JPH06100599B2 - How to measure body fluid components - Google Patents
How to measure body fluid componentsInfo
- Publication number
- JPH06100599B2 JPH06100599B2 JP60141090A JP14109085A JPH06100599B2 JP H06100599 B2 JPH06100599 B2 JP H06100599B2 JP 60141090 A JP60141090 A JP 60141090A JP 14109085 A JP14109085 A JP 14109085A JP H06100599 B2 JPH06100599 B2 JP H06100599B2
- Authority
- JP
- Japan
- Prior art keywords
- antigen
- reaction
- antibody
- body fluid
- carrier
- 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
- 210000001124 body fluid Anatomy 0.000 title claims description 34
- 239000010839 body fluid Substances 0.000 title claims description 34
- 239000000427 antigen Substances 0.000 claims description 83
- 102000036639 antigens Human genes 0.000 claims description 82
- 108091007433 antigens Proteins 0.000 claims description 82
- 238000006243 chemical reaction Methods 0.000 claims description 66
- 239000002245 particle Substances 0.000 claims description 34
- 238000000034 method Methods 0.000 claims description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims description 17
- 239000004816 latex Substances 0.000 claims description 12
- 229920000126 latex Polymers 0.000 claims description 12
- 239000007850 fluorescent dye Substances 0.000 claims description 9
- 239000000969 carrier Substances 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 229920001577 copolymer Polymers 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229920002223 polystyrene Polymers 0.000 description 4
- 230000004520 agglutination Effects 0.000 description 3
- 230000004931 aggregating effect Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 229920002845 Poly(methacrylic acid) Polymers 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- WRVRNZNDLRUXSW-UHFFFAOYSA-N acetic acid;prop-2-enoic acid Chemical compound CC(O)=O.OC(=O)C=C WRVRNZNDLRUXSW-UHFFFAOYSA-N 0.000 description 1
- XECAHXYUAAWDEL-UHFFFAOYSA-N acrylonitrile butadiene styrene Chemical compound C=CC=C.C=CC#N.C=CC1=CC=CC=C1 XECAHXYUAAWDEL-UHFFFAOYSA-N 0.000 description 1
- 229920000122 acrylonitrile butadiene styrene Polymers 0.000 description 1
- 239000004676 acrylonitrile butadiene styrene Substances 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920002689 polyvinyl acetate Polymers 0.000 description 1
- 239000011118 polyvinyl acetate Substances 0.000 description 1
- 229920002102 polyvinyl toluene Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- HJWLCRVIBGQPNF-UHFFFAOYSA-N prop-2-enylbenzene Chemical compound C=CCC1=CC=CC=C1 HJWLCRVIBGQPNF-UHFFFAOYSA-N 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 この発明は、抗原抗体反応を利用した体液成分の測定方
法に関するものである。TECHNICAL FIELD The present invention relates to a method for measuring a body fluid component using an antigen-antibody reaction.
従来の技術 粒状担体に抗体または抗原を付着させ抗原抗体反応を行
わせて体液成分を光学的に測定する方法としては、比濁
法と計数法とが知られている。前者は抗体（または抗
原）を付着（感作）させた担体を含有した溶液に抗原
（または抗体）を含む試料を加えて特異的な抗原抗体反
応に基づく免疫学的凝集作用によって粒子が凝集すると
きの溶液の濁度変化を透過および（または）散乱光の変
化で捉える方法であり、凝集が進むにつれて溶液の濁度
は減少する。一方、後者の方法は本出願人の出願になる
特願昭58−219753号，同59−100090号にみられる方法で
あり、前記と同様にして凝集反応を行わせた反応液をフ
ローセルに流して粒子検出曲線により凝集粒子数を凝集
していない粒子等と弁別しそれぞれの計数比率から試料
の測定値を得るものである。また、これら以外に免疫学
的な沈降反応を利用した一元放射状免疫拡散法（SRID
法），エンザイムイムノアッセイ法（EIA法）,RIA法な
どが従来よりよく知られている。2. Description of the Related Art As a method for optically measuring a body fluid component by allowing an antibody or an antigen to adhere to a granular carrier and causing an antigen-antibody reaction, a nephelometric method and a counting method are known. In the former method, a sample containing an antigen (or antibody) is added to a solution containing a carrier to which an antibody (or antigen) is attached (sensitized), and particles are aggregated by an immunological agglutination action based on a specific antigen-antibody reaction. This is a method of catching the change in the turbidity of the solution by the change in the transmitted and / or scattered light, and the turbidity of the solution decreases as the aggregation progresses. On the other hand, the latter method is the method found in Japanese Patent Application Nos. 58-219753 and 59-1009090 filed by the applicant of the present invention, and the reaction solution obtained by the agglutination reaction is flowed in the flow cell in the same manner as above. The particle detection curve is used to discriminate the number of agglomerated particles from non-aggregated particles and the like, and the measured value of the sample is obtained from the respective counting ratios. In addition to these, the single radial immunodiffusion method (SRID
Method), enzyme immunoassay method (EIA method), RIA method, etc. have been well known.
発明が解決しようとする問題点 上記比濁法や計数法のように粒子の凝集作用を利用する
方法では、必要とする抗原抗体反応が起きても、次に粒
子の凝集にまで至らなければ検知することができないた
め、感度が悪く、また他の要因で凝集が起こり測定誤差
を生じるおそれがあった。Problems to be Solved by the Invention In the method utilizing the aggregating action of particles such as the turbidimetric method and the counting method, even if the required antigen-antibody reaction occurs, it is detected unless the agglutination of particles occurs next time. Therefore, the sensitivity is poor, and aggregation may occur due to other factors to cause a measurement error.
また、その他の測定方法の場合は、測定に長時間を要す
るうえに、放射性物質，酵素などを使用するため測定前
後の処理に特別の注意を要するという問題があった。In addition, in the case of other measurement methods, there is a problem that the measurement takes a long time and special treatment is required before and after the measurement because a radioactive substance, an enzyme and the like are used.
この発明の主たる目的は、叙上の問題を排除し短時間に
かつ高精度で体液成分を定量することができる体液成分
の測定方法を提供することである。この発明の他の目的
は、同時に複数項目の体液成分を定量することができる
体液成分の測定方法を提供することである。A main object of the present invention is to provide a method for measuring a body fluid component which eliminates the above-mentioned problems and can quantify the body fluid component with high accuracy in a short time. Another object of the present invention is to provide a method for measuring a body fluid component, which can simultaneously quantify a plurality of body fluid components.
問題点を解決するための手段 第１の発明の体液成分の測定方法は、担体に抗体を付着
させた試薬を試料体液に加え試料体液中に含有される未
知濃度の抗原と第１の抗原抗体反応を行わせる工程と、
反応後，反応液に標識抗原を加え未反応抗体と第２の抗
原抗体反応を行わせる工程と、第２の抗原抗体反応で得
た反応液から前記標識抗原の前記試薬との反応量を求め
る工程と、得られた反応量を，前記標識抗原のみを前記
試薬と反応させて得られた基準反応量と比較してその変
化量から試料体液中の抗原濃度を定量する工程とを含
み、担体は粒子であり、前記担体に付着させた抗体はモ
ノクローナル抗体であり、前記標識抗原は前記担体に付
着させた抗体と特異的に反応する抗原に蛍光色素を付着
させたものであり、前記標識抗原の反応量は、反応液に
含まれる担体個々に散乱光強度と蛍光強度を検出して求
めた積算蛍光強度より算出されることを特徴とする。Means for Solving the Problems A method for measuring a body fluid component according to the first aspect of the present invention is a method in which a reagent in which an antibody is attached to a carrier is added to a sample body fluid, and an antigen having an unknown concentration contained in the sample body fluid and a first antigen-antibody A step of causing the reaction,
After the reaction, a step of adding a labeled antigen to the reaction solution to carry out a second antigen-antibody reaction with an unreacted antibody, and a reaction amount of the labeled antigen with the reagent is determined from the reaction solution obtained in the second antigen-antibody reaction And a step of comparing the obtained reaction amount with a reference reaction amount obtained by reacting only the labeled antigen with the reagent and quantifying the antigen concentration in the sample body fluid from the change amount thereof, Is a particle, the antibody attached to the carrier is a monoclonal antibody, the labeled antigen is a fluorescent dye attached to an antigen that specifically reacts with the antibody attached to the carrier, and the labeled antigen is The reaction amount is characterized by being calculated from the integrated fluorescence intensity obtained by detecting the scattered light intensity and the fluorescence intensity for each carrier contained in the reaction solution.
また、第２の発明の体液成分の測定方法は、粒径が異な
る複数種の担体にそれぞれ異なる抗原に対する抗体を付
着させた試薬を試料体液に加え試料体液中に含有される
未知濃度の複数種の抗原とそれぞれ第１の抗原抗体反応
を行わせる工程と、反応後，反応液に前記複数種の抗原
にそれぞれ対応する標識抗原を加え未反応抗体と第２の
抗原抗体反応を行わせる工程と、第２の抗原抗体反応で
得た反応液から前記各標識抗原の前記試薬との反応量を
求める工程と、得られた反応量を，前記標識抗原のみを
前記試薬と反応させて得られた基準反応量と比較してそ
の変化量から試料体液中の複数種の抗原濃度を定量する
工程とを含み、担体は粒子であり、前記担体に付着させ
た抗体はモノクローナル抗体であり、前記標識抗原は前
記担体に付着させた抗体と特異的に反応する抗原に蛍光
色素を付着させたものであり、前記標識抗原の反応量
は、反応液に含まれる担体個々に散乱光強度と蛍光強度
を検出して求めた積算蛍光強度より算出されることを特
徴とする。Further, the method for measuring a body fluid component according to the second aspect of the present invention is a method in which a reagent in which antibodies against different antigens are attached to a plurality of types of carriers having different particle sizes is added to a sample body fluid, and a plurality of types of unknown concentrations contained in the sample body fluid are added. And a second antigen-antibody reaction with the unreacted antibody after the reaction by adding labeled antigens respectively corresponding to the plurality of kinds of antigens to the reaction solution after the reaction. And a step of determining a reaction amount of each of the labeled antigens with the reagent from the reaction solution obtained in the second antigen-antibody reaction, and the obtained reaction amount was obtained by reacting only the labeled antigen with the reagent. Quantifying the concentration of a plurality of types of antigens in the sample body fluid from the amount of change compared to the reference reaction amount, the carrier is a particle, the antibody attached to the carrier is a monoclonal antibody, the labeled antigen Attached to the carrier A fluorescent dye is attached to an antigen that specifically reacts with an antibody, and the reaction amount of the labeled antigen is the integrated fluorescence intensity obtained by detecting the scattered light intensity and the fluorescence intensity of each carrier contained in the reaction solution. It is calculated by the following.
前記担体としては、均一な粒径を有する高分子ラテック
スがあげられ、このものは安定な分散媒に浮遊させて互
いに凝集しないようにする。ラテックスの粒径が不均一
なときは、後述のように蛍光強度と散乱光強度とから積
算蛍光強度を求める場合に粒子の径を示す前方散乱強度
がばらつき、データの解析が困難となる。これは、粒径
で項目情報を解析するためである。前記高分子ラテック
スとしては、ポリスチレン，カルボキシル化ポリスチレ
ン，アミノ基を有するカルボキシル化ポリスチテン，ポ
リビニルトルエン，スチレン−ブタジエン共重合体，カ
ルボキシル化スチレン−プタジエン共重合体，スチレン
−ジビニルベンゼン共重合体，ビニルトルエン−第三ブ
チルスチレン共重合体，ポリエステル，ポリアクリル
酸，ポリメタクリル酸，ポリアクリロニトリル，アクリ
ロニトリル−ブタジエン−スチレン共重合体，ポリ酢酸
ビニルアクリレート，ポリビニルピロリドン，塩化ビニ
ル−アクリレート共重合体等の合成高分子ラテックス粒
子からなるラテックスがあげられ、さらにこれらの合成
高分子ラテックス粒子の表面を非イオン界面活性剤等で
処理したものも使用可能である。また、ラテックスのほ
かに、類似の合成高分子物質（たとえばベントナイト，
カオリン，コロジオン等）も使用可能である。Examples of the carrier include a polymer latex having a uniform particle size, which is suspended in a stable dispersion medium so as not to aggregate with each other. When the particle size of the latex is non-uniform, the forward scattering intensity indicating the particle size varies when the integrated fluorescence intensity is calculated from the fluorescence intensity and the scattered light intensity as described later, and data analysis becomes difficult. This is to analyze the item information based on the particle size. Examples of the polymer latex include polystyrene, carboxylated polystyrene, carboxylated polystyrene having an amino group, polyvinyltoluene, styrene-butadiene copolymer, carboxylated styrene-ptadiene copolymer, styrene-divinylbenzene copolymer, vinyltoluene. -Synthesis of tertiary butyl styrene copolymer, polyester, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate acrylate, polyvinylpyrrolidone, vinyl chloride-acrylate copolymer, etc. Examples of the latex include molecular latex particles, and those obtained by treating the surface of these synthetic polymer latex particles with a nonionic surfactant can also be used. Also, in addition to latex, similar synthetic polymeric materials (eg bentonite,
Kaolin, collodion, etc.) can also be used.
担体粒子の表面に付着される抗体としては、特定の抗原
に対してのみ反応する抗体を使用する。かかる抗体とし
ては、モノクローナル抗体が好適に採用可能であり、こ
のものは抗体産生細胞と腫瘍細胞との細胞融合によって
得られた細胞を培養して得られる。このモノクローナル
抗体は単一の抗原決定基にのみ結合するので、ある抗体
が表面にその抗体と結合しうる決定基が１つしかない場
合には、すでにある抗原と結合した抗体はもはや他の抗
原と結合しない。そのため、担体の粒子表面にモノクロ
ーナル抗体を結合させたものは抗原を仲立ちとして粒子
同士が凝集することはない。As the antibody attached to the surface of the carrier particles, an antibody that reacts only with a specific antigen is used. As such an antibody, a monoclonal antibody can be preferably used, and this antibody can be obtained by culturing cells obtained by cell fusion of antibody-producing cells and tumor cells. This monoclonal antibody only binds to a single antigenic determinant, so if one antibody has only one determinant on its surface that can bind to that antibody, the antibody that already binds to that antigen will no longer bind to other antigens. Does not combine with. Therefore, in the carrier in which the monoclonal antibody is bound to the particle surface, the particles do not aggregate with the antigen as an intermediary.
担体表面の抗体が試料体液中の抗原と反応したのち、未
反応の抗体と結合する標識抗原としては、たとえばFITC
（フルオレッセインイソチオシアネート）などの蛍光色
素を付着させた抗原があげられる。ただし、標識の操作
においては、抗原性が失われないように注意することが
必要である。As the labeled antigen that binds to the unreacted antibody after the antibody on the carrier surface reacts with the antigen in the sample body fluid, for example, FITC
(Fluorescein isothiocyanate) and other fluorescent dyes are attached to the antigen. However, it is necessary to be careful not to lose the antigenicity in the operation of labeling.
作用 この発明によれは、担体に付着した抗体に試料体液に含
まれる抗原を反応させる第１の抗原抗体反応を行い、つ
いで試料中の抗原と反応せずに残った未反応抗体に標識
抗原を反応させる第２の抗原抗体反応を行わせるので、
この場合の標識抗原の抗体との反応量を測定し、標識抗
原のみを反応させたときの基準反応量との変化量（減少
量）を求めると、これが試料体液に含まれる抗原量と対
応しており、容易にかつ高精度に抗原濃度の定量を行う
ことができる。Effect According to the present invention, the antibody attached to the carrier is reacted with the antigen contained in the sample body fluid to perform the first antigen-antibody reaction, and then the unreacted antibody remaining without reacting with the antigen in the sample is treated with the labeled antigen. Since the second antigen-antibody reaction to react is performed,
In this case, the reaction amount of the labeled antigen with the antibody is measured, and the amount of change (decrease amount) from the reference reaction amount when only the labeled antigen is reacted is calculated. This corresponds to the amount of antigen contained in the sample body fluid. Therefore, the antigen concentration can be easily and accurately quantified.
反応量の測定は蛍光色素を付着させた標識抗原に基づく
蛍光強度と粒子の散乱光強度とから積算蛍光強度を求め
る。この積算蛍光強度は標識抗原の反応量に対応してお
り、これから試料中に抗原量を知ることができる。粒子
の散乱光強度と蛍光強度はたとえばフローサイトメータ
ーに反応液を流して測定することができる。フローサイ
トメータとしては、たとえば米国特許4325706号明細書
に記載のものが使用可能である。To measure the reaction amount, the integrated fluorescence intensity is obtained from the fluorescence intensity based on the labeled antigen to which the fluorescent dye is attached and the scattered light intensity of the particles. This integrated fluorescence intensity corresponds to the reaction amount of the labeled antigen, and the amount of antigen in the sample can be known from this. The scattered light intensity and fluorescence intensity of the particles can be measured, for example, by flowing the reaction solution through a flow cytometer. As the flow cytometer, for example, the one described in US Pat. No. 4,325,706 can be used.
次に第１図〜第３図に基づいてより詳細に説明する。第
１図は担体表面に結合した抗体に過剰量の標識抗原を作
用させて基準となる積算蛍光強度を求める説明図であ
る。同図において、６はこの発明における試薬であり、
このものはラテックスなどの担体１に抗体２を結合させ
たものである。抗体２は特定の抗原とのみ反応する。使
用する標識抗原３はあらかじめ蛍光色素４を結合させた
ものであり、それぞれが各抗体２と反応しすべての抗体
２と結合する。反応後、反応液をフローサイトメータに
流し蛍光強度，前方散乱光強度および側方散乱光強度を
それぞれ測定する。このとき、溶液は過剰の未反応標識
抗原３を含有しているので、背後蛍光の影響を無視でき
るほど反応液を希釈しなければならない。反応後は、担
体１表面の抗原３が遊離しないように、かつ担体１同士
が結合しないように注意する必要がある。測定された蛍
光強度および散乱光強度から蛍光強度の積算値を求め
る。この積算値は第１図に示す三次元のグラフのピーク
５で示され、このピーク５の体積が積算蛍光強度とな
る。この値は粒子のカウント数が一定ならば、ほぼ一定
である。すなわち、１個の担体１に結合する抗体２の量
は一定であり、これに結合する標識抗原３の量もほぼ一
定であるから、ある一定数の粒子をカウントすると、そ
の総蛍光量（総標識抗原量）もほぼ一定となり、個々の
担体１に結合する標識抗原３の量にばらつきがあって
も、充分に多い数をカウントすれば、総量としては一定
となるのである。なお、小ピーク９は抗体２と結合しな
い遊離の蛍光色素のピークである。Next, a more detailed description will be given with reference to FIGS. FIG. 1 is an explanatory diagram for obtaining an integrated fluorescence intensity as a reference by causing an excess amount of labeled antigen to act on an antibody bound to the surface of a carrier. In the figure, 6 is a reagent in the present invention,
This is one in which the antibody 2 is bound to a carrier 1 such as latex. Antibody 2 reacts only with specific antigens. The labeled antigen 3 to be used is one to which the fluorescent dye 4 has been bound in advance, and each reacts with each antibody 2 and binds to all the antibodies 2. After the reaction, the reaction solution is passed through a flow cytometer to measure the fluorescence intensity, forward scattered light intensity, and side scattered light intensity. At this time, since the solution contains an excess of unreacted labeled antigen 3, the reaction solution must be diluted so that the influence of background fluorescence can be ignored. After the reaction, it is necessary to take care so that the antigen 3 on the surface of the carrier 1 is not released and the carriers 1 are not bound to each other. An integrated value of fluorescence intensity is obtained from the measured fluorescence intensity and scattered light intensity. This integrated value is shown by peak 5 in the three-dimensional graph shown in FIG. 1, and the volume of this peak 5 becomes the integrated fluorescence intensity. This value is almost constant if the particle count is constant. That is, the amount of antibody 2 bound to one carrier 1 is constant, and the amount of labeled antigen 3 bound to this carrier 1 is also almost constant. Therefore, when a certain number of particles are counted, the total fluorescence amount (total The labeled antigen amount) is also substantially constant, and even if the amount of the labeled antigen 3 bound to each carrier 1 varies, the total amount becomes constant if a sufficiently large number is counted. The small peak 9 is a peak of a free fluorescent dye that does not bind to the antibody 2.
試料体液中の抗原濃度を求める場合、この試料体液に前
記試薬６を加え、第２図に示すように試料体液中に含有
される抗原７と第１の抗原抗体反応をおこさせる。つい
で、前記と同じ標識抗原３で第２の抗原抗体反応をおこ
させる。このため、標識抗原３の抗体２への結合量（反
応量，第２図に示すピーク８）は試料に含まれていた抗
原量だけ減少することになる。したがって、求められた
積算蛍光強度は試料中の抗原量に応じて変化し、その変
化量は抗原量と一定の関係にあるので、これから抗原濃
度を定量することができる。To obtain the antigen concentration in the sample body fluid, the reagent 6 is added to the sample body fluid to cause a first antigen-antibody reaction with the antigen 7 contained in the sample body fluid as shown in FIG. Then, a second antigen-antibody reaction is caused with the same labeled antigen 3 as described above. Therefore, the amount of the labeled antigen 3 bound to the antibody 2 (reaction amount, peak 8 shown in FIG. 2) is reduced by the amount of antigen contained in the sample. Therefore, the obtained integrated fluorescence intensity changes in accordance with the amount of antigen in the sample, and the amount of change has a fixed relationship with the amount of antigen, so that the antigen concentration can be quantified from this.
また、第３図に示すように、粒径の異なる複数種の担体
1a,1b,1c…にそれぞれ異なる抗原に対する抗体を付着さ
せると、１つの試料体液に含まれる多数の抗原8a,8b,8c
…を同時に測定することができる。第３図のグラフに示
す各ピーク5a,5b,5c…はそれぞれの抗原の変化量を示し
ている。Moreover, as shown in FIG. 3, a plurality of types of carriers having different particle sizes are used.
When antibodies against different antigens are attached to 1a, 1b, 1c ..., multiple antigens 8a, 8b, 8c contained in one sample body fluid
... can be measured simultaneously. Each peak 5a, 5b, 5c ... Shown in the graph of FIG. 3 shows the amount of change of each antigen.
なお、第１図〜第３図では、三次元のグラフで積算蛍光
強度を示したが、この場合、Ｘ方向の前方散乱光強度で
粒子の径を示しており、これとＺ方向の蛍光強度とで積
算蛍光強度を算出することもできるので、側方散乱強度
の測定は省略してもよい。側方散乱光強度は粒子の性
質，とくに粒子の表面状態と関係がある。In addition, in FIGS. 1 to 3, the integrated fluorescence intensity is shown in a three-dimensional graph. In this case, the particle diameter is shown by the forward scattered light intensity in the X direction, and this and the fluorescence intensity in the Z direction are shown. Since the integrated fluorescence intensity can also be calculated by and, the measurement of the side scatter intensity may be omitted. The side scattered light intensity is related to the properties of particles, especially the surface condition of particles.
以上では、担体に抗体を付着させて試料体液中の抗原量
を測定する場合について説明した。The case where the antibody is attached to the carrier to measure the amount of antigen in the sample body fluid has been described above.
上記説明とは、抗原と抗体の関係が逆になっているが、
以下では担体に抗原が付着され試料体液中の抗体量を測
定する場合について説明する。Although the relationship between the antigen and the antibody is reversed from the above explanation,
The case where the antigen is attached to the carrier and the amount of antibody in the sample body fluid is measured will be described below.
実施例 次に例をあげてこの発明の方法を詳細に説明する。EXAMPLES Next, the method of the present invention will be described in detail with reference to examples.
例：粒径が0.75μのポリスチレンラテックスにウサギの
イムノグロブリンＧ（ウサギIgG）を吸着させた。すな
わち、0.5％のラテックス溶液1ml当り40μｇのウサギIg
Gが吸着したものを調整した。ついで、これに検体中の
抗ウサギIgGを各濃度で反応させた後、蛍光色素（FIT
C）で標識した標識抗ウサギIgGを反応させた。ここで、
ウサギIgGは抗原となり、抗ウサギIgGは抗体となる。反
応後、反応液を第４図に示すフローサイトメーター（本
出願人の製造にかかるAR−II）によって蛍光強度を測定
し、抗ウサギIgG濃度に対する平均蛍光強度を求め、こ
れから第５図に示す検量線を作成した。平均蛍光強度と
はラテックス１個当りの蛍光強度であって、積算蛍光強
度÷カウント数で算出される。Example: Rabbit immunoglobulin G (rabbit IgG) was adsorbed on polystyrene latex having a particle size of 0.75μ. That is, 40 μg of rabbit Ig per ml of 0.5% latex solution
What adsorbed G was adjusted. Then, after reacting this with anti-rabbit IgG in the sample at each concentration, a fluorescent dye (FIT
A labeled anti-rabbit IgG labeled with C) was reacted. here,
Rabbit IgG serves as an antigen and anti-rabbit IgG serves as an antibody. After the reaction, the reaction solution was measured for fluorescence intensity by a flow cytometer (AR-II manufactured by the applicant of the present invention) shown in FIG. 4, and the average fluorescence intensity with respect to the anti-rabbit IgG concentration was determined, which is shown in FIG. A calibration curve was created. The average fluorescence intensity is the fluorescence intensity per latex, and is calculated by the cumulative fluorescence intensity / count number.
フローサイトメーターは、第４図に示すように、Arイオ
ン・レーザ（50mW）の光Ａをフローセル10に導びき、ピ
ンホール板11を経た前方散乱光Ｂを光検出器12で受け、
電気信号に変換して増幅器13へ送る。一方、フローセル
10から放出される蛍光および側方散乱光Ｃは第１および
第２のカラーフィルター14,15を経てピンホール板16を
通り、青反射ダイクロイックミラー17,赤反射（580nm）
ダイクロイックミラー18および前反射ミラー19でそれぞ
れ側方散乱光，赤蛍光および緑蛍光を反射させる。各反
射光は受光フィルタ20,21を通ってそれぞれの受光部22,
23,24（光増倍管,RMT）で受けられる。緑蛍光信号（540
〜580nm）Ｄは増幅器13へ送られる。赤蛍光信号および
側方散乱光信号も同様に、増幅器13へ送られる。増幅器
13へ送られた各信号はデータ解析部25により各粒子の蛍
光強度が測定される。As shown in FIG. 4, the flow cytometer guides the light A of the Ar ion laser (50 mW) to the flow cell 10 and receives the forward scattered light B passing through the pinhole plate 11 at the photodetector 12,
It is converted to an electric signal and sent to the amplifier 13. On the other hand, the flow cell
Fluorescence and side scattered light C emitted from 10 pass through the pinhole plate 16 through the first and second color filters 14 and 15, and are reflected by the blue dichroic mirror 17 and red (580 nm).
The dichroic mirror 18 and the front reflection mirror 19 reflect side scattered light, red fluorescence and green fluorescence, respectively. Each reflected light passes through the light receiving filters 20, 21, and the respective light receiving portions 22,
Received at 23, 24 (photomultiplier tube, RMT). Green fluorescent signal (540
~ 580 nm) D is sent to the amplifier 13. The red fluorescence signal and the side scattered light signal are similarly sent to the amplifier 13. amplifier
The fluorescence intensity of each particle of each signal sent to 13 is measured by the data analysis unit 25.
前記検量線を作成することにより、抗ウサギIgG量が未
知の試料の蛍光強度を検量線に照らし合わせてその濃度
を決定することができる。By preparing the calibration curve, the concentration can be determined by comparing the fluorescence intensity of the sample with an unknown amount of anti-rabbit IgG with the calibration curve.
発明の効果 第１の発明によれば、抗原抗体反応による凝集作用を利
用することなく簡単に、しかもモノクローナル抗体を使
用したことにより、担体同士が凝集することがなく、き
わめて高精度に試料中の抗原の定量を行うことができる
という効果がある。EFFECTS OF THE INVENTION According to the first aspect of the invention, the carrier is not aggregated with each other easily without utilizing the aggregating action due to the antigen-antibody reaction, and because the monoclonal antibody is used, the carrier in the sample can be highly accurately measured. There is an effect that the antigen can be quantified.
また、第２の発明によれば、さらに、粒径の異なる複数
種の担体にそれぞれ異なる抗原に対する抗体を付着させ
た試薬を用いることにより、同時に複数項目の体液成分
の測定が可能になり、しかもモノクローナル抗体を使用
したことにより、担体同士が凝集することがなく、ある
担体の凝集塊と別の未凝集の担体の分布がオーバーラッ
プするということが防止され、担体の種類に対応した各
抗原の濃度を正確に測定することができるという効果が
ある。Further, according to the second invention, by using a reagent in which antibodies against different antigens are attached to a plurality of types of carriers having different particle sizes, it is possible to simultaneously measure a plurality of body fluid components, and The use of a monoclonal antibody prevents the carriers from aggregating with each other and prevents the aggregates of one carrier from overlapping with the distribution of another unaggregated carrier. There is an effect that the concentration can be accurately measured.
第１図および第２図はこの発明の方法を示す説明図、第
３図はこの発明における他の方法を示す説明図、第４図
はこの発明の例におけるフローサイトメーターの説明
図、第５図は検量線を示すグラフである。 １……担体、２……抗体、３……標識抗原、６……試
薬、７……抗原1 and 2 are explanatory views showing a method of the present invention, FIG. 3 is an explanatory view showing another method of the present invention, and FIG. 4 is an explanatory view of a flow cytometer in an example of the present invention. The figure is a graph showing a calibration curve. 1 ... Carrier, 2 ... Antibody, 3 ... Labeled antigen, 6 ... Reagent, 7 ... Antigen
Claims (3)
加え試料体液中に含有される未知濃度の抗原と第１の抗
原抗体反応を行わせる工程と、反応後，反応液に標識抗
原を加え未反応抗体と第２の抗原抗体反応を行わせる工
程と、第２の抗原抗体反応で得た反応液から前記標識抗
原の前記試薬との反応量を求める工程と、得られた反応
量を，前記標識抗原のみを前記試薬と反応させて得られ
た基準反応量と比較してその変化量から試料体液中の抗
原濃度を定量する工程とを含み、担体は粒子であり、前
記担体に付着させた抗体はモノクローナル抗体であり、
前記標識抗原は前記担体に付着させた抗体と特異的に反
応する抗原に蛍光色素を付着させたものであり、前記標
識抗原の反応量は、反応液に含まれる担体個々に散乱光
強度と蛍光強度を検出して求めた積算蛍光強度より算出
されることを特徴とする体液成分の測定方法。1. A step of adding a reagent in which an antibody is attached to a carrier to a sample body fluid to cause a first antigen-antibody reaction with an antigen of an unknown concentration contained in the sample body fluid, and after the reaction, a labeled antigen is added to the reaction solution. And a step of causing a second antigen-antibody reaction with the unreacted antibody, a step of determining the reaction amount of the labeled antigen with the reagent from the reaction solution obtained by the second antigen-antibody reaction, and the obtained reaction amount A step of comparing the labeled antigen alone with the reference reaction amount obtained by reacting the reagent with the reagent and quantifying the antigen concentration in the sample body fluid from the change amount thereof, wherein the carrier is a particle, The attached antibody is a monoclonal antibody,
The labeled antigen is one in which a fluorescent dye is attached to an antigen that specifically reacts with the antibody attached to the carrier, and the reaction amount of the labeled antigen is the scattered light intensity and fluorescence of each carrier contained in the reaction solution. A method for measuring a body fluid component, which is calculated from the integrated fluorescence intensity obtained by detecting the intensity.
粒子である特許請求の範囲第（１）項記載の体液成分の
測定方法。2. The method for measuring a body fluid component according to claim 1, wherein the carrier is latex particles having a uniform particle size.
る抗原に対する抗体を付着させた試薬を試料体液に加え
試料体液中に含有される未知濃度の複数種の抗原とそれ
ぞれ第１の抗原抗体反応を行わせる工程と、反応後，反
応液に前記複数種の抗原にそれぞれ対応する標識抗原を
加え未反応抗体と第２の抗原抗原反応を行わせる工程
と、第２の抗原抗体反応で得た反応液から前記各標識抗
原の前記試薬との反応量を求める工程と、得られた反応
量を，前記標識抗原のみを前記試薬と反応させて得られ
た基準反応量と比較してその変化量から試料体液中の複
数種の抗原濃度を定量する工程とを含み、担体は粒子で
あり、前記担体に付着させた抗体はモノクローナル抗体
であり、前記標識抗原は前記担体に付着させた抗体と特
異的に反応する抗原に蛍光色素を付着させたものであ
り、前記標識抗原の反応量は、反応液に含まれる担体個
々に散乱光強度と蛍光強度を検出して求めた積算蛍光強
度より算出されることを特徴とする体液成分の測定方
法。3. A reagent in which antibodies against different antigens are attached to a plurality of types of carriers having different particle sizes is added to a sample body fluid, and a plurality of types of antigens of unknown concentration contained in the sample body fluid and a first antigen-antibody, respectively. The step of carrying out a reaction, the step of adding a labeled antigen corresponding to each of the plurality of kinds of antigens to the reaction solution after the reaction to carry out a second antigen-antigen reaction with an unreacted antibody, and obtaining the second antigen-antibody reaction Determining the reaction amount of each of the labeled antigens with the reagent from the reaction liquid, and comparing the obtained reaction amount with a reference reaction amount obtained by reacting only the labeled antigen with the reagent Quantifying the concentration of a plurality of types of antigen in the sample body fluid from the amount, the carrier is a particle, the antibody attached to the carrier is a monoclonal antibody, the labeled antigen is an antibody attached to the carrier Antigen that reacts specifically A fluorescent dye is attached, and the reaction amount of the labeled antigen is characterized by being calculated from an integrated fluorescence intensity obtained by detecting scattered light intensity and fluorescence intensity for each carrier contained in the reaction solution. Method for measuring body fluid components.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60141090A JPH06100599B2 (en) | 1985-06-27 | 1985-06-27 | How to measure body fluid components |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60141090A JPH06100599B2 (en) | 1985-06-27 | 1985-06-27 | How to measure body fluid components |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS622163A JPS622163A (en) | 1987-01-08 |
| JPH06100599B2 true JPH06100599B2 (en) | 1994-12-12 |
Family
ID=15283966
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60141090A Expired - Lifetime JPH06100599B2 (en) | 1985-06-27 | 1985-06-27 | How to measure body fluid components |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH06100599B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5407994A (en) * | 1992-10-30 | 1994-05-24 | Cetac Technologies Incorporated | Method for particulate reagent sample treatment |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IN142734B (en) * | 1975-04-28 | 1977-08-20 | Miles Lab | |
| DE2632478A1 (en) * | 1975-07-23 | 1977-02-24 | Coulter Electronics | METHOD FOR DETERMINING AND SEPARATING ANTIGEN AND ANTIBODY IN BLOOD AND OTHER SAMPLES |
| US4284412A (en) * | 1979-07-13 | 1981-08-18 | Ortho Diagnostics, Inc. | Method and apparatus for automated identification and enumeration of specified blood cell subclasses |
-
1985
- 1985-06-27 JP JP60141090A patent/JPH06100599B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS622163A (en) | 1987-01-08 |
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