JPS62285062A - Immuno assay apparatus - Google Patents
Immuno assay apparatusInfo
- Publication number
- JPS62285062A JPS62285062A JP61128110A JP12811086A JPS62285062A JP S62285062 A JPS62285062 A JP S62285062A JP 61128110 A JP61128110 A JP 61128110A JP 12811086 A JP12811086 A JP 12811086A JP S62285062 A JPS62285062 A JP S62285062A
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- sample
- membrane
- antibody
- voltage
- 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
- 238000003018 immunoassay Methods 0.000 title claims description 7
- 239000012528 membrane Substances 0.000 claims abstract description 24
- 238000001962 electrophoresis Methods 0.000 claims description 9
- 239000003792 electrolyte Substances 0.000 abstract description 14
- 238000005259 measurement Methods 0.000 abstract description 13
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 abstract description 7
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 239000008151 electrolyte solution Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- DGZSVBBLLGZHSF-UHFFFAOYSA-N 4,4-diethylpiperidine Chemical compound CCC1(CC)CCNCC1 DGZSVBBLLGZHSF-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 229940098197 human immunoglobulin g Drugs 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000000504 luminescence detection Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000005304 optical glass Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
３、発明の詳細な説明
〔産業上の利用分野〕
本発明はイムノアッセイ装置、特に試料導入の際の試料
拡散を防止し、精度を向」−させたイムノアッセイ装置
に関する。Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to an immunoassay device, and particularly to an immunoassay device that prevents sample diffusion during sample introduction and improves accuracy.
膜状の担体に抗体を固定化し、この膜の面に垂直に電位
勾配をかけることにより、この方向に被測定試料中の抗
原を電気泳動によって移動せしめ、上記固定化された抗
体と抗原抗体反応を起こさせて固定させ、さらに、上記
過程で固定化させた抗原に標識された抗体を電気泳動に
よって移動せしめて反応させるか、又は膜状の担体に固
定化された抗体の未反応部に標識された抗原を電気泳動
によって移動せしめて反応させるかのいずれかの反応に
より膜状の担体に標識物を固定化し、この標識物の濃度
を測定することにより、試料中の抗原の濃度を測定する
方法が提案されている（特開昭６Ｏ−５７２５７）。By immobilizing an antibody on a membrane-like carrier and applying a potential gradient perpendicular to the surface of this membrane, the antigen in the sample to be measured is moved in this direction by electrophoresis, and an antigen-antibody reaction occurs with the immobilized antibody. The antibody labeled with the antigen immobilized in the above process is moved by electrophoresis and reacted, or the unreacted portion of the antibody immobilized on the membrane carrier is labeled. The concentration of the antigen in the sample is determined by immobilizing the labeled substance on a membrane-like carrier by either moving the labeled antigen by electrophoresis and reacting it, and measuring the concentration of this labeled substance. A method has been proposed (Japanese Patent Laid-Open No. 6O-57257).
このイムノアッセイ法は、測定時間の短縮、装置化が容
易など多くの効果を有する方法であるが、」り定結果の
再現性、精度についてさらに配慮することが望ましい。Although this immunoassay method has many advantages, such as shortening measurement time and easy instrumentation, it is desirable to give further consideration to the reproducibility and accuracy of the assay results.
本発明の目的は、膜の面に垂直に電位勾配をかける電気
泳動法を利用したイムノアッセイ装置において、精度の
良好な結果が得られる改良されたイムノアッセイ装置を
提供することにある。An object of the present invention is to provide an improved immunoassay device that uses an electrophoresis method that applies a potential gradient perpendicular to the surface of a membrane and can provide highly accurate results.
上記目的は、試料注入時あるいはその直後における試料
と電解液との混合を極力おさえるようにした装置によっ
て達成される。具体的には、電気泳動させる担体上部に
直接試料を注ぎ込むものではなく、電気泳動担体に近接
して上部に細管を配置し、試料液をこの細管に入れた後
、細管内部に配置された電極と電気泳動担体下部に設け
られた電極との間に直流電圧を印加して試料を電気泳動
担体上部に導くものである。The above object is achieved by an apparatus that suppresses mixing of the sample and the electrolytic solution as much as possible during or immediately after sample injection. Specifically, rather than pouring the sample directly onto the top of the carrier to be electrophoresed, a thin tube is placed near the top of the electrophoresis carrier, and after the sample liquid is poured into this thin tube, an electrode is placed inside the tube. A DC voltage is applied between the sample and the electrode provided at the bottom of the electrophoresis carrier to guide the sample to the top of the electrophoresis carrier.
上記細管は試料と電解液との反応を極力おさえ、良好な
精度が得られる。The above-mentioned thin tube suppresses the reaction between the sample and the electrolyte as much as possible, and good accuracy can be obtained.
以下、本発明の実施例を第１図により説明する。 Embodiments of the present invention will be described below with reference to FIG.
実施例１゜
上部電解液注入ノズルｌ、及び下部電解液注入口２を介
して、上部電解槽３及び下部電解槽４にトリス・グリシ
ン緩衝液を注ぎ込み、上部電解液５及び下部電解液６と
する。試料導入用細管７に高濃度のトリス・グリシン緩
衝液を加えてｐ Ｈを調整した試料を１０μＱ吸入し、
上部電解槽３の内に移動させ、測定したい成分に対する
抗体を固定化したポリアクリルアミドゲルである抗体固
定化多孔質膜８の近傍で止める。試料導入細管７内部に
配置された試料導入用電極９及び下部電解槽４内の下部
電極１０との間に電源１１を用いて直流電圧を電極１０
が陽極、電極９が陰極になるように印加する。この時試
料中の各成分は抗体固定化多孔質膜８に導入される。電
源１１を切り、上部電極槽３内部に設けられた上部電極
１２と電極１０との間に電源１３を用いて直流電圧を電
極１０が陽極、電極１２が陰極となるように印加する。Example 1 Tris-glycine buffer solution is poured into the upper electrolyte tank 3 and the lower electrolyte tank 4 through the upper electrolyte injection nozzle l and the lower electrolyte injection port 2, and the upper electrolyte solution 5 and the lower electrolyte solution 6 are mixed. do. Inhale 10μQ of the sample whose pH has been adjusted by adding a high concentration Tris-glycine buffer into the sample introduction tube 7.
It is moved into the upper electrolytic cell 3 and stopped near the antibody-immobilized porous membrane 8, which is a polyacrylamide gel on which antibodies against the component to be measured are immobilized. A DC voltage is applied to the electrode 10 using a power source 11 between the sample introduction electrode 9 disposed inside the sample introduction tube 7 and the lower electrode 10 in the lower electrolytic cell 4.
The voltage is applied so that the electrode becomes the anode and the electrode 9 becomes the cathode. At this time, each component in the sample is introduced into the antibody-immobilized porous membrane 8. The power supply 11 is turned off, and a DC voltage is applied using the power supply 13 between the upper electrode 12 provided inside the upper electrode tank 3 and the electrode 10 so that the electrode 10 becomes the anode and the electrode 12 becomes the cathode.
次にルミノールを標識した抗体溶液（２０％の割合でし
ょ糖を含む）１０μＱをノズル１４を用いて抗体固定化
多孔質膜８上に静かに注入し。Next, 10 μQ of a luminol-labeled antibody solution (containing 20% sucrose) was gently injected onto the antibody-immobilized porous membrane 8 using the nozzle 14.
再び電極１２と電極１０との間に直流電圧を、電極１０
が陽極、電極１２が陰極となるように印加する。この特
売に膜８中にトラップされた測定成分の量に応じた量の
ルミノール標識抗体が膜８に残され、過剰のルミノール
標識抗体は下部電解液６に移動する。A DC voltage is again applied between the electrode 12 and the electrode 10, and the electrode 10
The voltage is applied so that the electrode 12 becomes the anode and the electrode 12 becomes the cathode. An amount of luminol-labeled antibody corresponding to the amount of the measurement component trapped in the membrane 8 is left on the membrane 8, and excess luminol-labeled antibody moves to the lower electrolyte 6.
次に上部電解液５を吸引ノズル１５を用いて上部電解槽
３から排除し、下部電解液５も下部電解液排出口１６を
用いて下部電解槽４から排出する。Next, the upper electrolytic solution 5 is removed from the upper electrolytic cell 3 using the suction nozzle 15, and the lower electrolytic solution 5 is also removed from the lower electrolytic cell 4 using the lower electrolytic solution outlet 16.
次に発光試薬注入ノズル１７を用いて０．１ＭのＨ２０
□水溶液１００ｐＱと、０．ＩＮのＮ ａ ＯＨ水溶液
に１０ｍＭの濃度で次亜塩素酸ナトリウムを含む溶液２
００μＱを膜８の上に注入し、その時の発光量を光学ガ
ラス窓１８を通して、その下に受光部１９を配置したフ
ォトカウンタ２０で測定する。Next, using the luminescent reagent injection nozzle 17, 0.1M H20 was added.
□Aqueous solution 100pQ and 0. Solution 2 containing sodium hypochlorite at a concentration of 10 mM in an aqueous NaOH solution of IN
00 μQ is injected onto the film 8, and the amount of light emitted at that time is measured through an optical glass window 18 using a photo counter 20 with a light receiving section 19 disposed below it.
測定結果の一例として測定対象物にヒト免疫グロブリン
Ｇを選び、試料導入、試料反応、及び標識抗体反応の直
流電圧の印加を印加電圧２５０Ｖとし、印加時間をそれ
ぞれ５分、２５分、３０分として測定した。同一試料を
本装置で２５回測定した結果、試料中濃度の平均で３．
３Ｘ１０−３ｇ／Ｑで標準偏差は０．２３ｘｌＯ−３ｇ
／Ｑであった。この測定と全く同一の試料に２０％の割
合でしょ糖を加え、これをルミノール標識抗体と同様の
方法で膜８上に注入することにより試料導入し他は実施
例記載と同様な装置で測定した。結果は、２５回測定の
平均で３．ＯＸ １０−８ｇＩＱ、標準偏差０．８５
Ｘ １０−”ｇ／ Ｑであった。このように試料導入細
管７を設けたことにより、大幅に精度が向上した測定結
果を得ることができた。As an example of the measurement results, human immunoglobulin G was selected as the measurement target, and the applied DC voltage was 250 V for sample introduction, sample reaction, and labeled antibody reaction, and the application times were 5 minutes, 25 minutes, and 30 minutes, respectively. It was measured. As a result of measuring the same sample 25 times with this device, the average concentration in the sample was 3.
3X10-3g/Q and standard deviation is 0.23xlO-3g
/Q. Sucrose was added at a rate of 20% to the same sample as in this measurement, and the sample was introduced by injecting it onto the membrane 8 in the same manner as the luminol-labeled antibody, and the other measurements were performed using the same equipment as described in the example. . The results are the average of 25 measurements. OX 10-8gIQ, standard deviation 0.85
X 10-''g/Q. By providing the sample introduction thin tube 7 in this way, it was possible to obtain measurement results with significantly improved accuracy.
実施例２゜ もう一つの実施例を第２図を用いて説明する。Example 2゜ Another embodiment will be explained using FIG. 2.
実施例１と同様の操作で、上部電解液５、及び下部電解
液６を注入した後、試料導入細管７に前に述べた実施例
と同様の試料を吸入し、上部電解槽３の内に移動させて
、抗体固定化多孔質膜８の直前で＋トめる。試料導入用
電極９と、上部電解槽３の内の抗体固定化多孔膜８の近
傍に設置された上部電極２１との間に、電源１１を用い
て電極２１が陽極、電極９が陰極となるように、直流電
圧を印加する。この時試料中の各成分は抗体固定化多孔
質膜−Ｆに移動する。After injecting the upper electrolyte 5 and the lower electrolyte 6 in the same manner as in Example 1, the same sample as in the previous example was sucked into the sample introduction capillary 7 and poured into the upper electrolytic cell 3. Move and top up just in front of the antibody-immobilized porous membrane 8. A power source 11 is used between the sample introduction electrode 9 and the upper electrode 21 installed near the antibody-immobilized porous membrane 8 in the upper electrolytic cell 3, so that the electrode 21 becomes an anode and the electrode 9 becomes a cathode. Apply a DC voltage. At this time, each component in the sample moves to the antibody-immobilized porous membrane-F.
なお、電極２１は先端部のみを残し他の部分には樹脂コ
ーティングを施すことによる絶縁を行った。電源１１を
切り、直ちに上部電極２１と下部電極１０との間に、電
極１０が陽極、電極２１が陰極となるように直流電源１
３を用いて直流電圧を印加する。次に、ルミノールを標
識した抗体溶液を同様の操作で抗体固定化多孔質膜上に
導入、さらに、膜内に移動２反応させる。Note that the electrode 21 was insulated by leaving only the tip portion and applying a resin coating to the other portions. Turn off the power supply 11, and immediately connect the DC power supply 1 between the upper electrode 21 and the lower electrode 10 so that the electrode 10 becomes the anode and the electrode 21 becomes the cathode.
3 to apply a DC voltage. Next, a luminol-labeled antibody solution is introduced onto the antibody-immobilized porous membrane in the same manner, and further transferred into the membrane for reaction.
実施例１と同様にルミノール発光検出を行なう。Luminol luminescence detection is performed in the same manner as in Example 1.
ヒト免疫グロブリンを測定対象として、試料導入。Introducing a sample to measure human immunoglobulin.
試料反応、標識抗体導入１ｍ識抗体反応、の直流電圧の
印加を、印加電圧をそれぞれ１５０Ｖ。The applied voltage was 150 V for sample reaction, labeled antibody introduction 1 m, and recognition antibody reaction for application of DC voltage.
２５０Ｖ、１５０Ｖ、２５０Ｖ、印加時間をそれぞれ、
２分、２５分、２分、３０分として測定した。実施例と
同じ同一試料を本装置で２５回測定した結果、試料中濃
度の平均で、３．３ＸＩＯ−３ｇ ／ Ｄで、標準偏差
は０．２４ Ｘｌ ０−３ｇ／Ｑであり、実施例１と同
等の高精度な測定結果を得ることができた。250V, 150V, 250V, application time respectively,
Measurements were made at 2 minutes, 25 minutes, 2 minutes, and 30 minutes. As a result of measuring the same sample as in Example 25 times with this device, the average concentration in the sample was 3.3XIO-3g/D, and the standard deviation was 0.24XIO-3g/Q, which was the same as in Example 1. We were able to obtain highly accurate measurement results equivalent to those of the previous method.
これらを実現する装置の一例の概略図を第３図に示す。A schematic diagram of an example of a device that realizes these is shown in FIG.
上部電解槽３、下部電解槽４、多孔質膜８、石英ガラス
窓１８、上部電極１２，２１．下部電極１０からなる反
応部分（以下反応セル２２という）を連続的に配列し、
各操作ごとに矢印２３の方向に進めながら反応処理し、
暗箱２８中で測定する。Upper electrolytic cell 3, lower electrolytic cell 4, porous membrane 8, quartz glass window 18, upper electrodes 12, 21. A reaction part (hereinafter referred to as reaction cell 22) consisting of the lower electrode 10 is continuously arranged,
Reaction processing is performed while proceeding in the direction of arrow 23 for each operation,
The measurement is carried out in a dark box 28.
以上で説明したように本発明は精度の高い測定結果が得
られるという効果を有する。As explained above, the present invention has the effect that highly accurate measurement results can be obtained.
第１図及び第２図は本発明を行なう反応セル部分を示す
概略図、第３図は、機構部分全体の構成を示す概念図で
ある。
１・・・上部電解液注入ノズル、２・・・下部電解液注
入口、３・・・上部電解槽、４・・・下部電解槽、５・
・・−り都電極液、６・・・下部電解液、７・・・試料
導入用細管、８・・・抗体固定化多孔質膜、９・・・試
料導入用電極、１０・・・下部電極、１１・・・直流電
源、１２・・・上部電極、１３・・・直流電源、１４・
・・ノズル、１５・・・吸引ノズル、１６・・・下部電
解液排出口、１７・・・発光試薬注入ノズル、１８・・
・石英ガラス窓、１９・・・受光７一
部、２０・・・フォトカウンタ、２１・・・上部電極、
２２・・・反応セル、２３・・・連なった反応セルの移
動方向を示す矢印、２４，２５，２６．２７・・・各種
のノズルの移動方向を示す矢印、２８・・・暗箱。1 and 2 are schematic diagrams showing a reaction cell portion in which the present invention is carried out, and FIG. 3 is a conceptual diagram showing the configuration of the entire mechanism portion. DESCRIPTION OF SYMBOLS 1... Upper electrolyte injection nozzle, 2... Lower electrolyte injection port, 3... Upper electrolytic tank, 4... Lower electrolytic tank, 5...
・・・-Limited electrode solution, 6... Lower electrolyte, 7... Thin tube for sample introduction, 8... Antibody immobilized porous membrane, 9... Electrode for sample introduction, 10... Lower part Electrode, 11... DC power supply, 12... Upper electrode, 13... DC power supply, 14.
... Nozzle, 15... Suction nozzle, 16... Lower electrolyte outlet, 17... Luminescence reagent injection nozzle, 18...
- Quartz glass window, 19... Part of light receiving 7, 20... Photo counter, 21... Upper electrode,
22...Reaction cell, 23...Arrow indicating the moving direction of a series of reaction cells, 24, 25, 26.27...Arrow indicating the moving direction of various nozzles, 28...Dark box.
Claims (1)
質的に全域に抗体を固定化した電気泳動用膜、上記上部
電解槽中に配置された上部電極及び上記下部電解槽中に
配置された下部電極よりなるイムノアッセイ装置におい
て、上記上部電解槽中にその先端を上記電気泳動用膜の
近傍に配置した細管を有することを特徴とするイムノア
ッセイ装置。1. An upper electrolytic cell, a lower electrolytic cell, an electrophoresis membrane with antibodies immobilized over substantially the entire area disposed between the two, an upper electrode disposed in the upper electrolytic cell, and an electrophoretic membrane arranged in the lower electrolytic cell. 1. An immunoassay device comprising lower electrodes arranged in the upper electrolytic tank, the immunoassay device comprising a thin tube with its tip disposed in the vicinity of the electrophoresis membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61128110A JPS62285062A (en) | 1986-06-04 | 1986-06-04 | Immuno assay apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61128110A JPS62285062A (en) | 1986-06-04 | 1986-06-04 | Immuno assay apparatus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62285062A true JPS62285062A (en) | 1987-12-10 |
| JPH0529065B2 JPH0529065B2 (en) | 1993-04-28 |
Family
ID=14976631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61128110A Granted JPS62285062A (en) | 1986-06-04 | 1986-06-04 | Immuno assay apparatus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62285062A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57100340A (en) * | 1980-12-15 | 1982-06-22 | Shimadzu Corp | Measuring apparatus of electrophoresis of cell |
| JPS6057257A (en) * | 1983-09-09 | 1985-04-03 | Hitachi Ltd | Immunoassay method |
| JPS61243353A (en) * | 1985-04-17 | 1986-10-29 | フオレシス トランスフア− システムズ，インコ−ポレ−テツド | Method and device for separating component of mixture |
-
1986
- 1986-06-04 JP JP61128110A patent/JPS62285062A/en active Granted
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57100340A (en) * | 1980-12-15 | 1982-06-22 | Shimadzu Corp | Measuring apparatus of electrophoresis of cell |
| JPS6057257A (en) * | 1983-09-09 | 1985-04-03 | Hitachi Ltd | Immunoassay method |
| JPS61243353A (en) * | 1985-04-17 | 1986-10-29 | フオレシス トランスフア− システムズ，インコ−ポレ−テツド | Method and device for separating component of mixture |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0529065B2 (en) | 1993-04-28 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |