JPH03216554A - Method and apparatus for particle labelling immunoassay - Google Patents
Method and apparatus for particle labelling immunoassayInfo
- Publication number
- JPH03216554A JPH03216554A JP1060990A JP1060990A JPH03216554A JP H03216554 A JPH03216554 A JP H03216554A JP 1060990 A JP1060990 A JP 1060990A JP 1060990 A JP1060990 A JP 1060990A JP H03216554 A JPH03216554 A JP H03216554A
- Authority
- JP
- Japan
- Prior art keywords
- fine particles
- substance
- particles
- measured
- particle
- 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.)
- Pending
Links
- 239000002245 particle Substances 0.000 title claims description 77
- 238000003018 immunoassay Methods 0.000 title claims description 10
- 238000000034 method Methods 0.000 title abstract description 17
- 238000002372 labelling Methods 0.000 title description 4
- 239000010419 fine particle Substances 0.000 claims abstract description 66
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- 239000000126 substance Substances 0.000 claims abstract description 34
- 238000001179 sorption measurement Methods 0.000 claims abstract description 15
- 239000011859 microparticle Substances 0.000 claims description 23
- 238000005259 measurement Methods 0.000 claims description 13
- 238000012545 processing Methods 0.000 claims description 12
- 239000012491 analyte Substances 0.000 claims description 10
- 238000000684 flow cytometry Methods 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 31
- 102000036639 antigens Human genes 0.000 abstract description 31
- 108091007433 antigens Proteins 0.000 abstract description 31
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000010521 absorption reaction Methods 0.000 abstract 1
- 101000848653 Homo sapiens Tripartite motif-containing protein 26 Proteins 0.000 description 6
- 102000046101 human AFP Human genes 0.000 description 6
- 230000005484 gravity Effects 0.000 description 4
- 239000004816 latex Substances 0.000 description 4
- 229920000126 latex Polymers 0.000 description 4
- 230000004520 agglutination Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- -1 antibodies Substances 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 101710085718 Defensin-like protein 3 Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 101000827785 Homo sapiens Alpha-fetoprotein Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 102000017761 Interleukin-33 Human genes 0.000 description 1
- 108010067003 Interleukin-33 Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000006096 absorbing agent Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
Abstract
Description
本発明は、微粒子を用いた免疫測定方法およびその装置
に関するものである。The present invention relates to an immunoassay method using microparticles and an apparatus therefor.
微粒子を使用した免疫測定方法として、表面に抗体を結
合させたラテックス粒子と抗原とを反応させ、抗原抗体
反応によって生成するラテックス粒子の凝集状態を吸光
度または散乱光強度により測定して抗原濃度を測定する
方法が知られている（ぶんせき，川， ６０５（１９８
７））。しかし，ラテックス粒子の凝集状態は一定では
なく分布を持つため，反応液全体の平均値を測定するこ
れらの方法では、抗原濃度の算出に精度的な問題があり
，極低濃度の抗原量の定量等が困難であった。
そこで、反応液をフローセルに導いて、セル内を流れる
微粒子の散乱光または蛍光強度を測定する方法が開発さ
れた（検査と技術， １６， ６０７（１９８８）、特
開昭６２−８１５６７、ジャーナル オブ イムノロジ
カル メソッズ（Ｊ． Ｉｎ＋＋＋ｕｎｏｌ． Ｍｅｔ
ｈｏｄｓ） ， ＩＬ３３（１９７７））。この方法に
よれば、個々の凝集塊の大きさを計測することができる
ことから、抗原濃度の算出精度を向上させることができ
る。
また、蛍光微粒子を用いて抗原抗体反応を起こさせ、そ
の凝集像を画像処理することにより凝集状態を解析し、
抗原濃度を算定する方法もある（特開昭６４−３５３７
３） ，
上記従来方法は、ホモジニアス系でラテックス粒子表面
の抗体（または抗原）と抗原（または抗体）とが反応し
て凝集をおこすことを利用している。そのため、抗原過
剰領域で抗原抗体反応が抑制される現象、いわゆるプロ
ゾーン現象が避けられない。また、試料中に共存する散
乱体や，色素等の吸収体・蛍光体の影響を完全に除去す
ることは困難である。さらに、抗原と抗体とが１対１に
結合するとは限らないため、凝集塊の数を計数する従来
方式では特に極低濃度領域での計数値の誤差が発生しや
すいという問題がある。
プロゾーン現象や共存物質の影響を受けない方法として
、特開昭５６−１５１３５７のようなヘテロジニアス系
の反応がある。しかし、この方法では簡便性・多項目測
定が可能になるが、非特異吸着について考慮されておら
ず、高感度化に対する配慮が成されていない。As an immunoassay method using microparticles, latex particles with antibodies bound to their surfaces are reacted with the antigen, and the aggregation state of the latex particles generated by the antigen-antibody reaction is measured by absorbance or scattered light intensity to measure the antigen concentration. There is a known method to do this (Bunseki, Kawa, 605 (198
7)). However, since the aggregation state of latex particles is not constant but has a distribution, these methods, which measure the average value of the entire reaction solution, have accuracy problems in calculating the antigen concentration, making it difficult to quantify the amount of antigen at extremely low concentrations. etc. were difficult. Therefore, a method was developed in which the reaction solution is introduced into a flow cell and the scattered light or fluorescence intensity of particles flowing through the cell is measured (Inspection and Technology, 16, 607 (1988), JP-A-62-81567, Journal of Immunological Methods (J. In++++unol. Met
hods), IL33 (1977)). According to this method, since the size of each aggregate can be measured, the accuracy of calculating the antigen concentration can be improved. In addition, the state of agglutination is analyzed by causing an antigen-antibody reaction using fluorescent particles and performing image processing on the agglutination image.
There is also a method to calculate the antigen concentration (Japanese Patent Application Laid-Open No. 64-3537
3) The above conventional method utilizes the fact that antibodies (or antigens) on the surface of latex particles react with antigens (or antibodies) to cause agglutination in a homogeneous system. Therefore, the so-called prozone phenomenon, in which the antigen-antibody reaction is suppressed in the antigen-excess region, cannot be avoided. Furthermore, it is difficult to completely eliminate the influence of scatterers, absorbers, and fluorescent substances such as dyes that coexist in the sample. Furthermore, since antigens and antibodies do not necessarily bind one-to-one, the conventional method of counting the number of aggregates has the problem that errors in counts are likely to occur, especially in extremely low concentration regions. As a method that is not affected by the prozone phenomenon or coexisting substances, there is a heterogeneous reaction as disclosed in JP-A-56-151357. However, although this method is simple and allows measurement of multiple items, it does not take into account non-specific adsorption and does not take into account high sensitivity.
本発明の目的は、丘記従来技術の問題点を解決し、微粒
子を利用した高感度測定ができる免疫測定方法およびそ
の装置を提供することにある。
［１１題を解決するための手段】
上記目的は、測定試料中の被測定物質と特異的に結合す
る物質を固定化した反応容器と，該被測定物質と特異的
に結合する物質を固定化した微粒子（微粒子ａ）と、該
被測定物質と特異的に結合する物質を固定化しない種類
の異なる微粒子（微粒子ｂ）を使用し、該反応容器と測
定試料と該微粒子（微粒子ａおよび微粒子ｂ）を接触さ
せることにより、微粒子を反応容器に捕捉し，捕捉した
微粒子の種類毎の微粒子数を計数し、微粒子ｂ数から、
微粒子ａ数中の非特異吸着によって反応容器に捕捉され
た微粒子数を推定することで、被測定物質のみに依存し
た微粒子数を算定することで達成できる，
微粒子を複数の種類に区別するには，蛍光性の微粒子ま
たは非蛍光性の微粒子を使用することで達成でき、粒径
の違い，または／および蛍光の有無、蛍光波長のちがい
によって識別することができる．例えば、直径が０．５
μｍの非蛍光性の微粒子と直径が０．５μｍの緑色蛍光
を発する蛍光微粒子を使うことで，２種類に微粒子を分
類することができる。また、０．４μｍと０．８μｍの
直径の非蛍光性の微粒子を使うことで，または、０．５
μｍの直径の緑色蛍光を発する蛍光微粒子と０．５μｍ
の直径の赤色蛍光を発する蛍光微粒子とを使うことでも
２種類に微粒子を分類することができる．
反応容器に捕捉した微粒子の種類と数を計測するには、
微粒子の像を画像化し、画像処理することで達成するこ
とができ，顕微鏡、画像入力装置（ＴＶカメラ等）、お
よび画像処理装置から成る装置によって達成できる。ま
ず、画像入力装置により得られた画像をもとに、個々の
微粒子の大きさを測定する。次に、目的の蛍光を検出す
るためのフィルター（例えば干渉フィルター）を通して
各微粒子の蛍光を識別する。このようにして粒径と蛍光
の違いにより複数の微粒子群に分類し、各分類毎の微粒
子数を計数する。
反応容器に捕捉した微粒子の種類と数を計測する別の方
法には、フローサイトメータを使用する方法がある。反
応容器に捕捉された微粒子の結合をはずして反応容器か
ら脱離し、フローサイトメータで微粒子の大きさと蛍光
強度等を測定し、微粒子の種類毎の微粒子数を計数する
。反応容器に捕捉された微粒子の結合をはずすには、機
械的な方法として、振動を与える方法やへら等で削り採
る方法、化学的な方法とレて、尿素で抗体等を変性させ
解離させる方法がある。
測定試料中の被測定物質および被測定物質と特異的に結
合する物質の組合せとしては、抗原（または抗体）と抗
体（または抗原）が代表的な組合せである。その他に、
例えばホルモンとレセブタ、糖とレクチン等の組合せも
可能である。
計数される微粒子としては、ボリスチレン，アクリル、
スチレンーブタジエン共重合体、スチレンーアクリル酸
共重合体等の比重が１〜１．４程度の材質の微粒子が適
当である。比重が１〜１．４程度の粒子のとき、反応が
効果的に行われる。
またその粒径は、５μｍ以下、特に０．１〜１μｍの範
囲が適当である。この粒子の表面に抗体（または抗原）
等を結合させるには、通常知られている物理吸着、化学
結合等が利用できる。
本発明によりヒトα−フェトプロテイン（ＡＦＰ），癌
胎児性抗原（ＣＥＡ）、フェリチン、風疹抗体、エイズ
ウィルス抗体，ヒト絨毛性ゴナドト口ピン（ＨＣＧ）等
種々の抗原、抗体，ホルモン等が測定できる。An object of the present invention is to solve the problems of the prior art and to provide an immunoassay method and apparatus that can perform highly sensitive measurements using microparticles. [Means for Solving Problem 11] The above purpose is to provide a reaction vessel in which a substance that specifically binds to the analyte in the measurement sample is immobilized, and a reaction vessel in which the substance that specifically binds to the analyte in the measurement sample is immobilized. microparticles (fine particles a) and different types of microparticles (fine particles b) that do not immobilize a substance that specifically binds to the analyte are used. ), the particles are captured in a reaction container, the number of particles for each type of captured particles is counted, and from the number of particles b,
Distinguishing fine particles into multiple types can be achieved by estimating the number of fine particles captured in the reaction vessel due to non-specific adsorption in the fine particle a number, and calculating the number of fine particles that depends only on the substance to be measured. , can be achieved by using fluorescent or non-fluorescent particles, and can be distinguished by differences in particle size, the presence or absence of fluorescence, and differences in fluorescence wavelength. For example, if the diameter is 0.5
By using non-fluorescent microparticles with a diameter of μm and fluorescent microparticles with a diameter of 0.5 μm that emit green fluorescence, it is possible to classify the microparticles into two types. In addition, by using non-fluorescent microparticles with diameters of 0.4 μm and 0.8 μm, or 0.5 μm
Fluorescent fine particles that emit green fluorescence with a diameter of μm and 0.5μm
Particles can also be classified into two types by using fluorescent particles that emit red fluorescence with a diameter of . To measure the type and number of particles trapped in the reaction vessel,
This can be achieved by creating an image of the particles and performing image processing, and can be achieved by a device consisting of a microscope, an image input device (such as a TV camera), and an image processing device. First, the size of each fine particle is measured based on an image obtained by an image input device. Next, the fluorescence of each particle is identified through a filter (for example, an interference filter) for detecting the target fluorescence. In this way, particles are classified into a plurality of groups based on differences in particle size and fluorescence, and the number of particles in each classification is counted. Another method for measuring the type and number of particles trapped in a reaction vessel is to use a flow cytometer. The particles captured in the reaction vessel are unbound and released from the reaction vessel, and the size and fluorescence intensity of the particles are measured using a flow cytometer, and the number of particles for each type of particle is counted. To remove the bonds of fine particles captured in the reaction vessel, there are mechanical methods such as applying vibration or scraping with a spatula, and chemical methods such as denaturing and dissociating antibodies etc. with urea. There is. A typical combination of a substance to be measured in a measurement sample and a substance that specifically binds to the substance to be measured is an antigen (or antibody) and an antibody (or antigen). Other,
For example, combinations of hormones and receptors, sugars and lectins, etc. are also possible. The fine particles counted include boristyrene, acrylic,
Fine particles of a material having a specific gravity of about 1 to 1.4, such as styrene-butadiene copolymer or styrene-acrylic acid copolymer, are suitable. The reaction is effectively carried out when the particles have a specific gravity of about 1 to 1.4. The particle size is suitably 5 μm or less, particularly in the range of 0.1 to 1 μm. Antibodies (or antigens) on the surface of this particle
Generally known physical adsorption, chemical bonding, etc. can be used for bonding the like. According to the present invention, various antigens, antibodies, hormones, etc. such as human α-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, rubella antibody, AIDS virus antibody, and human chorionic gonadotopin (HCG) can be measured.
被測定物質と特異的に結合する物質を固定化した微粒子
と，被測定物質と特異的に結合する物質を固定化しない
別の微粒子を同一容器内で反応させることにより、被測
定物質と特異的に結合する物質を固定化した微粒子の非
特異吸着等によるバックグランド微粒子数を容器毎に算
定することができ、被測定物質の量のみに依存した微粒
子数を正確に測定することができ、高感度に被測定物質
を測定することができる。By reacting in the same container microparticles with immobilized substances that specifically bind to the analyte and other particles that do not immobilize the substance that specifically binds with the analyte, the analyte and The number of background particles due to non-specific adsorption of particles immobilized with substances that bind to the substance can be calculated for each container, and the number of particles that depends only on the amount of the substance to be measured can be accurately measured. The substance to be measured can be measured with high sensitivity.
以下、本発明の実施例を、ＡＦＰの測定を例にして説明
する。
［実施例１］
（固定化抗体の調製）
マイクロプレートのウェルを反応容器とし、内面にＡＦ
Ｐに対する抗体を固定化する。平底のマイクロプレート
のウェルに濃度４μｇ ／ ｍ Ｑの抗ヒトＡＦＰ抗体
溶液５０μＱ注入し、２時間間欠的に攪拌して反応させ
て抗ヒトＡＦＰ抗体固定化した。
（微粒子の調製）
標識用微粒子として、表面にカルボキシル基を有し、直
径が０．５μｍで蛍光性（最大蛍光波長が５４０ｎｍ）
のラテックス微粒子と、直径が同じ０．５μｍで非蛍光
性のラテックス微粒子を使用する６蛍光性の微粒子の表
面に抗ヒトＡＦＰ抗体をカルボジイミド法により固定化
した（固定化量約０．７ｍｇ／ｇ）−また、非蛍光性の
微粒子には特に何も固定化しないものを使用した。
（反応手順）
測定抗原であるＡＦＰを含む試料血清５０μＱを抗ヒト
ＡＦＰ抗体を固定化した反応容器（ウェル）に注入して
、２時間反応させ，測定抗原（ＡＦＰ）を反応容器（ウ
ェル）に捕捉する．その後、０．５％ＢＳＡを含むりん
酸緩衝液（０．５％ＢＳＡ−ＰＢＳ）で洗浄する。次に
，抗ヒトＡＦＰ抗体を固定化した蛍光微粒子溶液（微粒
子濃度０．３％）６０μΩと何も固定化しない非蛍光性
の微粒子溶液（微粒子濃度０．３％）６０μＱを注入し
、５時間静置して反応させる。次に，０．５％ＢＳＡ−
ＰＢＳで静かに洗浄し、未反応の微粒子を除去する。
このときの抗原と抗体および微粒子の結合状態は、第１
図のような模式図で表すことができる．反応容器（ウェ
ル）１上に固定化された抗ヒトＡＦＰ抗体２に抗原のヒ
トＡＦＰ３が抗原抗体反応により結合し、さらにヒトＡ
ＦＰ３には，抗ヒトＡＦＰ抗体４を介して蛍光性微粒子
５が結合する．また、抗原を介さないで微粒子が反応容
器（ウェル）上に結合する現象、すなわち非特異吸着も
おこる。例えば、蛍光性微粒子５や非蛍光性微粒子６が
直接、反応容器（ウェル）１または反応容器（ウェル）
１上に固定化された抗ヒトＡＦＰ抗体２等に結合すると
考えられる。抗原に依存する微粒子数は，全体の粒子数
から非特異吸着の粒子数を減じた数になる。
（粒子計測手順）
第２図に平底のマイクロプレートのウェルに捕捉した微
粒子の径と数を計測する装置の概略図を示す。装置は、
（倒立型の）蛍光顕微鏡７、顕微鏡像を読み取るＴＶカ
メラ８、像を解析する画像処理装置９，蛍光顕微鏡の蛍
光検出用フィルタを測定対象に応じて切り替えるフィル
タ変換装［１０、画像処理装置９とフィルタ変換装置１
０等を制御するコントローラー１１、画像を出力するモ
ニターテレビ１２、処理結果を出力する出力装置１３と
で構成される。
微粒子の捕捉されたマイクロプレートのウェル１を蛍光
顕微鏡７のステージに載せ，ウェル１内の微粒子１５の
顕微鏡像をＴＶカメラ８で観察し，画像処理して粒子の
種類を識別し、粒子数を計数する。
まず、透過光でウェル内の粒子像をｉ［する。
ＴＶカメラ８で検出している像を８ビットにディジタル
化して、画像処理装置９の画像メモリに蓄積する。この
操作を６４フレーム行い，像のＳ／Ｎを良くする。得ら
れた画像データから微粒子を識別する。径が０．４μｍ
〜０．６μｍの範囲の像を微粒子として認識し、その総
数を測定する。
なお、０．４μｍより小さいものおよび０．６μｍより
大きいものは、ゴミ等と判断して計数から除外した。測
定された微粒子の総数は蛍光性および非蛍光性の微粒子
の総数であり、抗原と結合した微粒子と非特異吸着によ
って結合している微粒子の総数である。この総数をＮ１
とする。
次に、５４０ｎｍの蛍光を発する顕微鏡像を測定する。
フィルタ変換装置１０で、５４０ｎｍの光を透過し、そ
れ以外の光を遮断する干渉フィルターを顕微鏡にセット
する。そのときの蛍光像をＴＶカメラ８で検出し、像の
強度を８ビットにディジタル化して画像処理装置９の画
像メモリに蓄積する。なお、この操作を６４フレーム行
い、蛍光像のＳ／Ｎを良くした。この画像より，蛍光を
発している微粒子の総数を計算する。この総数をＮ２
とする。Ｎ２は、測定抗原ヒトＡＦＰに結合している蛍
光微粒子（総数をＮ，とする）と非特異吸着で結合して
いる蛍光微粒子（総数をＮ４とする）の和であり，
Ｎ２＝Ｎ，＋Ｎ４
となる。
また、
？，＝Ｎ■−Ｎ２
は、非特異吸着によって結合している非蛍光性微粒子の
総数となる。Ｎ４ （非特異吸着で結合している蛍光微
粒子の総数）はＮ５と強い相関があり、あらかじめＮ４
とＮ，との関係を測定抗原ヒトＡＦＰがＯのときの値か
ら求めておくことで、Ｎ，＝ｋＸＮ， （ｋは定
数）
と計算できる，つまり、測定抗原であるヒトＡＦＰに結
合している蛍光微粒子の総数Ｎ，は，Ｎｆｆ ＝ Ｎ！
ｋ ｘ （ ＮＩ Ｎ，）となり、このようにす
ることで、非特異吸着の影響を受けずに、測定抗原量の
みに依存する微粒子数を測定することができる，
本方法により測定抗原（ヒトＡＦＰ）濃度を定量するに
は、あらかじめ濃度が既知の試料で検量線をつくり、そ
れぞれの粒子数から濃度を算定すれば良い。
［実施例２］
実施例１と同じようにして、固定化抗体を調製し、微粒
子を調製し、反応させて、反応容器上に微粒子を結合さ
せる。
微粒子は、フローサイトメータで計測する。そこで、微
粒子の結合している反応容器に濃度８Ｍの尿素０．２ｍ
ｌを注入して微粒子の結合をはがし、微粒子懸濁液を得
る。この微粒子懸濁液をフローサイトメータで解析する
。散乱光強度と蛍光？度を同時に測定することで、微粒
子の有無，微粒子の大きさ、および微粒子が蛍光性か非
蛍光性かを識別する。散乱光強度より計算された微粒子
径が０．３μｍ相当以下のものは、ゴミ等の可能性が高
く、計数から除外した。本実施例で使用した標識用の微
粒子の大きさは蛍光性と非蛍光性のどちらも０．５μｍ
径であるため、微粒子の種類の判別はその蛍光強度で行
う。
第３図は、検出された微粒子の蛍光強度に対する数量を
示すヒストグラムである。ヒストグラムは２つの分布を
示す。蛍光強度の小さい方が非蛍光性の微粒子を示して
おり、その総数をＮ■とする。蛍光強度の大きい方が蛍
光微粒子を示し，その総数をＮ２ とする。実施例１と
同じように、測定抗原であるヒＩ− Ａ Ｆ Ｐのみに
結合している蛍光微粒子の総数（Ｎ．とする）は、
Ｎ，＝Ｎ２−ｋＸ （Ｎ，−Ｎ２）
となり、このようにすることで、非特異吸着の影響を受
けずに、測定抗原量のみに依存する微粒子数を測定する
ことができる。
本方法により測定抗原（ヒトＡＦＰ）濃度を定量するに
は、あらかじめ濃度が既知の試料で検量線をつくり，そ
れぞれの粒子数から濃度を算定すれば良い。
なお、実施例エないし実施例２での微粒子標識抗体溶液
の微粒子の濃度は、０．０１％から５％程度が適当であ
る。比重のやや大きいアクリル系等の微粒子を使用した
場合は、その微粒子濃度は、０．０１％から１％程度が
適当となる。その他の微粒子についても、微粒子濃度は
微粒子の比重の大きさや粒径等によって決定される．
本実施例によれば、測定抗原と標識物である微粒子との
結合比率が一定となるため、精度が高く、高感度な定量
が可能になる。また、抗原過剰領域で抗原抗体反応が抑
制される現象、いわゆるプロゾーン現象が生じないため
、高濃度の抗原濃度域での定量も可能である。Examples of the present invention will be described below using AFP measurement as an example. [Example 1] (Preparation of immobilized antibody) A well of a microplate was used as a reaction container, and AF was placed on the inner surface.
Immobilize antibodies against P. 50 μQ of an anti-human AFP antibody solution at a concentration of 4 μg/mQ was injected into the wells of a flat-bottomed microplate, and reacted with intermittent stirring for 2 hours to immobilize the anti-human AFP antibody. (Preparation of fine particles) Fine particles for labeling have carboxyl groups on the surface, have a diameter of 0.5 μm, and are fluorescent (maximum fluorescence wavelength is 540 nm).
An anti-human AFP antibody was immobilized on the surface of fluorescent microparticles using the carbodiimide method (immobilized amount of approximately 0.7 mg/g). )-Furthermore, non-fluorescent fine particles on which nothing was immobilized were used. (Reaction procedure) Inject 50μQ of sample serum containing AFP, which is the antigen to be measured, into a reaction container (well) on which anti-human AFP antibody has been immobilized, and let it react for 2 hours, and then add the antigen to be measured (AFP) into the reaction container (well). Capture. Thereafter, it is washed with a phosphate buffer containing 0.5% BSA (0.5% BSA-PBS). Next, 60 μΩ of a fluorescent microparticle solution immobilized with anti-human AFP antibody (particle concentration 0.3%) and 60 μQ of a non-fluorescent microparticle solution (particle concentration 0.3%) without any immobilization were injected for 5 hours. Let stand and react. Next, 0.5% BSA-
Gently wash with PBS to remove unreacted particles. At this time, the binding state of the antigen, antibody, and microparticle is
It can be represented schematically as shown in the figure. The antigen human AFP3 binds to the anti-human AFP antibody 2 immobilized on the reaction container (well) 1 through an antigen-antibody reaction, and further human AFP
Fluorescent microparticles 5 bind to FP3 via anti-human AFP antibody 4. In addition, a phenomenon in which fine particles bind to the reaction container (well) without the intermediary of the antigen, ie, non-specific adsorption, also occurs. For example, the fluorescent fine particles 5 and the non-fluorescent fine particles 6 are directly connected to the reaction vessel (well) 1 or the reaction vessel (well).
It is thought that the antibody binds to the anti-human AFP antibody 2 etc. immobilized on 1. The number of particles depending on the antigen is the total number of particles minus the number of non-specifically adsorbed particles. (Particle measurement procedure) Figure 2 shows a schematic diagram of an apparatus for measuring the diameter and number of particles captured in the wells of a flat-bottomed microplate. The device is
An (inverted) fluorescence microscope 7, a TV camera 8 that reads the microscope image, an image processing device 9 that analyzes the image, a filter conversion device [10] that changes the fluorescence detection filter of the fluorescence microscope according to the measurement target, and an image processing device 9 and filter conversion device 1
It is composed of a controller 11 that controls the 0, etc., a monitor television 12 that outputs images, and an output device 13 that outputs processing results. The well 1 of the microplate containing the captured particles is placed on the stage of the fluorescence microscope 7, the microscopic image of the particles 15 in the well 1 is observed with the TV camera 8, the type of particles is identified through image processing, and the number of particles is calculated. Count. First, an image of the particles in the well is captured using transmitted light. The image detected by the TV camera 8 is digitized into 8 bits and stored in the image memory of the image processing device 9. This operation is performed for 64 frames to improve the image S/N. Identify particles from the obtained image data. Diameter is 0.4μm
Images in the range of ~0.6 μm are recognized as fine particles, and the total number thereof is measured. Note that particles smaller than 0.4 μm and particles larger than 0.6 μm were judged to be dust and the like and were excluded from counting. The total number of microparticles measured is the total number of fluorescent and non-fluorescent microparticles, and the total number of microparticles bound to antigen and microparticles bound by non-specific adsorption. This total number is N1
shall be. Next, a microscope image emitting fluorescence at 540 nm is measured. Using the filter conversion device 10, an interference filter that transmits 540 nm light and blocks other light is set in the microscope. The fluorescent image at that time is detected by the TV camera 8, and the intensity of the image is digitized into 8 bits and stored in the image memory of the image processing device 9. Note that this operation was performed for 64 frames to improve the S/N of the fluorescent image. From this image, the total number of particles emitting fluorescence is calculated. This total number is N2
shall be. N2 is the sum of fluorescent particles bound to the measurement antigen human AFP (total number N) and fluorescent particles bound by non-specific adsorption (total number N4), N2 = N, +N4 becomes. Also, ? ,=N■-N2 is the total number of non-fluorescent fine particles bound by non-specific adsorption. N4 (total number of fluorescent fine particles bound by non-specific adsorption) has a strong correlation with N5.
By calculating the relationship between and N, from the value when the antigen to be measured, human AFP, is O, it is possible to calculate N, = kXN, (k is a constant). The total number of fluorescent particles, N, is Nff = N!
k ) To quantify the concentration, create a calibration curve with samples whose concentrations are known in advance, and calculate the concentration from the number of particles for each. [Example 2] In the same manner as in Example 1, an immobilized antibody is prepared, fine particles are prepared, and reacted to bind the fine particles onto a reaction vessel. Fine particles are measured using a flow cytometer. Therefore, 0.2 m of urea with a concentration of 8 M was added to the reaction vessel containing the fine particles.
1 is injected to detach the bonds between the fine particles and obtain a fine particle suspension. This fine particle suspension is analyzed using a flow cytometer. Scattered light intensity and fluorescence? Simultaneously measuring the particle size identifies the presence or absence of particles, the size of the particles, and whether the particles are fluorescent or non-fluorescent. Particles with a diameter of 0.3 μm or less calculated from the intensity of scattered light were likely to be dust, and were excluded from counting. The size of the labeling particles used in this example was 0.5 μm for both fluorescent and non-fluorescent particles.
Since it is based on the diameter, the type of particle can be determined based on its fluorescence intensity. FIG. 3 is a histogram showing the quantity of detected fine particles relative to their fluorescence intensity. The histogram shows two distributions. The one with lower fluorescence intensity indicates non-fluorescent fine particles, and the total number thereof is assumed to be N■. The one with higher fluorescence intensity indicates fluorescent fine particles, and the total number thereof is set as N2. As in Example 1, the total number of fluorescent particles (denoted as N) that bind only to human I-AFP, which is the antigen to be measured, is N,=N2-kX (N,-N2), By doing so, it is possible to measure the number of microparticles that depends only on the amount of antigen to be measured without being affected by non-specific adsorption. In order to quantify the concentration of the antigen to be measured (human AFP) using this method, it is sufficient to prepare a calibration curve using samples whose concentrations are known in advance, and calculate the concentration from the number of particles of each sample. In addition, the concentration of fine particles in the fine particle-labeled antibody solution in Examples D to 2 is suitably about 0.01% to 5%. When using fine particles of acrylic or the like having a rather large specific gravity, the appropriate concentration of the fine particles is about 0.01% to 1%. Regarding other fine particles, the fine particle concentration is determined by the specific gravity and particle size of the fine particles. According to this example, since the binding ratio between the antigen to be measured and the fine particles as the labeling substance is constant, highly accurate and sensitive quantification is possible. Furthermore, since the so-called prozone phenomenon, in which the antigen-antibody reaction is suppressed in an antigen-excess region, does not occur, quantification in a high antigen concentration range is also possible.
本発明によれば、反応容器毎に非特異吸着等に基づいて
結合する微粒子数を計数することができることから、非
特異吸着等に影響されずに測定物質に結合した微粒子数
を正確に計数することができ、高感度に抗原濃度を定量
することができる。According to the present invention, since it is possible to count the number of fine particles bound to each reaction vessel based on non-specific adsorption, etc., it is possible to accurately count the number of fine particles bound to the measurement substance without being affected by non-specific adsorption, etc. It is possible to quantify the antigen concentration with high sensitivity.
第１図は本発明の一実施例における抗原と微粒子標識抗
体の結合状態を示す模式図、第２図は本発明の一実施例
になる顕微画像処理装置の構成のを示すブロック図、第
３図は本発明の他の実施例において、フローサイトメー
タで測定した微粒子の、蛍光強度に対する数量を示すヒ
ストグラムである。
符号の説明
１・・・反応容器（ウェル），２・・・抗ヒトＡＦＰ抗
体、３・・・ヒトＡＦＰ、４・・・抗ヒトＡＦＰ抗体、
５・・・微粒子、６・・・微粒子（例えば０．５μｍ径
の非蛍光性微粒子）、７・・（倒立型）蛍光顕微鏡，８
・・・ＴＶカメラ、９・・・画像処理装置、１０・・・
フィルタ変換装置． １１・・・コントローラー、ｌ２
・・モニターテレビ、第
躬
第
？
閉
ｌ．ダFIG. 1 is a schematic diagram showing the binding state of an antigen and a particle-labeled antibody in one embodiment of the present invention, FIG. 2 is a block diagram showing the configuration of a microscopic image processing apparatus in one embodiment of the present invention, and FIG. The figure is a histogram showing the quantity of microparticles measured with a flow cytometer with respect to fluorescence intensity in another example of the present invention. Explanation of symbols 1... Reaction container (well), 2... Anti-human AFP antibody, 3... Human AFP, 4... Anti-human AFP antibody,
5... Fine particles, 6... Fine particles (for example, non-fluorescent particles with a diameter of 0.5 μm), 7... (Inverted) fluorescence microscope, 8
...TV camera, 9...Image processing device, 10...
Filter conversion device. 11...controller, l2
...Monitor TV, first episode? Closed l. da
Claims (1)
固定化した反応容器と、該被測定物質と特異的に結合す
る物質を固定化した微粒子と、該被測定物質と特異的に
結合する物質を固定化しない該微粒子と種類の異なる微
粒子を使用し、該反応容器と測定試料と該微粒子を接触
させることにより、微粒子を反応容器に捕捉し、捕捉し
た微粒子の種類毎の微粒子数を計数し、該被測定物質と
特異的に結合する物質を固定化しない微粒子の数から、
該被測定物質と特異的に結合する物質を固定化した微粒
子の非特異吸着数を推定し、被測定物質濃度を定量する
粒子標識免疫測定方法。 ２、蛍光性の微粒子または非蛍光性の微粒子を使用する
ことを特徴とする特許請求の範囲第１項記載の粒子標識
免疫測定方法。 ３、微粒子の大きさによって、または／および微粒子の
蛍光波長によって、複数の種類に識別することのできる
微粒子を使用することを特徴とする特許請求の範囲第１
項または第２項記載の粒子標識免疫測定方法。 ４、画像入力装置と、画像入力装置により得られる微粒
子の像を画像処理する装置とからなる粒子標識免疫測定
装置。 ５、フローサイトメトリにより、微粒子を解析処理する
ことを特徴とする特許請求の範囲第１項または第２項ま
たは第３項記載の粒子標識免疫測定方法。 ６、個々の微粒子の大きさ、または／および蛍光波長を
計測して複数の種類に分類し、同じ種類の微粒子毎にそ
の数を計数する処理を行うことを特徴とする特許請求の
範囲第４項または第５項記載の粒子標識免疫測定方法ま
たは装置。[Scope of Claims] 1. A reaction container on which a substance that specifically binds to the analyte in a measurement sample is immobilized, fine particles on which a substance that specifically binds to the analyte is immobilized, and the analyte. By using fine particles of a different type from the fine particles that do not immobilize a substance that specifically binds to the measurement substance, and by bringing the reaction vessel and the measurement sample into contact with the fine particles, the fine particles are captured in the reaction vessel, and the captured fine particles are captured. The number of particles of each type is counted, and from the number of particles that do not immobilize a substance that specifically binds to the analyte,
A particle-labeled immunoassay method for quantifying the concentration of a substance to be measured by estimating the number of non-specific adsorption of microparticles on which a substance that specifically binds to the substance to be measured is immobilized. 2. The particle-labeled immunoassay method according to claim 1, characterized in that fluorescent fine particles or non-fluorescent fine particles are used. 3. Claim 1, which uses fine particles that can be distinguished into a plurality of types depending on the size of the fine particles and/or the fluorescence wavelength of the fine particles.
The particle label immunoassay method according to item 1 or 2. 4. A particle label immunoassay device consisting of an image input device and a device that performs image processing on a particle image obtained by the image input device. 5. The particle label immunoassay method according to claim 1, 2, or 3, characterized in that the microparticles are analyzed by flow cytometry. 6. Claim 4, characterized in that the size and/or fluorescence wavelength of each fine particle is measured, the particles are classified into a plurality of types, and the number of fine particles of the same type is counted. 6. The particle-labeled immunoassay method or device according to item 5.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1060990A JPH03216554A (en) | 1990-01-22 | 1990-01-22 | Method and apparatus for particle labelling immunoassay |
| DE19904036288 DE4036288A1 (en) | 1989-11-15 | 1990-11-14 | Immunological dye - using marker particles carrying antigen or antibody reactive with analyte previously immobilised on vessel wall |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1060990A JPH03216554A (en) | 1990-01-22 | 1990-01-22 | Method and apparatus for particle labelling immunoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03216554A true JPH03216554A (en) | 1991-09-24 |
Family
ID=11754986
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1060990A Pending JPH03216554A (en) | 1989-11-15 | 1990-01-22 | Method and apparatus for particle labelling immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03216554A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011517775A (en) * | 2008-04-02 | 2011-06-16 | アボット ポイント オブ ケア インコーポレイテッド | Methods for serological agglutination immunoassays and other immunoassays performed on thin film body fluid samples |
| WO2013146694A1 (en) * | 2012-03-28 | 2013-10-03 | コニカミノルタ株式会社 | Method for detection biological substance |
| JP2022521672A (en) * | 2019-01-30 | 2022-04-12 | スチョウ アストラバイオ テクノロジー カンパニー リミテッド | Single molecule quantitative detection method and detection system |
-
1990
- 1990-01-22 JP JP1060990A patent/JPH03216554A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011517775A (en) * | 2008-04-02 | 2011-06-16 | アボット ポイント オブ ケア インコーポレイテッド | Methods for serological agglutination immunoassays and other immunoassays performed on thin film body fluid samples |
| US8569076B2 (en) | 2008-04-02 | 2013-10-29 | Abbott Point Of Care, Inc. | Method for serologic agglutination and other immunoassays performed in a thin film fluid sample |
| WO2013146694A1 (en) * | 2012-03-28 | 2013-10-03 | コニカミノルタ株式会社 | Method for detection biological substance |
| US9632081B2 (en) | 2012-03-28 | 2017-04-25 | Konica Minolta, Inc. | Detection method for biological substance |
| JP2022521672A (en) * | 2019-01-30 | 2022-04-12 | スチョウ アストラバイオ テクノロジー カンパニー リミテッド | Single molecule quantitative detection method and detection system |
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