JPH03195953A - Optical reaction measurement - Google Patents
Optical reaction measurementInfo
- Publication number
- JPH03195953A JPH03195953A JP33607089A JP33607089A JPH03195953A JP H03195953 A JPH03195953 A JP H03195953A JP 33607089 A JP33607089 A JP 33607089A JP 33607089 A JP33607089 A JP 33607089A JP H03195953 A JPH03195953 A JP H03195953A
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- optical component
- optical
- measurement
- fine particles
- 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.)
- Granted
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 44
- 230000003287 optical effect Effects 0.000 title claims abstract description 40
- 238000005259 measurement Methods 0.000 title claims abstract description 16
- 239000010419 fine particle Substances 0.000 claims abstract description 19
- 238000000691 measurement method Methods 0.000 claims description 12
- 239000011859 microparticle Substances 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 6
- 239000011324 bead Substances 0.000 claims description 4
- 238000011481 absorbance measurement Methods 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 239000004816 latex Substances 0.000 claims description 2
- 229920000126 latex Polymers 0.000 claims description 2
- 238000004020 luminiscence type Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000000758 substrate Substances 0.000 description 6
- 102000003992 Peroxidases Human genes 0.000 description 5
- 238000003018 immunoassay Methods 0.000 description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000000696 magnetic material Substances 0.000 description 2
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
Description
【発明の詳細な説明】
［発明の目的］
（産業上の利用分野）
本発明は、免疫測定や生化学分析等で反応によって最終
的に得られた物を光学的に測定する光学的反応測定法に
関する。[Detailed Description of the Invention] [Object of the Invention] (Industrial Application Field) The present invention is an optical reaction measurement method for optically measuring a product finally obtained by a reaction in immunoassay, biochemical analysis, etc. Regarding the law.
（従来の技術）
最近ＥＩＡ（エンザイムイムノアッセイ）法によって免
疫測定を行う場合や生化学反応を行う場合、反応を促進
するために分離を目的とした抗体又は酵素を担体である
微粒子上に固定化することが行われている。第４図（ａ
）、（ｂ）はこのような各反応を示すもので、第４図（
ａ）は固定化酵素反応、第４図（ｂ）は免疫測定反応を
示すものである。(Prior art) Recently, when performing immunoassays or biochemical reactions using the EIA (enzyme immunoassay) method, antibodies or enzymes for the purpose of separation are immobilized on microparticles that are carriers to promote the reaction. things are being done. Figure 4 (a
) and (b) show these reactions, and Figure 4 (
Figure 4(a) shows the immobilized enzyme reaction, and Figure 4(b) shows the immunoassay reaction.
第４図（ａ）では先ず磁性材料等の微粒子担体１上に酵
素２を固定した後、基質３を反応させることにより酵素
反応で生じた分解産物（プロダクト）４を光学的に測定
して抗原、酵素、基質等の定量を行うようにしたもので
ある。また第４図（ｂ）は微粒子担体１上に抗体５を固
定した後、抗原６を反応させさらに酵素標識抗体７を反
応させ、続いて基質３を反応させることにより生じたプ
ロダクト４を光学的に測定して同様な定量を行うように
したものである。この光学的測定は例えば吸光度測定、
蛍光度測定１発光度測定等によって行うことができる。In FIG. 4(a), first, an enzyme 2 is immobilized on a particulate carrier 1 such as a magnetic material, and then a substrate 3 is reacted to optically measure the decomposition products (products) 4 generated in the enzymatic reaction. , enzymes, substrates, etc. FIG. 4(b) shows a product 4 produced by immobilizing an antibody 5 on a microparticle carrier 1, reacting with an antigen 6, further reacting with an enzyme-labeled antibody 7, and then reacting with a substrate 3. It was designed to perform a similar quantitative determination. This optical measurement is, for example, absorbance measurement,
Fluorescence measurement 1 This can be carried out by measuring luminescence intensity or the like.
ここで各反応は微粒子存在下で進行するが、基質３又は
プロダクト４を光学的に測定する場合、微粒子担体１に
よって光の散乱が生じるという問題がある。従って従来
においてはこの散乱の影響を除（ために、遠心分離、濾
過、磁力吸着等の対策を施して微粒子を除いた状態で測
定が行われていた。これによれば微粒子担体が除かれる
ことにより、光学的に反応を測定する場合清澄な状態で
測定を行うことができる。Here, each reaction proceeds in the presence of fine particles, but when the substrate 3 or product 4 is optically measured, there is a problem in that the fine particle carrier 1 causes light scattering. Therefore, in the past, in order to eliminate the influence of this scattering, measurements were performed after removing fine particles by taking measures such as centrifugation, filtration, and magnetic adsorption. According to this method, the fine particle carriers were removed. Therefore, when measuring a reaction optically, the measurement can be carried out in a clear state.
（発明が解決しようとする課題）
ところで従来の光学的反応測定法では、微粒子を除くた
めの手段及び時間を余分に必要とすると共に、連続的な
反応経過を追求することができないので測定効率が低下
するという問題がある。(Problems to be Solved by the Invention) However, the conventional optical reaction measurement method requires extra means and time to remove fine particles, and it is not possible to pursue a continuous reaction progress, so the measurement efficiency is low. There is a problem with the decline.
例えばエンド法のように反応経過の一点を測定する場合
は可能であるが、レイト法のように連続的な反応経過を
測定する場合には微粒子が残ってないため適用できない
ので用途が制約されることになる。For example, it is possible to measure a single point in the reaction process as in the end method, but it cannot be applied to measure a continuous reaction process as in the late method because there are no particles left, which limits its application. It turns out.
本発明は以上のような問題に対処してなされたもので、
余分な手段及び時間を不要にすると共に測定用途を拡大
することができる光学的反応測定法を提供することを目
的とするものである。The present invention has been made in response to the above-mentioned problems.
It is an object of the present invention to provide an optical reaction measurement method that eliminates the need for extra means and time and can expand measurement applications.
［発明の構成コ
（課題を解決するための手段）
上記目的を達成するために本発明は、微粒子上又は微粒
子共存下での反応を光学的に追跡する光学的反応測定法
において、反応由来の光学的成分と微粒子散乱由来の光
学的成分を同時に測定し、前者の光学的成分から後者の
光学的成分を差引くことにより微粒子共存下で反応を光
学的に測定することを特徴とするものである。[Configuration of the Invention (Means for Solving the Problems)] In order to achieve the above object, the present invention provides an optical reaction measurement method for optically tracking reactions on microparticles or in the coexistence of microparticles. This method is characterized by simultaneously measuring the optical component and the optical component derived from particle scattering, and subtracting the latter optical component from the former optical component to optically measure the reaction in the presence of particles. be.
（作 用）
反応由来の光学的成分と微粒子散乱由来の光学的成分を
例えば異なる波長によって同時に測定し、前者の光学的
成分から後者の光学的成分を差引くことにより反応を測
定する。これによって微粒子の影響を除去して反応に関
係する物質由来の光学的変化を追跡することができる。(Function) The reaction is measured by simultaneously measuring the optical component derived from the reaction and the optical component derived from particle scattering, for example using different wavelengths, and subtracting the latter optical component from the former optical component. This makes it possible to remove the influence of fine particles and track optical changes originating from substances involved in the reaction.
（実施例）
以下本発明の光学的反応測定法の実施例について説明す
る。(Example) Examples of the optical reaction measurement method of the present invention will be described below.
１、材料
先ず酵素としてＰＯＤ （パーオキシダーゼ）を０．１
■／ｍｌ用い、これを０．ＩＭＰＢ （リン酸バッファ
、ＰＨ７，０）で４．５Ｘ１０−５倍に希釈した基準液
Ａを用意する。またこのＡ液を各々０、ＩＭＰＢ （Ｐ
Ｈ７，０）で３倍に希釈したＢ液。1. Materials First, use POD (peroxidase) as an enzyme at 0.1
■/ml, and add this to 0. Prepare standard solution A diluted 4.5×10 −5 times with IMPB (phosphate buffer, pH 7,0). In addition, this A liquid was added to 0, IMPB (P
Solution B diluted 3 times with H7,0).
１０倍に希釈したＣ液、３０倍に希釈したＤ液。Solution C diluted 10 times, Solution D diluted 30 times.
５５倍に希釈したＥ液、１００倍に希釈したＦ液を用意
する。Prepare solution E diluted 55 times and solution F diluted 100 times.
次に基質としてＴＮＢＺ（トリニトロペンゼニン）をＨ
２Ｏ２（過酸化水素）で２：１で混合したものを用意し
、また０、ＩＭＰＢ（ＰＨ７，０）で置換した磁性微粒
子（マグネチックパーティクル）を２．２■／ｍｌ用意
する。Next, TNBZ (trinitropenzenin) was used as a substrate.
A 2:1 mixture of 2O2 (hydrogen peroxide) was prepared, and 2.2 μ/ml of magnetic particles substituted with 0 and IMPB (PH7,0) were prepared.
さらに分光光度計を用意し前記混合液を３７℃に恒温し
た状態で、６５０ｎｍ及び７５ ［１ｎｍの波長で追跡
するようにする。Further, a spectrophotometer is prepared, and the mixture is kept at a constant temperature of 37° C., and the mixture is monitored at wavelengths of 650 nm and 75 [1 nm].
２、実験例
前記ＰＯＤ液を４５０μｌ用い、５分間インキュベート
した後、基質１３５０μ！＋磁性微粒子４５０ｇの計１
８００μｌを前記ＰＯＤ液に加える。2. Experimental Example Using 450μl of the above POD solution and incubating for 5 minutes, 1350μl of substrate was added! +Magnetic fine particles 450g total 1
Add 800 μl to the POD solution.
続いてこの混合液を３０℃に保存し攪拌した状態で、５
分間タイムコースに移して異なる２波長例えば６５０ｎ
ｍ及び７５０ｎｍで吸光度を測定する。Next, this mixed solution was stored at 30°C and stirred for 5 minutes.
Two different wavelengths, for example 650n, are transferred to a minute time course.
Measure the absorbance at m and 750 nm.
第１図はこのような測定における吸光度の波長依存性を
示しており、波形の異なる波長におけるピーク値とベー
ス値との各吸光度を測定し両者の差を求めることにより
、第２図のように吸光度の時間依存性を求めることがで
きる。例えばピーク値を６５０ｎｍの波長で測定し、ベ
ース値を７５０ｎｍの波長で測定する。第３図は各波長
の差に対応した吸光度とＰＯＤ （酵素）の濃度との関
係を示しており、ＰＯＤの濃度を１／１００程度（前記
Ｆ液に相当し実際にはさらにこれを４．５Ｘ１０−５倍
に希釈した濃度）までとっても測定が可能であることを
示している。Figure 1 shows the wavelength dependence of absorbance in such measurements.By measuring the absorbance of the peak value and the base value at different wavelengths of the waveform and finding the difference between the two, as shown in Figure 2, The time dependence of absorbance can be determined. For example, the peak value is measured at a wavelength of 650 nm, and the base value is measured at a wavelength of 750 nm. Figure 3 shows the relationship between the absorbance and the concentration of POD (enzyme) corresponding to the difference in each wavelength. This shows that it is possible to measure up to a concentration of 5x10-5 times diluted.
このような反応測定法によれば、従来のように微粒子を
除去しなくとも測定が可能なので、微粒子を除くための
手段及び時間が不要となる。またこれと共に微粒子の存
在下で測定ができるので、エンド法に限らずレイト法の
ように連続的な反応経過を測定する場合にも適用するこ
とができるようになる。According to such a reaction measurement method, measurement can be performed without removing particulates as in the conventional method, so there is no need for means and time for removing particulates. In addition, since measurement can be performed in the presence of fine particles, it can be applied not only to the end method but also to the case of measuring a continuous reaction progress, such as the late method.
このような本発明方法によって、反応を促進できるとい
う微粒子担体を用いた場合の利点を損ねることなく反応
の測定を行うことができるようになり、微粒子共存反応
液の比色測定の効率を向上することができる。According to the method of the present invention, the reaction can be measured without sacrificing the advantage of using a fine particle carrier that can promote the reaction, and the efficiency of colorimetric measurement of a reaction solution in which fine particles coexist can be improved. be able to.
微粒子は磁性材料に限らずラテックス、各種ビーズ、ガ
ラスビーズ等の他の材料を用いることができる。The fine particles are not limited to magnetic materials, but other materials such as latex, various beads, and glass beads can be used.
［発明の効果コ
以上述べたように本発明によれば、反応由来の光学的成
分から微粒子散乱由来の光学的成分を差引いて微粒子共
存下で反応を光学的に測定するようにしたので、余分な
手段及び時間を不要にすると共に、連続的な反応経過も
測定できるようになる。[Effects of the Invention] As described above, according to the present invention, the optical component derived from particle scattering is subtracted from the optical component derived from the reaction to optically measure the reaction in the coexistence of fine particles. In addition to eliminating the need for additional means and time, it also becomes possible to measure the continuous course of the reaction.
第１図は本発明の光学的反応測定法の実施例によって得
られた波長と吸光度の関係を示す特性図、第２図は本実
施例によって得られた時間と吸光度の関係を示す特性図
、第３図は本実施例によって得られた酵素濃度と吸光度
の関係を示す特性図、第４図（ａ）、 （ｂ）は固定
化酵素反応及び免疫測定反応における反応経過を示す説
明図である。
１・・・微粒子担体、２・・・酵素、４・・・プロダク
ト、５・・・抗体、 ６・・・抗原、７・・・酵素
標識抗体。
ＬＯＧ（Ａ６５０−Ａ７５０１／ＭＩＮ（０）百口１こ
イヒニ畔濁〔ｇ乙沁３
埼繕（分］−FIG. 1 is a characteristic diagram showing the relationship between wavelength and absorbance obtained by an example of the optical reaction measurement method of the present invention, FIG. 2 is a characteristic diagram showing the relationship between time and absorbance obtained by this example, Figure 3 is a characteristic diagram showing the relationship between enzyme concentration and absorbance obtained in this example, and Figures 4 (a) and (b) are explanatory diagrams showing the reaction progress in the immobilized enzyme reaction and immunoassay reaction. . DESCRIPTION OF SYMBOLS 1... Fine particle carrier, 2... Enzyme, 4... Product, 5... Antibody, 6... Antigen, 7... Enzyme-labeled antibody. LOG (A650-A7501/MIN(0) 1 hundred mouths 1 koihini 畔收〔g 沁沁3 缼繕(minutes) -
Claims (3)
跡する光学的反応測定法において、反応由来の光学的成
分と微粒子散乱由来の光学的成分を同時に測定し、前者
の光学的成分から後者の光学的成分を差引くことにより
微粒子共存下で反応を光学的に測定することを特徴とす
る光学的反応測定法。(1) In an optical reaction measurement method that optically tracks reactions on microparticles or in the coexistence of microparticles, the optical component derived from the reaction and the optical component derived from microparticle scattering are simultaneously measured, and the optical component derived from the former is An optical reaction measurement method characterized by optically measuring a reaction in the presence of fine particles by subtracting the latter optical component.
定のいずれかから成る請求項１記載の光学的反応測定法
。(2) The optical reaction measurement method according to claim 1, wherein the optical measurement comprises any one of absorbance measurement, fluorescence measurement, and luminescence measurement.
ク値を検出し、微粒子散乱由来の光学的成分を第２の波
長によってベース値を検出する請求項１記載の光学的反
応測定法。（４）微粒子がラテックス、磁性微粒子、多
種ビーズ、ガラスビーズのいずれかから成る請求項１記
載の光学的反応測定法。(3) The optical reaction measurement method according to claim 1, wherein the peak value of the optical component derived from the reaction is detected using the first wavelength, and the base value of the optical component derived from particle scattering is detected using the second wavelength. (4) The optical reaction measurement method according to claim 1, wherein the fine particles are made of latex, magnetic fine particles, various types of beads, or glass beads.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33607089A JP2938485B2 (en) | 1989-12-25 | 1989-12-25 | Optical response measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33607089A JP2938485B2 (en) | 1989-12-25 | 1989-12-25 | Optical response measurement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03195953A true JPH03195953A (en) | 1991-08-27 |
| JP2938485B2 JP2938485B2 (en) | 1999-08-23 |
Family
ID=18295384
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33607089A Expired - Fee Related JP2938485B2 (en) | 1989-12-25 | 1989-12-25 | Optical response measurement method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2938485B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994029722A1 (en) * | 1993-06-08 | 1994-12-22 | Chronomed, Inc. | Two-phase optical assay method and apparatus |
| WO2008090645A1 (en) * | 2007-01-23 | 2008-07-31 | Olympus Corporation | Analytical method and analytical apparatus |
| WO2010041736A1 (en) * | 2008-10-10 | 2010-04-15 | コニカミノルタホールディングス株式会社 | Assay method using surface plasmon |
| JP2010181323A (en) * | 2009-02-06 | 2010-08-19 | Konica Minolta Holdings Inc | Assay method using surface plasmon |
-
1989
- 1989-12-25 JP JP33607089A patent/JP2938485B2/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994029722A1 (en) * | 1993-06-08 | 1994-12-22 | Chronomed, Inc. | Two-phase optical assay method and apparatus |
| WO2008090645A1 (en) * | 2007-01-23 | 2008-07-31 | Olympus Corporation | Analytical method and analytical apparatus |
| JP2008180537A (en) * | 2007-01-23 | 2008-08-07 | Olympus Corp | Analysis method and analyzer |
| WO2010041736A1 (en) * | 2008-10-10 | 2010-04-15 | コニカミノルタホールディングス株式会社 | Assay method using surface plasmon |
| JP2010181323A (en) * | 2009-02-06 | 2010-08-19 | Konica Minolta Holdings Inc | Assay method using surface plasmon |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2938485B2 (en) | 1999-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0464120B1 (en) | Assay method using surface plasmon resonance spectrometry | |
| EP0137678B1 (en) | Ultrasonic enhanced immuno-reactions | |
| JPH0145027B2 (en) | ||
| JPS6112224B2 (en) | ||
| EP0247796A1 (en) | Solid phase immunoassay method | |
| CA1247523A (en) | Method for reducing non-specific interferences in agglutination immunoassays | |
| EP0245981B1 (en) | Signal enhancement in immunoassay by modulation of chemical catalysis | |
| US4693970A (en) | Immunoassay using complement | |
| DE3781643D1 (en) | METHOD AND TEST CARRIER FOR DETERMINING AN ANALYT. | |
| JPH03195953A (en) | Optical reaction measurement | |
| Ohkuma et al. | Detection of luciferase having two kinds of luminescent colour based on optical filter procedure: application to an enzyme immunoassay | |
| JPS62502633A (en) | Enzyme assay of body fluids | |
| CA2355294A1 (en) | A method of detecting and/or quantifying a specific ige antibody in a liquid sample | |
| US5783455A (en) | Regenerable solid phase for carrying out specific binding reactions | |
| JPH0531743B2 (en) | ||
| JPS59176675A (en) | Reagent for measuring enzyme immune | |
| JPS5847491A (en) | Suspension of beta-d-galactosidase action | |
| NO884878L (en) | QUALITATIVE AND QUANTITATIVE DETERMINATION OF SPECIAL ANTIGENS, SPECIAL TRACKS OF CLOSTRIDIUM TYROBUTYRICUM. | |
| JPH0694713A (en) | Method for measuring antithrombin iii activity and measurement kit of antithrombin iii activity using it | |
| Maggio et al. | Immunology: current and future technology assessment | |
| JP5143046B2 (en) | Test substance measurement method and kit for carrying out the measurement method | |
| ES2052451A1 (en) | Particle agglutination assay | |
| RAYEV et al. | Design and Application of Multi-Purpose Test System using Non-Enzymatic Diagnostic Sets for Non-Instrumental Assay of Specific Antibodies | |
| JPH0315759A (en) | Immunoassay | |
| ES2004066A6 (en) | A kit for qualitative and quantitative analysis of antigens, antibodies and/or microorganisms or other cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |