JP3283078B2 - Immunological measurement device - Google Patents
Immunological measurement deviceInfo
- Publication number
- JP3283078B2 JP3283078B2 JP32503292A JP32503292A JP3283078B2 JP 3283078 B2 JP3283078 B2 JP 3283078B2 JP 32503292 A JP32503292 A JP 32503292A JP 32503292 A JP32503292 A JP 32503292A JP 3283078 B2 JP3283078 B2 JP 3283078B2
- Authority
- JP
- Japan
- Prior art keywords
- antigen
- antibody
- sample
- immunological measurement
- measurement device
- 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
- 238000005259 measurement Methods 0.000 title claims description 41
- 230000001900 immune effect Effects 0.000 title claims description 19
- 239000002245 particle Substances 0.000 claims description 46
- 239000000523 sample Substances 0.000 claims description 41
- 239000000427 antigen Substances 0.000 claims description 39
- 102000036639 antigens Human genes 0.000 claims description 39
- 108091007433 antigens Proteins 0.000 claims description 39
- 238000006243 chemical reaction Methods 0.000 claims description 23
- 239000012488 sample solution Substances 0.000 claims description 17
- 230000004520 agglutination Effects 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000004816 latex Substances 0.000 claims description 12
- 229920000126 latex Polymers 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 9
- 230000002776 aggregation Effects 0.000 claims description 6
- 238000003018 immunoassay Methods 0.000 claims description 6
- 239000012472 biological sample Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 238000005054 agglomeration Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000005096 rolling process Methods 0.000 claims 1
- 229940027941 immunoglobulin g Drugs 0.000 description 26
- 210000004027 cell Anatomy 0.000 description 24
- 210000002966 serum Anatomy 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 6
- 238000004220 aggregation Methods 0.000 description 4
- 230000001678 irradiating effect Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000004065 semiconductor Substances 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000001268 chyle Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By Optical Means (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、抗原抗体反応を利用し
て血液や尿中の体液成分などを測定する免疫学的測定装
置に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay apparatus for measuring a body fluid component in blood or urine using an antigen-antibody reaction.
【０００２】[0002]
【従来の技術】免疫学的測定は、検体中の抗原または抗
体と反応する試薬として抗原に対する抗体、抗体に対す
る抗原を加えて特異的な抗原抗体反応を起こさせ、高い
選択性を碓保する事ができる。従来、この抗原抗体反応
により形成される凝集塊（抗原抗体複合体）を検出する
免疫学的測定法として、 １）比濁法（turbitometric immunoassay）：抗原抗体
反応による凝集塊を含む溶液に光を照射し、その透過光
強度を検出して測定する方法、 ２）比ろう法（nephelometric immunoassay）：抗原抗
体反応の凝集塊を含む溶液に光を照射し、その散乱光強
度を検出する方法、 ３）ラテックス免疫凝集法（latex agglutination immu
noassay）：反応試薬としての抗体をポリスチロールな
どの固相表面に固定化して、凝集感度を上昇させたもの
で、検出測定法は比濁法、比ろう法と同じ方法がある。
さらに、フローセル中に試料を流し凝集塊の数を計測す
る方法もある。2. Description of the Related Art In immunological measurement, a specific antigen-antibody reaction is caused by adding an antibody to an antigen or an antigen to an antibody as a reagent that reacts with an antigen or an antibody in a sample to ensure a high selectivity. Can be. Conventionally, as an immunoassay for detecting an aggregate (antigen-antibody complex) formed by the antigen-antibody reaction, 1) Turbometric immunoassay: irradiating light to a solution containing the aggregate by the antigen-antibody reaction Irradiation and detection and measurement of the transmitted light intensity. 2) Nephelometric immunoassay: A method of irradiating a solution containing an aggregate of an antigen-antibody reaction with light and detecting its scattered light intensity. ) Latex agglutination immu
noassay): An antibody as a reaction reagent is immobilized on a solid phase surface such as polystyrene to increase the agglutination sensitivity, and the detection measurement method is the same as the turbidimetric method or the iris method.
Furthermore, there is a method of flowing a sample through a flow cell and measuring the number of aggregates.
【０００３】[0003]
【発明が解決しようとする課題】ところが、従来の技術
の比濁法、比ろう法およびラテックス免疫凝集法では多
くの凝集塊（抗原抗体複合体）を含む広い領域からの透
過光或いは散乱光の強度を測定しているため、試料中に
含まれる乳び、ビリルビン、へモグロビン等による光吸
収や光多重散乱の影響を受け易く精度の高い測定ができ
ない。However, in the conventional turbidimetric method, turbidimetric method, and latex immunoagglutination method, the transmitted light or scattered light from a large area including a large number of aggregates (antigen-antibody complex) is not used. Since the intensity is measured, the measurement is susceptible to light absorption and light multiple scattering by chyle, bilirubin, hemoglobin, and the like contained in the sample, and high-precision measurement cannot be performed.
【０００４】特に、ラテックス免疫凝集法においては、
未凝集のラッテクス粒子が散乱体として大きな背景光と
なる点や、多重散乱光を測定している点で、精度の高い
高感度測定ができない。これらの課題を解決する従来技
術として、レーザ光を照射したフローセル中に凝集塊を
含む試料を流し、凝集塊個々の散乱光強度から凝集塊の
大きさとその数を計測する方法がある。[0004] In particular, in the latex immunoagglutination method,
High-accuracy and high-sensitivity measurement cannot be performed because unagglomerated latex particles serve as a large background light as a scatterer and multi-scattered light is measured. As a conventional technique for solving these problems, there is a method of flowing a sample containing aggregates into a flow cell irradiated with laser light, and measuring the size and number of aggregates from the scattered light intensity of each aggregate.
【０００５】しかしながら、この方法はフローセル中に
試料を流して測定するために、凝集塊の形成を時系列的
に測定することができず、凝集反応が終了する時点での
１点計測のみがなされている。すなわち、フローセル法
では、凝集反応終了後の試料をフローセル中に流し、凝
集率と測定対象の生体成分の量との相関から試料中の生
体成分の量を測定しており、定量測定に重要なパラメー
タであるところの、試料に抗体或いは抗原を加えてから
凝集塊が発現するまでの時間（遅延時間）や凝集塊が形
成されて行く速度を測定することができない。However, in this method, since the measurement is carried out by flowing the sample in the flow cell, the formation of agglomerates cannot be measured in time series, and only one point measurement at the time when the agglutination reaction is completed is performed. ing. That is, in the flow cell method, the sample after the agglutination reaction is flowed into the flow cell, and the amount of the biological component in the sample is measured from the correlation between the agglutination rate and the amount of the biological component to be measured, which is important for quantitative measurement. As parameters, it is impossible to measure the time (delay time) from the addition of the antibody or antigen to the sample to the appearance of the aggregate or the speed at which the aggregate is formed.
【０００６】また、フローセル中に個々の凝集塊を分離
して流すために試料の希釈が必要であり、測定時に凝集
塊が解離する可能性があり、精度の高い測定ができない
点や、フローセル中に試料を流すために複雑な制御装置
が必要で、装置の容量が大きくなる等の欠点がある。Further, it is necessary to dilute the sample in order to separate and flow the individual aggregates in the flow cell, and the aggregates may be dissociated at the time of measurement. However, there is a disadvantage that a complicated control device is required to flow the sample through the device, and the capacity of the device becomes large.
【０００７】そこで本発明では、上記のような従来技術
の欠点を解消し、凝集塊の大きさと数とを時系列で測定
できる免疫学的凝集測定装置を提供することにある。Accordingly, an object of the present invention is to provide an immunological agglutination measuring apparatus capable of solving the above-mentioned drawbacks of the prior art and measuring the size and number of aggregates in time series.
【０００８】[0008]
【課題を解決するための手段】上記の課題を解決するた
めに、本発明によれば、生体試料中に含まれる抗原もし
くは抗体と特異的に反応する抗体もしくは抗原と、或い
は、抗体もしくは抗原を付けたラテックス粒子を含む試
料溶液を混合して抗原抗体凝集反応を生じさせることに
より、生体試料中の諸成分の量を測定する免疫学的測定
装置において、前記試料溶液を収容する試料セルと、レ
ーザ光源と、レーザ光源からのレーザ光束をコリメート
して前記試料セルに照射する手段と、前記試料セル中の
抗原及び抗体を含む前記試料溶液を回転撹拌することに
より、抗原抗体凝集反応をほぼ均一に発現させ、且つ、
前記回転撹拌による流れにより凝集塊をレーザ光束中に
通過させる手段と、前記試料セル中の散乱体からの多重
散乱光の受光を防ぐためレーザ光束を前記試料セル内壁
近傍に通過させる手段と、前記試料セルからの散乱光を
受光する受光素子と、受光素子からの信号を評価して抗
原抗体反応による凝集塊の粒子径とその数を時系列的に
計測する手段と、計測された凝集塊の粒子径とその数を
表示する手段とを備えた構成を採用した。According to the present invention, there is provided an antibody or antigen specifically reacting with an antigen or an antibody contained in a biological sample, or an antibody or an antigen. trial, including with latex particles
By causing charge solution by mixing an antigen-antibody agglutination reaction, in immunological measurement apparatus for measuring the amount of the components of the biological sample, a sample cell for accommodating the sample solution, a laser light source, a laser light source means for irradiating a laser beam into collimated to the sample cell from by rotating agitating the sample solution containing the antigen and antibodies in the sample cell, substantially uniformly to express antigen-antibody agglutination reaction, and,
Said means for passing the flow by the rotary stirring aggregates during the laser beam, and means for passing the laser beam into the sample cell near the inner wall to prevent reception of multiple scattered light from scatterers in the sample cell, the A light-receiving element for receiving scattered light from the sample cell; a means for evaluating a signal from the light-receiving element to measure the particle diameter and the number of the aggregates due to the antigen-antibody reaction in a time series; and A configuration including means for displaying the particle diameter and the number thereof was adopted.
【０００９】[0009]
【作用】このような構成によれば、抗原抗体反応による
粒子（凝集塊）の大きさと数とを時系列的に測定でき、
抗原抗体反応によって生ずる凝集塊の粒子の特性を多角
的に測定することが可能になるとともに、高感度の抗原
抗体凝集反応測定が可能になる。According to this configuration, the size and number of particles (agglomerates) due to the antigen-antibody reaction can be measured in time series,
The characteristics of the particles of the aggregate generated by the antigen-antibody reaction can be measured from various angles, and the antigen-antibody agglutination measurement can be performed with high sensitivity.
【００１０】好ましい実施例では、試料セル内壁近傍を
レーザ光束で照射し、試料溶液を撹拌し、計測手段が、
粒子径に対応した上方しきい値と下方しきい値を有し受
光素子からの信号を各しきい値と比較して粒子径を識別
する複数の比較手段と、各比較手段から信号を計測する
カウンタからなり、比較手段の数に対応した粒子径とそ
の数が時系列的に計測される。In a preferred embodiment, the vicinity of the inner wall of the sample cell is irradiated with a laser beam to agitate the sample solution, and the measuring means comprises:
A plurality of comparing means having an upper threshold value and a lower threshold value corresponding to the particle diameter and comparing the signal from the light receiving element with each threshold value to identify the particle diameter; and measuring the signal from each comparing means. It consists of a counter, and the particle diameter and the number corresponding to the number of the comparison means are measured in time series.
【００１１】また、受光素子が測定粒子のほぼ１個から
の散乱光を受光するように構成され、それにより測定精
度を向上させることができる。Further, the light receiving element is configured to receive the scattered light from almost one of the measurement particles, whereby the measurement accuracy can be improved.
【００１２】また、複数個の受光素子が設けられ、その
受光素子に対応した散乱光が同時に測定され、その場
合、受光素子の一対からの出力を減算して有効信号の割
合を増大させることができ、それにより測定時のＳ／Ｎ
比を向上させることができる。Also, a plurality of light receiving elements are provided, and scattered light corresponding to the light receiving elements is measured simultaneously. In this case, the output from a pair of light receiving elements is subtracted to increase the ratio of the effective signal. S / N at the time of measurement
The ratio can be improved.
【００１３】このような免疫学的凝集測定装置は、従来
技術の比濁法および比ろう法による免疫凝集測定装置に
比ベ遥かに測定感度が高く、微量成分の測定が可能とな
る。且つ、フローセル法と比べ、凝集塊粒子の大きさと
数と時系列的測定が可能なため、抗原抗体反応を観察す
る上に多くのパラメータを提供することが可能であり、
装置は簡素で小型軽量化が可能である。[0013] Such an immunological agglutination measuring apparatus has a much higher measurement sensitivity than that of the conventional immunoagglutination measuring apparatus using a turbidimetric method and a turbidimetric method, and can measure a trace component. And, compared to the flow cell method, since the size and number of the aggregated particles can be measured in time series, it is possible to provide many parameters in observing the antigen-antibody reaction,
The device is simple and can be reduced in size and weight.
【００１４】[0014]
【実施例】以下、図面に示す実施例に基づき、本発明を
詳細に説明する。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail based on embodiments shown in the drawings.
【００１５】図１に、本発明を採用した免疫学的凝集測
定装置の構成を示す。図１において、散乱光強度測定の
ための半導体レーザ光源は、駆動回路９により駆動され
てレーザ光を発生する。このレーザ光は、集光レンズ２
によってシート光にコリメートされ、抗原および抗体を
含む試料ガラスセル内の内壁近傍に照射される。FIG. 1 shows the configuration of an immunological agglutination measuring apparatus employing the present invention. In FIG. 1, a semiconductor laser light source for measuring scattered light intensity is driven by a drive circuit 9 to generate laser light. This laser light is transmitted to the condenser lens 2
Is collimated into sheet light, and is irradiated near the inner wall in the sample glass cell containing the antigen and the antibody.
【００１６】試料セル３内の溶液は３７℃の一定温度に
保たれ、スターラーバー（撹拌棒）４とマグネチックス
ターラ５によって１０００ｒｐｍで回転撹拌される。The solution in the sample cell 3 is kept at a constant temperature of 37 ° C., and is rotationally stirred at 1000 rpm by a stir bar (stirring bar) 4 and a magnetic stirrer 5.
【００１７】試料溶液中の凝集塊からの散乱光は、受光
レンズ６を介して複数の受光素子のフォトダイオード８
（８ａ〜８ｄ）によって電気信号として測定される。各
々のフォトダイオードの前部に、統計的に凝集塊１個の
みが測定できる観察領域からの散乱光を受光するために
ピンホール７が配置される。フォトダイオード８の出力
は増幅器１０により電流電圧変換、増幅後、ＡＤ変換器
１１によりＡＤ変換されてコンピュータ１２に入力され
る。The scattered light from the aggregate in the sample solution is passed through a light receiving lens 6 to a plurality of photodiodes 8 of light receiving elements.
(8a to 8d) are measured as electric signals. A pinhole 7 is arranged at the front of each photodiode to receive scattered light from an observation area where only one aggregate can be statistically measured. The output of the photodiode 8 is subjected to current-voltage conversion and amplification by the amplifier 10, then AD converted by the AD converter 11, and input to the computer 12.
【００１８】ＡＤ変換された信号は、図２に示すよう
に、コンピュータにより信号処理される。２個のフォト
ダイオード８ａ、８ｂからの信号は、減算回路１３によ
り一方から他方を減算することによりバックグランド信
号が相殺され、符号１４で示すように凝集塊のみからの
散乱光信号が測定される。このバックグランド信号を除
去された信号１４は絶対値回路１５により絶対値化さ
れ、符号１６のようなバックグランドのないピークのみ
の信号となる。The AD-converted signal is processed by a computer as shown in FIG. The signals from the two photodiodes 8a and 8b are subtracted from one another by the subtraction circuit 13 to cancel the background signal, and the scattered light signal only from the aggregate is measured as indicated by reference numeral 14. . The signal 14 from which the background signal has been removed is converted into an absolute value by an absolute value circuit 15, and becomes a signal having only a peak without a background as indicated by reference numeral 16.
【００１９】この絶対値化された信号１６はぞれぞれ複
数個のウィンドコンパレータ１７に入力され、そのレベ
ルが識別される。各コンパレータは凝集塊の粒径に対応
したレベル比較を行なうので、コンパレータの各出力は
凝集塊の粒径に対応した信号となっている。この信号が
それぞれカウンタ１８でカウントされ、その粒径の凝集
塊の数が計数される。この計数されたデータは演算回路
１９に入力され、後述するように凝集塊の粒径とその数
を表示するためのデータ演算を行なう。なお、コンパレ
ータ、カウンタ、演算回路は、図１のコンピュータ１２
により実装することができる。The absolute value of the signal 16 is input to each of a plurality of window comparators 17 and their levels are identified. Since each comparator performs a level comparison corresponding to the particle diameter of the aggregate, each output of the comparator is a signal corresponding to the particle diameter of the aggregate. Each of these signals is counted by the counter 18, and the number of agglomerates having the particle size is counted. The counted data is input to the arithmetic circuit 19, which performs a data operation for displaying the particle size of the aggregate and the number thereof as described later. The comparator, the counter, and the arithmetic circuit are the same as those of the computer 12 shown in FIG.
Can be implemented.
【００２０】また、図３では、フォトダイオード８ａ、
８ｂの出力信号の処理を説明したが、他のフォトダイオ
ード８ｃ、８ｄについても同様な処理が行われる。この
ような一対の受光素子の凝集塊の計測確率を高めるため
に複数組の受光素子が用いられる。In FIG. 3, the photodiodes 8a,
Although the processing of the output signal of 8b has been described, similar processing is performed for the other photodiodes 8c and 8d. A plurality of sets of light receiving elements are used in order to increase the measurement probability of the aggregate of such a pair of light receiving elements.
【００２１】図１のコンピュータにより凝集塊の粒径と
その数を計数するとともに、式The computer shown in FIG. 1 is used to count the particle size and the number of agglomerates,
【００２２】[0022]
【数２】 (Equation 2)
【００２３】（ただし、Ｋは凝集数、ωｋはｋ個の凝集
塊に付加する重み係数、Ｐｋは凝集数ｋの粒子数、ｎは
２以上の任意の整数）から時間ｔ当たりの凝集塊粒子数
Ｘｔを求める演算を行ない、不図示のディスプレイによ
り結果を表示する。(Where K is the number of agglomerates, ωk is a weighting factor added to the k agglomerates, Pk is the number of particles of the number of agglomerations k, and n is any integer of 2 or more). An operation for calculating the number Xt is performed, and the result is displayed on a display (not shown).
【００２４】次に試料液中の測定対象成分と特異的に結
合する抗体もしくは抗原、あるいは抗体もしくは抗原を
付けたラテックス粒子を試料液中に添加した時点から一
定以上の凝集塊粒子数Ｘｔが計測されるまでの時間ＴL
（遅延時間）と、このＴLと試料中の測定対象成分であ
る抗体もしくは抗原の量（濃度）との相関より目的の成
分の濃度をコンピュータ１２により演算し、表示する。Next, the number Xt of aggregated particles equal to or more than a certain number is measured from the time when the antibody or antigen specifically binding to the component to be measured in the sample solution, or the latex particles with the antibody or antigen added to the sample solution. Time TL
The computer 12 calculates and displays the concentration of the target component from the correlation between the (delay time), the TL and the amount (concentration) of the antibody or antigen which is the component to be measured in the sample.
【００２５】さらに、試料液中の測定対象成分と特異的
に結合する抗体もしくは抗原、あるいは抗体もしくは抗
原を付けたラテックス粒子とを混合して生ずる凝集塊粒
子の生成速度Ｖ（Ｖ＝ｄＸｔ／ｄｔ）の最大値Ｖmax
と、Ｖmax と試料液中の測定対象成分である抗体もしく
は抗原の量（濃度）との相関より目的の成分の濃度をコ
ンピュータ１２により演算し、表示する。Furthermore, the formation rate V of aggregated particles formed by mixing an antibody or antigen specifically binding to the component to be measured in the sample solution or latex particles to which the antibody or antigen is attached (V = dXt / dt) ) Maximum value Vmax
And the computer 12 calculates and displays the concentration of the target component from the correlation between Vmax and the amount (concentration) of the antibody or antigen which is the component to be measured in the sample solution.
【００２６】次に、試料液中の測定対象成分と特異的に
結合する抗体もしくは抗原、あるいは抗体もしくは抗原
を付けたラテックス粒子とを混合して生ずる凝集塊粒子
の数Ｘｔが最大になった時の値Ｘmax と、Ｘmax と試料
液中抗体もしくは抗原の量（濃度）との相関により目的
の成分の濃度をコンピュータ１２により演算し、表示す
る。Next, when the number Xt of agglomerated particles formed by mixing an antibody or antigen specifically binding to the component to be measured in the sample solution, or latex particles to which the antibody or antigen is attached is maximized. The concentration of the target component is calculated by the computer 12 based on the correlation between the value Xmax and the correlation between the Xmax and the amount (concentration) of the antibody or antigen in the sample solution, and displayed.
【００２７】図３に本発明を採用した測定装置によって
イミュノグロブリンＧ（ＩｇＧ）を測定した実施例を示
す。FIG. 3 shows an example in which immunoglobulin G (IgG) was measured by a measuring apparatus employing the present invention.
【００２８】同図の（Ａ）には１．２ｍｇ／ｌのＩｇＧ
を含む血清の測定結果を、（Ｂ）には０．２５ｍｇ／ｌ
のＩｇＧを含む血清の測定結果を、（Ｃ）には、０．０
１３ｍｇ／ｌのＩｇＧを含む血清の測定結果を、（Ｄ）
には、０．０６ｍｇ／ｌのＩｇＧを含む血清の測定結果
を、（Ｅ）には、０．０３ｍｇ／ｌのＩｇＧを含む血清
の測定結果を示してある。FIG. 2A shows 1.2 mg / l of IgG.
(B) shows 0.25 mg / l
(C) shows the results of measurement of serum containing IgG
The measurement results of the serum containing 13 mg / l of IgG were determined by (D)
Shows the measurement results of the serum containing 0.06 mg / l IgG, and (E) shows the measurement results of the serum containing 0.03 mg / l IgG.
【００２９】以上の測定においては、ＩｇＧを含む血清
を反応試料セルに入れ、続いてＩｇＧの抗体を添加して
測定を開始する。図３に示すように、本発明を採用した
測定装置では、コンパレータ１７により粒径がそれぞれ
凝集塊粒度として識別され、各カウンタによりカウント
された粒度の数が凝集塊数として時系列的に時間経過に
したがって表示されている。ディスプレイでの表示は、
図示のようなグラフ形式により行なうことが考えられ
る。In the above measurement, a serum containing IgG is put into a reaction sample cell, and then an IgG antibody is added to start the measurement. As shown in FIG. 3, in the measuring apparatus employing the present invention, the particle size is identified as the aggregate size by the comparator 17, and the number of the particle sizes counted by each counter is time-series as the aggregate number. It is displayed according to. The indication on the display is
It is conceivable to use a graph format as shown.
【００３０】本発明を採用した測定装置では、０．０１
ｍｇ／ｌの極めて低濃度のＩｇＧを含む血清での測定が
可能であり、従来技術の比濁法、比ろう法に比ベ約１０
０倍の高感度測定ができる。In the measuring apparatus employing the present invention, 0.01
It is possible to measure with serum containing an extremely low concentration of IgG of 1 mg / l.
High sensitivity measurement of 0 times is possible.
【００３１】図４に、上記のＩｇＧ測定において演算、
表示された凝集塊総粒子数Ｘｔ、凝集生成速度Ｖｔ、遅
延時間ＴL、最大凝集生成速度Ｖmaxと最大凝集総粒子数
Ｘmax を示す。同図（Ａ）は、０．２５ｍｇ／ｌのＩｇ
Ｇを、（Ｂ）は、０．１２ｍｇ／ｌのＩｇＧを、（Ｃ）
は、０．０６ｍｇ／ｌのＩｇＧを、（Ｄ）は、０．０３
ｍｇ／ｌのＩｇＧをそれぞれ測定した際のＸｔおよびＶ
ｔの変化を示している。FIG. 4 shows the calculation in the above IgG measurement,
The displayed total number of aggregated particles Xt, aggregation generation speed Vt, delay time TL, maximum aggregation generation speed Vmax and maximum aggregated total particle number Xmax are shown. The figure (A) shows 0.25 mg / l of Ig.
G, (B) 0.12 mg / l of IgG, (C)
Represents 0.06 mg / l of IgG, and (D) represents 0.03 mg / l of IgG.
Xt and V when each mg / l of IgG was measured
The change of t is shown.
【００３２】また、図５の（Ａ）は、遅延時間の逆数１
／ＴLとＩｇＧ濃度との相関を、（Ｂ）は最大凝集生成
速度ＶmaxとＩｇＧ濃度との相関を、（Ｃ）は最大凝集
総粒子数Ｘmax のそれぞれとＩｇＧ濃度との相関を示
す。FIG. 5A shows the reciprocal 1 of the delay time.
/ TL and the IgG concentration, (B) shows the correlation between the maximum aggregation generation rate Vmax and the IgG concentration, and (C) shows the correlation between each of the maximum aggregate total particle number Xmax and the IgG concentration.
【００３３】図５の（Ａ）より明らかなように、遅延時
間ＴLの逆数１／ＴLＩｇＧ濃度とは１次の相関を示し、
式 ＴL＝ｋ／［ＩｇＧ］ （ただし、ｋは正の定数、［ＩｇＧ］はＩｇＧの濃度）
により示される。As is apparent from FIG. 5A, the reciprocal of the delay time TL, 1 / TL IgG concentration, shows a first-order correlation,
Formula TL = k / [IgG] (where k is a positive constant and [IgG] is the concentration of IgG)
Is indicated by
【００３４】また、図５の（Ｂ）より明らかなように、
最大凝集生成速度ＶmaxとＩｇＧ濃度は、式As is clear from FIG. 5B,
The maximum aggregation generation rate Vmax and the IgG concentration are calculated by the following equation.
【００３５】[0035]
【数３】 (Equation 3)
【００３６】（ただし、Ｖおよびｋは正の定数）により
示される。(Where V and k are positive constants).
【００３７】また、図５の（Ｃ）より明らかなように、
最大凝集総粒子数Ｘmax のそれぞれとＩｇＧ濃度は、式 Ｘmax ＝ ｋ・log ［ＩｇＧ］ （ただし、ｋは正の定数）により示される。As is clear from FIG. 5C,
Each of the maximum aggregated total particle number Xmax and the IgG concentration is represented by the formula Xmax = klog [IgG] (where k is a positive constant).
【００３８】以上の式のいずれかを用いて、コンピュー
タで演算を行なうことにより、測定されるＴL、Ｖmax、
もしくはＸmaxより血清中のＩｇＧの濃度を算出するこ
とができる。Using any one of the above equations and performing a calculation with a computer, the measured TL, Vmax,
Alternatively, the concentration of IgG in serum can be calculated from Xmax.
【００３９】[0039]
【発明の効果】以上から明らかなように、本発明では、
レーザ光源からのレーザ光束をコリメートして試料セル
に照射する手段と、試料セル中の抗原及び抗体を含む試
料溶液を回転撹拌することにより、抗原抗体凝集反応を
ほぼ均一に発現させ、且つ、前記回転撹拌による流れに
より凝集塊をレーザ光束中に通過させる手段と、試料セ
ル中の散乱体からの多重散乱光の受光を防ぐためレーザ
光束を試料セル内壁近傍に通過させる手段を設け、試料
セルからの散乱光を受光し、受光信号を評価するように
しているので、試料セル中の試料溶液を回転撹拌し、レ
ーザ光束を試料セル内壁近傍に通過させることにより、
同一試料溶液を繰り返し測定し、その試料溶液中の抗原
抗体反応による凝集塊の粒子径とその数を時系列的に計
測することが可能となる。したがって、抗原抗体凝集反
応を測定する場合、高感度に、且つ、時間経過に応じて
生じる凝集塊の大きさと数とを時系列で測定することが
でき、医療分野において血液や尿など生体成分の分析に
有用な免疫学的測定装置を提供することができる。特
に、本発明の免疫学的測定装置は、従来技術の比濁法お
よび比ろう法による免疫凝集測定装置に比ベ遥かに測定
感度が高く、微量成分の測定が可能となる。且つ、フロ
ーセル法と比べ、凝集塊粒子の大きさと数と時系列的測
定が可能なため、抗原抗体反応を観察する上に多くのパ
ラメータを提供することが可能であり、装置は簡素で小
型軽量化が可能である。 As is clear from the above, according to the present invention ,
Sample cell by collimating the laser beam from the laser light source
Means for irradiating the sample and the sample containing the antigen and antibody in the sample cell.
The antigen-antibody agglutination reaction is performed by rotating and stirring the sample solution.
Developed almost uniformly, and in the flow by the rotary stirring
A means for allowing the aggregate to pass through the laser beam,
Laser to prevent the reception of multiple scattered light from scatterers in the
A means for passing a light beam near the inner wall of the sample cell is provided.
Receives scattered light from the cell and evaluates the received light signal
The sample solution in the sample cell is rotated and stirred.
By passing the laser beam near the inner wall of the sample cell,
The same sample solution is repeatedly measured, and the antigen in the sample solution is measured.
Aggregate particle size and number due to antibody reaction are measured in time series
It becomes possible to measure. Therefore, when measuring the antigen antibody agglutination reaction, a high sensitivity, and can be measured in time series and the number and size of agglomerates produced in accordance with the elapsed time, the biological components such as blood and urine in the medical field The present invention can provide an immunological measurement device useful for the analysis of lipase. Special
In addition, the immunological measurement device of the present invention is compatible with the conventional turbidimetry and
Far more than immunoagglutination measuring device
High sensitivity enables measurement of trace components. And flow
Size and number of aggregate particles and time series measurement
Because it is possible to determine the
Parameters can be provided, the equipment is simple and small
The mold can be reduced in weight.
【図１】本発明による免疫学的測定装置の全体構成を示
す構成図である。FIG. 1 is a configuration diagram showing the overall configuration of an immunological measurement device according to the present invention.
【図２】図１の受光素子から得られる散乱強度の変化を
示す波形図である。FIG. 2 is a waveform chart showing a change in scattering intensity obtained from the light receiving element of FIG.
【図３】信号処理の詳細な構成を示すブロック図であ
る。FIG. 3 is a block diagram illustrating a detailed configuration of signal processing.
【図４】本発明装置による免疫凝集反応測定データを表
示した説明図である。FIG. 4 is an explanatory view showing the data of the immunoagglutination reaction measured by the apparatus of the present invention.
【図５】本発明装置による免疫凝集反応測定データを表
示した説明図である。FIG. 5 is an explanatory diagram displaying the data of the immunoagglutination reaction measured by the apparatus of the present invention.
１ 半導体レーザ ２ 集光レンズ ３ 試料セル ４ スターラバー（撹拌棒） ５ マグネチックスターラ ６ 受光レンズ ７ ピンホール ８ フォトダイオード ９ 駆動回路 １０ 増幅器 １１ ＡＤ変換器 １２ コンピュータ １３ 減算回路 １７ ウィンドコンパレータ １８ カウンタ １９ 演算回路 REFERENCE SIGNS LIST 1 semiconductor laser 2 condenser lens 3 sample cell 4 stir bar (stir bar) 5 magnetic stirrer 6 light receiving lens 7 pinhole 8 photodiode 9 drive circuit 10 amplifier 11 AD converter 12 computer 13 subtraction circuit 17 window comparator 18 counter 19 Arithmetic circuit
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭60−243565（ＪＰ，Ａ) 特開 平４−77670（ＪＰ，Ａ) 特開 昭57−175957（ＪＰ，Ａ) 特開 昭59−173759（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 33/543 G01N 15/06 E G01N 15/06 C ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-60-243565 (JP, A) JP-A-4-77670 (JP, A) JP-A-57-175957 (JP, A) JP-A-59-175957 173759 (JP, A) (58) Fields investigated (Int. Cl. 7 , DB name) G01N 33/543 G01N 15/06 E G01N 15/06 C
Claims (9)
と特異的に反応する抗体もしくは抗原と、或いは、抗体
もしくは抗原を付けたラテックス粒子を含む試料溶液を
混合して抗原抗体凝集反応を生じさせることにより、生
体試料中の諸成分の量を測定する免疫学的測定装置にお
いて、前記試料溶液を収容する試料セルと、 レーザ光源と、 レーザ光源からのレーザ光束をコリメートして前記試料
セルに照射する手段と、前記 試料セル中の抗原及び抗体を含む前記試料溶液を回
転撹拌することにより、抗原抗体凝集反応をほぼ均一に
発現させ、且つ、前記回転撹拌による流れにより凝集塊
をレーザ光束中に通過させる手段と、前記 試料セル中の散乱体からの多重散乱光の受光を防ぐ
ためレーザ光束を前記試料セル内壁近傍に通過させる手
段と、前記 試料セルからの散乱光を受光する受光素子と、 受光素子からの信号を評価して抗原抗体反応による凝集
塊の粒子径とその数を時系列的に計測する手段と、 計測された凝集塊の粒子径とその数を表示する手段とを
備えたことを特徴とする免疫学的測定装置。1. An antigen-antibody agglutination reaction is produced by mixing an antigen or an antibody specifically reacting with an antibody or an antigen contained in a biological sample, or a sample solution containing latex particles to which the antibody or the antigen is attached. by irradiation, the immunoassay apparatus for measuring the amount of the components of the biological sample, a sample cell for accommodating the sample solution, a laser light source, the sample cell the laser beams collimated from a laser light source means for, the sample solution containing the antigen and antibodies in the sample cell times
By stirring rolling, antigen-antibody agglutination substantially uniformly to express, and, means for passing the agglomerates <br/> the flow by the rotary stirring during the laser beam, from scatterers in the sample cell Prevents reception of multiple scattered light
Means for passing a laser beam into the sample cell vicinity of the inner wall for a light receiving element for receiving scattered light from the sample cell, the particle size and number of agglomerates evaluates the signals from the light receiving element by antigen-antibody reaction An immunological measurement device comprising: means for measuring the number of particles in time series; and means for displaying the measured particle diameter and the number of aggregates.
した上方しきい値と下方しきい値を有し受光素子からの
信号を各しきい値と比較して凝集塊の粒子径を識別する
複数の比較手段と、各比較手段からの信号を計測するカ
ウンタからなり、比較手段の数に対応した凝集塊の粒子
径とその数が時系列的に計測されることを特徴とする請
求項１に記載の免疫学的測定装置。2. The method according to claim 1, wherein the measuring unit has an upper threshold value and a lower threshold value corresponding to the particle diameter of the aggregate, and compares a signal from the light receiving element with each threshold to determine the particle diameter of the aggregate. A plurality of comparing means for discriminating, and a counter for measuring a signal from each comparing means, wherein the particle diameter and the number of agglomerates corresponding to the number of the comparing means are measured in time series. Item 2. The immunological measurement device according to item 1.
の散乱光を受光するように構成されることを特徴とする
請求項１または請求項２に記載の免疫学的測定装置。3. The immunological measurement device according to claim 1, wherein the light receiving element is configured to receive scattered light from substantially one of the measurement particles.
素子に対応した散乱光が同時に測定されることを特徴と
する請求項１から請求項３までのいずれか１項に記載の
免疫学的測定装置。4. The immunology according to claim 1, wherein a plurality of light receiving elements are provided, and scattered light corresponding to the light receiving elements is measured at the same time. Measuring device.
算して有効信号の割合を増大させることを特徴とする請
求項４に記載の免疫学的測定装置。5. The immunological measurement apparatus according to claim 4, wherein the ratio of the effective signal is increased by subtracting a pair of outputs from the light receiving element.
重み係数、Ｐｋは凝集数ｋの粒子数、ｎは２以上の任意
の整数）から時間ｔ当たりの凝集塊粒子数Ｘｔを求める
ことを特徴とする請求項１から請求項５までのいずれか
１項に記載の免疫学的測定装置。6. The measuring means according to the following equation: (Where K is the number of agglomerates, ωk is a weighting factor added to k agglomerates, Pk is the number of particles with the number of agglomeration k, and n is any integer of 2 or more) The immunological measurement device according to any one of claims 1 to 5, wherein the immunological measurement device is obtained.
らと特異的に結合する抗体もしくは抗原、或いは、抗体
もしくは抗原を付けたラテックス粒子を添加した時点か
ら一定以上の凝集塊粒子数Ｘｔが計測されるまでの時間
ＴＬ（遅延時間）を求め、ＴＬと試料中の抗原もしくは
抗体の量（濃度）との相関から目的の生体成分の濃度を
求めることを特徴とする請求項１から請求項６までのい
ずれか１項に記載の免疫学的測定装置。7. The number Xt of agglutinated particles, which is equal to or more than a certain number, is measured from the time when an antibody or an antigen specifically binding to the antigen or an antibody or latex particles having the antibody or the antigen is added to a sample containing the antigen or the antibody. 7. A time TL (delay time) until the detection is performed, and a concentration of a target biological component is calculated from a correlation between the TL and the amount (concentration) of the antigen or antibody in the sample. The immunological measurement device according to any one of the above.
らと特異的に結合する抗体もしくは抗原、或いは、抗体
もしくは抗原を付けたラテックス粒子とを混合して生ず
る凝集塊粒子の生成速度Ｖ（Ｖ＝ｄＸｔ／ｄｔ）の最大
値Ｖmaxを求め、Ｖmaxと試料中の抗原もしくは抗体の量
（濃度）との相関から目的の生体成分の濃度を求めるこ
とを特徴とする請求項１から請求項７までのいずれか１
項に記載の免疫学的測定装置。8. A production rate V (V) of agglomerated particles formed by mixing an antibody or an antigen specifically binding to the antigen or an antibody or a latex particle to which the antibody or the antigen is attached in a sample containing the antigen or the antibody. = DXt / dt), and the concentration of the target biological component is determined from the correlation between Vmax and the amount (concentration) of the antigen or antibody in the sample. Any one of
Item 14. The immunological measurement device according to Item 1.
いて、抗原もしくは抗体とを含む試料中にそれらと特異
的に結合する抗体もしくは抗原、或いは、抗体もしくは
抗原を付けたラテックス粒子とを混合して生ずる凝集塊
粒子の数Ｘｔが最大になった時の値Ｘmaxを求め、Ｘmax
と試料中の抗原もしくは抗体の量（濃度）との相関から
目的の生体成分の濃度を求めることを特徴とする請求項
１から請求項８までのいずれか１項に記載の免疫学的測
定装置。9. The immunological measurement device according to claim 1, wherein the sample containing the antigen or the antibody is combined with an antibody or an antigen specifically binding thereto or a latex particle provided with the antibody or the antigen. A value Xmax when the number Xt of the agglomerated particles generated by mixing is maximized is determined, and Xmax is determined.
The immunological measurement apparatus according to any one of claims 1 to 8, wherein a concentration of a target biological component is obtained from a correlation between the amount and the amount (concentration) of the antigen or antibody in the sample. .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32503292A JP3283078B2 (en) | 1992-12-04 | 1992-12-04 | Immunological measurement device |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32503292A JP3283078B2 (en) | 1992-12-04 | 1992-12-04 | Immunological measurement device |
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| Publication Number | Publication Date |
|---|---|
| JPH06174724A JPH06174724A (en) | 1994-06-24 |
| JP3283078B2 true JP3283078B2 (en) | 2002-05-20 |
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ID=18172393
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|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101486196B1 (en) * | 2014-07-03 | 2015-01-26 | 고려대학교 산학협력단 | Antibody tissue penetration acceleration technique using pressure |
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| DE69832413T2 (en) * | 1997-11-28 | 2006-07-27 | Compagnie Générale des Etablissements Michelin-Michelin & Cie., Clermont-Ferrand | ALUMINUM-CONTAINING, REINFORCING FILLER AND COMPOSITION COMPRISING THEREOF |
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| AU2009221393B2 (en) * | 2008-03-03 | 2014-01-09 | Koninklijke Philips Electronics N.V. | High resolution classification |
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| TWI708052B (en) * | 2011-08-29 | 2020-10-21 | 美商安美基公司 | Methods and apparati for nondestructive detection of undissolved particles in a fluid |
| JP6488973B2 (en) * | 2015-10-02 | 2019-03-27 | 株式会社島津製作所 | Aggregation measuring apparatus and aggregation measuring method |
| JP2017150963A (en) * | 2016-02-25 | 2017-08-31 | 株式会社島津製作所 | Aggregate measuring method and aggregate measuring apparatus |
| CN110609139B (en) * | 2018-06-14 | 2023-06-30 | 深圳市理邦精密仪器股份有限公司 | Antigen concentration excess detection method, device and storage medium |
-
1992
- 1992-12-04 JP JP32503292A patent/JP3283078B2/en not_active Expired - Lifetime
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101486196B1 (en) * | 2014-07-03 | 2015-01-26 | 고려대학교 산학협력단 | Antibody tissue penetration acceleration technique using pressure |
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|---|---|
| JPH06174724A (en) | 1994-06-24 |
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