JP2591089B2 - Immunological detection reagents - Google Patents
Immunological detection reagentsInfo
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- JP2591089B2 JP2591089B2 JP63184951A JP18495188A JP2591089B2 JP 2591089 B2 JP2591089 B2 JP 2591089B2 JP 63184951 A JP63184951 A JP 63184951A JP 18495188 A JP18495188 A JP 18495188A JP 2591089 B2 JP2591089 B2 JP 2591089B2
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- Japan
- Prior art keywords
- substance
- immunological detection
- antibody
- quenching
- fluorescence
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Description
【発明の詳細な説明】 産業上の利用分野 本発明は、主として臨床検査における病原体、あるい
は疾患マーカー等の検出、さらには広く産業上の極微量
検出分野に用いられる免疫的検出用試薬に関する。Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the detection of pathogens or disease markers in clinical tests, and more particularly to an immunological detection reagent widely used in the field of ultra-trace detection in industry.
従来の技術 天然に存在する、あるいは人工的に作製した抗体を用
いた検出方法は、高特異性及び高感度の点に特徴を有
し、極小量の存在割合の目的物質を検出する目的で現在
用いられている。このような目的には、例えば、血液中
から病原体、あるいは腫瘍、心筋梗塞、脳血栓等の疾患
時に特異的に分泌されるいわゆる疾患マーカーなどの臨
床検査業務や、体内および大気中から生理活性のなる極
微量の物質を検出する目的などがある。近年このような
目的で、例えば石川栄治、河合忠、宮井潔著「酸素免疫
測定法第３版」（医学書院1987年、31〜54頁）に記載さ
れているように多くの種類の免疫測定法が開発されてい
る。この方法は、検出物質が高分子（タンパク質）か低
分子（ハプテン）かによって２分される。本発明では主
としてハプテン系なかでも精神疾患用医薬品でありかつ
覚醒剤として習慣性の高いメタンフェタミン及びその類
似物を対象としている。以下、ハプテン系の免疫的測定
方法の代表例である、抗原固相酵素免疫測定法（以下EL
ISA法と称する）の実験手順を段階を追って説明する。2. Description of the Related Art Detection methods using naturally occurring or artificially produced antibodies are characterized by high specificity and high sensitivity, and are currently used to detect extremely small amounts of target substances. Used. For this purpose, for example, pathological agents, or tumors, myocardial infarction, clinical test work such as so-called disease markers secreted specifically at the time of diseases such as cerebral thrombosis, and biological activity from the body and the atmosphere The purpose is to detect trace amounts of substances. In recent years, many types of immunoassays have been used for such purposes, as described in, for example, "Oxygen Immunoassay 3rd Edition" by Eiji Ishikawa, Tadashi Kawai and Kiyoshi Miyai (Medical Shoin 1987, pp. 31-54). A law is being developed. This method is divided into two types depending on whether the detection substance is a macromolecule (protein) or a low molecule (hapten). The present invention is mainly directed to methamphetamine and its analogs, which are mainly hapten-based drugs for psychiatric disorders and are highly addictive as stimulants. The following is a representative example of a hapten immunoassay, which is an enzyme-linked immunosorbent assay (hereinafter referred to as EL).
The experimental procedure will be described step by step.
（Ａ）抗原のコーティング キャリアー蛋白、例えばウシ血清アルブミン（BSA）
に、検出物質あるいはこれに官能基を導入した誘導体を
結合したコンジュゲートをバッファーに溶解して抗原溶
液とする。(A) Coating of antigen Carrier protein such as bovine serum albumin (BSA)
Then, a conjugate in which a detection substance or a derivative having a functional group introduced thereto is bound to a buffer is dissolved in a buffer to prepare an antigen solution.
マイクロプレート（塩化ビニルあるいはポリスチレン
製96ウェルプレート）に抗原溶液を100μL/ウェル注入
し、20℃で１夜保存する。Inject 100 μL / well of the antigen solution into a microplate (96-well plate made of vinyl chloride or polystyrene) and store it at 20 ° C. overnight.
（Ｂ）ブロッキング BASのバッファー溶液を250μL/ウェル注入し、0.5−
２時間室温で放置する。その後、バッファーまたは純水
で３−５回洗浄する。(B) Blocking Inject 250 µL / well of BAS buffer solution and add 0.5-
Leave at room temperature for 2 hours. Then, it is washed 3-5 times with a buffer or pure water.
（Ｃ）抗体の反応 検出物質溶液を注入し、振とうしながら抗体溶液をさ
らに加える。常温で３−５時間保存した後、アスピレー
タで抗体溶液を除去し、バッファーまたは純水で３−５
回洗浄する。(C) Reaction of antibody A detection substance solution is injected, and the antibody solution is further added while shaking. After storing at room temperature for 3-5 hours, the antibody solution is removed with an aspirator, and the buffer solution or pure water is used for 3-5 hours.
Wash twice.
（Ｄ）第２抗体の反応 酵素、例えばペルオキシダーゼで標識した抗体に対す
る抗体（第２抗体）の溶液を注入し、常温で0.5−２時
間放置する。その後、バッファーまたは純水で３−５回
洗浄する。(D) Reaction of Second Antibody A solution of an antibody (second antibody) for an enzyme, for example, an antibody labeled with peroxidase is injected, and left at room temperature for 0.5 to 2 hours. Then, it is washed 3-5 times with a buffer or pure water.
（Ｅ）基質の反応と停止 発色剤、例えばｏ−フェニレンジアミン（セレン検出
用）をバッファーに溶解し、使用直前に30％過酸化水素
水を加えた溶液（基質溶液）を注入し、室温で発色反応
を行う。５−20分後、硫酸で反応を停止する。(E) Substrate reaction and termination A coloring agent, for example, o-phenylenediamine (for selenium detection) is dissolved in a buffer, and a solution (substrate solution) to which 30% hydrogen peroxide solution is added just before use is injected, and the mixture is allowed to stand at room temperature. Perform a color reaction. After 5-20 minutes, stop the reaction with sulfuric acid.
（Ｆ）測定 マイクロプレート用吸光光度系を用いて492nmの吸光
度を測定する。最終的に検出物質が多いほど吸光度が弱
いことから、物質の検出を行う。(F) Measurement The absorbance at 492 nm is measured using an absorption spectrophotometer for a microplate. Finally, since the absorbance is weaker as the amount of the detected substance is larger, the substance is detected.
発明が解決しようとする課題 上述のように、従来の免疫的検出方法は多くの手順を
必要とし、また反応が固相と液相の共存する不均一系で
進行、最終的な検出に酵素反応を用いているため、本質
的に長時間を要していた。本発明は、免疫的検出に要す
る時間を短縮することを目的とする。Problems to be Solved by the Invention As described above, the conventional immunological detection method requires many steps, and the reaction proceeds in a heterogeneous system in which a solid phase and a liquid phase coexist. Because of the use of, it essentially took a long time. An object of the present invention is to reduce the time required for immunological detection.
課題を解決するための手段 免疫的な検出を行う際の被検出物質と、抗体の有する
蛍光を消光する機能を有した蛍光消光物質が化学的に結
合した免疫的検出用試薬を構成する。Means for Solving the Problems An immunodetection reagent in which a substance to be detected when performing immunological detection and a fluorescence quenching substance having a function of quenching the fluorescence of an antibody are chemically bonded is constituted.
作用 上記構成によれば、反応がすべて均一系（液相）で進
行し、さらに酵素反応も用いないため検出時間の著しい
短縮を行うことが可能となる。Operation According to the above configuration, the reaction can all proceed in a homogeneous system (liquid phase), and the enzymatic reaction is not used, so that the detection time can be significantly shortened.
実施例 本発明は、例えばニトロベンゼン、ジニトロベンゼ
ン、トリニトロベンゼンまたはそれらの誘導体と被検出
物質を化学的に結合したプローブ物質、即ち免疫的検出
用試薬を提供する。このプローブ物質を用いた免疫的検
出方法においては、あらかじめ抗体とプローブ物質を混
合することによって抗体とプローブ物質を結合させ、抗
体が本来有している栄光を消光しておく。目的検出物質
を導入することによって、抗体とプローブ物質を解離さ
せ、蛍光が再び増大する現象を用いて免疫的検出を行な
う。EXAMPLES The present invention provides a probe substance in which, for example, nitrobenzene, dinitrobenzene, trinitrobenzene, or a derivative thereof and a substance to be detected are chemically bonded, that is, an immunodetection reagent. In the immunological detection method using the probe substance, the antibody and the probe substance are mixed in advance by mixing the antibody and the probe substance, and the glory inherent to the antibody is extinguished. By introducing the target detection substance, the antibody and the probe substance are dissociated, and immunodetection is performed using the phenomenon that the fluorescence increases again.
一般的に抗体は280nmの励起で340nm付近に蛍光を発す
る。この蛍光は、種々の消光物質により消光されるが、
特によく知られた消光物質としてはニトロベンゼン、ジ
ニトロベンゼン、トリニトロベンゼン等があり、蛋白あ
るいはハプテンと結合するためにスルフォン酸基、ハロ
ゲン、アミノ基、カルボキシル基、水酸基、チオール基
などの官能基が導入されていても同様の効果がある。な
かでも2,4−ジニトロフルオロベンゼンはサンガー法に
よるアミノ酸配列決定の試薬として容易に入手でき、ハ
プテン、蛋白を問わずアミノ基に容易に結合するので望
ましいと思われる。Generally, an antibody emits fluorescence at around 340 nm when excited at 280 nm. This fluorescence is quenched by various quenchers,
Particularly well-known quenching substances include nitrobenzene, dinitrobenzene, trinitrobenzene, etc., and functional groups such as sulfonic acid groups, halogens, amino groups, carboxyl groups, hydroxyl groups, and thiol groups are introduced to bind to proteins or haptens. The same effect is obtained even if it is performed. Above all, 2,4-dinitrofluorobenzene is easily available as a reagent for amino acid sequencing by the Sanger method, and is considered to be desirable because it easily bonds to an amino group regardless of hapten or protein.
特に、メタンフェタミンおよびアンフェタミン、エフ
ェドリン等の覚醒剤検出については、下記の構造式の免
疫的検出試薬が良好である。In particular, for detection of stimulants such as methamphetamine, amphetamine, and ephedrine, the immunological detection reagents having the following structural formulas are preferable.
ただし、ｎは０もしくは１以上の整数、R1は水素原子
もしくは水酸基、R2は水素原子もしくはアルキル基、Ｘ
は水素原子もしくはメトキシ基を表わすものとする。 Wherein n is 0 or an integer of 1 or more; R1 is a hydrogen atom or a hydroxyl group; R2 is a hydrogen atom or an alkyl group;
Represents a hydrogen atom or a methoxy group.
以下、本発明の原理確認のため実験液に行った実施例
について説明する。Hereinafter, examples performed on experimental liquids for confirming the principle of the present invention will be described.
実施例１ まず、プローブ物質の合成方法を簡単に説明する。玉
置、福田、岸田、高橋、日本法医学雑誌（Jpn,J.,Legal
Med.,37（４）,417,1983）に記載された方法で、以下
の構造式を有するＮ−（４−アミノブチル）メタンフェ
タミン、（MANH2）を合成した。Example 1 First, a method for synthesizing a probe substance will be briefly described. Tamaki, Fukuda, Kishida, Takahashi, Japanese Forensic Magazine (Jpn, J., Legal
Med., 37 (4), 417, 1983), N- (4-aminobutyl) methamphetamine (MANH2) having the following structural formula was synthesized.
アセトン中でMANH2と2,4−ジニトロフルオロベンゼン
の等モル混合し１時間撹はんをおこなったのち分取用薄
層クロマトグラフィーで精製した。この結果、以下の構
造式を有するプローブ物質、MANH2DNPを得た。 MANH2 and 2,4-dinitrofluorobenzene were equimolarly mixed in acetone, stirred for 1 hour, and purified by preparative thin-layer chromatography. As a result, a probe substance MANH2DNP having the following structural formula was obtained.
本実施例で用いた抗体は、発明者らによって作製され
た抗メタンフェタミンモノクローナル抗体（αMAMAB1）
である。この抗体はメタンフェタミン（MA）に対し、約
107のアフィニティーを有していた。 The antibody used in this example was an anti-methamphetamine monoclonal antibody (αMAMAB1) prepared by the inventors.
It is. This antibody reacts against methamphetamine (MA)
Had an affinity of 10 7 .
以下、実験的な検出の方法について手順を述べる。 Hereinafter, a procedure of an experimental detection method will be described.
バッファー（pH7のリン酸バッファーを0.45μのフィ
ルターに通したもの）でαMAMAB1を溶解して１×10-7M
の濃度とした。この溶液360μＬを蛍光測定用ミクロセ
ル波長280nm（バンドパス5nm）の励起光、蛍光波長340n
m（バンドパス10nm）で強度約45（FL0）の蛍光を発した
（第１図、ａ部分）。Dissolve αMAMAB1 in a buffer (pH7 phosphate buffer passed through a 0.45μ filter) and add 1 × 10 −7 M
Concentration. 360 μL of this solution was used for excitation light having a microcell wavelength of 280 nm (band pass 5 nm) for fluorescence measurement and a fluorescence wavelength of 340 nm.
Fluorescence of about 45 (FL0) was emitted at m (bandpass 10 nm) (FIG. 1, part a).
MANH2DNP3×1017Mのバッファー溶液20μＬを加える
と、消光が起こり蛍光強度が28（FL1）に減少した。こ
の消光反応は約15秒で平衡状態に達した。（第１図、ｂ
部分） 上記の溶液にMAの３×10-2Mバッファー溶液20μＬ
（最終濃度１×10-4M）を加えると、抗体とMAが結合
し、MANH2DNPが脱離したため消光が阻害され、その結果
蛍光強度が約40（FLx）に増大した。この反応は約30秒
で平衡に達した。（第１図、ｃ部分） 以上記載のように、の段階の溶液にMAを導入し、蛍
光強度の増大からMAを検出することができた。この実験
条件での検出感度を確かめるため、で各濃度のMAを用
いたときの蛍光強度の変化を第２図に示す。なお、第２
図でMA濃度は最終濃度で示した。縦軸は で示した。When 20 μL of MANH2DNP3 × 10 17 M buffer solution was added, quenching occurred and the fluorescence intensity was reduced to 28 (FL1). The quenching reaction reached an equilibrium state in about 15 seconds. (Fig. 1, b
Part) 20 μL of 3 × 10 -2 M buffer solution of MA in the above solution
When a final concentration of 1 × 10 −4 M was added, the antibody and MA bound, and quenching was inhibited due to the elimination of MANH2DNP, resulting in an increase in the fluorescence intensity to about 40 (FLx). The reaction reached equilibrium in about 30 seconds. (FIG. 1, part c) As described above, MA was introduced into the solution in step (2), and MA was detected from the increase in fluorescence intensity. FIG. 2 shows the change in fluorescence intensity when using MA at each concentration in order to confirm the detection sensitivity under these experimental conditions. The second
In the figure, the MA concentration is shown as the final concentration. The vertical axis is Indicated by
第２図の結果から約10-6Mの濃度のMAが本発明の方法
により検出できたことが証明された。From the results shown in FIG. 2, it was proved that MA at a concentration of about 10 −6 M could be detected by the method of the present invention.
実施例２ 実施例１のメタンフェタミンの代わりにアンフェタミ
ンを用いて同様の合成を行った結果、以下に示す構造式
の化合物（APNH2DNP）が得られた。APNH2DNPをプローブ
物質として用いて実施例１と同様の操作を行った結果、
検出感度は10−6.5に落ちたものの、同様の効果が得ら
れた。Example 2 A similar synthesis was performed using amphetamine instead of methamphetamine of Example 1, and as a result, a compound having the following structural formula (APNH2DNP) was obtained. As a result of performing the same operation as in Example 1 using APNH2DNP as a probe substance,
Although the detection sensitivity dropped to 10-6.5 , the same effect was obtained.
実施例３ 実施例１におけるMANH2の代わりにアンフェタミンを
用いて同様の合成方法をとった結果、以下に示す構造式
の化合物（APDNP）を得た。APDNPをプローブ物質として
用いて実施例１と同様の操作を行った結果、検出感度は
実施例２と同じく10−6.5に落ちたものの、同様の効果
が得られた。 Example 3 A similar synthesis method was performed using amphetamine instead of MANH2 in Example 1, and as a result, a compound (APDNP) having the following structural formula was obtained. APDNP the results of performing the same operations as in Example 1 by using as the probe substance, the detection sensitivity despite fell well 10 -6.5 Example 2, similar effects were obtained.
以上、主としてMAの検出を例にとって本発明の説明を
行ったが、もちろんその他の化学物質すべてに応用可能
な一般的方法である。また、実施の手軽さから蛍光消光
物質として、ジニトロベンゼンの誘導体を用いたが、ト
リニトロベンゼン、ニトロベンゼンであっても同様の効
果が得られることは容易に考えられる。 Although the present invention has been described above mainly with reference to the detection of MA, it is a general method applicable to all other chemical substances. Although a derivative of dinitrobenzene was used as the fluorescence quenching substance for ease of implementation, it is easily conceivable that the same effect can be obtained with trinitrobenzene or nitrobenzene.
発明の効果 本発明によれば、従来５時間以上要していた免疫的検
出を１分以下に短縮することが可能となる。Effects of the Invention According to the present invention, it is possible to reduce the time required for immunological detection, which has conventionally required 5 hours or more, to 1 minute or less.
第１図は、本発明の一実施例における免疫的検出用試薬
を用いた測定の際の蛍光強度の時間変化を示すグラフ、
第２図は各MA濃度における蛍光消光阻害の変化を示した
グラフである。FIG. 1 is a graph showing a time change of a fluorescence intensity at the time of measurement using an immunological detection reagent in one example of the present invention,
FIG. 2 is a graph showing the change in fluorescence quenching inhibition at each MA concentration.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭60−233555（ＪＰ，Ａ) 特開 昭59−141066（ＪＰ，Ａ) 米国特許4199559（ＵＳ，Ａ) 米国特許4277437（ＵＳ，Ａ) 米国特許4261968（ＵＳ，Ａ) ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-60-233555 (JP, A) JP-A-59-141066 (JP, A) US Pat. US Patent 4261968 (US, A)
Claims (4)
学標識を施していない抗体が、元来有する蛍光を消光す
る機能を有した蛍光消光物質が化学的に結合したことを
特徴とする免疫的検出用試薬。The present invention is characterized in that a substance to be detected at the time of immunological detection is chemically bound to a fluorescent quenching substance having a function of quenching the fluorescence inherent in an antibody without chemical labeling. For immunological detection.
が、ニトロベンゼン、ジニトロベンゼン、トリニトロベ
ンゼンまたはそれらの誘導体である特許請求の範囲第１
項記載の免疫的検出用試薬。2. The fluorescent quencher having a function of quenching fluorescence is nitrobenzene, dinitrobenzene, trinitrobenzene or a derivative thereof.
Item 7. The reagent for immunological detection according to Item 1.
ェタミン、あるいはエフェドリンである特許請求の範囲
第１項記載の免疫的検出用試薬。3. The reagent for immunological detection according to claim 1, wherein the substance to be detected is methamphetamine, amphetamine or ephedrine.
有している特許請求の範囲第１項記載の免疫的検出用試
薬。 ただし、ｎは０もしくは１以上の正数、R1は水素原子も
しくは水酸基、R2は水素原子もしくはアルキル基、Ｘは
水素原子もしくはメトキシ基を表わすものとする。4. The reagent for immunological detection according to claim 1, wherein the analogous substance which binds to the antibody has the following structural formula. Here, n is 0 or a positive number of 1 or more, R1 is a hydrogen atom or a hydroxyl group, R2 is a hydrogen atom or an alkyl group, and X is a hydrogen atom or a methoxy group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63184951A JP2591089B2 (en) | 1988-07-25 | 1988-07-25 | Immunological detection reagents |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63184951A JP2591089B2 (en) | 1988-07-25 | 1988-07-25 | Immunological detection reagents |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0235359A JPH0235359A (en) | 1990-02-05 |
| JP2591089B2 true JP2591089B2 (en) | 1997-03-19 |
Family
ID=16162208
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63184951A Expired - Fee Related JP2591089B2 (en) | 1988-07-25 | 1988-07-25 | Immunological detection reagents |
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| Country | Link |
|---|---|
| JP (1) | JP2591089B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3628332B2 (en) | 1995-02-02 | 2005-03-09 | 中外製薬株式会社 | Method of measuring the test substance by adjusting the amount of chemiluminescence |
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| US4261968A (en) | 1979-05-10 | 1981-04-14 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
| US4277437A (en) | 1978-04-05 | 1981-07-07 | Syva Company | Kit for carrying out chemically induced fluorescence immunoassay |
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|---|---|---|---|---|
| EP0104926A3 (en) * | 1982-09-27 | 1986-11-12 | Unilever Plc | Assay processes and materials therefor |
| JPS60233555A (en) * | 1984-01-05 | 1985-11-20 | オ−ソ・ダイアグノステイツク・システムズ・インコ−ポレ−テツド | Anti-gene type check using movement of fluorescent energy |
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1988
- 1988-07-25 JP JP63184951A patent/JP2591089B2/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4199559A (en) | 1974-08-12 | 1980-04-22 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
| US4277437A (en) | 1978-04-05 | 1981-07-07 | Syva Company | Kit for carrying out chemically induced fluorescence immunoassay |
| US4261968A (en) | 1979-05-10 | 1981-04-14 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0235359A (en) | 1990-02-05 |
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| LAPS | Cancellation because of no payment of annual fees |