質の大部分
の反応活性基に反応させた後、ついで被測定物質と免疫
反応する物質または被測定物質と同一の免疫反応を示す
物質と反応させて製造する。In the methods (2) and (3), the substance which immunoreacts with the substance to be measured or the substance which shows the same immune reaction as the substance to be measured binds to the substance having a plurality of reactive groups, and the reaction The active group bound to avidin labeled with a fluorescent substance through biotin can be produced by the following method. That is, after reacting biotin with most of the reactive groups of a substance having a plurality of reactive groups, it is then reacted with a substance that immunoreacts with the substance to be measured or a substance that shows the same immune reaction as the substance to be measured, Further, biotin is modified with avidin labeled with a fluorescent substance to produce.
Further, the fluorescent dye may be directly bonded to most of the reaction active groups of the substance having a plurality of reaction active groups. In this case, after reacting the fluorescent substance with most of the reaction-active groups of the substance having a plurality of reaction-active groups, then a substance that immunoreacts with the substance to be measured or a substance that shows the same immune reaction as the substance to be measured. It is manufactured by reacting with.
【００２１】本発明の方法（２）及び方法（３）の分析
法においては、図１及び図２に示すように、小型光源
（６）及び励起光又は蛍光を伝播するための光ファイバ
ー（１）と、その一方の端面のコア表面（３）を露出さ
せ、その表面に被測定物質と特異的に結合する抗原
（４）などを固定化した検出部、並びに検出部で励起さ
れた蛍光の強度を測定するための検出器（８）を用いる
ことができる。In the analysis methods of the methods (2) and (3) of the present invention, as shown in FIGS. 1 and 2, a small light source (6) and an optical fiber (1) for propagating excitation light or fluorescence. And a core surface (3) of one end face thereof is exposed, and an antigen (4) that specifically binds to the substance to be measured is immobilized on the surface, and the intensity of fluorescence excited by the detection unit. A detector (8) for measuring can be used.
【００２２】前記光ファイバーは、樹脂の方が低価格で
あり、使用しやすいため、通常、アクリル酸メチル、ア
クリル酸エチル、メタクリル酸メチルなどのモノマーと
スチレンなどのモノマーとの共重合体である樹脂製ファ
イバーが用いられる。前記樹脂性光ファイバーの表面に
免疫物質を結合させるには、反応活性基としてホルミル
基を導入して免疫物質と共有結合させ固定化させる。The optical fiber is usually a copolymer of a monomer such as methyl acrylate, ethyl acrylate and methyl methacrylate and a monomer such as styrene because the resin is cheaper and easier to use. Fibers are used. In order to bind the immunological substance to the surface of the resinous optical fiber, a formyl group is introduced as a reactive group to covalently bond with the immunogenic substance and immobilize it.
【００２３】[0023]
【発明の効果】本発明では、６００〜１２００ｎｍの半
導体レーザーの波長領域で励起できるので、小型の装置
を用いることができ、フィルターによる吸収損失が少な
いので高感度化が実現できる。According to the present invention, since it is possible to excite in the wavelength region of the semiconductor laser of 600 to 1200 nm, it is possible to use a small device, and it is possible to realize high sensitivity because the absorption loss by the filter is small.
【００２４】[0024]
【実施例】以下に本発明の実施例を示すが、本発明はこ
れらの実施例に限られるものでなく、広い範囲で適用可
能である。EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited to these examples and can be applied in a wide range.
【００２５】実施例１（肝臓中のビタミンＢ2 の測定） （１）肝臓片をリン酸緩衝生理食塩水中ですりつぶし
た。遠心で沈殿除去後、上澄を薄膜クロマトプレートに
スポットした。これを水／ブタノール／ピリジンの体積
比１／１／２の混合溶液（pH９）で展開した。 （２）ビタミンＢ2 の最大励起波長（３６０ｎｍ）の２
倍の波長である７２０ｎｍの光をクロマトプレートに照
射し、蛍光（５１０ｎｍ）を検出し、その保持位置から
肝臓中のビタミンＢ2 を確認した。Example 1 (Measurement of Vitamin B 2 in Liver) (1) Liver pieces were ground in phosphate buffered saline. After removing the precipitate by centrifugation, the supernatant was spotted on a thin film chromatography plate. This was developed with a mixed solution (pH 9) of water / butanol / pyridine in a volume ratio of 1/1/2. (2) Maximum excitation wavelength of vitamin B 2 (360 nm) 2
The chromatographic plate was irradiated with light having a double wavelength of 720 nm, fluorescence (510 nm) was detected, and vitamin B 2 in the liver was confirmed from the holding position.
【００２６】実施例２（サンドイッチ法による抗マウス
ＩｇＧ抗体の測定） （１）水１００μｌに炭酸ナトリウム３mgとビオチン４
mgを溶かし、ついで、１．８μM のキトサン（アミノ基
の数は１分子あたり４０００個）溶液２mlに、上記のビ
オチン溶液を添加した。さらに水１００μｌを添加した
後、水溶性カルボジイミド５０mgを添加し、さらに撹拌
しながら一晩室温で反応させ、酢酸を３滴滴下して反応
を停止させた。ついで、０．３ｇ／mlの炭酸ナトリウム
と０．３ｇ／mlの塩化ナトリウム混合液４mlを加えて、
ビオチン化キトサンを沈殿させた。この沈殿を遠心分離
して回収した後、０．３ｇ／mlの塩化ナトリウムと０．
１ｇ／ml炭酸ナトリウム混合液で沈殿を洗浄した。この
沈殿を１０ｍM のカリウム−リン酸緩衝液（pH７）２ml
に懸濁し、さらに同緩衝液５００mlで４℃一晩透析し
て、ビオチン化キトサン（以下、ＢＣと略す）溶液を得
た。 （２）上記（１）のＢＣ懸濁液２mlにヒツジ由来抗ウサ
ギＩｇＧ抗体（以下ａＲＧと略す）溶液１mgと、水溶性
カルボジイミド１０mgを添加し、４℃で１夜反応させ
た。反応終了後、リン酸緩衝生理食塩水で１２時間透析
を行い、さらに、陰イオン交換カラムを用いて未反応物
を除去し、ａＲＧが結合したビオチン化キトサン（以
下、ａＲＧ−ＢＣと略す）溶液を得た。 （３）アビジン１mg及びトリエチルアミン０．２mlをエ
タノール１mlに溶解し、さらに７−ヒドロキシクマリン
−３−カルボン酸２mgを加えて充分に溶解し、さらにジ
シクロヘキシルカルボジイミド１４mgを加えて、室温で
４時間反応させた。反応終了後、アスピレータでエタノ
ールとトリエチルアミンを減圧除去し、生じた残留物
を、０．０１M 酢酸緩衝液（pH６．５）２mlに懸濁した
後、５０００rpm で１０分間遠心分離して上澄を採取
し、再度遠心分離して７−ヒドロキシクマリン−３−カ
ルボン酸で修飾されたアビジン（以下、ＦＡと略す）溶
液を得た。 （４）ポリメタクリル酸メチルを主成分とする直径１mm
の光ファイバー（三菱レイヨン製、商品名：エスカ）の
先端を酢酸エチルに浸して拭きとり、クラッド層を１cm
剥離し、水洗した。 （５）水０．５mlに硫酸ニッケル１０mgを溶解し、つい
でエタノール２．５mlを加えた。このとき生じた白色沈
殿を３０００rpm で遠心分離して上澄液を採取し、これ
をＮｉ−エタノール溶液とした。５０mM水酸化カリウム
−エタノール溶液０．４mlにＮｉ−エタノール溶液０．
１mlを加え、さらに５０％グルタルアルデヒド５０μl
を添加して反応液とした。 （６）上記（５）で調製した反応液に、上記（４）の光
ファイバーを５０℃で１０分間浸漬した後、水洗した。
ついで、２０mMの塩酸溶液に上記光ファイバーを１０分
間浸漬した後、水で洗浄し、光ファイバーのコア部分表
面にホルミル基を導入した。 （７）Bacillus１６−３Ｆ株が産生する耐熱性α−アミ
ラーゼに対するモノクローナル抗体であるマウスＩｇＧ
（以下、ＭＧと略す）１mgをリン酸緩衝生理食塩水（pH
＝７．５）１mlに溶かした。この溶液に上記（６）の光
ファイバーを４℃で１２時間浸漬した。光ファイバーを
溶液から取り出し、水で洗浄した後、１％ホウ素化水素
ナトリウム水溶液に１５分間浸漬した後、水で洗浄して
ＭＧをブロック化し、ＭＧ固定化センサーとした。この
ようにして製造した光ファイバーのコア部分を図２に示
すセンサーチップとした。 （８）濃度既知のウサギ由来抗マウスＩｇＧ抗体（以
下、ａＭＧと略す）溶液に、上記（７）のセンサーチッ
プを浸漬（ＭＧを抗原として免疫反応を起こす）した
後、リン酸緩衝生理食塩水で洗浄した。 （９）次に、上記（２）で得たａＲＧ−ＢＣ溶液にセン
サーチップを浸漬（ａＭＧを抗原として免疫反応を起こ
す）して、リン酸緩衝生理食塩水で洗浄した。 （１０）次に、上記（３）のＦＡ溶液に上記（９）のセ
ンサーチップを浸漬して、リン酸緩衝生理食塩水で洗浄
し、蛍光標識抗体（ａＲＧ−ＢＣ−ＦＡ）が結合したセ
ンサーチップを得た（結合形式が、ファイバー−ＭＧ−
ａＭＧ−ａＲＧ−ＢＣ−ＦＡ）。 （１１）次に、上記（１０）のセンサーチップを２wt％
の炭酸水素ナトリウム溶液（pH１２）に浸漬し、図１に
示す装置にて、７−ヒドロキシクマリン−３−カルボン
酸の最大励起波長（３８７ｎｍ）の約２倍の波長（７７
０ｎｍ）を有する半導体レーザー光学系で蛍光を検出器
（８）を用いて測定した。 （１２）ａＭＧの濃度を変え、上記（８）〜（１１）と
同様の測定を繰り返し、ａＭＧの濃度と蛍光強度の関係
を調べ検量線を作成した。検量線から、検出限界は０．
６×１０-4（mg／ml）であった。Example 2 (Measurement of anti-mouse IgG antibody by sandwich method) (1) Sodium carbonate 3 mg and biotin 4 in 100 μl of water
The above biotin solution was added to 2 ml of a 1.8 μM chitosan (the number of amino groups is 4000 per molecule) solution. After further adding 100 μl of water, 50 mg of water-soluble carbodiimide was added, and the mixture was further reacted overnight at room temperature with stirring, and 3 drops of acetic acid was added dropwise to stop the reaction. Then add 4 g of 0.3 g / ml sodium carbonate and 0.3 g / ml sodium chloride mixture,
Biotinylated chitosan was precipitated. The precipitate was centrifuged and collected, and then 0.3 g / ml of sodium chloride and 0.
The precipitate was washed with a 1 g / ml sodium carbonate mixture. 2 ml of this precipitate was added to 10 mM potassium-phosphate buffer (pH 7).
Then, the solution was suspended in the above and dialyzed against 500 ml of the same buffer overnight at 4 ° C. to obtain a biotinylated chitosan (hereinafter abbreviated as BC) solution. (2) To 2 ml of the BC suspension obtained in (1) above, 1 mg of a sheep-derived anti-rabbit IgG antibody (hereinafter referred to as aRG) solution and 10 mg of a water-soluble carbodiimide were added and reacted overnight at 4 ° C. After completion of the reaction, dialyzing with phosphate buffered saline for 12 hours, further removing unreacted substances using anion exchange column, aRG-bound biotinylated chitosan (hereinafter referred to as aRG-BC) solution Got (3) Avidin (1 mg) and triethylamine (0.2 ml) were dissolved in ethanol (1 ml), 7-hydroxycoumarin-3-carboxylic acid (2 mg) was further added and dissolved sufficiently, and further dicyclohexylcarbodiimide (14 mg) was added and the mixture was reacted at room temperature for 4 hours. It was After the reaction was completed, ethanol and triethylamine were removed under reduced pressure using an aspirator, the resulting residue was suspended in 2 ml of 0.01M acetate buffer (pH 6.5), and then centrifuged at 5000 rpm for 10 minutes to collect the supernatant. Then, the solution was centrifuged again to obtain a solution of avidin (hereinafter, abbreviated as FA) modified with 7-hydroxycoumarin-3-carboxylic acid. (4) Diameter of 1mm with polymethylmethacrylate as the main component
The optical fiber (made by Mitsubishi Rayon, brand name: ESCA) is dipped in ethyl acetate and wiped off, and the clad layer is 1 cm.
It was peeled off and washed with water. (5) 10 mg of nickel sulfate was dissolved in 0.5 ml of water, and then 2.5 ml of ethanol was added. The white precipitate generated at this time was centrifuged at 3000 rpm to collect a supernatant, which was used as a Ni-ethanol solution. 0.4 ml of 50 mM potassium hydroxide-ethanol solution was added with Ni-ethanol solution of 0.
Add 1 ml, 50% glutaraldehyde 50 μl
Was added to obtain a reaction solution. (6) The optical fiber of (4) above was immersed in the reaction solution prepared in (5) above at 50 ° C. for 10 minutes and then washed with water.
Then, the above optical fiber was immersed in a 20 mM hydrochloric acid solution for 10 minutes and then washed with water to introduce a formyl group on the surface of the core portion of the optical fiber. (7) Mouse IgG which is a monoclonal antibody against thermostable α-amylase produced by Bacillus 16-3F strain
1 mg (hereinafter abbreviated as MG) of phosphate buffered saline (pH
= 7.5) Dissolved in 1 ml. The optical fiber of (6) above was immersed in this solution at 4 ° C. for 12 hours. The optical fiber was taken out of the solution, washed with water, immersed in a 1% sodium borohydride aqueous solution for 15 minutes, and then washed with water to block MG to obtain an MG-immobilized sensor. The core portion of the optical fiber thus manufactured was used as the sensor chip shown in FIG. (8) After immersing the sensor chip of (7) above (causing an immune reaction with MG as an antigen) in a rabbit-derived anti-mouse IgG antibody (hereinafter abbreviated as abbreviated) solution of known concentration, phosphate buffered saline Washed with. (9) Next, the sensor chip was immersed in the aRG-BC solution obtained in (2) above (an immunoreaction is caused by using aMG as an antigen), and washed with phosphate buffered saline. (10) Next, the sensor chip of the above (9) is immersed in the FA solution of the above (3), washed with phosphate buffered saline, and a sensor to which a fluorescent labeled antibody (aRG-BC-FA) is bound. A chip was obtained (coupling type is fiber-MG-
aMG-aRG-BC-FA). (11) Next, add 2 wt% of the sensor chip of (10) above.
1 was immersed in a sodium hydrogen carbonate solution (pH 12), and a wavelength (77 nm) about twice the maximum excitation wavelength (387 nm) of 7-hydroxycoumarin-3-carboxylic acid was measured using the apparatus shown in FIG.
Fluorescence was measured using a detector (8) with a semiconductor laser optical system having 0 nm). (12) The concentration of aMG was changed and the same measurements as in (8) to (11) above were repeated to examine the relationship between the concentration of aMG and the fluorescence intensity to prepare a calibration curve. From the calibration curve, the detection limit was 0.
It was 6 × 10 −4 (mg / ml).
【００２７】実施例３（競合法による抗マウスＩｇＧ抗
体の測定） （１）実施例２の（１）〜（２）と同様の方法でａＭＧ
が結合したビオチン化キトサン（以下、ａＭＧ−ＢＣと
略す）溶液を得た。 （２）アビジン１mg及びフルオレセインイソチオシアネ
ート１．８mgを０．５Mの炭酸ナトリウム−炭酸水素ナ
トリウム緩衝液（pH９．０）からなる塩基性溶媒５mlに
溶解し、４℃で光を遮断して撹拌を続け、２０時間反応
させた。反応終了後、アスピレータを用いて減圧下で溶
媒を除去し、この残留物を０．０５M リン酸緩衝液（pH
４．０）５mlに懸濁した後、５０００rpm で１０分間遠
心分離し、未反応物質を除去して、上澄を採取した。上
記操作をさらに２回繰り返し、フルオレセインイソチオ
シアナートで修飾されたアビジン（以下、Ｆ2 Ａと略
す）溶液を得た。 （３）実施例２の（４）〜（７）と同様の方法でＭＧ固
定化センサーチップを製造した。 （４）濃度既知のａＭＧ溶液と、上記（１）のａＭＧ−
ＢＣ溶液を１：１の体積比で混合し、次いで上記（３）
のセンサーチップを浸漬した後、リン酸緩衝生理食塩水
で洗浄した。 （５）上記（２）のＦ2 Ａ溶液に上記（４）のセンサー
チップを浸漬し、リン酸緩衝生理食塩水で洗浄した。 （６）次に、上記（５）のセンサーチップをトリス緩衝
液（pH１２）に浸漬し、図１に示す装置にて、フルオレ
セインの最大励起波長（４９６ｎｍ）の約２倍の波長
（９９４ｎｍ）を有する半導体レーザ光学系で蛍光を検
出器を用いて測定した。 （７）ａＭＧの濃度を変え、上記（４）〜（６）と同様
の測定を繰り返し、ａＭＧの濃度と蛍光強度の関係を調
べ検量線を作成した。検量線から、検出限界は０．６×
１０-4（mg／ml）であった。Example 3 (Measurement of anti-mouse IgG antibody by competitive method) (1) aMG in the same manner as in (2) of Example 2
A biotinylated chitosan (hereinafter, abbreviated as aMG-BC) solution having bound thereto was obtained. (2) Dissolve 1 mg of avidin and 1.8 mg of fluorescein isothiocyanate in 5 ml of a basic solvent consisting of 0.5M sodium carbonate-sodium hydrogen carbonate buffer (pH 9.0) and stir at 4 ° C with light blocked. The reaction was continued for 20 hours. After the reaction was completed, the solvent was removed under reduced pressure using an aspirator, and the residue was washed with 0.05M phosphate buffer (pH
4.0) After suspending in 5 ml, the mixture was centrifuged at 5000 rpm for 10 minutes to remove unreacted substances, and the supernatant was collected. The above operation was repeated twice more to obtain a solution of avidin (hereinafter abbreviated as F 2 A) modified with fluorescein isothiocyanate. (3) An MG-immobilized sensor chip was manufactured in the same manner as in (4) to (7) of Example 2. (4) aMG solution of known concentration, and aMG- of (1) above
The BC solution was mixed at a volume ratio of 1: 1 and then the above (3)
After immersing the sensor chip of No. 1, it was washed with phosphate buffered saline. (5) The sensor chip of (4) above was immersed in the F 2 A solution of (2) above and washed with phosphate buffered saline. (6) Next, the sensor chip of (5) above is immersed in Tris buffer (pH 12), and a wavelength (994 nm) about twice the maximum excitation wavelength (496 nm) of fluorescein is applied in the apparatus shown in FIG. Fluorescence was measured using a semiconductor laser optical system having a detector. (7) The concentration of aMG was changed and the same measurements as in (4) to (6) above were repeated to examine the relationship between the concentration of aMG and the fluorescence intensity to prepare a calibration curve. From the calibration curve, the detection limit is 0.6 ×
It was 10 −4 (mg / ml).
【００２８】実施例４（サンドイッチ法による抗マウス
ＩｇＧ抗体の測定） （１）実施例２の（２）と同様の方法で、ａＲＧ−ＢＣ
溶液を得、実施例２の（３）と同様の方法でＦＡ溶液を
得た。次いで、ａＲＧ−ＢＣ溶液とＦＡ溶液を混合し、
ａＲＧ−ＢＣ−ＦＡからなる測定試薬を調製した。 （２）実施例２の（４）〜（７）と同様の方法でＭＧ固
定化センサーチップを製造した。 （３）濃度既知のａＭＧ溶液に、上記（２）のセンサー
チップを浸漬した後、リン酸緩衝生理食塩水で洗浄し
た。 （４）次に、上記（１）で得た測定試薬に上記（３）の
センサーチップを浸漬して、リン酸緩衝生理食塩水で洗
浄した。測定試薬中のａＲＧは、ａＭＧを抗原として免
疫反応を起こす。 （５）次に、上記（４）のセンサーチップを２wt％炭酸
水素ナトリウム溶液（pH１２）に浸漬し、図１に示す装
置にて、７−ヒドロキシクマリン−３−カルボン酸の最
大励起波長（３８７ｎｍ）の２倍の波長（７７０ｎｍ）
を有する半導体レーザ光学系で蛍光を検出器（８）を用
いて測定した。 （６）ａＭＧの濃度を変え、上記（３）〜（５）と同様
の測定を繰り返し、ａＭＧの濃度と蛍光強度の関係を調
べ検量線を作成した。検量線から、検出限界は０．６×
１０-4（mg／ml）であった。Example 4 (Measurement of anti-mouse IgG antibody by sandwich method) (1) In the same manner as in Example 2, (2), aRG-BC.
A solution was obtained, and an FA solution was obtained in the same manner as in (2) of Example 2. Then, the aRG-BC solution and the FA solution are mixed,
A measurement reagent consisting of aRG-BC-FA was prepared. (2) An MG-immobilized sensor chip was manufactured by the same method as (4) to (7) of Example 2. (3) The sensor chip of (2) above was immersed in an aMG solution of known concentration, and then washed with phosphate buffered saline. (4) Next, the sensor chip of (3) above was immersed in the measurement reagent obtained in (1) above, and washed with phosphate buffered saline. ARG in the measurement reagent causes an immune reaction with aMG as an antigen. (5) Next, the sensor chip of (4) above was dipped in a 2 wt% sodium hydrogen carbonate solution (pH 12), and the maximum excitation wavelength (387 nm) of 7-hydroxycoumarin-3-carboxylic acid was measured using the apparatus shown in FIG. ) Twice the wavelength (770 nm)
Fluorescence was measured using a semiconductor laser optical system having a detector (8). (6) The concentration of aMG was changed and the same measurements as in (3) to (5) above were repeated to examine the relationship between the concentration of aMG and the fluorescence intensity to prepare a calibration curve. From the calibration curve, the detection limit is 0.6 ×
It was 10 −4 (mg / ml).
【００２９】実施例５（競合法による抗マウスＩｇＧ抗
体の測定） （１）キトサンの代わりにポリガラクトサミン（アミノ
基の数は１分子あたり５０００個）を用い、実施例３の
（１）と同様の方法で、ａＭＧが結合したビオチン化ポ
リガラクトサミン（以下、ａＭｇ−ＢＧと略す）溶液を
作成した。 （２）実施例３の（２）と同様の方法でＦ2 Ａ溶液を得
た。 （３）次いで、上記（１）のａＭＧ−ＢＧ溶液と上記
（２）のＦ2 Ａ溶液を混合し、結合形式が、ａＭｇ−Ｂ
Ｇ−Ｆ2 Ａからなる測定試薬を調製した。 （４）実施例２の（４）〜（７）と同様の方法でＭＧ固
定化センサーチップを作成した。 （５）濃度既知のａＭＧ溶液と上記（３）の測定試薬を
１：１の体積比で混合し、次いで上記（４）のセンサー
チップを浸漬した後、リン酸緩衝生理食塩水で洗浄し
た。 （６）次に、上記（５）のセンサーチップを０．１mM水
酸化ナトリウム−炭酸水素ナトリウム水溶液（pH１２）
に浸漬し、図１に示す装置にて、フルオレセインの最大
励起波長（４９６ｎｍ）の２倍の波長（９９４ｎｍ）を
有する半導体レーザー光学系で蛍光を検出器（８）を用
いて測定した。 （７）ａＭＧの濃度を変え、上記（５）〜（６）と同様
の測定を繰り返し、ａＭＧの濃度と蛍光強度の関係を調
べ検量線を作成した。検量線から、検出限界は０．５×
１０-4（mg／ml）であった。Example 5 (Measurement of anti-mouse IgG antibody by competitive method) (1) As in Example 3 (1), using polygalactosamine (the number of amino groups is 5000 per molecule) instead of chitosan By the method described above, a biotinylated polygalactosamine (hereinafter abbreviated as aMg-BG) solution to which aMG was bound was prepared. (2) An F 2 A solution was obtained in the same manner as in (2) of Example 3. (3) Then, the aMG-BG solution of (1) above is mixed with the F 2 A solution of (2) above, and the binding form is aMg-B.
A measurement reagent consisting of G-F 2 A was prepared. (4) An MG-immobilized sensor chip was prepared by the same method as (4) to (7) of Example 2. (5) An aMG solution of known concentration and the measurement reagent of (3) above were mixed at a volume ratio of 1: 1 and then the sensor chip of (4) above was dipped and then washed with phosphate buffered saline. (6) Next, the sensor chip of (5) above is treated with a 0.1 mM sodium hydroxide-sodium hydrogen carbonate aqueous solution (pH 12)
Then, fluorescence was measured using a detector (8) with a semiconductor laser optical system having a wavelength (994 nm) twice the maximum excitation wavelength (496 nm) of fluorescein in the apparatus shown in FIG. (7) The concentration of aMG was changed and the same measurements as in (5) to (6) above were repeated to examine the relationship between the concentration of aMG and the fluorescence intensity to prepare a calibration curve. From the calibration curve, the detection limit is 0.5 ×
It was 10 −4 (mg / ml).
【００３０】実施例６（２種類の波長のレーザ光を励起
光源とする抗マウスＩｇＧ抗体の測定） （１）実施例２の（２）と同様の方法でａＲＧ−ＢＣ溶
液を作成した。 （２）実施例２の（３）と同様の方法でＦＡ溶液を得
た。 （３）実施例２の（４）〜（７）と同様の方法でＭＧ固
定化センサーチップを作成した。 （４）濃度既知のａＭＧ溶液に上記（３）のセンサーチ
ップを浸漬した後、リン酸緩衝生理食塩水で洗浄した。 （５）次に、上記（１）のａＲＧ−ＢＣ溶液に上記
（４）のセンサーチップを浸漬して、リン酸緩衝生理食
塩水で洗浄した。 （６）次に、上記（２）のＦＡ溶液に上記（５）のセン
サーチップを浸漬して、リン酸緩衝生理食塩水で洗浄し
た。 （７）次に、上記（６）のセンサーチップを２wt％炭酸
水素ナトリウム溶液（pH１２）に浸漬し、図３及び図４
に示す装置にて、７−ヒドロキシクマリン−３−カルボ
ン酸の最大励起波長（３８７ｎｍ）の約２倍の波長であ
る７７０ｎｍと７８０ｎｍの２種類の波長の半導体レー
ザー光学系で蛍光を検出器（８）を用いて測定した。 （８）ａＭＧの濃度を変え、上記（４）〜（７）と同様
の測定を繰り返し、ａＭＧの濃度と蛍光強度の関係を調
べ検量線を作成した。検量線から、検出限界は０．４×
１０-4（mg／ml）であった。Example 6 (Measurement of anti-mouse IgG antibody using laser light of two kinds of wavelengths as excitation light sources) (1) An aRG-BC solution was prepared in the same manner as in (2) of Example 2. (2) An FA solution was obtained by the same method as in (2) of Example 2. (3) An MG-immobilized sensor chip was prepared in the same manner as in (4) to (7) of Example 2. (4) The sensor chip of (3) above was immersed in an aMG solution of known concentration, and then washed with phosphate buffered saline. (5) Next, the sensor chip of (4) above was immersed in the aRG-BC solution of (1) above and washed with phosphate buffered saline. (6) Next, the sensor chip of (5) above was immersed in the FA solution of (2) above and washed with phosphate buffered saline. (7) Next, the sensor chip of (6) above was dipped in a 2 wt% sodium hydrogen carbonate solution (pH 12), and
In the device shown in FIG. 1, fluorescence is detected by a semiconductor laser optical system having two wavelengths of 770 nm and 780 nm, which are about twice the maximum excitation wavelength (387 nm) of 7-hydroxycoumarin-3-carboxylic acid. ) Was used for the measurement. (8) The concentration of aMG was changed, and the same measurements as those in (4) to (7) were repeated to examine the relationship between the concentration of aMG and the fluorescence intensity to prepare a calibration curve. From the calibration curve, the detection limit is 0.4 ×
It was 10 −4 (mg / ml).
【００３１】実施例７（サンドウィッチ法による耐熱性
α−アミラーゼに対するマウスＩｇＧの測定） （１）実施例２の（３）と同様の方法でＦＡ溶液を得
た。 （２）上記（１）で得たＦＡ溶液の充分量に市販のビオ
チン化ａＭＧ（フナコシ薬品）を添加して、蛍光標識さ
れたａＭＧ−Ｂ−ＦＡ溶液を得た。 （３）実施例２の（４）〜（６）と同様の方法で光ファ
イバーのコア部分表面にホルミル基を導入した。 （４）Bacillus由来の耐熱性α−アミラーゼを２mg/ml
となるようにリン酸緩衝生理食塩水（pH７．５）に溶解
した。この溶液に光ファイバーを４℃で１２時間浸漬し
た。光ファイバーを溶液から取り出し、水で洗浄した
後、１％ホウ素水素ナトリウム水溶液に１５分間浸漬し
た後、水で洗浄して、耐熱性アミラーゼ固定化センサー
チップとした。これを図２に示すセンサーチップとし
た。 （５）濃度既知の耐熱性α−アミラーゼに対するＭＧ溶
液に、上記（４）のセンサーチップを浸漬して、免疫反
応させた。 （６）上記（５）のセンサーチップを取り出し、０．０
５％トゥイーン２０含有リン酸緩衝生理食塩水で洗浄し
た後、上記（２）のａＭＧ−Ｂ−ＦＡ溶液に浸漬して、
トゥイーン２０含有リン酸緩衝生理食塩水で洗浄し、蛍
光標識抗体が結合したセンサーチップを得た。 （７）次いで、上記（６）のセンサーチップを２wt％炭
酸水素ナトリウム溶液（pH１２）に浸漬し、図１に示す
装置にて、７−ヒドロキシクマリン−３−カルボン酸の
最大励起波長（３８７ｎｍ）の約２倍の波長（７８０ｎ
ｍ）を有する半導体レーザー系で蛍光を検出器（８）を
用いて測定した。 （８）耐熱性α−アミラーゼに対するＭＧ溶液の濃度を
変え、上記（５）〜（７）と同様の測定を繰り返し、耐
熱性α−アミラーゼに対するＭＧ溶液の濃度と蛍光強度
の関係を調べ、検量線を作成した。検量線から、検出限
界は１．０×１０-4（mg／ｍｌ）であった。Example 7 (Measurement of mouse IgG against heat-resistant α-amylase by sandwich method) (1) An FA solution was obtained by the same method as in Example 2 (3). (2) Commercially available biotinylated aMG (Funakoshi Chemical Co., Ltd.) was added to a sufficient amount of the FA solution obtained in (1) above to obtain a fluorescently labeled aMG-B-FA solution. (3) A formyl group was introduced on the surface of the core portion of the optical fiber in the same manner as in (4) to (6) of Example 2. (4) Bacillus-derived thermostable α-amylase 2 mg / ml
It was dissolved in phosphate buffered saline (pH 7.5) so that The optical fiber was immersed in this solution at 4 ° C. for 12 hours. The optical fiber was taken out of the solution, washed with water, immersed in a 1% sodium borohydride aqueous solution for 15 minutes, and then washed with water to obtain a thermostable amylase-immobilized sensor chip. This was used as the sensor chip shown in FIG. (5) The sensor chip of (4) above was immersed in an MG solution for thermostable α-amylase of known concentration to cause an immune reaction. (6) Take out the sensor chip of (5) above and
After washing with phosphate buffered saline containing 5% Tween 20, it was immersed in the aMG-B-FA solution of (2) above,
It was washed with phosphate buffered saline containing Tween 20 to obtain a sensor chip to which a fluorescent labeled antibody was bound. (7) Then, the sensor chip of the above (6) was immersed in a 2 wt% sodium hydrogen carbonate solution (pH 12), and the maximum excitation wavelength (387 nm) of 7-hydroxycoumarin-3-carboxylic acid was measured by the apparatus shown in FIG. About twice the wavelength (780n
Fluorescence was measured with a detector (8) in a semiconductor laser system with m). (8) The concentration of the MG solution for the thermostable α-amylase is changed, and the same measurement as in (5) to (7) above is repeated to examine the relationship between the concentration of the MG solution for the thermostable α-amylase and the fluorescence intensity for calibration. Created a line. From the calibration curve, the detection limit was 1.0 × 10 −4 (mg / ml).
【００３２】実施例８（サンドイッチ法によるマウスＩ
ｇＧの測定） （１）実施例２の（１）と同様の方法でＢＣ溶液を得
た。 （２）上記（１）のＢＣ溶液２mlにヒトアルブミン（フ
ナコシ薬品製、以下、ＨＡＬと略す）溶液１mgと、水溶
性カルボジイミド１０mgを添加し、４℃で一晩反応させ
た。反応終了後、リン酸緩衝生理食塩水で１２時間透析
を行い、さらに、陰イオン交換カラムを用いて未反応物
を除去し、ＨＡＬが結合したビオチン化キトサン（以
下、ＨＡＬ−ＢＣと略す）を得た。 （３）実施例２の（３）と同様の方法でＦＡ溶液を得
た。 （４）実施例２の（４）〜（６）と同様の方法で、光フ
ァイバーのコア部分表面にホルミル基を導入した。 （５）ヤギ由来マウスＩｇＧ（フナコシ薬品製、以下、
ＹＩＧと略す）１mgをリン酸緩衝生理食塩水（pH７．
５）１mlに溶かした。この溶液に上記（４）の光ファイ
バーを４℃で１２時間浸漬した。光ファイバーを溶液か
ら取り出し、水で洗浄した後、１％ホウ素化水素ナトリ
ウム水溶液に１５分間浸漬した後、水で洗浄してＹＩＧ
をブロック化し、ＹＩＧ固定化センサーとした。このよ
うにして製造した光ファイバーのコア部分を図２に示す
センサーチップとした。 （６）濃度既知のマウス由来抗ＨＡＬモノクローナル抗
体であるマウスＩｇＧ（フナコシ薬品製、以下、ＭＩＧ
と略す）溶液に、上記（５）のセンサーチップを浸漬
（ＭＩＧがＹＩＧの抗原となり免疫反応を起こす）した
後、リン酸緩衝生理食塩水で洗浄した。 （７）次に、上記（２）のＨＡＬ−ＢＣ溶液に上記
（６）のセンサーチップを浸漬（ＭＩＧが抗体として免
疫反応を起こす）して、リン酸緩衝生理食塩水で洗浄し
た。 （８）次に、上記（３）のＦＡ溶液に上記（７）のセン
サーチップを浸漬して、リン酸緩衝生理食塩水で洗浄
し、蛍光標識抗体が結合したセンサーチップを得た（結
合形式が、ファイバー−ＹＩＧ−ＭＩＧ−ＨＡＬ−ＢＣ
−ＦＡ）。 （９）次に、上記（８）のセンサーチップを２wt％炭酸
水素ナトリウム溶液（pH１２）に浸漬し、図１に示す装
置にて、７−ヒドロキシクマリン−３−カルボン酸の最
大励起波長（３８７ｎｍ）の約２倍の波長（７７０ｎ
ｍ）を有する半導体レーザー光学系で蛍光を検出器
（８）を用いて測定した。 （１０）ＭＩＧの濃度を変え、上記（６）〜（９）と同
様の測定を繰り返し、ＭＩＧの濃度と蛍光強度の関係を
調べ、検量線を作成した。検出限界は０．６×１０
-4（mg/ml)であった。Example 8 (mouse I by sandwich method)
Measurement of gG) (1) A BC solution was obtained in the same manner as in (1) of Example 2. (2) To 2 ml of the BC solution of the above (1), 1 mg of a human albumin (manufactured by Funakoshi Chemical Co., Ltd., hereinafter abbreviated as HAL) solution and 10 mg of a water-soluble carbodiimide were added and reacted overnight at 4 ° C. After completion of the reaction, dialysis was performed for 12 hours against phosphate buffered saline, and unreacted substances were removed using an anion exchange column, and biotinylated chitosan (hereinafter abbreviated as HAL-BC) having HAL bound thereto was removed. Obtained. (3) An FA solution was obtained by the same method as in (3) of Example 2. (4) In the same manner as in (4) to (6) of Example 2, a formyl group was introduced on the surface of the core portion of the optical fiber. (5) Goat-derived mouse IgG (Funakoshi Chemical Co., Ltd.,
1 mg of a phosphate buffered saline (abbreviated as YIG) (pH 7.
5) Dissolved in 1 ml. The optical fiber of (4) above was immersed in this solution at 4 ° C. for 12 hours. Remove the optical fiber from the solution, wash it with water, immerse it in a 1% sodium borohydride aqueous solution for 15 minutes, then wash it with water and use YIG.
Was blocked into a YIG-immobilized sensor. The core portion of the optical fiber thus manufactured was used as the sensor chip shown in FIG. (6) Mouse IgG, which is a mouse-derived anti-HAL monoclonal antibody of known concentration (manufactured by Funakoshi Yakuhin, hereinafter referred to as MIG
The sensor chip of (5) above was immersed in a solution (MIG acts as an antigen of YIG to cause an immune reaction), and then washed with phosphate buffered saline. (7) Next, the sensor chip of (6) above was immersed in the HAL-BC solution of (2) above (MIG causes an immune reaction as an antibody) and washed with phosphate buffered saline. (8) Next, the sensor chip of (7) above was immersed in the FA solution of (3) above, and washed with phosphate buffered saline to obtain a sensor chip to which a fluorescent labeled antibody was bound (bonding format But fiber-YIG-MIG-HAL-BC
-FA). (9) Next, the sensor chip of (8) above was immersed in a 2 wt% sodium hydrogen carbonate solution (pH 12), and the maximum excitation wavelength (387 nm) of 7-hydroxycoumarin-3-carboxylic acid was measured using the apparatus shown in FIG. About twice the wavelength (770n
Fluorescence was measured using a detector (8) with a semiconductor laser optical system having m). (10) The concentration of MIG was changed and the same measurements as in (6) to (9) above were repeated to examine the relationship between the concentration of MIG and the fluorescence intensity to prepare a calibration curve. Detection limit is 0.6 × 10
-4 (mg / ml).
【００３３】実施例９（サンドイッチ法によるマウスＩ
ｇＧの測定） （１）実施例８の（２）と同様の方法でＨＡＬ−ＢＣ溶
液を作成した。 （２）実施例２の（３）と同様の方法でＦＡ溶液を得
た。 （３）ついで、上記（１）のＨＢＬ−ＢＣ溶液と上記
（２）のＦＡ溶液を混合し、ＨＡＬ−ＢＣ−ＦＡの結合
形式からなる測定試薬を調製した。 （４）実施例８の（４）〜（５）と同様の処理を行い、
ＹＩＧ固定化センサーチップを作成した。 （５）濃度既知のＭＩＧ溶液に、上記（４）のセンサー
チップを浸漬（ＭＩＧがＹＩＧの抗原となり免疫反応を
起こす）した後、リン酸緩衝生理食塩水で洗浄した。 （６）次に、上記（３）のＨＡＬ−ＢＣ−ＦＡ溶液に上
記（５）のセンサーチップを浸漬（ＭＩＧが抗体として
免疫反応を起こす）して、リン酸緩衝生理食塩水で洗浄
した。 （７）次に、上記（６）のセンサーチップを２wt％炭酸
水素ナトリウム溶液（pH１２）に浸漬し、図１に示す装
置にて、７−ヒドロキシクマリン−３−カルボン酸の最
大励起波長（３８７ｎｍ）の約２倍の波長（７７０ｎ
ｍ）を有する半導体レーザー光学系で蛍光を検出器
（８）を用いて測定した。 （８）ＭＩＧの濃度を変え、上記（５）〜（７）と同様
の測定を繰り返し、ＭＩＧの濃度と蛍光強度の関係を調
べ、検量線を作成した。検出限界は０．６×１０-4（mg
/ml)であった。Example 9 (mouse I by sandwich method)
Measurement of gG) (1) A HAL-BC solution was prepared in the same manner as in (2) of Example 8. (2) An FA solution was obtained by the same method as in (2) of Example 2. (3) Then, the HBL-BC solution of (1) above was mixed with the FA solution of (2) above to prepare a measurement reagent in the HAL-BC-FA binding form. (4) The same processes as (4) to (5) of Example 8 are performed,
A YIG-immobilized sensor chip was created. (5) The sensor chip of (4) above was immersed in a MIG solution of known concentration (MIG acts as an antigen of YIG to cause an immune reaction), and then washed with phosphate buffered saline. (6) Next, the sensor chip of (5) above was immersed in the HAL-BC-FA solution of (3) above (MIG causes an immune reaction as an antibody) and washed with phosphate buffered saline. (7) Next, the sensor chip of (6) above was immersed in a 2 wt% sodium hydrogen carbonate solution (pH 12), and the maximum excitation wavelength (387 nm) of 7-hydroxycoumarin-3-carboxylic acid was measured using the apparatus shown in FIG. About twice the wavelength (770n
Fluorescence was measured using a detector (8) with a semiconductor laser optical system having m). (8) The concentration of MIG was changed, and the same measurement as the above (5) to (7) was repeated to examine the relationship between the concentration of MIG and the fluorescence intensity to prepare a calibration curve. The detection limit is 0.6 × 10 -4 (mg
/ ml).
【００３４】比較例１ （１）実施例２の（１）と同様の方法で、ＢＣ懸濁液を
得た。 （２）このＢＣ懸濁液２mlに、ウサギ由来抗ＨＡＬ抗体
（以下、ＵＡＧと略す）溶液１mgと、水溶性カルボジイ
ミド１０mgを添加し、４℃で一晩反応させた。反応終了
後、リン酸緩衝生理食塩水で１２時間透析を行い、さら
に、陰イオン交換カラムを用いて未反応物を除去し、Ｕ
ＡＧが結合したビオチン化キトサン（以下ＵＡＧ−ＢＣ
と略す）を得た。 （３）実施例３の（２）と同様の方法により、Ｆ2 Ａ溶
液を得た。 （４）実施例２の（４）〜（６）と同様の方法で、光フ
ァイバーのコア部分表面にホルミル基を導入した。 （５）ＭＩＧ１mgをリン酸緩衝生理食塩水（pH７．５）
１mlに溶かした溶液に、上記（４）の光ファイバーを４
℃で１２時間浸漬した。光ファイバーを溶液から取り出
し、水で洗浄した後、１％ホウ素化水素ナトリウム水溶
液に１５分間浸漬した後、水で洗浄してＭＩＧをブロッ
ク化し、ＭＩＧ固定化センサーとした。このようにして
製造した光ファイバーのコア部分を図２に示すセンサー
チップとした。 （６）濃度既知のＨＡＬ溶液に、上記（５）のセンサー
チップを浸漬（ＨＡＬがＭＩＧの抗原となり免疫反応を
起こす）した後、リン酸緩衝生理食塩水で洗浄した。 （７）次に、上記（２）で得たＵＡＧ−ＢＣ溶液に上記
（６）のセンサーチップを浸漬（ＵＡＧが抗体として免
疫反応を起こす）して、リン酸緩衝生理食塩水で洗浄し
た。 （８）次に、上記（３）のＦ2 Ａ溶液に上記（７）のセ
ンサーチップを浸漬して、リン酸緩衝生理食塩水で洗浄
し、蛍光標識抗体が結合したセンサーチップを得た（結
合形式が、ファイバー−ＭＩＧ−ＨＡＬ−ＵＡＧ−ＢＣ
−Ｆ2 Ａ）。 （９）次に、上記（８）のセンサーチップを２wt％炭酸
水素ナトリウム溶液（pH１２）に浸漬し、図１に示す装
置にて、フルオレセインイソシアナートの最大励起波長
（４９６ｎｍ）とほぼ同波長（４８６ｎｍ）を有するＡ
ｒレーザー光学系で蛍光を検出器（８）を用いて測定し
た。 （１０）ＨＡＬの濃度を変え、上記（６）〜（９）と同
様の測定を繰り返し、ＭＩＧの濃度と蛍光強度との関係
を調べ、検量線を作成した。検出限界は０．５×１０-3
（mg/ml)となり、検出限界がほぼ１／１０となった。ま
た、励起光源としてＡｒレーザーを用いたために、測定
装置が大型になり、同時に光学系の調整が難しくなり使
用困難なものとなった。Comparative Example 1 (1) A BC suspension was obtained in the same manner as in (1) of Example 2. (2) To 2 ml of this BC suspension, 1 mg of a rabbit-derived anti-HAL antibody (hereinafter abbreviated as UAG) solution and 10 mg of a water-soluble carbodiimide were added, and reacted at 4 ° C. overnight. After the reaction was completed, the solution was dialyzed against phosphate buffered saline for 12 hours, and the unreacted material was removed using an anion exchange column.
Biotinylated chitosan to which AG is bound (hereinafter UAG-BC
Abbreviated). (3) An F 2 A solution was obtained by the same method as in (2) of Example 3. (4) In the same manner as in (4) to (6) of Example 2, a formyl group was introduced on the surface of the core portion of the optical fiber. (5) 1 mg of MIG is phosphate buffered saline (pH 7.5)
Add the optical fiber of (4) above to the solution dissolved in 1 ml.
It was soaked for 12 hours at ° C. The optical fiber was taken out of the solution, washed with water, immersed in a 1% sodium borohydride aqueous solution for 15 minutes, and then washed with water to block the MIG to obtain a MIG-immobilized sensor. The core portion of the optical fiber thus manufactured was used as the sensor chip shown in FIG. (6) The sensor chip of (5) above was immersed in HAL solution of known concentration (HAL acts as an antigen of MIG to cause an immune reaction), and then washed with phosphate buffered saline. (7) Next, the sensor chip of (6) above was immersed in the UAG-BC solution obtained in (2) above (UAG causes an immune reaction as an antibody) and washed with phosphate buffered saline. (8) Next, the sensor chip of (7) above was immersed in the F 2 A solution of (3) above and washed with phosphate buffered saline to obtain a sensor chip to which a fluorescent labeled antibody was bound ( The connection type is fiber-MIG-HAL-UAG-BC
-F 2 A). (9) Next, the sensor chip of (8) above is immersed in a 2 wt% sodium hydrogen carbonate solution (pH 12), and the same wavelength (496 nm) as the maximum excitation wavelength (496 nm) of fluorescein isocyanate ( A with 486 nm)
Fluorescence was measured using a detector (8) with r laser optics. (10) The HAL concentration was changed and the same measurements as in (6) to (9) above were repeated to examine the relationship between the MIG concentration and the fluorescence intensity to prepare a calibration curve. Detection limit is 0.5 × 10 -3
(Mg / ml), the detection limit was almost 1/10. Further, since the Ar laser is used as the excitation light source, the measuring device becomes large in size, and at the same time, adjustment of the optical system becomes difficult, which makes it difficult to use.
【００３５】比較例２ （１）実施例８の（２）と同様の方法でＨＡＬ−ＢＣ溶
液を作成した。 （２）実施例３の（２）と同様の方法でＦ2 Ａ溶液を得
た。 （３）上記（１）のＨＢ−ＬＢＣ溶液と上記（２）のＦ
2 Ａ溶液を混合し、ＨＡＬ−ＢＣ−Ｆ2 Ａの結合形式か
らなる測定試薬（以下、ＨＡＬ−ＢＣ−Ｆ2 Ａと略す）
溶液を調製した。 （４）比較例１の（４）〜（５）と同様の処理を行い、
ＭＩＧ固定化センサーチップを作成した。 （５）濃度既知のＨＡＬ溶液と上記（３）のＨＡＬ−Ｂ
Ｃ−Ｆ2 Ａ溶液を１：１の体積比で混合し、ついで上記
（４）のセンサーチップを浸漬（ＭＩＧが抗体として免
疫反応を起こす）した後、リン酸緩衝生理食塩水で洗浄
した。 （６）次に、上記（５）のセンサーチップを２wt％炭酸
水素ナトリウム溶液（pH１２）に浸漬し、図１に示す装
置にて、フルオレセインイソシアナートの最大励起波長
（４９６ｎｍ）とほぼ同波長（４８６ｎｍ）を有するＡ
ｒレーザー光学系で蛍光を検出器（８）を用いて測定し
た。 （７）ＨＡＬの濃度を変え、上記（５）〜（６）と同様
の測定を繰り返し、ＭＩＧの濃度と蛍光強度の関係を調
べ検量線を作成した。検出限界は０．５×１０-3（mg/m
l)となり、検出限界がほぼ１／１０となった。また、励
起光源としてＡｒレーザーを用いたために、測定装置が
大型になり、同時に光学系の調整が難しくなり使用困難
なものとなった。Comparative Example 2 (1) A HAL-BC solution was prepared in the same manner as in (2) of Example 8. (2) An F 2 A solution was obtained in the same manner as in (2) of Example 3. (3) HB-LBC solution of (1) above and F of (2) above
2 A solution was mixed, consisting of binding form of HAL-BC-F 2 A measurement reagent (hereinafter, abbreviated as HAL-BC-F 2 A)
A solution was prepared. (4) Perform the same processes as (4) to (5) of Comparative Example 1,
A MIG-immobilized sensor chip was prepared. (5) HAL solution of known concentration and HAL-B of (3) above
The C—F 2 A solution was mixed at a volume ratio of 1: 1 and then the sensor chip of (4) above was immersed (MIG causes an immune reaction as an antibody) and then washed with phosphate buffered saline. (6) Next, the sensor chip of the above (5) is immersed in a 2 wt% sodium hydrogen carbonate solution (pH 12), and the same wavelength as the maximum excitation wavelength (496 nm) of fluorescein isocyanate ( A with 486 nm)
Fluorescence was measured using a detector (8) with r laser optics. (7) The HAL concentration was changed and the same measurements as in (5) to (6) above were repeated to examine the relationship between the MIG concentration and the fluorescence intensity to prepare a calibration curve. The detection limit is 0.5 × 10 -3 (mg / m
l) and the detection limit was almost 1/10. Further, since the Ar laser is used as the excitation light source, the measuring device becomes large in size, and at the same time, adjustment of the optical system becomes difficult, which makes it difficult to use.
【図１】半導体レーザーを使用する蛍光測定系を示す。FIG. 1 shows a fluorescence measurement system using a semiconductor laser.
【図２】上記装置における蛍光検出部を示す。FIG. 2 shows a fluorescence detection unit in the above device.
【図３】７７０ｎｍ及び７８０ｎｍの２種類の半導体レ
ーザーを使用した蛍光測定装置を示す。FIG. 3 shows a fluorescence measuring device using two types of semiconductor lasers of 770 nm and 780 nm.
【図４】図３のセンサーチップの拡大図を示す。FIG. 4 shows an enlarged view of the sensor chip of FIG.
１ 光ファイバー ３ コア表面 ４ 抗原 ６ Ｈｅ−Ｎｅレーザー発生装置 ７ フィルター ８ 分光光度計 ９ センサーチップ １０ プレート １１ 光軸合せのためのガイドレール Ｙ 蛍光標識抗体 ｙ 抗体 １２ ７８０ｎｍ半導体レーザー １３ ７７０ｎｍ半導体レーザー １４ 反射鏡 １５ ハーフミラー １６ ７７０ｎｍレーザー １７ ７８０ｎｍレーザー １８ 蛍光 １９ クラッド層 ２０ ミラーコーティング 1 optical fiber 3 core surface 4 antigen 6 He-Ne laser generator 7 filter 8 spectrophotometer 9 sensor chip 10 plate 11 guide rail for optical axis alignment Y fluorescent labeled antibody y antibody 12 780 nm semiconductor laser 13 770 nm semiconductor laser 14 reflection Mirror 15 Half mirror 16 770nm laser 17 780nm laser 18 Fluorescence 19 Cladding layer 20 Mirror coating
Claims (1)
基性条件下で該蛍光性被測定物質の最大励起波長の約２
倍の波長のレーザー光で励起し、蛍光を測定することを
特徴とする蛍光分析法。 【請求項２】 光ファイバーのコア表面に免疫物質を固
定化し、（ａ）該コア表面の免疫物質に対して、被測定
物質及び塩基に可溶性の蛍光物質で標識された被測定物
質と同一の免疫反応を示す物質を競合的に反応させる
か、或いは、（ｂ）該コア表面の免疫物質と被測定物質
を反応させ、次いで塩基に可溶性の蛍光色素で標識され
た被測定物質と免疫反応する物質を反応させた後、塩基
性条件下で該蛍光物質の最大励起波長の約２倍の波長の
レーザー光で励起し、蛍光を測定することを特徴とする
蛍光免疫分析法。 【請求項３】 前記被測定物質と同一の免疫反応を示す
物質又は被測定物質と免疫反応する物質が、ビオチンと
結合し、該ビオチンは塩基に可溶性の蛍光物質で標識さ
れたアビジンが結合している請求項２記載の蛍光免疫分
析法。 【請求項４】 前記被測定物質と同一の免疫反応を示す
物質又は被測定物質と免疫反応する物質が、複数の反応
活性基を有する物質と結合し、該複数の反応活性基には
ビオチンを介して塩基に可溶性の蛍光物質で標識された
アビジンが結合している請求項２記載の蛍光免疫分析
法。 【請求項５】 光ファイバーのコア表面に免疫物質を固
定化し、（ａ）該コア表面の免疫物質に対して、被測定
物質及びビオチンが結合した被測定物質と同一の免疫反
応を示す物質を競合的に反応させるか、或いは、（ｂ）
該コア表面の免疫物質と被測定物質を反応させ、次い
で、ビオチンが結合した被測定物質と免疫反応する物質
を反応させた後、塩基に可溶性の蛍光物質で標識したア
ビジンを反応させ、塩基性条件下で該蛍光物質の最大励
起波長の約２倍の波長のレーザー光で励起し、蛍光を測
定することを特徴とする蛍光免疫分析法。 【請求項６】 被測定物質と同一の免疫反応を示す物質
又は被測定物質と免疫反応する物質が、複数の反応活性
基を有する物質と結合し、該複数の反応活性基にビオチ
ンが結合している請求項５記載の蛍光免疫分析法。Claim: What is claimed is: 1. A fluorescent substance to be measured which is soluble in a base is added under a basic condition to a maximum excitation wavelength of about 2 of the maximum excitation wavelength of the substance to be measured.
A fluorescence analysis method characterized by measuring fluorescence by exciting with a laser beam having a double wavelength. 2. An immunity substance is immobilized on the core surface of an optical fiber, and (a) the same immunity against the immunity substance on the core surface as the substance to be measured and the substance to be measured labeled with a fluorescent substance soluble in a base. A substance that reacts competitively, or (b) a substance that reacts with the substance to be measured on the surface of the core and the substance to be measured, and then immunoreacts with the substance to be measured labeled with a base-soluble fluorescent dye Is reacted with the fluorescent substance under a basic condition and then excited with a laser beam having a wavelength about twice the maximum excitation wavelength of the fluorescent substance, and the fluorescence is measured. 3. A substance which shows the same immunological reaction as the substance to be measured or a substance which immunoreacts with the substance to be measured is bound to biotin, and the biotin is bound to avidin labeled with a fluorescent substance soluble in a base. The fluorescent immunoassay method according to claim 2, wherein 4. A substance which shows the same immune reaction as the substance to be measured or a substance which immunoreacts with the substance to be measured binds to a substance having a plurality of reactive groups, and biotin is added to the plurality of reactive groups. The fluorescent immunoassay method according to claim 2, wherein avidin labeled with a base-soluble fluorescent substance is bound via the base. 5. An immunological substance is immobilized on the core surface of an optical fiber, and (a) a substance that shows the same immunological reaction as the substance to be measured and the substance to be measured bound with biotin competes with the immunological substance on the surface of the core. Reaction, or (b)
The immunological substance on the surface of the core is reacted with the substance to be measured, and then the substance to be immunologically reacted with the substance to be measured bound with biotin is reacted with avidin labeled with a base-soluble fluorescent substance to form a basic substance. A fluorescence immunoassay method, which comprises exciting under a condition with a laser beam having a wavelength about twice the maximum excitation wavelength of the fluorescent substance and measuring fluorescence. 6. A substance which shows the same immune reaction as the substance to be measured or a substance which immunoreacts with the substance to be measured binds to a substance having a plurality of reactive groups, and biotin binds to the plurality of reactive groups. The fluorescent immunoassay method according to claim 5, wherein
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3287858A JP3025078B2 (en) | 1990-11-26 | 1991-11-01 | Fluorescence analysis |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2-317994 | 1990-11-26 | ||
| JP31799490 | 1990-11-26 | ||
| JP3287858A JP3025078B2 (en) | 1990-11-26 | 1991-11-01 | Fluorescence analysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH055742A true JPH055742A (en) | 1993-01-14 |
| JP3025078B2 JP3025078B2 (en) | 2000-03-27 |
Family
ID=26556914
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3287858A Expired - Fee Related JP3025078B2 (en) | 1990-11-26 | 1991-11-01 | Fluorescence analysis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3025078B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07110330A (en) * | 1993-09-01 | 1995-04-25 | Bio Sensor Kenkyusho:Kk | Optical sensor |
| JP2012519850A (en) * | 2009-03-03 | 2012-08-30 | アクセス メディカル システム カンパニー，リミティド | Detection system and method for high sensitivity fluorescence analysis |
-
1991
- 1991-11-01 JP JP3287858A patent/JP3025078B2/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07110330A (en) * | 1993-09-01 | 1995-04-25 | Bio Sensor Kenkyusho:Kk | Optical sensor |
| JP2012519850A (en) * | 2009-03-03 | 2012-08-30 | アクセス メディカル システム カンパニー，リミティド | Detection system and method for high sensitivity fluorescence analysis |
| JP2015166750A (en) * | 2009-03-03 | 2015-09-24 | アクセス メディカル システムズ，リミティド | Detection system and method for high sensitivity fluorescent assays |
| US9434789B2 (en) | 2009-03-03 | 2016-09-06 | Access Medical Systems, Ltd. | Crosslinked polysaccharide |
| US10379116B2 (en) | 2009-03-03 | 2019-08-13 | Access Medical Systems, Ltd. | Detection system for high sensitivity fluorescent assays |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3025078B2 (en) | 2000-03-27 |
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