JPH0348765A - Material for immunological detection and immunological detection method - Google Patents
Material for immunological detection and immunological detection methodInfo
- Publication number
- JPH0348765A JPH0348765A JP18551989A JP18551989A JPH0348765A JP H0348765 A JPH0348765 A JP H0348765A JP 18551989 A JP18551989 A JP 18551989A JP 18551989 A JP18551989 A JP 18551989A JP H0348765 A JPH0348765 A JP H0348765A
- Authority
- JP
- Japan
- Prior art keywords
- substance
- antibody
- detected
- fluorescence
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 22
- 230000001900 immune effect Effects 0.000 title claims description 8
- 239000000463 material Substances 0.000 title abstract 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 230000002860 competitive effect Effects 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims description 45
- 229960001252 methamphetamine Drugs 0.000 claims description 18
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 14
- 238000003018 immunoassay Methods 0.000 claims description 8
- 230000005284 excitation Effects 0.000 claims description 6
- 239000013076 target substance Substances 0.000 claims description 5
- KWGRBVOPPLSCSI-WPRPVWTQSA-N (-)-ephedrine Chemical compound CN[C@@H](C)[C@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 claims description 4
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 claims description 4
- 239000007850 fluorescent dye Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 3
- KWTSXDURSIMDCE-QMMMGPOBSA-N (S)-amphetamine Chemical compound C[C@H](N)CC1=CC=CC=C1 KWTSXDURSIMDCE-QMMMGPOBSA-N 0.000 claims description 2
- 229940025084 amphetamine Drugs 0.000 claims description 2
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 claims description 2
- 229960002179 ephedrine Drugs 0.000 claims description 2
- 239000012456 homogeneous solution Substances 0.000 claims 1
- 238000006911 enzymatic reaction Methods 0.000 abstract description 3
- 239000007791 liquid phase Substances 0.000 abstract description 3
- 230000001268 conjugating effect Effects 0.000 abstract 2
- 239000000203 mixture Substances 0.000 abstract 2
- 238000000034 method Methods 0.000 description 6
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- -1 dinitrophenyl Chemical group 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- UCAHJECAHMOSHI-UHFFFAOYSA-N n-[(5-bromo-2-methoxyphenyl)methyl]adamantan-1-amine Chemical compound COC1=CC=C(Br)C=C1CNC1(C2)CC(C3)CC2CC3C1 UCAHJECAHMOSHI-UHFFFAOYSA-N 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明Ｃ−ｔ 主として臨床検査における病原体ある
いは疾病マーカーなどの検と さらには広〈産業上の極
微量検出に利用される免疫的検出用物質及び免疫的測定
法に関すも
従来の技術
天然に存在すゑ あるいは人工的に作製した抗体を用い
た測定法（よ 高特異性及び高感度の点に特徴を有し
極微量の存在割合の被検出物質を検出する目的で現在用
いられていも 例えば 血液中から病原体 あるいは腫
戚 心筋梗寡 脳血栓などの疾病時に特異的に分泌され
ているいわゆる疾患マーカーなどの臨床検査業務や、大
気中から極微蛍の物質を検出する場合などに用いられも
近年この様な目的で、例えば石川栄治河合忠 宮井潔著
［“酵素免疫測定法第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（飄 検出物質が
高分子（タンパク質）か低分子（ハブテン）かによって
２分されも 本発明では主としてハプテン系を対象とし
ており、このための代表例であゑ ＥＬＩＳＡ法の実験
手順を段階を追って説明すも
（Ａ、）抗原のコーティング
キャリアー蛋巨 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に溶解して抗原溶液
とす４
マイクロプレート（塩化ビニル或はポリスチレン製９６
ウエルプレート）に抗原を１００μＬ／ウェル注入り、
２０℃で１夜保存すａ
（Ｂ）ブロッキング
ＢＳＡのバッファー溶液を２５０μｌ／ウエル注入し０
、５−２時間室温で放置すム その後、バッファーまた
は純水で３−５回洗浄する。DETAILED DESCRIPTION OF THE INVENTION Industrial Application Fields of the Invention C-t Mainly used for the detection of pathogens or disease markers in clinical tests, and also for the wide range of immunological detection substances and immunological detection used for industrial trace amount detection. Regarding conventional measurement methods, there are also methods using naturally occurring or artificially produced antibodies (which are characterized by high specificity and high sensitivity).
Although it is currently used to detect extremely small amounts of substances to be detected, for example, it is used for clinical testing of so-called disease markers that are secreted specifically during diseases such as pathogens, tumors, myocardial infarction, and cerebral thrombosis in the blood. In recent years, it has also been used for such purposes, such as when detecting minute fluorescent substances from the atmosphere.
Many types of immunoassays have been developed, as described on pages 31-54). Although this method is divided into two depending on whether the substance to be detected is a high molecule (protein) or a low molecule (habten), the present invention mainly targets hapten-based substances, and the experimental procedure of the ELISA method is described in steps as a representative example for this purpose. (A) Coating of antigen with carrier protein macromolecules such as bovine serum albumin (BSA)
Dissolve the conjugate containing the detection substance or a derivative with a functional group introduced into it in a buffer to obtain an antigen solution.4 Microplate (made of vinyl chloride or polystyrene 96
Inject 100 μL/well of antigen into a well plate).
Store at 20°C overnight. (B) Inject 250 μl/well of blocking BSA buffer solution.
, leave at room temperature for 5-2 hours, then wash 3-5 times with 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
に加えも 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Ｕ バッファーまたは純水で３−５回
洗浄すａ
（Ｄ）第２抗体の反応
（Ｃ）項の抗体に対する抗体を酵泰 例えばペルオキシ
ダーゼで標識したちのく第２抗体）の溶液を注入し 常
温で０．５−２時間放置すａ その後、バッファーまた
は純水で３−５回洗浄すム（Ｅ）基質の反応と停止
発色剋 例えば０−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 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＆４Ｎ硫酸で反応を停止すム
（Ｆ）測定
マイクロプレート用吸光光度系を用いて４９２ｎｍの吸
光度を測定すも 最終的に被検出物質が多いほど吸光度
が小さいことを利用して、被検出物質の検出を行う。(C) Inject the antibody reaction detection substance solution and add more antibody solution while shaking. After storing at room temperature for 3-5 hours, remove the antibody solution with an aspirator. Wash 3-5 times with buffer or pure water. a (D) Reaction of the second antibody. Inject a solution of the antibody against the antibody in (C) (for example, the second antibody labeled with peroxidase) and leave it at room temperature for 0.5-2 hours. After that, Wash with buffer or pure water 3-5 times. Substrate solution) is injected and the color reaction is carried out at room temperature. Stop the reaction with 4N sulfuric acid for 5-20 minutes. Measure the absorbance at 492 nm using a microplate absorbance system. Finally, using the fact that the more the substance to be detected, the lower the absorbance, Detects the substance to be detected.
発明が解決しようとする課題
従来の免疫的検出方法は 抗原抗体結合物と未結合物と
を分離するために多くの手順を必要としてい九 また反
応系が固相と液相め共存する不均一系であり、最終的な
検出に酵素反応を用いているたべ 通常５時間程度を要
してい九
本発明は このような従来技術の課題を解決することを
目的とすム
課題を解決するための手段
本発明（よ 被検出物質あるいはその類似物質に蛍光物
質を結合させたものであって、これが被検出物質に対す
る抗体を結合したとき抗体の発する蛍光を励起光として
蛍光が増強するものであることを特徴とする免疫的検出
用物質であもまた 本発明（よ 被検出物質に対する抗
体溶液にあらかじめその免疫的検出用物質を混合してお
き、その混合溶液に被検出物質を加えると免疫的検出用
物質と被検出物質との間の競合反応に伴う蛍光強度の変
化から被検出物質の存在量を測定することを特徴とする
免疫的測定法であも作用
本発明は 被検出物質に対する抗体溶液艮 あらかじめ
被検出物質あるいはその類似物質に蛍光物質を結合させ
たものを混合しておき、その混合溶液に被検出物質を加
えると、その混合物質と被検出物質との間の競合反応に
伴い蛍光強度が変化すも この変化を利用して、被検出
物質の存在量を測定すも
このようにして、反応がすべて均−系（液相）で進行し
さらに酵素反応を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ん実施例
以下へ 本発明の実施例について図面を参照しながら説
明すも
発明者ら１よ メタンフェタミン、アンフエタミンある
いはエフェドリン家 それ自身蛍光性に蛍光消光性も有
していない物質と蛍光プローブの結合物質が抗体水溶液
中で抗体と結合した胤 抗体自身が本質的に有している
蛍光が、 蛍光プローブ物質の励起波長となり蛍光が増
強する事実を発児し この蛍光プローブ物質の合成を
し、この原理を検出方法に適用したものであａ
一般に抗体は２８０Ｉｍの励起光で３４０Ｉｍ付近に蛍
光を発する。この蛍光（よ 構成アミノ酸の一つである
Ｉ、トリプトファン残基などに由来するものであるが、
この蛍光の性質（よ 抗原が結合することによって変
化するこ七があム 例えばジニトロフェニルやジアゾ色
素へ 各々の抗体が結合すると抗体分子の持つトリプト
ファン残基の蛍光は消光されてしまう。この蛍光消光（
よ 抗原分子へのエネルギー転移の結果起こるものであ
ａ 一方 ダンシル基に対する抗体とダンシル基との抗
原抗体反応物を２８０ｎｌｌＩで励起すると５３０Ｉｍ
付近に蛍光を発することは知られていも この現象（上
結合部位の環境などに情報を与えるものであっム こ
れ（上２８０ｎｍで励起された抗体分子の３４０Ｉｍの
蛍光がダンシル基の励起波長となった結果５３０ｎｍ付
近に蛍光を発するものであム
そこで我々はそれ自信蛍光性を持たないメタンフェタミ
ン（ＭＡ）とダンシル基の結合物（ＤＮＳ−ＡＢＭＡ）
を合成し九 このＤＮＳ−ＡＢＭＡｉｉ メタンフェ
タミンに対する抗体に特異的に結合しその結果２８０ｎ
ｍで励起すると５３０Ｉｍ付近の蛍光が増強し九 この
混合溶液に遊離のメタンフェタミンを加えるとメタンフ
ェタミンとＤＮＳ−ＡＢＭＡとの間に競合反応が起こり
５３０ｎａ＋付近の蛍光強度が変化し九
この蛍光強度の変化に基づいて遊離のメタンフェタミン
の存在量を測定することができ４ 以下、ＤＮＳ、−Ａ
ＢＭＡの合成及び実験的な検出の方法について手順を述
べも
（１）ダンシル化メタンフェタミン（ＤＮＳ−ＡＢＭＡ
）の合成
Ｎ・（４−アミノブチル）メタンフェタミン（ＡＢＭＡ
）は＋−・アオキ、ワイ・クロイワ（Ｋ、 Ａｎｋｉ
、 Ｙ、 Ｋｕｒｏｉｗａ）の方法により合成し九
次にダンジルクロライドを加え、ｐＨ！１１．８で反
応させａＮｓ−ＡｎＭ″Ａを合成した。ＤＮＳ−ＡＢＭ
Ａの構造を次に示す。Problems to be Solved by the Invention Conventional immunodetection methods require many steps to separate antigen-antibody bound substances from unbound substances.9 In addition, the reaction system is heterogeneous in that solid and liquid phases coexist. The present invention aims to solve the problems of the prior art, and uses an enzymatic reaction for final detection, which usually takes about 5 hours. Means The present invention is a substance in which a fluorescent substance is bound to a substance to be detected or a substance similar thereto, and when this substance binds to an antibody against the substance to be detected, the fluorescence is enhanced by using the fluorescence emitted by the antibody as excitation light. The present invention also provides a substance for immunological detection characterized by The present invention is also applicable to an immunoassay method characterized by measuring the amount of a target substance present from a change in fluorescence intensity due to a competitive reaction between a target substance and a target substance.艮 When a substance to be detected or a similar substance bound to a fluorescent substance is mixed in advance, and the substance to be detected is added to the mixed solution, fluorescence is generated due to a competitive reaction between the mixed substance and the substance to be detected. Although the intensity changes, this change is used to measure the amount of the target substance present.In this way, the reaction proceeds in a homogeneous system (liquid phase), and since no enzymatic reaction is used, the detection time can be reduced. Embodiments of the present invention will be described below with reference to the drawings.Methamphetamine, amphetamine, or ephedrine itself has fluorescence quenching properties. When a substance binds to an antibody in an aqueous antibody solution, the fluorescence that the antibody itself inherently has becomes the excitation wavelength of the fluorescent probe substance, and the fluorescence is enhanced.This fluorescent probe This method synthesizes a substance and applies this principle to a detection method.A Generally, antibodies emit fluorescence at around 340 Im when excited by 280 Im. It is derived from
The nature of this fluorescence changes when antigen binds. For example, when each antibody binds to dinitrophenyl or diazo dye, the fluorescence of the tryptophan residue in the antibody molecule is quenched. This fluorescence quenching (
This occurs as a result of energy transfer to the antigen molecule.A On the other hand, when an antigen-antibody reaction product between an antibody against a dansyl group and a dansyl group is excited with 280nllI, 530Im
Although it is known that fluorescence is emitted in the vicinity, this phenomenon (above) gives information about the environment of the binding site. As a result, we found that it emits fluorescence at around 530 nm. Therefore, we developed a compound of methamphetamine (MA) and a dansyl group (DNS-ABMA), which does not have fluorescence.
This DNS-ABMAii specifically binds to the antibody against methamphetamine, and as a result, 280n
When excited with m, the fluorescence around 530Im increases.9 When free methamphetamine is added to this mixed solution, a competitive reaction occurs between methamphetamine and DNS-ABMA, and the fluorescence intensity around 530na+ changes. The amount of free methamphetamine present can be determined based on 4, below, DNS, -A
We also describe the procedures for the synthesis and experimental detection of BMA (1) dansylated methamphetamine (DNS-ABMA).
) Synthesis of N.(4-aminobutyl)methamphetamine (ABMA
) is +- Aoki, Wai Kuroiwa (K, Anki
Next, danzyl chloride was added, and the pH! 11.8 to synthesize aNs-AnM″A. DNS-ABM
The structure of A is shown below.
して５３０ｎａ＋の蛍光強度は減少しなその減少率 Ｉ
（％）を、次式を用いて算出しｔもＣＨ，ＣＨ。The fluorescence intensity of 530na+ does not decrease, and the rate of decrease is I
(%) is calculated using the following formula, and t is also CH, CH.
（２）検出方法
（Ａ）バッファー（ｐＨ７のリン酸バッファーを０．４
５μｍのフィルターでろ過したもの）でメタンフェタミ
ンに対する抗体を溶解して２Ｘ１０−７Ｍの濃度とした
。この溶液３ＧＯμｌを蛍光測定用恒温ミクロセル（２
０℃に設定）に入れ、さらにｉｘｌＯ−ｅＭのＤＮＳ−
五Ｂ１１１Ａを２０μｌ加えた。この混合溶液を２８
Ｏｎ、ｔｓで励起し５３０Ｉの蛍光強度を測定すると蛍
光強度は約８０であった。(2) Detection method (A) Buffer (pH 7 phosphate buffer at 0.4
Antibodies against methamphetamine were dissolved in a 5 μm filter (filtered through a 5 μm filter) to a concentration of 2×10 −7 M. Add 3 GO μl of this solution to a thermostatic microcell for fluorescence measurement (2
ixlO-eM DNS-
20 μl of 5B111A was added. Add this mixed solution to 28
When the fluorescence intensity of 530I was measured with excitation on and ts, the fluorescence intensity was about 80.
（Ｂ）上記（Ａ）の溶液に種々の濃度のメタンフェタミ
ン溶液２０μｍを加え２８０Ｉｍ励起の５３０Ｉｍの蛍
光強度を測定するとメタンフェタミンの濃度に依存ここ
で、Ｆは抗体溶液にＤＮＳ−ＡＢＭＡを加えたときの蛍
光強塞 また）’ ｏｂｓは上記の溶液にメタンフェタ
ミン溶液を加えたときの蛍光強度を示す。(B) When adding 20 μm of methamphetamine solution of various concentrations to the solution of (A) above and measuring the fluorescence intensity of 530 Im after excitation at 280 Im, it depends on the concentration of methamphetamine. Here, F is the difference when DNS-ABMA is added to the antibody solution. Fluorescence intensification (also)' obs indicates the fluorescence intensity when a methamphetamine solution is added to the above solution.
メタンフェタミンの濃度に対する蛍光強度の減少率を図
に示す。図の結果から約１０−″・６Ｍの濃度のメタン
フェタミンが本発明により検出できたことが証明された
ま？＝ ＤＮＳ−ＡＢＭＡの代わりにＤＮＳ−ＡＢＡ
Ｐを用いても同様の結果を得ることができ島
さらに 蛍光測定温度を１０℃とすると、抗体の蛍光強
度は低温の方が大きいので、精度よく測定することがで
きた 以Ｌ 主としてメタンフェタミンの検出を例にと
って本発明の説明を行なったが、 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も
このようにして、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っ九
発明の詳細
な説明したようＪＱ 本発明は 全反応が均−系で行
われ 抗原抗体結合物と結合物の分離操作を含まず、ま
た抗原抗体反応による抗体の蛍光を直接利用し蛍光プロ
ーブの蛍光の変化に基づく免疫的測定法及びそれに利用
する免疫的検出用物質を提供できもThe figure shows the rate of decrease in fluorescence intensity with respect to the concentration of methamphetamine. The results shown in the figure prove that methamphetamine at a concentration of about 10-''6M can be detected by the present invention. = DNS-ABA instead of DNS-ABMA
Similar results were obtained using P. Furthermore, when the fluorescence measurement temperature was set to 10°C, the fluorescence intensity of the antibody was greater at low temperatures, so it was possible to measure with high accuracy.L Mainly methamphetamine detection The present invention has been explained by taking the example of a chemical substance, but of course it is also a general method that can be applied to all other chemical substances.In this way, immunological detection, which conventionally required more than 5 hours, can be shortened to less than 1 minute. As explained in detail, the present invention enables the entire reaction to be carried out in a homogeneous system, does not involve separation of antigen-antibody conjugates, and eliminates the fluorescence of antibodies caused by antigen-antibody reactions. It is possible to provide an immunoassay method based on changes in the fluorescence of a fluorescent probe that can be used directly, and an immunological detection substance used therein.
図は各メタンフェタミン濃度における蛍光減少率を示し
たグラフであムThe figure is a graph showing the fluorescence reduction rate at each methamphetamine concentration.
Claims (7)
合させたものであって、これが被検出物質に対する抗体
と結合したときの抗体の発する蛍光を励起光として蛍光
が増強するものであることを特徴とする免疫的検出用物
質。(1) A fluorescent substance is bound to a substance to be detected or a substance similar to it, and when it binds to an antibody against the substance to be detected, the fluorescence emitted by the antibody is used as excitation light to enhance the fluorescence. Characteristic immunodetection substances.
光物質が、ダンシル基であることを特徴とする請求項１
記載の免疫的検出用物質。(2) Claim 1, characterized in that the fluorescent substance bound to the substance to be detected or its analogous substance is a dansyl group.
Substances for immunological detection as described.
１記載の免疫的検出用物質を混合しておき、その混合溶
液に被検出物質を加えると免疫的検出用物質と被検出物
質との間の競合反応に伴う蛍光強度の変化から被検出物
質の存在量を測定することを特徴とする免疫的測定法。(3) If the immunodetection substance according to claim 1 is mixed in advance with an antibody solution against the detection substance and the detection substance is added to the mixed solution, the difference between the immunodetection substance and the detection substance is An immunoassay method characterized by measuring the amount of a target substance present from changes in fluorescence intensity associated with competitive reactions.
ンあるいはエフェドリンである請求項３記載の免疫的測
定法。(4) The immunoassay method according to claim 3, wherein the substance to be detected is methamphetamine, amphetamine, or ephedrine.
蛍光が蛍光プローブの励起波長となることを特徴とする
請求項３記載の免疫的測定法。(5) The immunoassay method according to claim 3, wherein the measurement is a reaction in a homogeneous solution, and the fluorescence of the antibody itself serves as the excitation wavelength of the fluorescent probe.
徴とする請求項３記載の免疫的測定法。(6) The immunoassay method according to claim 3, characterized in that a monoclonal antibody is used as the antibody.
徴とする請求項３記載の免疫的測定法。(7) The immunoassay method according to claim 3, characterized in that a polyclonal antibody is used as the antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18551989A JPH0348765A (en) | 1989-07-18 | 1989-07-18 | Material for immunological detection and immunological detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18551989A JPH0348765A (en) | 1989-07-18 | 1989-07-18 | Material for immunological detection and immunological detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0348765A true JPH0348765A (en) | 1991-03-01 |
Family
ID=16172212
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18551989A Pending JPH0348765A (en) | 1989-07-18 | 1989-07-18 | Material for immunological detection and immunological detection method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0348765A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004027424A1 (en) * | 2002-09-19 | 2004-04-01 | Hamamatsu Photonics K.K. | Fluorescence analysis method with the use of fluorescent antibody |
| US8574925B2 (en) | 2002-09-19 | 2013-11-05 | Hamamatsu Photonics K.K. | Fluorescence analysis method using fluorescent-activating antibodies |
-
1989
- 1989-07-18 JP JP18551989A patent/JPH0348765A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004027424A1 (en) * | 2002-09-19 | 2004-04-01 | Hamamatsu Photonics K.K. | Fluorescence analysis method with the use of fluorescent antibody |
| US8574925B2 (en) | 2002-09-19 | 2013-11-05 | Hamamatsu Photonics K.K. | Fluorescence analysis method using fluorescent-activating antibodies |
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