JPH0581860B2 - - Google Patents
Info
- Publication number
- JPH0581860B2 JPH0581860B2 JP58235132A JP23513283A JPH0581860B2 JP H0581860 B2 JPH0581860 B2 JP H0581860B2 JP 58235132 A JP58235132 A JP 58235132A JP 23513283 A JP23513283 A JP 23513283A JP H0581860 B2 JPH0581860 B2 JP H0581860B2
- Authority
- JP
- Japan
- Prior art keywords
- sample
- antigen
- antibody
- concentration
- flow line
- 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.)
- Expired - Lifetime
Links
- 239000003153 chemical reaction reagent Substances 0.000 claims description 23
- 238000004458 analytical method Methods 0.000 claims description 13
- 230000001900 immune effect Effects 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims 1
- 239000000427 antigen Substances 0.000 description 29
- 102000036639 antigens Human genes 0.000 description 29
- 108091007433 antigens Proteins 0.000 description 29
- 239000004816 latex Substances 0.000 description 11
- 229920000126 latex Polymers 0.000 description 11
- 239000002245 particle Substances 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 239000006185 dispersion Substances 0.000 description 7
- 238000000149 argon plasma sintering Methods 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000001507 sample dispersion Methods 0.000 description 3
- 239000013543 active substance Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- -1 blood cells Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Description
【発明の詳細な説明】
技術分野
本発明は、免疫学的分析方法に関するものであ
る。DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to an immunological analysis method.
従来技術
免疫学的反応を利用する生理活性物質の分析法
には種々の方法があり、その一つに抗原抗体反応
による凝集物を光学的に検出する方法がある。ま
た、この抗原抗体反応による分析法には、抗原と
抗体との複合体を検出するものと、抗体（抗原）
を人工粒子や赤血球等の粒子の固相化し、分析す
べき抗原（抗体）との反応による粒子凝集物を検
出するものとがある。Prior Art There are various methods for analyzing physiologically active substances using immunological reactions, one of which is a method of optically detecting aggregates due to antigen-antibody reactions. In addition, there are two types of analytical methods based on this antigen-antibody reaction: one that detects a complex between an antigen and an antibody, and one that detects a complex between an antigen and an antibody.
There is a method in which particles such as artificial particles or red blood cells are solidified, and particle aggregates are detected by reaction with the antigen (antibody) to be analyzed.
このような抗原抗体反応による生理活性物質の
分析は、一般には抗原抗体反応によつて生成され
る凝集物に由来する光散乱強度や透過光量に基い
て同定、定量される。しかしながら、サンプル中
の所定の抗原（抗体）を分析するために、一定量
のサンプルと、試薬として目的とする抗原（抗
体）に対する一定量の抗体（抗原）またはこれを
固相化した人工粒子や赤血球等の粒子とを反応さ
せると、そのときの抗原抗体反応による凝集物の
生成程度は、目的とする抗原（抗体）量が多けれ
ば多い程その程度が上昇するのではなく、第１図
に示すように、目的とする抗原（抗体）量が試薬
である抗体（抗原）量に対して非常に多いと反対
に凝集物の生成程度が低下してしまう。この現象
は、いわゆる抗原過剰と呼ばれているが、抗原過
剰においては見掛上目的とする抗原（抗体）量が
少なくなるため、分析結果は実際とは非常にかけ
離れたものとなる。 In the analysis of physiologically active substances based on such antigen-antibody reactions, identification and quantification are generally performed based on the light scattering intensity and amount of transmitted light derived from aggregates produced by the antigen-antibody reaction. However, in order to analyze a given antigen (antibody) in a sample, a certain amount of sample and a certain amount of antibody (antigen) against the target antigen (antibody) or artificial particles immobilized thereon as reagents are used. When reacting with particles such as red blood cells, the degree of aggregate formation due to the antigen-antibody reaction does not increase as the amount of target antigen (antibody) increases; As shown, if the amount of the target antigen (antibody) is too large compared to the amount of the antibody (antigen) used as a reagent, the degree of aggregate formation will be reduced. This phenomenon is called antigen excess, but in antigen excess, the amount of target antigen (antibody) appears to be small, so the analysis results are very different from reality.
従来は、このような抗原過剰による不具合を解
決するため、二度に分けて試薬を添加したり、デ
イスクリートタイプの分析法でサンプルの希釈の
度合を変えたものを複数テストしたりする方法が
とられているが、これらの方法は分析に長時間を
要すると共に、操作も面倒であり、また不経済で
ある。 Conventionally, in order to solve problems caused by antigen excess, methods were used such as adding reagents in two batches or conducting multiple tests with different degrees of sample dilution using discrete analysis methods. However, these methods require a long time for analysis, are troublesome to operate, and are uneconomical.
発明の目的
本発明の目的は、上述した不具合を解決し、抗
原過剰の影響を受けることなく、被検物質を簡単
かつ迅速に、しかも高精度で安定して分析できる
免疫学的分析方法を提供しようとするものであ
る。Purpose of the Invention The purpose of the present invention is to solve the above-mentioned problems and provide an immunological analysis method that can easily and quickly analyze a test substance with high accuracy and stability without being affected by antigen excess. This is what I am trying to do.
発明の概要
本発明においては、サンプルをフローライン中
で緩衝液中に適度に分散させる。このようにする
と、フローラインのあるポイントにおける被測定
物の濃度と時間との関係は第２図に曲線あるい
はで示すようになる。この曲線あるいは
は、分散の様式で異なり、様式が決まればサンプ
ルに関係なくほゞ一定となる。本発明では、この
ようにフローラインを輸送されるサンプルが所定
の濃度勾配を形成しながら分散している状態で、
間欠的に空気等の気体を注入することにより濃度
の異なる複数個のセグメント化されたサンプルを
得て、これらサンプルのそれぞれに対して一定濃
度の試薬と反応させることにより、抗原過剰をチ
エツクすると共に、抗原過剰領域においても高精
度の分析を行ない得るようにしたものである。な
お、フローラインでの緩衝液中へのサンプルの分
散の程度は、フローラインの長さ、径を変化させ
たり、ミキシング室を設定したりすること等によ
り容易にコントロールすることができる。SUMMARY OF THE INVENTION In the present invention, a sample is appropriately dispersed in a buffer solution in a flow line. In this way, the relationship between the concentration of the substance to be measured and time at a certain point on the flow line becomes as shown by a curve in FIG. This curve or curve differs depending on the mode of dispersion, and once the mode is determined, it becomes almost constant regardless of the sample. In the present invention, in a state where the sample transported through the flow line is dispersed while forming a predetermined concentration gradient,
By injecting gas such as air intermittently, multiple segmented samples with different concentrations are obtained, and each of these samples is reacted with a reagent of a fixed concentration to check for antigen excess. This makes it possible to perform highly accurate analysis even in antigen-rich areas. Note that the degree of dispersion of the sample into the buffer solution in the flow line can be easily controlled by changing the length and diameter of the flow line, setting the mixing chamber, etc.
このように、フローラインを分散して輸送され
るサンプルを濃度の異なる複数のセグメントに分
けて一定濃度の試薬と反応させ、その凝集物の程
度を光散乱強度あるいは透過光量の変化から検出
すると、第３図あるいは第４図に示すような信号
が得られる。すなわち、サンプル中の目的とする
抗原（抗体）が抗原過剰領域以下の濃度では、第
３図に示すようにピーク状の曲線が得られ、その
ピークはサンプル中の目的とする抗原（抗体）の
濃度が高くなるに従つて高くなり、抗原過剰領域
に近づくに従つて伸びなくなるが、第４図に示す
ようにピークの立ち上がりの傾きが大きく変化す
るから、その傾きから目的とする抗原（抗体）が
抗原過剰領域付近まで存在することがわかると共
に、その光散乱強度からその量を高精度で定量す
ることができる。また、抗原過剰領域において
は、反対にピークは低くなるものの、立ち上がり
の傾きが大きくなるから、同様にその傾きと光散
乱強度とから抗原過剰をチエツクすることができ
ると共に、その量を高精度で定量することができ
る。 In this way, if a sample transported through a flow line is divided into multiple segments with different concentrations and reacted with a reagent of a constant concentration, and the extent of the aggregates is detected from changes in light scattering intensity or transmitted light amount, A signal as shown in FIG. 3 or 4 is obtained. In other words, when the concentration of the target antigen (antibody) in the sample is below the antigen-excess region, a peak-like curve is obtained as shown in Figure 3, and the peak corresponds to the concentration of the target antigen (antibody) in the sample. It increases as the concentration increases, and stops increasing as it approaches the antigen-excess region, but as shown in Figure 4, the slope of the rise of the peak changes greatly, so the target antigen (antibody) can be determined from the slope. It can be seen that the antigen exists up to the vicinity of the antigen-excess region, and its amount can be quantified with high precision from the light scattering intensity. In addition, in the antigen excess region, on the contrary, although the peak becomes lower, the rising slope becomes larger, so it is possible to check the antigen excess from the slope and the light scattering intensity, and also to measure the amount with high precision. Can be quantified.
実施例
第５図は本発明に先立つて開発した免疫学的分
析装置の一例の構成を示すものである。この免疫
学的分析装置は、緩衝液タンク１に収容した緩衝
液２をポンプ３によりフローライン４中に輸送
し、その中にサンプルを分散させて、表面に目的
とする抗原（抗体）に対する抗体（抗原）を固相
化したラテツクス試薬を一定濃度で反応させて、
サンプル中の目的とする抗原（抗体）を分析する
ものである。Example FIG. 5 shows the configuration of an example of an immunological analyzer developed prior to the present invention. This immunological analyzer transports a buffer solution 2 stored in a buffer solution tank 1 into a flow line 4 using a pump 3, disperses the sample therein, and coats the surface with antibodies against the target antigen (antibody). By reacting a latex reagent immobilized with (antigen) at a constant concentration,
This is to analyze the target antigen (antibody) in a sample.
サンプルはサンプルインジエクタ５でフローラ
イン４内に注入し、分散室６で第２図に示したよ
うに分散させる。分散室６はフローライン４内に
スペースを設定するだけでよいし、またフローラ
イン４の一部の内径を調整することで実施すれば
特別必要ない。分散したサンプルがミキシングポ
イント７に達するのに同期して、試薬タンク８に
収容されたラテツクス試薬９をポンプ１０により
注入し、これらを混合器１１にて完全に混合す
る。この分散したサンプルに対するラテツクス試
薬９の注入は、第６図に破線で示すように、実線
で示すサンプル濃度変化に対して一定の濃度とな
るようにする。なお、このラテツクス試薬９は必
ずしもサンプル濃度変化全域に亘つて注入する必
要はなく、その一部、例えば濃度ピーク部分を含
む前半分あるいは後半分だけでもよい。 The sample is injected into the flow line 4 using the sample injector 5 and dispersed in the dispersion chamber 6 as shown in FIG. The dispersion chamber 6 only needs to be provided as a space within the flow line 4, and is not particularly necessary if the dispersion chamber 6 is implemented by adjusting the inner diameter of a part of the flow line 4. In synchronization with the dispersed sample reaching the mixing point 7, the latex reagent 9 contained in the reagent tank 8 is injected by the pump 10 and mixed completely in the mixer 11. The latex reagent 9 is injected into the dispersed sample so that the concentration remains constant as shown by the broken line in FIG. 6 with respect to the change in sample concentration shown by the solid line. Note that the latex reagent 9 does not necessarily need to be injected over the entire range of sample concentration changes, but may be injected only over a portion thereof, for example, the first half or the second half including the concentration peak portion.
このようにして、フローライン４内を輸送しな
がら分散したサンプルと一定濃度のラテツクス試
薬９とを反応させ、検出器１２においてその反応
の程度を光散乱強度もしくは吸光度より検出して
サンプル中の目的とする抗原（抗体）量を求め
る。なお、検出器１２を通過した反応液は排液タ
ンク１３に排出する。 In this way, the dispersed sample reacts with the latex reagent 9 at a constant concentration while being transported through the flow line 4, and the extent of the reaction is detected by the detector 12 from the light scattering intensity or absorbance. Determine the amount of antigen (antibody) to be used. Note that the reaction liquid that has passed through the detector 12 is discharged into a drain tank 13.
しかしながら、第５図に示した免疫学的分析装
置にあつては、ラテツクス試薬９を添加する際の
サンプルの分散の乱れを考慮しないと安定な分析
ができないという問題がある。 However, the immunological analyzer shown in FIG. 5 has a problem in that stable analysis cannot be performed unless disturbances in sample dispersion are taken into account when adding the latex reagent 9.
第７図はかかる問題をも解決した本発明を実施
する免疫学的分析装置の一例の構成を示す線図で
ある。本例では、分散室６とラテツクス試薬９の
ミキシングポイント７との間のフローライン４に
セグメントポイント１４を設け、このセグメント
ポイント１４においてポンプ１５によりフローラ
イン４に間欠的に空気を注入して、分散して輸送
されるサンプルを第８図および第９図に示すよう
に各濃度毎にセグメント化し、その各セグメント
S1〜Soにラテツクス試薬９を一定濃度で反応させ
るようにしたもので、その他の構成は第５図と同
じである。このようにすれば、ラテツクス試薬９
を添加する際のサンプルの分散の乱れを考慮する
ことなく、より安定な分析が可能となる。 FIG. 7 is a diagram showing the configuration of an example of an immunological analyzer implementing the present invention which also solves this problem. In this example, a segment point 14 is provided in the flow line 4 between the dispersion chamber 6 and the mixing point 7 of the latex reagent 9, and air is intermittently injected into the flow line 4 by a pump 15 at this segment point 14. The sample transported in a dispersed manner is segmented for each concentration as shown in Figures 8 and 9, and each segment is
The latex reagent 9 is reacted with S 1 to S o at a constant concentration, and the other configuration is the same as that in FIG. 5. In this way, latex reagent 9
More stable analysis is possible without considering the disturbance of sample dispersion when adding .
なお、上述した各実施例においては反応液を一
箇所で測光するようにしたが、これを複数箇所で
測光してレイトアツセイを行なうこともできる。
また、本発明はラテツクス粒子を用いる試薬のみ
でなく、その他の粒子、例えば血球を用いる試薬
や、あるいは粒子に固相化しない抗原または抗体
からなる試薬による免疫学的反応に基く分析にも
有効に適用できることは勿論である。 In each of the above-mentioned Examples, the reaction solution was photometered at one location, but it is also possible to perform a late assay by photometry at multiple locations.
Furthermore, the present invention is effective not only for reagents using latex particles, but also for analyzes based on immunological reactions with reagents using other particles, such as blood cells, or reagents consisting of antigens or antibodies that are not immobilized on particles. Of course, it can be applied.
発明の効果
以上述べたように、本発明によれば、分散して
輸送されるサンプルに間欠的に空気等を注入して
一つのサンプルを濃度の異なる複数のセグメント
に分け、これらのセグメントに対して一定濃度の
試薬を反応させることにより、サンプルの分散の
乱れや抗原過剰の影響を受けることなく、被検物
質を簡単かつ迅速に、しかも高精度で安定して分
析することができる。Effects of the Invention As described above, according to the present invention, one sample is divided into a plurality of segments with different concentrations by intermittently injecting air etc. into a sample that is being transported in a dispersed manner, and these segments are By reacting a reagent at a constant concentration with a sample, a test substance can be easily and quickly analyzed with high precision and stability without being affected by disturbances in sample dispersion or excess antigen.
第１図は抗原過剰現象を説明するための線図、
第２図、第３図および第４図は本発明の免疫学的
分析方法を説明するための線図、第５図は本発明
に先立つて開発した免疫学的分析装置の一例の構
成を示す線図、第６図はその動作を説明するため
の線図、第７図は本発明を実施する免疫学的分析
装置の一例の構成を示す線図、第８図および第９
図はその動作を説明するための線図である。
１……緩衝液タンク、２……緩衝液、３……ポ
ンプ、４……フローライン、５……サンプルイン
ジエクタ、６……分散室、７……ミキシングポイ
ント、８……試薬タンク、９……ラテツクス試
薬、１０……ポンプ、１１……混合器、１２……
検出器、１３……排液タンク、１４……セグメン
トポイント、１５……ポンプ。
Figure 1 is a diagram to explain the antigen excess phenomenon.
Figures 2, 3, and 4 are diagrams for explaining the immunological analysis method of the present invention, and Figure 5 shows the configuration of an example of an immunological analyzer developed prior to the present invention. 6 is a diagram for explaining its operation, FIG. 7 is a diagram showing the configuration of an example of an immunological analyzer implementing the present invention, and FIGS. 8 and 9 are diagrams.
The figure is a diagram for explaining the operation. 1... Buffer tank, 2... Buffer solution, 3... Pump, 4... Flow line, 5... Sample injector, 6... Dispersion chamber, 7... Mixing point, 8... Reagent tank, 9 ... Latex reagent, 10 ... Pump, 11 ... Mixer, 12 ...
Detector, 13... Drain tank, 14... Segment point, 15... Pump.
Claims (1)
応する試薬を用いて免疫学的に分析するにあた
り、希釈液で満たされたフローライン中に所定濃
度のサンプルを導入して輸送することにより該サ
ンプルを分散させてフローライン中に該サンプル
の濃度勾配を形成する工程と、前記濃度勾配を形
成したサンプルに対して間欠的に空気等の気体を
注入することにより濃度の異なる複数個のセグメ
ント化されたサンプルを得る工程と、得られた複
数のセグメントのそれぞれを一定濃度の試薬と反
応させる工程と、混合後の反応結果を測定する工
程とを有する免疫学的分析方法。1. When performing immunological analysis using a test substance in a sample and a reagent that specifically reacts with it, the sample at a predetermined concentration is introduced into a flow line filled with diluent and transported. A step of dispersing the sample to form a concentration gradient of the sample in the flow line, and intermittently injecting a gas such as air into the sample that has formed the concentration gradient to create a plurality of segments with different concentrations. 1. An immunological analysis method comprising the steps of: obtaining a converted sample; reacting each of the obtained plurality of segments with a reagent at a fixed concentration; and measuring the reaction result after mixing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23513283A JPS60128368A (en) | 1983-12-15 | 1983-12-15 | Immunological analysis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23513283A JPS60128368A (en) | 1983-12-15 | 1983-12-15 | Immunological analysis method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60128368A JPS60128368A (en) | 1985-07-09 |
| JPH0581860B2 true JPH0581860B2 (en) | 1993-11-16 |
Family
ID=16981524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23513283A Granted JPS60128368A (en) | 1983-12-15 | 1983-12-15 | Immunological analysis method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60128368A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5324015A (en) * | 1976-08-16 | 1978-03-06 | Teikoku Hormone Mfg Co Ltd | Method and apparatus for detection of antigen-antibody reaction |
-
1983
- 1983-12-15 JP JP23513283A patent/JPS60128368A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5324015A (en) * | 1976-08-16 | 1978-03-06 | Teikoku Hormone Mfg Co Ltd | Method and apparatus for detection of antigen-antibody reaction |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60128368A (en) | 1985-07-09 |
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