JPH01272970A - Laser magnetic immunoassay method and measuring instrument and labeling superparamagnetic material used for laser magnetic immunoassay and its production - Google Patents

Abstract

PURPOSE:To enable the inspection of an antigen-antibody reaction with good efficiency and high sensitivity by identifying an unreacted labeling superparamagnetic material and the labeling superparamagnetic material-specimen complex after the antigen-antibody reaction by a difference in the arrival time of laser light at a concn. position. CONSTITUTION:The labeling superparamagnetic material 7 is obtd. by immobilizing an antibody 2 consisting of an immune globulin antibody to protein A coated on the surface of the superfine superparamagnetic material particles 1. On the other hand, the surface of nonmagnetic microspheres 5 consisting of an acrylic polymer having 1mum grain size is activated and thereafter, the antibody 4 consisting of the immune globulin antibody similar to the immune globulin antibody of the antibody 2 is adsorbed on the surface thereof to obtain a nonmagnetic antibody complex 8. Influenza virus 6 existing in a patient's mouth wash is used as a specimen and the antigen-antibody reaction is effected in the liquid contg. this virus 6, the labeling material 7 and the complex 8. A ferromagnetic field is applied to the liquid during this reaction to uniformize the directions of easy magnetization of the complex 9 of the labeling material 7, the complex 8 and the virus 6. The concn. position is then irradiated by the laser light and the labeling material 7 and the complex 9 are identified from the difference in the arrival time thereof.

Description

【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、抗原抗体反応を利用し、極めて微量の検体か
ら特定の抗体または抗原を定量的に検出することのでさ
゛るレーザ磁気免疫測定方法及び測定’ＩＡ　Ｆｌ　ｉ
ｌＤびにレー膏ア磁気免疫測定に用いる超常磁性体標識
体及びその製造り法に関づるものである。Detailed Description of the Invention [Industrial Application Field] The present invention relates to a laser magnetic immunoassay method and a method for quantitatively detecting a specific antibody or antigen from an extremely small amount of a sample by utilizing an antigen-antibody reaction. Measurement'IA Fl i
The present invention relates to a superparamagnetic label used in ID and laser magnetic immunoassays and a method for producing the same.

（従来技術およびその課題〕 （（天性免疫不全症候群、成人子細胞白血病等のような
新型ウィルス竹疾病、あるいは各種ガンの〒期検査払と
して、抗原抗体反応を利用した免疫測定法の開発が、現
在、Ｉ１１界的規模Ｃ′推進されている。(Prior art and its problems) ((The development of an immunoassay method using antigen-antibody reactions as a preliminary test for new viral bamboo diseases such as congenital immunodeficiency syndrome, adult cell leukemia, etc., and various cancers. Currently, I11 is being promoted on an international scale C'.

従来から知られる微ｆ７＋免疫測定方法としては、ラジ
オイムノアラレイ（以Ｆ、ＲＩＡ法と記′？ｌ）、酸素
イムノアラレイ（［ＩΔ）、蛍光イムノアラレイ（「１
△）法等が既に実用化されている。これらの方法は、そ
れぞれアイソ１〜−ブ、酵素、蛍光物質を標識としτ伺
加しＩご抗原または抗体を用い、これと特巽的に反応Ｊ
−る抗体または抗原の有無を検出づる方法である。Conventionally known fine f7+ immunoassay methods include radioimmunoarrray (hereinafter referred to as RIA method), oxygen immunoarray ([IΔ), and fluorescent immunoarray ([IΔ]).
△) Laws, etc. have already been put into practical use. These methods use antigens or antibodies labeled with iso-1, enzymes, or fluorescent substances, respectively, and are specifically reacted with the antigen or antibody.
This method detects the presence or absence of antibodies or antigens.

ところが、ＲＩＡ払は高い検出感度を有しているものの
、標識に放射性物質を使用するために、その実施につい
ては多くの制約がある。Ｊ、た、ＥＩＡ法及びＦ７Ａθ
、はいずれし実施についての制約がＲｒＡ法に比べて少
イ；　＜　、その実施は容易であるが、検１Ｊ１感瓜が
低く、精密イｆ定早的測定が周到であった。However, although RIA detection has high detection sensitivity, there are many restrictions on its implementation because radioactive substances are used as labels. J, T, EIA method and F7Aθ
However, there are fewer restrictions on implementation than the RrA method; it is easy to implement, but test 1J1 sensitivity was low, and precise and early measurements were required.

本発明者らは、上記の免疫測定方法の欠点を克服Ｊべく
、上記方法とは原理を異にりるレーザ磁気免疫測定方法
等の研究を行ない、その成果を先に特願昭６１−２２４
５６７．６１−２５２／１２７．６１−２５４１６＝１
．６２−２２０６２．６２−２２０６３．６２’−１５
２７９１，６２−１５２７９２，６２−１８／ｌ　９０
２．６２−２６／１３１９．６２−２６７４８１として
特許出願している。これらの新しい免疫測定方法は抗原
抗体反応の有無の検出にレーザ光を利用し、標識材料と
してＩ６磁性微粒子を用いる点に特徴があり、アイソ１
〜−プを用いないでピコグラムの超微量検出が可能であ
る。In order to overcome the drawbacks of the above-mentioned immunoassay methods, the present inventors conducted research on a laser magnetic immunoassay method that differs in principle from the above-mentioned methods, and the results were first published in Japanese Patent Application No. 61-224.
567.61-252/127.61-25416=1
．． 62-22062.62-22063.62'-15
2791,62-152792,62-18/l 90
A patent application has been filed as No. 2.62-26/1319.62-267481. These new immunoassay methods are characterized by the use of laser light to detect the presence or absence of antigen-antibody reactions, and the use of I6 magnetic particles as the labeling material.
It is possible to detect ultra-trace amounts of picograms without using a ~-p.

ところで、このようなレーＩｆ磁気免疫測定ｆｊＦＡに
おいては、標識材料としての磁性体微粒子に１つの抗原
あるいは抗体をイ」加した磁性体標識体と、この磁性体
標識体と検体たる抗体あるいは抗原とを抗原抗体反応さ
せた磁性体標識検体複合体とを磁力などによって識別し
、未反応の磁性体標識体を分離・除去づるにうにしてい
る。この分離・除去操作は比較的煩わしいものであり、
この操作を行く【わずに磁性体標識検体複合体のみを識
別して検出できれば、イのメリッ１〜は甜り知れない。By the way, in such a RayIf magnetic immunoassay fjFA, a magnetic label in which one antigen or antibody is added to magnetic fine particles as a labeling material, and this magnetic label and an antibody or antigen as a specimen are used. The antigen-antibody reaction is performed on the magnetically labeled sample complex, which is distinguished by magnetic force, and the unreacted magnetically labeled substance is separated and removed. This separation/removal operation is relatively troublesome;
If only the magnetically labeled specimen complex could be identified and detected without performing this operation, the advantages of step (1) would be immeasurable.

また、上）ホした磁性体微粒子を細胞の分離や薬剤の運
搬に用いる方ｄ１は以前から知られている。Furthermore, the method d1 of using magnetic fine particles as described in (a) above for separating cells and transporting drugs has been known for a long time.

例エバ、ｊ′Ｓ＜ハ、１９７５年出ＪＷｉのｃ：ａｅｖ
ｅｒの発明ににる米国性ｇ’＋　！’！　３９７０５１
８　”　Ｍａｇｎｅｔｉｃｓｅｐａｒａｔｉｏｎ　ｏｆ
　ｂｉｏｌｏｇｉｃａｌ　ｐａｒｔｉｃｌｅｓ　”には
、コロイド粒子リイズから１０μｍの大ぎざの、フ丁ラ
イ１〜、ペロブスカイ１〜、クロマイ１へ、マグネトプ
ランバイ］〜等のフＪＤ磁性、フ１り磁性月利に抗体を
被覆し一０１細胞等を分離する方法が開示されている。Example Eva, j′S<ha, c: aev of JWi released in 1975
American g'+ in the invention of er! '! 397051
8” Magnetic separation of
"Biological particles" include antibodies to colloidal particles with large 10 μm serrations, F-JD magnetic, F-1, F-1, etc. A method for coating and isolating 101 cells and the like is disclosed.

しかし、現在までのところ、細胞の分離や薬剤の運搬（
，１研究段階であり、はとんど実用化されていない。However, until now, cell separation and drug delivery (
, 1 is at the research stage and has hardly been put into practical use.

さて、細胞の分離・１′）薬剤の運搬等に用いられるこ
の種の磁性標識材料として、今までなされた発明とイの
概要を月代順に組節的に列挙すると、Ｉ’記のごとくで
ある。Now, if we list in chronological order the summaries of the inventions and A's that have been made so far as this type of magnetic labeling material used for cell separation, 1') drug transport, etc., it will be as shown in I'. be.

（１）　Ｄａｖｉｅｓら米国特ｆｌ第４１７７２５３“
Ｍａｇｎｅｔｉｃ　ｐａｒｔｉｃｌｅ　ｆｏｒ　ｉｍｍ
ＵＩ（ＯａＳＳａｙ”には低密度の＝１７月１′８１の
表面にＮＬ等の金属磁性材料と、抗１ｊｒｊ、抗体なと
の生物的に活性な物質とを被覆した、粒径が１μｍから
１　ｃｍの複合磁性粒子が記載されている。(1) Davies et al. US Pat. No. 4,177,253"
Magnetic particle for imm
UI (OaSSay) has a particle size ranging from 1 μm to 1 μm, coated with a metal magnetic material such as NL and biologically active substances such as anti-1jrj and antibodies on the surface of low-density = 17/1'81. cm composite magnetic particles are described.

（２）　Ｈｏ１ｄａＶの米国特許用４４５２７７３”Ｍ
ａｇｎｅｔｉｃ　１ｒｏｎ−ｄｅｘｔｒａｎ　ｍ１ｃｒ
ｏｓｐｈｃｒｅｓ”にはデキス１〜ラン等で被覆された
フＪＯ磁性体のマグネタイ１ル微粒子であって、好まし
い粒径どして３０〜４ＱＩｌｌｌｌの−ｂのが記載され
ている。(2) Ho1daV US Patent No. 4452773”M
agnetic 1ron-dextran m1cr
osphcres" describes magnetite 1 fine particles of FJO magnetic material coated with dex 1 to run, etc., with a preferred particle size of 30 to 4 QIllll.

（３）　Ｃｚｅｒｌｉｎｓｋｉの米国特許用４４５４２
３　’１“Ｃｏ１１ｌｅｄ　ｍａｇｎｅｔｉｚａｂｌｅ
　ｍ１ｃｒｏｐａｒｔｉｃｌｅｓ。(3) Czerlinski U.S. Patent No. 44542
3'1“Co11led magnetizable
m1croparticles.

ｒｅｖｅｒｓｉｂｌｅ　５ｕｓｐｅｎｓｉｏｎｓ　ｔｈ
ｅｒｅｏｆ、　ａｎｄｐｒｏｃｅｓｓｅｓ　ｒｅｌａｔ
ｉｎｇ　ｔｂｅｒＯｔｏ″′にはキ］り一温度が５〜６
５℃の範囲にあるフエライ１〜、イソ１ヘリウム鉄ガー
ネッ１〜等の強磁性材料の微粒子の表面をアクリルアミ
ドをベースにした」を重合ポリマー等で被覆され、磁区
の人きさから０．１μｍＦ？度まで・の磁性微粒子が記
載されている。reversible 5uspensions th
ereof, and processes relat
ing tberOto''' has a temperature of 5 to 6
The surface of fine particles of ferromagnetic material such as ferrite 1 ~, iso 1 helium iron garnet 1 ~, etc. in the range of 5℃ is coated with a polymeric polymer etc. based on acrylamide, and the magnetic domain is 0.1 μm F ? Magnetic particles of up to 100% have been described.

（４）　Ｉｋｅｄａらの米国特許用４５８２６２２“Ｍ
ａｇｎｅｔｉｃ　ｐａｒｔｉｃｕｌａｔｃ　ｆｏｒ　ｉ
ｍｍｏｂｉｌｉｚａｔｉｏｎ　ｏｆｂｉｏｌｏｇｉｃａ
ｌ　ｐｒｏｔｅｉｎ　ａｎｄ　ｐｒｏｃｅｓｓ、　ｏｆ
　ｐｒｏｄｕｃｔｉｎｇｔｌｌｅ　Ｓａｍｅ　”にはじ
ラブンを主成分とし、０．００００１〜２％のフ］−ラ
イｌ−からなるフｘＤ磁性体を含有する粒径３μ７ｎ稈
度の粒子が記載されている。(4) Ikeda et al. U.S. Patent No. 4582622“M
agnetic particulate for i
mmmobilizationofbiologica
l protein and process, of
"Producing Same" describes particles having a grain size of 3 μ7n and a culm degree containing Nijilabun as a main component and a FxD magnetic material consisting of 0.00001 to 2% fly l-.

（５）　Ｈａｒｇｅｌの米国特許用１１３２４９２３“
Ｍｅｔａｌ　ｃｏａｔｅｄ　ｐｏｌｙａｌｄｅｈｙｄｅ
　ｍ１ｃｒｏｓｐｈｅｒｅｓ”には遷移金属で被覆され
たボリアルアヒト微小球であって、＋１ｆｉｌ性材料ど
して強■竹体の鉄、ニッケル、コバルトが記載されてい
る。(5) Hargel's U.S. Patent No. 11324923
Metal coated polyaldehyde
"m1crospheres" describes boreal atom microspheres coated with transition metals, and strong iron, nickel, and cobalt as +1fil materials.

以上（１）〜（５）の各磁性相お１は小さくとも３Ｑｎ
ｍ以上の粒径をもつノ■−ロ磁性あるいはフＪり磁性の
もので、これらはいずれも強磁性月利に分類されるもの
である。この強磁性月利はその材料種にＪン）で異なる
が、通常数トｎｍ以上の粒径で、外部磁界を取り去った
後も残留磁化がみられるものである。一方、磁性材料に
は、外部磁界を取り去ると磁化が残らない性質をもち、
上記強磁性材料より粒径が小さい私営磁性月ｒ１がある
。そして、これら超富磁性祠籾と強磁性材料とは、ヒス
テリシス曲線ヤ）帯磁率の測定、あるいはメスバウワー
効果などが全＜巽イ【るものであり、従来の細胞の分離
や薬剤の運搬には、標識に用いる磁性粒子は弱い磁力で
６効果的に誘導される必要があるので、この目的には強
磁性月利が最も適しく−いるが、以小に詳述りる本発明
に従う新しい非分＃を法によるレー（ア磁気免疫測定法
では磁性標識体は単独では磁力で誘導されにくいことが
必要であるので、この目的のためには私営磁竹材利が最
も適している。Each of the magnetic phases (1) to (5) above is at least 3Qn
They are ferromagnetic or ferromagnetic with a particle size of m or more, and are classified as ferromagnetic. This ferromagnetic yield varies depending on the type of material, but the grain size is usually several tons or more, and residual magnetization is observed even after the external magnetic field is removed. On the other hand, magnetic materials have the property that no magnetization remains when the external magnetic field is removed.
There is a private magnetic moon r1 which has a smaller particle size than the above ferromagnetic material. These super-magnetic grains and ferromagnetic materials are highly sensitive to hysteresis curves, magnetic susceptibility measurements, and the Mössbauer effect, and are not suitable for conventional cell separation or drug delivery. Since the magnetic particles used in the labeling need to be effectively guided by weak magnetic forces, ferromagnetic particles are most suitable for this purpose, but the new magnetic particles according to the present invention, which will be described in detail below, are suitable for this purpose. In the magnetic immunoassay method, it is necessary that the magnetic label alone is difficult to be induced by magnetic force, so privately-produced porcelain bamboo materials are most suitable for this purpose.

本発明は、上記の事情に鑑みてなされたちのひ、その目
的とするところはＲＩＡ法以上の検出感度を右しながら
、検体調製が簡便で、かつ実施１の制限のない新規なレ
ーザ磁気免疫測定方法及び測定装首ｊ１９びにレーザ磁
気免疫測定に好適に用いることのできる超常磁性体標識
体及びその製造方法を提供づることにある３゜ 〔課題を解決するための手段〕 本発明の第１の発明に従うと、超常磁性体超微粒子に１
つの抗原あるいは抗体をイ・」加した超常磁性体標識体
と、検体たる抗体あるいは抗原とを抗原抗体反応ざゼる
第１−１皿稈と、該第１］−稈後の超＝　　９　− 常磁性体標識体と検体との複合体である超常磁性体標識
体（Ａ複合体を含む溶液に磁界を作用さｔ！（該超常磁
性体標識検体複合体を定められた位置に誘導・濃縮ざ甘
る第２■稈と、未反応の該超常磁性体標識体ど抗原抗体
反応後の該超常磁性体標識検体複合体とを濃縮（ｌ／首
への到）ヱ１時間差により識別刀る」二稈を少なくとも
含むことを特徴どづるレーザ磁領免疫測定方法が提供さ
れる。The present invention was made in view of the above circumstances, and its purpose is to provide a novel laser magnetic immunotherapy that has detection sensitivity higher than that of the RIA method, has simple specimen preparation, and does not have the limitations of Embodiment 1. 3. [Means for Solving the Problems] The first aspect of the present invention is to provide a superparamagnetic label that can be suitably used for a measurement method, a measurement neck j19, and a laser magnetic immunoassay, and a method for producing the same. According to the invention, superparamagnetic ultrafine particles have 1
A superparamagnetic labeled substance to which one antigen or antibody has been added, and a 1-1 plate culm in which an antigen-antibody reaction occurs between the antibody or antigen as a specimen, and the superparamagnetic substance after the culm = 9 - A magnetic field is applied to a solution containing a superparamagnetic label (complex A), which is a complex of a paramagnetic label and an analyte (the superparamagnetic label analyte complex is guided to a predetermined position and concentrated). Distinguish the second culm from the unreacted superparamagnetic substance-labeled substance and the superparamagnetic substance-labeled sample complex after the antigen-antibody reaction by concentrating (l/reaching to the neck) 1 hour difference. '' A laser magnetic domain immunoassay method is provided, which comprises at least two culms.

また、第２の発明に従うと、第１の発明の第１■稈にお
い−Ｃ１抗原抗体反応を磁界中で行ない、抗原抗体反応
後の超ＩＩけ１体標識検体複合体の磁化方向を揃える処
Ｊシ１をＭづことを特徴と一す゛るレー（ｆ磁気免疫測
定方法が提供される。Further, according to the second invention, the first culm-C1 antigen-antibody reaction of the first invention is carried out in a magnetic field, and the magnetization direction of the super II single-labeled specimen complex is aligned after the antigen-antibody reaction. A magnetic immunoassay method is provided which is characterized in that J and M are combined.

これら第１または第２の発明において、超常磁性体標識
体と超常磁性体標識検体複合体との濃縮位置への到）ヱ
時間差は、レーザ光を濃縮位置へ照則し、濃縮位置から
の散乱光、透過光、反射光、干渉光、回折光〜゛の出用
光を検出することにより得られる３、このとぎ、検出感
度の向−にのために、レーザ光の走引周波数に同期した
信号成分を選択−１〇　− 的に検出力る方法を用いてもＪ：い。まＩＣ１第２丁稈
にｄ３いて濃縮位置と非濃縮位置とにレーリ゛光を同１
１、“ｌあるいは時系列的に照則し、イの出射光を検出
し、非濃縮位置からのア′−夕を比較対照として検体ｆ
ｊ’ｊを定Ｈ＋づる方法を用いてもよい３゜さらに、第
３の発明に従うと、超常磁性体超微粒子に１′つの抗原
あるいは抗体を伺加した超常磁性体標識体と、該超常磁
性体標識体と検体たる抗体あるい【３１抗原とを抗ハ５
（抗体反応さけｌＣ超富磁刺体標識検体複合体とを含む
溶液を収容づる検査容器と、該超常磁性体標識検体複合
体を検査容器の１点に誘導・濃縮する傾斜磁界発生装置
と、シー１ア光を検査容器の超常磁性体標識検体複合体
のＷＡ縮位首へ導く入射光学系と、超常磁性体標識検体
複合体からの出射光及び超常磁性体標識検体複合体を含
まない溶液からの出射光をそれぞれ受光する光学系とを
少なくとも含むレーザ磁気免疫測定装置であって、上記
超常磁性体標識検体複合体と未反応の超常磁性体標識体
とを識別づる機構を具備したことを特徴とＪるレーザ磁
気免疫測定装置が提供される。In these first or second inventions, the time difference between the superparamagnetic label and the superparamagnetic labeled sample complex to reach the concentration position is determined by directing the laser beam to the concentration position and scattering from the concentration position. This is obtained by detecting the output light of light, transmitted light, reflected light, interference light, diffracted light. It is not possible to use a method that selectively detects signal components. Also, d3 is placed in the second culm of IC1, and the same ray light is applied to the concentration position and non-concentration position.
1. Detect the emitted light of ``1'' or chronologically, detect the emitted light of
Furthermore, according to the third invention, a superparamagnetic label in which 1' antigen or antibody is added to superparamagnetic ultrafine particles, and the superparamagnetic The antibody labeled body and the sample antibody or [31 antigen] are
(A test container containing a solution containing a superparamagnetic target labeled specimen complex for antibody reaction; a gradient magnetic field generator that guides and concentrates the superparamagnetic labeled specimen complex to one point in the test container; An entrance optical system that guides the SE1A light to the WA neck of the superparamagnetically labeled specimen complex in the test container, the exit light from the superparamagnetically labeled specimen complex, and a solution that does not contain the superparamagnetically labeled specimen complex. A laser magnetic immunoassay device comprising at least an optical system for receiving the emitted light from each of A laser magnetic immunoassay device having the following characteristics is provided.

なお、抗原抗体反応としては、検体ど超常磁性体標識体
とを直接反応させる直接法、あるいは既知の抗原あるい
は抗体を非磁性微小球の表面に固定化しておき、Ｊ１磁
性微小球と検体とを反応させたのら、超常磁性体標識体
を加えて検体と超常磁性体標識体とを反応させる（ノン
ドイツｙ法などを用いることができる。The antigen-antibody reaction can be carried out by a direct method in which the sample or superparamagnetic label is reacted directly with the sample, or by immobilizing a known antigen or antibody on the surface of a non-magnetic microsphere and then combining the J1 magnetic microspheres with the sample. After the reaction, a superparamagnetic label is added and the sample is reacted with the superparamagnetic label (a non-German method or the like can be used).

また、第３の発明にａ３いて、識別機構【ま、未反応の
超常磁性体標識体からの出射光の経時変化を基準どじで
反応後の超常磁性体標識検体複合体からの出射光の経時
変化を変化づる電子回路部により構成されるのが望まし
い。また、傾斜磁界発生装置Ｎ　ＬＪ、’　、電磁石と
、この電１ｆ（’″ｉに対向しかつ検査容器を挟むよう
に配置された磁極Ｊ４とから構成され、電磁石ど磁極片
のいずれか又は検査容器が水平面内で移動可能に構成さ
れるのが好ましい。In addition, in a3 of the third invention, the identification mechanism [well, the time course of the emitted light from the superparamagnetic material labeled sample complex after the reaction is based on the time course of the emitted light from the unreacted superparamagnetic material labeled material. It is preferable to include an electronic circuit section that changes the change. In addition, the gradient magnetic field generator N LJ,' is composed of an electromagnet and a magnetic pole J4 which is arranged to face the electric field 1f ('''i and sandwich the inspection container between them, and either of the magnetic pole pieces of the electromagnet or the inspection Preferably, the container is configured to be movable in a horizontal plane.

また、第４の発明に従うと、第１または第２の発明に従
うレーザ磁気免疫測定方法に用いる標識体であって、超
常磁性体からなる超微粒子の表面が生物的に活性な物質
で被覆され、該被覆層に抗原あるいは抗体が固定化され
Ｃなることを特徴とするレー１１磁気免疫測定用超常磁
竹体標識体が提供される。Further, according to a fourth invention, the surface of ultrafine particles made of a superparamagnetic substance is coated with a biologically active substance, in the label used in the laser magnetic immunoassay method according to the first or second invention, There is provided a superparamagnetic bamboo label for Ray-11 magnetic immunoassay, characterized in that an antigen or antibody is immobilized on the coating layer.

ざらに、第５の発明に従うと、超常磁性体超微粒子の表
面にｔ１５性な物質からイ≧る被覆層を形成・］る王稈
と、被覆層が形成された磁個体超微粒子のうち私営性体
のみを分画１・回収づる工程と、超常磁性体超微粒子の
被覆層に抗原あるいは抗体を固定化−する工程とを少な
くとも含むことを特徴とり−るレー量ア磁気免疫測定用
超常磁性体標識体の製造方法が提供される。。Roughly speaking, according to the fifth invention, among the magnetic solid ultrafine particles on which the coating layer has been formed, there is a king culm that forms a coating layer of t15 material on the surface of the superparamagnetic ultrafine particles, and a privately produced magnetic solid ultrafine particle on which the coating layer is formed. A superparamagnetic material for magnetic immunoassays characterized by comprising at least a step of fractionating and collecting only the magnetic substance, and a step of immobilizing an antigen or antibody on a coating layer of superparamagnetic ultrafine particles. A method for producing a labeled body is provided. .

ここで、上記の超常磁性体標識体に用いられる超常磁性
体超微粒子（，１、放射線あるいはａＩ性等の問題を有
しないことはいうよでもなく、これを利用づることに格
別の制約は＜＞い。また、この超常磁性体超微粒子は、
あらゆる強磁性体を超微粒子に１）ることにＪ：つて得
ることが出来る。例えば、強！ｉ性体としては、マグネ
タイ１〜やγ−フエライ１〜等の各種化合物磁性体、あ
るいは鉄、ニラクル、＝　１３− ＝Ｉパル１−等の金属磁性体の中から適宜選択できる。Here, it goes without saying that the superparamagnetic ultrafine particles used in the above-mentioned superparamagnetic label (1) do not have any problems such as radiation or aI properties, and there are no particular restrictions on the use of these particles. > Yes. Also, these superparamagnetic ultrafine particles are
Any ferromagnetic material can be obtained by converting it into ultrafine particles (1). For example, strong! The i-type material can be appropriately selected from various compound magnetic materials such as magnetite 1- and γ-ferrite 1-, or metal magnetic materials such as iron, niracle, and =13-=I-pal 1-.

これらの強磁性＋、ｌ利を超微粒子にする方法どしては
、機械的粉砕法を除く、従来から知られている、各種の
気相法、液相法を用いることが出来る。例えば、ガス中
蒸介θいシー１蒸発熱蒸発法、共沈法等が適用できる１
、これらの気相法、液相法で作成した超微粒子【３１、
超常磁性体と強磁竹林粒子が混在しており、超常磁性体
のみを分離・回収づる必要がある。分離・回収づるには
、機械的、化学的、物理的な各種方法が適用できる。例
えば、遠心分離、液体クロマ１〜グラフイ、磁気フィル
タ等の方法などがある。超常磁性体超微粒子の粒径は強
磁性祠料ににって異なるが、単磁区粒子の限界寸法以下
で４丁ければならない。例えば、マグネタイ１〜、γ−
フエライ１−の場合（。ｔｉｏｎｍ以下、純鉄の場合は
３ｎｍ以下が好ましい。As a method for making these ferromagnetic materials into ultrafine particles, various conventionally known gas phase methods and liquid phase methods can be used, excluding mechanical pulverization methods. For example, heat of evaporation in gas, evaporation method, co-precipitation method, etc. can be applied.
, ultrafine particles created by these gas phase methods and liquid phase methods [31,
Superparamagnetic material and ferromagnetic bamboo forest particles coexist, and it is necessary to separate and collect only the superparamagnetic material. Various mechanical, chemical, and physical methods can be applied to separation and recovery. For example, there are methods such as centrifugation, liquid chromatography, and magnetic filters. The particle size of the superparamagnetic ultrafine particles differs depending on the ferromagnetic abrasive, but it must be less than the critical size of single-domain particles and must be 4 particles. For example, magnetite 1~, γ-
In the case of Ferrite 1- (. tionm or less, in the case of pure iron, it is preferably 3 nm or less.

このような超常磁性体超微粒子は、その表面に抗原ある
いは抗体を固定化するため、生物的に活性な物質で被覆
され−Ｃいる。生物的に活性な物質としては例えばデキ
ス１〜ラン等の糖や、プロティー１／ｌ・　− ンＡ４９の蛋白あるいはメヂルメタクリレ−１〜のよう
な高分子皮膜などが用いられる３、そして、抗原あるい
は抗体は生物的に活性な物質の表面に固定化される。Such superparamagnetic ultrafine particles are coated with a biologically active substance in order to immobilize antigens or antibodies on their surfaces. Examples of biologically active substances that can be used include sugars such as dex1-ran, proteins such as protein 1/l-A49, and polymeric coatings such as medyl methacrylate. Antibodies are immobilized on the surface of biologically active substances.

そし−Ｃ１」二記超常磁性体標識体は、凍結乾燥しＣ長
ＪＪｌ保存ηることがｕ１来る。標識試桑としで用いる
場合、標識体を界面活性イオ判を添加した水溶液中に分
散さμればよい１．また、凝集を抑制りる界面活性剤を
添加して、溶液状態で冷所保存り−ることもできる。界
面活性剤としては、例えば「［ＩＳＡの洗浄液としても
用いられるＴｗｃｅｎ　２０．あるいはビッタ−図形法
で磁区観察に用いられるマグネタイト磁性コロイド分散
用のラウリルアミン、セラー１−ル（Ｓｏｄｉｕｍ　ｃ
ａｒｂｏｘｙ　ｍｅｔｈｙｌ　ｃｅｌｌｕｌｏｓｅ　）
、あるい（よ写真フィルムの水切り乾燥用とじて用いら
れるドライウェル等が有効である１゜〔作用〕 公知のように、強磁性体粒子は、その粒径が非常に小さ
くなると容易磁化方向が熱運動のためレンダ１１になり
、超常磁性体どなる。例えば、マグネタイ１〜の場合、
粒径が１０ｎｍ以下になると、強磁性体ど超常磁性体に
変化づることが知られている。強磁性体と私営磁ｆ１体
とはヒステリシス曲線や帯磁率の測定、あるいはメスバ
ウワー効果から容易に見分けることが出来る。即ち、超
常磁性体は保持ツノが０であり、帯磁率は強磁性体から
超常磁性体に変化する臨界粒径を境にして、帯（ｔ１率
に及ぼづ粒径の効果が反Φλし、粒径が小さくなるほど
減少づる。また、強磁性では鉄のメスバウヮースペク１
〜ルは６本に分かれ−Ｃいるが、超常磁性になると、中
央に２木の吸収線が現われることから、超常磁性の定量
が出来る。熱１９乱によって磁化の反転が起こる熱磁気
緩和時間は鉄の超微粒子の場合、外部磁界がな【−）れ
ぼ、室温て・は粒径２．９ｎｍでは１秒、粒径３．６ｎ
ｍでは約３０汗どｈ１算される。わずか１１１ｍの粒径
の違い（・磁気的物質は大きく変化ザる。Then, the superparamagnetic substance labeled with C1 can be lyophilized and stored at a C length. When used as a labeled test sample, the labeled substance may be dispersed in an aqueous solution containing a surfactant iodine.1. It is also possible to add a surfactant to suppress aggregation and store the solution in a cool place. Examples of surfactants include Twcen 20, which is also used as a cleaning solution for ISA;
arboxy methyl cellulose)
(A dry well, etc. used for draining and drying photographic film is effective.) As is known, ferromagnetic particles can easily change their magnetization direction when their particle size becomes very small. Due to thermal motion, it becomes a renderer 11 and becomes a superparamagnetic material.For example, in the case of magnetite 1~,
It is known that when the particle size becomes 10 nm or less, the material changes to a ferromagnetic material or a superparamagnetic material. Ferromagnetic materials and private magnetic f1 materials can be easily distinguished from hysteresis curves, magnetic susceptibility measurements, or the Mössbauer effect. In other words, the retention horn of a superparamagnetic material is 0, and the magnetic susceptibility becomes a band (the effect of the grain size is anti-Φλ on the t1 ratio, with the critical grain size changing from a ferromagnetic material to a superparamagnetic material as a boundary). It decreases as the particle size becomes smaller.Also, in the case of ferromagnetism, the female bow width of iron is 1.
-C is divided into six lines, and when it becomes superparamagnetic, two absorption lines appear in the center, so superparamagnetism can be quantified. In the case of ultrafine iron particles, the thermomagnetic relaxation time at which magnetization is reversed due to thermal disturbance is 1 second at room temperature for a particle size of 2.9 nm, and 3.6 nm for a particle size of 3.6 nm at room temperature.
In m, it is calculated to be about 30 sweats per h1. The difference in particle size is only 111 m (magnetic substances vary greatly).

本発明の超常磁性体標識体は、上記のような超常磁性体
からなる磁性体微粒子を核として、その表面が抗原ある
いは抗体で被覆され−Ｃいるから、ウィルスや癌等の標
的細胞及び抗体と特賃的に反応して、これらの検体を捕
捉し、磁気標識することができる。この超常磁性体標識
体は癌やリンパ球等の標的細胞ｊミリも２桁以十小さく
、ウィルスと同等以下の大ぎざである。一般に、これら
の検体の抗原抗体結合部位（よ多数存在づるので、検体
よりも過剰に本発明の私営１１性体標識体を加えれば、
例えば、ウィルス１個に対して複数個の超常磁性体標識
体が結合することになる。このような結合により磁気的
増幅効果と体積的増幅効果を勺じることから、反応前は
超常磁性である超常磁性体標識体（Ｊ、抗原抗体反応後
の検体と結合した複合体は強磁性体的に振舞う。したが
って、本発明に従うレーザ磁気免疫測定方法において、
磁気標識として用いる超常磁性体標識体は、超常磁性体
性質を承りから、この標識体を傾斜＠界中で濃縮しよう
どしでも、標識体の容易磁化方向がランダムで、ブラウ
ン運動が活発であるため、標識体のｍ線位置への到達に
は長時間を要Ｊ−ることになる。The superparamagnetic label of the present invention has magnetic fine particles made of the above-mentioned superparamagnetic material as a core, and the surface thereof is coated with an antigen or an antibody. These analytes can be selectively reacted to be captured and magnetically labeled. This superparamagnetic labeled material is smaller than target cells such as cancer and lymphocytes by more than two orders of magnitude, and has the same or smaller serrations than viruses. Generally, the antigen-antibody binding sites of these specimens (there are many of them), so if the privately-produced 11-mer labeled compound of the present invention is added in excess of the specimen,
For example, multiple superparamagnetic labels will bind to one virus. Because such binding exhibits magnetic amplification effects and volumetric amplification effects, the superparamagnetic label (J) is superparamagnetic before the reaction, and the complex bound to the sample after the antigen-antibody reaction is ferromagnetic. Therefore, in the laser magnetic immunoassay method according to the present invention,
Due to the superparamagnetic properties of superparamagnetic labels used as magnetic labels, even if the labels are concentrated in a gradient field, the direction of easy magnetization of the labels is random and Brownian motion is active. Therefore, it takes a long time for the marker to reach the m-line position.

一方、超常磁性体標識体と検体との複合体である超常磁
性体標識検体複合体は、超常磁性体標識体よりも粒径が
大きいため、強磁性体的になり、ブラウン運動が不活発
となるから、傾斜磁界中に８３いて一ト記超常磁性体標
識体よりも類ｎ間で濃縮位置へ到達づることができる。On the other hand, the superparamagnetic labeled analyte complex, which is a composite of the superparamagnetic label and the analyte, has a larger particle size than the superparamagnetic label, so it becomes ferromagnetic and exhibits inactive Brownian motion. Therefore, when placed in a gradient magnetic field, the superparamagnetic label can reach the concentration position more quickly than the superparamagnetic label.

このため、濃縮位［行への到達時間差ににす、未反応の
超常磁性体標識体と反応後の超常磁性体標識検体複合体
とを識別できる。Therefore, the unreacted superparamagnetic label and the reacted superparamagnetic label analyte complex can be distinguished based on the time difference in arrival at the concentration level.

したがって、本発明に従うレーザ磁気免疫測定方法によ
れば、標識どして上記超常磁性体標識体を用いることか
ら、濃縮位置への到達時間差により、未反応の超常磁性
体標識体と反応後の超常磁性体標識体（Ａ複合体とを両
者の混在づる液中で容易に識別できるから、両者の分離
操伯が不用どなり、ＩＩＡ法以上の高い検出感度の測定
を極めて簡便に行なうこと／＋（でさる。したがって、
本発明にＪ：れば、検体たる抗原あるいは抗体の定量を
自動的に行なうことも可能どなる。。Therefore, according to the laser magnetic immunoassay method according to the present invention, since the above superparamagnetic label is used as a label, due to the difference in arrival time to the concentration position, the unreacted superparamagnetic label and the superparamagnetic label after the reaction are Since the magnetic labeled substance (complex A) can be easily identified in a liquid containing both, there is no need to separate the two, and measurements with higher detection sensitivity than the IIA method can be carried out extremely easily. A monkey. Therefore,
According to the present invention, it becomes possible to automatically quantify antigens or antibodies as specimens. .

また、本発明に従う免疫測定方法１こよれば、第１」−
稈にお４Ｊる抗原抗体反応中に超常磁性体標識検体複合
体の容易磁化方向を揃えるようにしたので、抗原抗体反
応後の−に記複合体を強磁性体とし、濃縮位置に複合体
を効率よく短時間のうらに誘導し、その位置に濃縮する
ことができる。Moreover, according to the immunoassay method 1 according to the present invention,
During the antigen-antibody reaction in the culm, the directions of easy magnetization of the superparamagnetic material-labeled specimen complex were aligned, so the complex shown in - after the antigen-antibody reaction was made into a ferromagnetic material, and the complex was placed at the concentration position. It can be efficiently guided to the backside for a short time and concentrated there.

さらに、本発明に従う免疫測定り法装首にＪ、れぽ、−
１−記の免疫測定方法を好適に実施することができる。Furthermore, the immunoassay method neck device according to the present invention includes J.
The immunoassay method described in 1- can be suitably carried out.

。 これら本発明の特徴的な構成によって、従来のＲ１Δ法
、［ＩΔｖ１、「■△法及び本発明者らが先に出願した
レーザ磁気免疫測定法等で、不可避であった未反応の標
識体を洗浄・分離するＴ稈が不用になる。このＪ、うな
特徴にＪζす、検出感度を低下さＵること【【＜測定の
自動化を極めて容易に仕ることが可能となる。. These characteristic configurations of the present invention eliminate unreacted labeled substances that were unavoidable in the conventional R1Δ method, [IΔv1, "■Δ method, and laser magnetic immunoassay method that the present inventors previously applied for. The T culm to be washed and separated becomes unnecessary. Due to this feature, the detection sensitivity is reduced, making it possible to automate the measurement extremely easily.

以１・に図面を参照して本発明をより具体的に訂述づ−
るが、以下に示すものは本発明の一実施例に過ぎず、本
発明の技術的範囲を何等制限するものではない。In the following, the present invention will be described in more detail with reference to the drawings.
However, what is shown below is only one example of the present invention, and does not limit the technical scope of the present invention in any way.

まず、本発明に従うレーザ磁気免疫測定方法の一実施例
について説明づる。First, an embodiment of the laser magnetic immunoassay method according to the present invention will be described.

〔実施例１〕 第１図・〜第４図は、本発明に従うレーザ磁気免疫測定
方法の一実施例を模式的に説明するためのもので、第１
図は分散溶媒中に分散した状態の超常磁性体標識体を示
し、第２図は非磁性微小球表面に固定された既知の抗体
を示し、第３図はサンドイツチ法による抗原抗体反応中
にかけられた５ＫＧの磁界により容易磁化方向が揃えら
れた状態の超常磁性体標識検体複合体を示し、第４図は
第３図の複合体を傾斜磁界中で濃縮し、複合体を定量的
に検出する工程を示したものである。[Example 1] Figures 1 to 4 are for schematically explaining an example of the laser magnetic immunoassay method according to the present invention.
The figure shows a superparamagnetic label dispersed in a dispersion medium, Figure 2 shows a known antibody immobilized on the surface of a non-magnetic microsphere, and Figure 3 shows a superparamagnetic label dispersed in a dispersion medium. Figure 4 shows the superparamagnetic substance-labeled specimen complex with its easy magnetization direction aligned by a 5 KG magnetic field. Figure 4 shows the complex shown in Figure 3 concentrated in a gradient magnetic field and the complex detected quantitatively. This shows the process.

図中符月１は超常磁性体超微粒子、２は抗体、３は超常
磁性体超微粒子の容易磁化方向を示り矢印、４は抗体、
５は非磁性微小球、６はウィルス（抗原）　、７１．Ｌ
超常磁性体標識体、８（よ非磁性体抗体複合体、９は超
常磁性体標識検体複合体、１０はレーザ入射光線、１１
は出射光線、１２は磁極片、１３は検査容器、１／Ｉは
電磁石である。In the figure, 1 is a superparamagnetic ultrafine particle, 2 is an antibody, 3 is an arrow indicating the direction of easy magnetization of the superparamagnetic ultrafine particle, 4 is an antibody,
5 is a non-magnetic microsphere, 6 is a virus (antigen), 71. L
superparamagnetic label, 8 (non-magnetic antibody complex, 9 superparamagnetic label sample complex, 10 laser incident beam, 11
is an emitted light beam, 12 is a magnetic pole piece, 13 is a test container, and 1/I is an electromagnet.

超常磁性超微粒子１は平均粒径２ｎｍの鉄からなる超微
粒子であつ−（、その表面はプロティンΔで被覆されて
いる。この鉄超微粒子は、公知の真空蒸発法℃・作製し
、超常磁性体のみを回収するため、磁場フィルタで強磁
ゼ１体と私営磁４Ｉ［体とを分離し！ζものを用いた。Superparamagnetic ultrafine particles 1 are ultrafine particles made of iron with an average particle size of 2 nm, and their surfaces are coated with protein Δ. In order to collect only the body, we used a magnetic field filter to separate the ferromagnetic body and the privately produced magnetic body 4I!ζ.

第１図に示づように、超常磁性体超微粉子１の表面に被
覆されたプロティン八にＴ＜］Ｇ（免疫グ［１プリン）
抗体からなる抗体２を固定化して超常磁性体標識体７を
得た。抗体２の固定化には、プロデインＡにインフルエ
ン１Ｆウイルス（Ｂ　／　％城／２／８５）に対重る兎
高度免疫血清を作用させる方法を用いた。As shown in FIG. 1, T
A superparamagnetic material label 7 was obtained by immobilizing the antibody 2 consisting of an antibody. For immobilization of Antibody 2, a method was used in which rabbit hyperimmune serum, which is anti-influenza 1F virus (B/% Shiro/2/85), was allowed to act on Prodein A.

一方、第２図に示すように、粒径１μｍのアクリルポリ
マからなる非磁性微小球５の表面を活性化したのち、そ
の表面に抗体２と同様のＩｇＧ抗体からなる抗体／Ｉを
吸着させて非磁性体抗体複合体８を栂だ。On the other hand, as shown in FIG. 2, after activating the surface of nonmagnetic microspheres 5 made of acrylic polymer with a particle size of 1 μm, antibody/I made of an IgG antibody similar to antibody 2 was adsorbed onto the surface. Non-magnetic antibody complex 8 is toga.

次に、第３図に示すように、患当のうがい液中に存在す
るインフルエンザウィルス６を検体とし、このウィルス
６と、前記私営１ｉ竹体標識体７及び非ｖＪ１＋１体抗
体複合体８とを含む液中で抗原抗体反＝　　２１　− 応を行／ｉつだ３．この反応中、強磁界をか（プて、標
識体７及び非磁性体抗体複合体８とウィルス６との複合
体９の容易磁化方向をｊ前えて複合体９を強磁性体どじ
だ。Next, as shown in FIG. 3, the influenza virus 6 present in the patient's gargle fluid was used as a sample, and this virus 6, the privately produced 1i bamboo label 7 and the non-vJ1+1 antibody complex 8 were combined. 3. Carry out antigen-antibody reaction = 21 - reaction in the solution containing /i. During this reaction, a strong magnetic field is applied, and the direction of easy magnetization of the complex 9 of the labeled body 7, the non-magnetic antibody complex 8, and the virus 6 is shifted forward, and the complex 9 is moved away from the ferromagnetic material.

次いで、複合体９を含む溶液を検査容器１３内に収容し
た。この検査容器１３は、第４図に示づように、互いに
対向する磁極片１２と電磁石１／１とに挟まれており、
検査容器１３の表面には、直径８ＩｎＩｎのロッド状の
もので、ぞの先端が鋭利な純鉄製の磁極片１２の真下位
ｎの磁界が最も高くなるように、磁極片１２と電磁石１
４とからなる傾斜磁界発生装置により傾斜磁界がかかる
構成と＜’にっている。この傾斜磁界を用いて磁極片１
２の真下位置の液面上に複合体９を誘導・濃縮した。こ
の濃縮位置に、波長６３２．８ｎｍのＨｅ　−Ｎ　ｅレ
ーザ入射光１０を照射し、イの散乱光を出射光１１とし
て検出した。出射光１１の経時変化を検出したので、濃
縮位置への到達時間の短い複合体９と、到達時間の遅い
未反応の超常磁性体標識体７とを確実に識別できた。な
お、出射光として濃縮位置からの散乱光、透過〉に、反
射光、干渉光、回折光を選択して検出したところ、同様
に標識体７と複合体９とを識別できた。Next, a solution containing the complex 9 was placed in the test container 13. As shown in FIG. 4, this test container 13 is sandwiched between a magnetic pole piece 12 and an electromagnet 1/1 that face each other.
On the surface of the test container 13, a magnetic pole piece 12 and an electromagnet 1 are arranged so that the magnetic field directly below the pole piece 12, which is made of pure iron and has a rod shape with a diameter of 8 InIn and has a sharp tip, is highest.
The configuration is such that a gradient magnetic field is applied by a gradient magnetic field generator consisting of . Using this gradient magnetic field, the magnetic pole piece 1
Complex 9 was induced and concentrated on the liquid surface directly below 2. This concentrated position was irradiated with incident He-Ne laser light 10 with a wavelength of 632.8 nm, and the scattered light (A) was detected as the output light 11. Since the change over time of the emitted light 11 was detected, it was possible to reliably distinguish between the complex 9, which took a short time to reach the concentration position, and the unreacted superparamagnetic label 7, which took a long time to reach the concentration position. Incidentally, by selecting and detecting reflected light, interference light, and diffracted light in addition to scattered light from the concentration position and transmitted light as emitted light, labeled body 7 and complex 9 could be similarly identified.

次に、本発明に従う免疫測定力ン六を好適に実施りるこ
とのぐさる測定装置の一例を説明づ−る。。Next, an example of a measuring device that can suitably carry out the immunoassay according to the present invention will be explained. .

〔実施例２〕 第５図は、本発明に従う免疫測定装置の−・例を示１１
１略構成図であって、図中符＞２１０は入射光束、１１
ａ及び１１ｂは散乱光束、１２ａ及び１２ｂは磁極片、
１３ａ及び１３ｂμ検査容器１３のつ」ル、１４は電磁
石、１５はレーザ光源、１６は偏向器、１７ａ及び１７
ｂは集光レンズ、１８ａ及び１８ｂはスリット、１９ａ
及び１９　ｂ　１．；Ｌ光電子増倍管、２０は検査容器
１３の水平移動装置、２１は識別機構をＲ４成する電子
回路部である。[Example 2] FIG. 5 shows an example of the immunoassay device according to the present invention.
1 is a schematic configuration diagram, where the reference mark >210 in the figure represents an incident light flux, and 11
a and 11b are scattered light fluxes, 12a and 12b are magnetic pole pieces,
13a and 13b μ test container 13, 14 is an electromagnet, 15 is a laser light source, 16 is a deflector, 17a and 17
b is a condenser lens, 18a and 18b are slits, 19a
and 19 b 1. ; 20 is a horizontal movement device for the test container 13; 21 is an electronic circuit section forming an identification mechanism R4;

レーリ゛光源１５は、イのレーザ入射光束１０が偏向器
１６により平面内を走引して両ウェル１３ａ及び１３ｂ
を順次、照射するＪ、うに水平面から３０度の角瓜で取
イ」りられている。両ウェル１３ａ及び１３ｂの照ｑ１
位置の真上には両ｖＡ極片１２ａ及び１２ｂが配置され
ている。また、散乱光束１１ａ及び１１ｂは、集光レン
ズ１７ａ及び１７ｂ、スリッ１〜１８ａ及び１８ｂを通
って光電子増倍管１９ａ及び１９ｂでそれそ′れ検出さ
れるＪ、うになっている、イしＣ１つＪル１３ａ及び１
３ｂにそれぞれ複合体９を含む溶液を収容し、両つＪル
１３ａ及び１３ｂからの散乱光束１１ａ及び１１［）を
光電子増倍管１９ａ及び１９ｂで検出し、゛重子回路部
２１て・分析づることで、二つの検体の測定を効率よく
行なうことができた。そして、水平移動装置２０にＪ：
り検査容器１３を順次移動させてゆくことでざらに多く
の検体の測定が可能であ−）た。また、ウェル１３ａ及
び１３ｂのうＪ）、一方に検体、他方に検体対照を収容
して比較測定りることｂｒきた１゜ なＪ３、入射光束１０をビームスプリッタで分割して複
数のウェルを同時照射するようにしてもＪ、い。この場
合、言うまでもなく、分割されたビームは濃縮位置を含
むＪ、うに照射される必要がある。In the Rayleigh light source 15, the laser incident light beam 10 is caused to travel within a plane by a deflector 16, and is directed to both wells 13a and 13b.
The sea urchin is taken at a angle of 30 degrees from the horizontal plane. Light q1 of both wells 13a and 13b
Directly above the position are both vA pole pieces 12a and 12b. Further, the scattered light beams 11a and 11b pass through condensing lenses 17a and 17b, slits 1 to 18a and 18b, and are respectively detected by photomultiplier tubes 19a and 19b. 13a and 1
3b respectively contain a solution containing the complex 9, and the scattered light beams 11a and 11[) from both the tubes 13a and 13b are detected by photomultiplier tubes 19a and 19b, and the multiplex circuit section 21 is analyzed. This made it possible to efficiently measure two samples. Then, the horizontal movement device 20 has J:
By sequentially moving the test containers 13, it was possible to measure a larger number of specimens. In addition, the wells 13a and 13b can be used for comparative measurements by storing a sample in one side and a sample control in the other. Even if I try to irradiate it, it still doesn't work. In this case, needless to say, the divided beams need to be irradiated onto J, which includes the concentration position.

また、バックグランド光の影響を除くため、ビー−２／
ｌ　　− へ分割直前に上述の偏向器１６を挿入し、各つＴル１３
ａ及び１３ｂの該濃縮位置と：１１ｍ縮位置の間を同時
に走引づることが望ましい。走引周波数に同期した検体
からのイハ尼をロックイン増幅り゛れば更にＳ／Ｎが改
善できる。In addition, in order to eliminate the influence of background light,
The above-mentioned deflector 16 is inserted just before dividing into T-13, and each
It is desirable to simultaneously run between the condensation positions a and 13b and the 11m contraction position. The S/N ratio can be further improved by lock-in amplification of the signal from the sample synchronized with the scanning frequency.

第６図及び第７図は本発明に従うレーザ磁気免疫測定装
置を用いて測定した結果の−・例を示Ｊグラフて゛あっ
て、第６図は検体、即ノ）、ウィルスが存在りる場合、
第７図は検体対照、即ち、検体と同一の磁界中処理等を
５　＜ｒつだものの、ウィルスが存在しない場合、の散
乱光部の経時変化を示したしのである。散乱光部の測定
（ま、光電子増幅管１９と１及び１９ｂの出力をロック
インアンプで増幅し、入射光束１０の走引周波数に同期
した信号を記録リ−る方法を採った。検体が存在する場
合、検体からの散乱光ｆｉｔは電磁石を励磁すると耐ら
に増大し、一定値を示づ。これに対して、検体対照から
の散乱光部は時間経過とともに極わずか増大するのみで
ある。このように、散乱光部の経時変化を検出し、検体
対照の散乱光部を差し引くことによって、検体のみを定
ｆｆ１−Ｊることが出来る。ｋお、検体対照は、検体ご
とに同■、１に測定する必要は必ずしもなく、検体の測
定前に一度測定しておき、この結果をメ七り−十に蓄え
ておく方法ももらろん可能である。Figures 6 and 7 are graphs showing examples of measurement results using the laser magnetic immunoassay device according to the present invention. ,
FIG. 7 shows the change over time in the scattered light area for a specimen control, that is, when the specimen was subjected to the same magnetic field treatment as the specimen but no virus was present. Measurement of the scattered light part (well, a method was adopted in which the outputs of the photoelectron amplifier tubes 19, 1, and 19b were amplified by a lock-in amplifier, and a signal synchronized with the scanning frequency of the incident light beam 10 was recorded and read. In this case, when the electromagnet is excited, the scattered light part from the specimen increases steadily and shows a constant value.On the other hand, the scattered light part from the specimen control increases only slightly over time. In this way, by detecting the change over time in the scattered light area and subtracting the scattered light area of the sample control, it is possible to determine only the sample ff1-J. It is not always necessary to measure the sample at once, and it is of course possible to measure it once before measuring the sample and store the results in a memory.

本発明によれば、従来検出前にウィルス培養が必要−（
゛あ−）たインフル１ンリ゛ウイルスを培養づることな
しに直接検出でき、うがい液中に１０個程度存在する各
種のインフルエンザウィルスを測定りることが出来た。According to the present invention, virus culture is required before conventional detection.
It was possible to directly detect influenza viruses without culturing them, and it was possible to measure about 10 different influenza viruses present in the gargle fluid.

ちなみに、従来のＲＩＡ法の場合、ウィルスが数千個／
ｄ以上存在しな（プれば検出できなかった。By the way, in the case of the conventional RIA method, several thousand viruses/
There are no more than d (couldn't be detected if pulled).

次に、本発明に従う免疫測定方法の他の例について説明
する、。Next, another example of the immunoassay method according to the present invention will be explained.

〔実施例３〕 ウィルスど標識体との抗原抗イホ反応を直接法で行なっ
てウィルスの検出を試みた。[Example 3] An attempt was made to detect a virus by conducting an antigen-anti-Iho reaction with a labeled virus by a direct method.

標識体に用いた超１ル磁性体超微粒子は平均粒径９ｎｍ
のマグネタイ１ル超微粒子であって、その表面はデキス
１〜ランが被覆されている。このングネタイ１〜超微粒
子はＨＯｌｄａｙらの方法（Ｊ、　Ｉｍｍ、　Ｈｅｔｈ
、　５２巻、３５３−３６７頁、１９８２）を参考にし
て作製し、強磁性体粒子を除去するため、遠心分離にか
（づ上清に残るものを用いた。１次に、上記微粒子表面
のデキス１ヘランにインフルエン１アウイルスに対する
兎高度免疫血清から単＃］シたＩＱＧ抗体を共有結合さ
せて、私営１！竹体標識体を得た。The ultra-fine magnetic particles used for the label have an average particle size of 9 nm.
These are ultrafine particles of Magnetile 1, the surface of which is coated with Dex 1 to Ran. These Ngunnetai 1 to ultrafine particles were prepared using the method of Holday et al. (J, Imm, Heth
, Vol. 52, pp. 353-367, 1982), and in order to remove the ferromagnetic particles, they were centrifuged (the supernatant remaining in the supernatant was used). An IQG antibody obtained from a rabbit hyperimmune serum against influenza 1 virus was covalently bound to Dex 1 Heran to obtain a privately available 1! bamboo body labeled substance.

な お、超常磁性体と超常磁性体とを分離する方法どしては
、公知の液体クロマトグラフィや磁気フィルターを単独
あるいは併用して用いる方法でもよい。Note that the method for separating the superparamagnetic material and the superparamagnetic material may be a method using known liquid chromatography or a magnetic filter alone or in combination.

次に、患者のうがい液から採取した検体１ｄに前記超常
磁性体標識体１Ｘ１０’ｇを加え、検体と超常磁性体標
識体とを直接抗原抗体反応させて超常磁性体標識検体複
合体を得、この複合体に対して本発明者らが先に出願し
た特願昭６２−１８／１．９０２ｒレーザ磁気免疫測定
方法及び装置」に記載の干渉法による測定を行なっｌｃ
。すなわら、この測定方法は、複合体からの出射光をス
クリー−２７＝ ン上に投影して干渉縞を観察づるものである。Next, 1 x 10'g of the superparamagnetic substance labeled substance is added to the specimen 1d collected from the patient's gargling fluid, and a superparamagnetic substance labeled specimen complex is obtained by directly causing an antigen-antibody reaction between the specimen and the superparamagnetic substance labeled substance, This complex was measured by the interferometry method described in Japanese Patent Application No. 18/1983, ``Laser Magnetic Immunoassay Method and Apparatus'' previously filed by the present inventors.
. In other words, this measurement method projects the light emitted from the composite onto a screen and observes interference fringes.

検体からの干渉光からは明瞭な干渉縞が観察され、検体
（・νイルス）が存在しない検体対照からの干渉光から
は干渉縞がわずかしか観察されなかった。Clear interference fringes were observed from the interference light from the specimen, and only a few interference fringes were observed from the interference light from the specimen control in which the specimen (·ν virus) was not present.

これら干渉縞の３ｆｉいＩＪ上、ウィルスの大ぎさが１
２Ｑｎｍぐあるのに対して、超常磁性体標識体の大きさ
は９０…程度であるので、ウィルスと超常磁性体標識体
との抗原抗体複合体は超常磁性体標識体よりも約１桁大
きいため、濃縮の程度が異なることににる。′？ｌ’／
：Ｉわち、上述したように、超常磁性体標識体はブラウ
ン運動が活発なため濃縮に時間がかかるが、抗原抗体複
合体はブラウン運動Ｊζりも傾斜磁界による磁気吸引が
効果的に作用するためである。。On the 3fi IJ of these interference fringes, the size of the virus is 1
2 Qnm, whereas the size of the superparamagnetic label is about 90 nm, so the antigen-antibody complex between the virus and the superparamagnetic label is about one order of magnitude larger than the superparamagnetic label. , resulting in different degrees of enrichment. ′? l'/
:I As mentioned above, superparamagnetic labels take time to concentrate due to active Brownian motion, but antigen-antibody complexes have Brownian motion and magnetic attraction by gradient magnetic fields is effective. It's for a reason. .

また、変形例として、前記検体と前記超常磁性体標識体
との抗原抗体反応を１０ｋＧの磁界中で行なった後、上
記と同様の干渉法で測定した。その結果、検体対照の干
渉縞は磁界を加えずに抗原抗体反応させた場合と同じで
あったが、検体の千渉縞は磁界を加えずに抗原抗体反応
させた場合よりも速く出現し、測定時間の短縮が図られ
た。In addition, as a modification, the antigen-antibody reaction between the specimen and the superparamagnetic label was performed in a magnetic field of 10 kG, and then measured by the same interference method as above. As a result, the interference fringes of the specimen control were the same as when the antigen-antibody reaction was performed without applying a magnetic field, but the interference fringes of the specimen appeared faster than when the antigen-antibody reaction was performed without applying a magnetic field. Measurement time was reduced.

なお、比較のために、磁性標識体どして、平均粒径５Ｑ
ｎｍのマグネタイト強磁性体粒子に上述の方法でウィル
ス抗体を結合して、上述と同様な実験を行なったが、抗
原抗体複合体と未反応のマグネタ１１〜強磁性標識体の
間には濃縮時間差が生じないため、検体を識別できなか
った。For comparison, the average particle size of the magnetic label was 5Q.
The same experiment as above was carried out by binding virus antibodies to nanomagnetite ferromagnetic particles using the method described above, but there was a difference in concentration time between the antigen-antibody complex and the unreacted magnetite 11 to ferromagnetic label. The specimen could not be identified because no

本発明は上述の干渉法に限られるものではなく、検体か
らの出射光が前記抗原抗体複合体と未反応の超常磁性体
標識体との間で識別が容易になるような、測定条ｆＩ（
磁界、時間等）を選び、電磁石励ＩＩｊｌ後一定時間経
った時点での散乱光間、透過光Ｗ、反（ト）光ｍ等を測
定づる方法ももらろん可能ひある。The present invention is not limited to the above-mentioned interference method, and the measurement strip fI (
Of course, it is also possible to select a method (magnetic field, time, etc.) and measure the scattered light, transmitted light W, reflected light m, etc. at a certain time after electromagnet excitation IIjl.

〔実施例４〕 白血病や癌への適用の可能性を調べるために、標的細胞
検出のモデル実験を行なった。粒径３μｍのアクリル粒
子（東し製、商品名トレスフユア）を標的細胞に見立て
、該アクリル粒子の表面を活Ｍ化した後、抗原としてイ
ンフルＴンザウイルスのエーテル処理抗原を感作した。[Example 4] In order to investigate the possibility of application to leukemia and cancer, a model experiment for target cell detection was conducted. Acrylic particles with a particle size of 3 μm (manufactured by Toshi, trade name: Tresfuure) were used as target cells, and after the surface of the acrylic particles was activated, they were sensitized with an ether-treated antigen of influenza T virus as an antigen.

超常磁性体Ｗ、識休体は前記実施例３のマグネタイト超
帛゛磁竹体を用いた。前記抗原感作粒子と超常磁性体標
識体とを直接、抗原抗体反応させ、未反応の超常磁性体
標識体を分離し４Ｔいで、前記干渉法で測定したどころ
、抗原感作粒子は１個でも検出することが出来た。従っ
て、本発明のレーザ磁気免疫測定法は、エイズに感染し
た一丁すンパ球ヤ〕癌等の異常細胞の早期診断に適用で
きる。As the superparamagnetic material W and the magnetic material, the magnetite superwoven magnetic bamboo material of Example 3 was used. Although the antigen-sensitized particles and the superparamagnetic label were directly subjected to an antigen-antibody reaction, the unreacted superparamagnetic label was separated, and the unreacted superparamagnetic label was separated and measured using the interference method described above, even one antigen-sensitized particle was detected. I was able to detect it. Therefore, the laser magnetic immunoassay method of the present invention can be applied to early diagnosis of abnormal cells such as cancer cells infected with AIDS.

次に、本発明に従う免疫測定方法を実施するうえで好適
に用いられる超常磁性体標識体の製造方法の一例につい
て説明する。Next, an example of a method for producing a superparamagnetic label suitably used in carrying out the immunoassay method according to the present invention will be described.

〔実施例５〕 第８図は、本発明に従う超常磁性体標識体の製造工程を
示づものである。[Example 5] FIG. 8 shows the manufacturing process of a superparamagnetic label according to the present invention.

塩化第一６９　（ＦｅＣｆ２・４　＋−１２０）２．ｏ
ｙと塩化第二Ｒ（ＦｅＣｆ３　・６Ｈ２０）５．４ｇを
蒸留水に溶かし、ざらに、５０％デキス１〜ラン水溶液
５０ｍＱを加え、液温を８５℃に保ら、よくかきまぜな
がら、苛性ソーダ５９１５０ａｅの溶液を一定の速さく
毎分２５ｄ）で少しずつ滴下した。沈澱が沈Ｅｆ　１ノ
たら再び上澄み液を捨てる洗浄操作を８回はど繰り返し
た。次に、分散安定剤として、０゜５％の１ｗｅｅｎ　
２０水溶液を加え、全量を１００ｄとした後、遠心管に
１０ｍ毎に分別採集した。遠心管に採取した該溶液を毎
分２万回転、３０分間の遠心にか（）、強磁性体が含ま
れた沈澱物は捨で、ト澄み液から超常磁性体超微粒子を
（９た。このようにして作製した私営強磁性体超微粒子
は平均粒子９０…であり、ぞの表ｍ１はデキストランで
被覆されていた。Daiichichloride 69 (FeCf2.4 +-120)2. o
Dissolve y and 5.4 g of ferric chloride (FeCf3 .6H20) in distilled water, add 50 mQ of 50% dex1-ran aqueous solution to a colander, keep the liquid temperature at 85°C, and while stirring well, add 59150 ae of caustic soda. The solution was added dropwise at a constant rate of 25 d/min. When the precipitate had settled to Ef 1, the supernatant liquid was discarded again, and the washing operation was repeated 8 times. Next, as a dispersion stabilizer, 0°5% 1ween
After adding 20 m of aqueous solution to bring the total volume to 100 m, the samples were collected in centrifugal tubes at intervals of 10 m. The solution collected in a centrifuge tube was centrifuged at 20,000 revolutions per minute for 30 minutes (), the precipitate containing the ferromagnetic material was discarded, and the superparamagnetic ultrafine particles were collected from the clear liquid (9). The privately produced ultrafine ferromagnetic particles produced in this way had an average particle size of 90, and the surface m1 was coated with dextran.

私営強磁性体超微粒子を分散した溶液に兎面清から単離
したインフルエン１アウイルスＩＱＧ抗体を加え、デキ
ス１〜ランとＩｇＧ抗体とを共有結合させることによっ
て超常磁性体標識体を得た。A superparamagnetic label was obtained by adding influenza 1 virus IQG antibody isolated from rabbit serum to a solution in which privately produced ultrafine ferromagnetic particles were dispersed, and covalently bonding Dex 1-Ran with the IgG antibody. .

この超常磁性体標識体を浮遊さけた液（０，１ｄ）は４
０１−１△のインフルエンザウィルスを吸名゛すること
ができた。The liquid (0,1d) in which this superparamagnetic label was suspended was 4
I was able to catch the 01-1△ influenza virus.

〔実施例６〕 第９図は、本発明に従う超常磁性体標識体を製造するの
に好適に用いられる製造装置の一例を示すものである。[Example 6] FIG. 9 shows an example of a manufacturing apparatus suitably used for manufacturing the superparamagnetic label according to the present invention.

図中１′：１月３１は雰囲気槽、３２は　′超常磁性体
超微粒子原料、３３はレーザ光源、３４はレーザビーム
、３５はミラー、３６は真空ポンプ、３７は蒸発粒子の
捕集管、３８は超常磁性体超微粒子の回収槽、３９（よ
真空ポンプ、４０は分散溶液、４１は磁気フィルター、
４２は雰囲気ガス、４３はリークバルブ、４．４は液体
窒素容器、４５は液体窒素、４６及び４７はバルブであ
る。In the figure, 1': January 31 is an atmosphere tank, 32 is a superparamagnetic ultrafine particle raw material, 33 is a laser light source, 34 is a laser beam, 35 is a mirror, 36 is a vacuum pump, 37 is a collection tube for evaporated particles, 38 is a recovery tank for superparamagnetic ultrafine particles, 39 is a vacuum pump, 40 is a dispersion solution, 41 is a magnetic filter,
42 is an atmospheric gas, 43 is a leak valve, 4.4 is a liquid nitrogen container, 45 is liquid nitrogen, and 46 and 47 are valves.

まず、回収Ｗ４３８の内部に１０％のデキストラン水溶
液（Ｐｈａｒｍａｃｉａ礼製、ｆｆ１ｌｊ）平均分子量
７７００）を５０蛇入れ、次に、液体窒素容器４４に液
体窒素４５を注いで、上記デキストラン水溶液を凍結さ
せた。次いで、ターボ七しキューラーボンプ３６にＪ：
つて、雰囲気槽３１の内部を２×１Ｑ−７Ｔｏｒｒまで
真空υ１気した後、バルブ４６を閉じ、アルゴンガス４
２をリークバルブ４３を開いて導入した。次に、バルブ
４７を開いて油回転ポンプ３９でアルゴンガスを排気し
た。このとぎ、り一クバルブ４３を調節することによっ
て雰囲気槽３１の圧力を５　ｘ　１０’　Ｔｏｒｒに保
った。この結果、アルゴンガス／１２は定常的に雰囲気
槽３１から捕集慎・３７を通して回収槽３８へ導かれ、
油回転ポンプ３９で外に排気された。First, 50% of a 10% dextran aqueous solution (manufactured by Pharmacia Rei, FF1LJ, average molecular weight 7700) was poured into the recovered W438, and then 45% of liquid nitrogen was poured into the liquid nitrogen container 44 to freeze the dextran aqueous solution. . Next, turbo seven and Kueller Bonp 36 J:
Then, after evacuating the inside of the atmosphere tank 31 to 2×1Q-7 Torr, the valve 46 was closed, and the argon gas was
2 was introduced by opening the leak valve 43. Next, the valve 47 was opened and the oil rotary pump 39 was used to exhaust the argon gas. At this point, the pressure in the atmosphere tank 31 was maintained at 5 x 10' Torr by adjusting the leak valve 43. As a result, argon gas/12 is constantly guided from the atmosphere tank 31 to the collection tank 38 through the collection tank 37,
It was exhausted to the outside by an oil rotary pump 39.

次に、電磁石からなる磁気フィルタ４１を作動させた。Next, the magnetic filter 41 consisting of an electromagnet was activated.

磁気フィルタ４１の作動時には前記捕集管３７の内部に
１０００ガウスの磁界を捕集管３７ど直交方向にかけた
。以上の操作の後、出力１０ＷのＹＡＧレーザ３３のレ
ーザ光線３４をミラー３５にＪ：って、雰囲気槽３１に
導き、純度９９゜９％の純鉄からなる蒸発原料３２を加
熱蒸発させた。蒸発した鉄微粒子はアルゴンガスと共に
、前記磁気フィルタ４１を通り、回収槽３８へ達した。When the magnetic filter 41 was in operation, a magnetic field of 1000 Gauss was applied inside the collection tube 37 in a direction perpendicular to the collection tube 37. After the above operations, the laser beam 34 of the YAG laser 33 with an output of 10 W was guided to the atmosphere tank 31 by the mirror 35, and the evaporation raw material 32 made of pure iron with a purity of 99.9% was heated and evaporated. The evaporated iron particles together with argon gas passed through the magnetic filter 41 and reached the collection tank 38.

この時、強磁性微粒子は磁気フィルタ４１で捕捉される
ので、回収槽３８へ到達づるの【よ超常磁性体超微粒子
のみである。回収槽３８の内部は上述の操作にＪ：って
、デキス１〜ラン溶液が凍結されているので、超常磁性
体超微粒子は凍結した該デキストラン溶液の表面に堆積
する。所定の量を蒸発させた後、前記液体窒素容器４４
を下げ、回収槽３８を室温に戻す。凍結した溶液の表面
に堆積した鉄の超常磁性体超微粒子は、デキス１〜ラン
溶液の融解にともなって、溶液中に分散する。この時、
鉄の表面にデキス１〜ランが吸着し、デキストランで被
覆した平均粒径１０ｎｍの超常磁性体超微粒子が得られ
た。At this time, since the ferromagnetic particles are captured by the magnetic filter 41, only the superparamagnetic ultrafine particles reach the collection tank 38. Since the dextran to run solutions are frozen in the interior of the recovery tank 38 by the above-described operation, the superparamagnetic ultrafine particles are deposited on the surface of the frozen dextran solution. After evaporating a predetermined amount, the liquid nitrogen container 44
is lowered and the recovery tank 38 is returned to room temperature. The iron superparamagnetic ultrafine particles deposited on the surface of the frozen solution are dispersed in the solution as the Dex 1-Ran solution is melted. At this time,
Dex 1-Ran was adsorbed on the surface of iron, and superparamagnetic ultrafine particles coated with dextran and having an average particle diameter of 10 nm were obtained.

次いで、レーザ出力、アルゴンガス圧、磁気フィルタ４
１の磁界等を調節することによって、平均粒径５ｎｍ〜
２０ｎｍの超常磁性体超微粒子を用いて、前記実施例５
と同じ方法でインフルエンザウィルスＡ、Ｂ型に対する
ＩｇＧ抗体をそれぞれ共有結合さゼて超常磁性体標識体
を得た。Next, the laser output, argon gas pressure, magnetic filter 4
By adjusting the magnetic field etc. of 1, the average particle size is 5 nm ~
Using 20 nm superparamagnetic ultrafine particles, the above Example 5
In the same manner as above, IgG antibodies against influenza virus types A and B were covalently bonded to obtain superparamagnetic labeled substances.

本発明者らが先に出願した干渉法を用い、かつ患者のう
がい液から採取した検体１威に上記超常磁性標識体を１
Ｘ１０’Ｓ？を加える直接法でウィルスの検出を試みた
結果、従来の血球凝集法よりも１０万倍の検出感度でウ
ィルスのＡ、Ｂ型を特定することが出来た。Using the interference method previously applied by the present inventors, the above superparamagnetic label was added to one sample collected from the patient's gargle fluid.
X10'S? As a result of trying to detect the virus using a direct method in which the blood cells are added, it was possible to identify virus types A and B with a detection sensitivity 100,000 times higher than that of the conventional hemagglutination method.

〔発明の効果〕〔Effect of the invention〕

以上詳述したＪ：うに、本発明に従うレーザ磁気免疫測
定方法及び測定装置ににれば、抗原抗体反応後の磁性体
標識検体複合体と未反応の超常磁性体標識体とを濃縮位
置への到達時間差により識別するようにしたので、上記
複合体の検出に際して複合体と標識体とを分離する必要
がなく、極めて効率よ＜ＲＩＡ法以上の検出感度で抗原
抗体反応検査を実施できる。As described in detail above, the laser magnetic immunoassay method and measuring device according to the present invention can move the magnetically labeled sample complex after antigen-antibody reaction and the unreacted superparamagnetic label to the concentration position. Since discrimination is made based on the arrival time difference, there is no need to separate the complex and the label when detecting the complex, and the antigen-antibody reaction test can be carried out with extremely high efficiency and a detection sensitivity higher than that of the RIA method.

また、本発明に従う超常磁性体標識体は、ウィルスと同
程度以下の大きさであり、またリンパ球や癌等のＩＩＩ
胞よりも３稍小さい。従って、例えば、ウィルス等に感
染したリンパ球を特異的に捕捉するｔツクローナル抗体
を本発明の超常磁性体標識体に用いた場合、リンパ球１
個の回りに多数個の超常磁性体標識体が結合するために
、磁気的増幅効果が生じる。例えば、散乱光、干渉光、
反射光、回折光など光学的に検出すれば、リンパ球と超
常磁性体標識体との抗原抗体複合体は、体積が著しく増
加するために、体積的増幅効果も生じる。これらの増幅
効果のため、超常磁性体標識体の検出限度はおおにそ、
ｌＸ１０−１２ｇであるが、これよりも１桁以上改善さ
れるから、ウィルスやリンパ球が１個でも検出できる。In addition, the superparamagnetic labeled substance according to the present invention has a size comparable to or smaller than a virus, and also has a size similar to that of a virus,
It is 3 mm smaller than a cyst. Therefore, for example, when a t-clonal antibody that specifically captures lymphocytes infected with a virus or the like is used in the superparamagnetic label of the present invention, lymphocytes 1
A magnetic amplification effect occurs because a large number of superparamagnetic labels bind around the individual. For example, scattered light, interference light,
When optically detected using reflected light or diffracted light, the volume of the antigen-antibody complex of lymphocytes and superparamagnetic label increases significantly, resulting in a volumetric amplification effect. Due to these amplification effects, the detection limits of superparamagnetic labels are generally
Although it is 1X10-12g, it is improved by more than one order of magnitude, so even one virus or lymphocyte can be detected.

したがって、本発明によれば、従来ｒｌｌＡ法が適用さ
れていたペブヂドホルモン等の種々のホルモンあるいは
種々の酵素、ビタミン、薬剤なとの測定にも応用するこ
とが可能である。従って、従来は限定された茄設でＲＩ
　Ａ　’１１＜によらなければ実施できなかった精密な
測定を、一般的な環境で広〈実施することが可能となる
。集団検診等の」；うな一般的な状況で、各種のウィル
ス、癌等のスクリーニング検査等の精密な測定が広〈実
施できれば、癌あるいはウィルス性疾患等の早期診断が
可能となり、有効ｔ【〒期治療を的確に実施することが
可能となる。このように、本発明が医学。医療の分野で
果たす効果は８１り知れない。Therefore, the present invention can be applied to the measurement of various hormones such as peptide hormone, various enzymes, vitamins, and drugs, to which the rllA method has conventionally been applied. Therefore, in the past, RI was performed with limited capacity.
Precise measurements that could not be carried out otherwise can now be carried out in a general environment. If precise measurements such as screening tests for various viruses and cancers can be carried out widely in common situations such as mass medical examinations, early diagnosis of cancer or viral diseases, etc. will be possible and effective. It becomes possible to carry out period treatment accurately. In this way, the present invention is medical. The effects it will have in the medical field are immeasurable.

【図面の簡単な説明】[Brief explanation of the drawing]

第１図へ・第４図は、本発明に従うシー１ｆ磁気免疫測
定方法の一例を説明するだめのもので、第１図は分散溶
媒中に分散した状態の超常磁性体標識体を示す模式図、
第２図は非磁性微小球表面に固定された抗体を示す模式
図、第３図は抗原抗体反応中に磁界より容易磁化方向が
揃えられた状態の超常磁性体標識検体複合体を示す模式
図、第４図は第３図の複合体を濃縮し検出する工程を示
す模式図である。 第５図は、本発明に従うレーザ磁気免疫測定装置の一例
を示ず概略構成図、ｆｆｒ６図及び第７図は第５図に示
した測定装置を用いて得た測定結果を示すもので、第６
図は検体が存在する場合の散乱光量の時間依存性を示す
グラフ、第７図は検体が存在しない場合の散乱光１の時
間依存性を示すグラフ、第８図は本発明に従う超常磁性
体標識体の製造方法の一例を示す工程図、第９図は本発
明に従う超常磁性体Ｗ！識体の製造方法を実施するうえ
で好適に用いられる製造装置の一例を示す概略断面図で
ある。 １・・・超常磁性体超微粒子、２・・・抗体、３・・・
超常磁性体超微粒子の容易磁化方向を示す矢印、４・・
・抗体、５・・・非磁性微小球、６・・・ウィルス（抗
原）、７・・・超常磁性体標識体、８・・・非磁性体抗
体複合体、９・・・超常磁性体標識検体複合体、１０・
・・レーザ入射光線、１１・・・出射光線、１１ａ、１
１ｂ・・・散乱光束、１２．１２ａ、１２ｂ・・・磁極
片（傾斜磁界発生装置）、１３・・・検査容器、１３ａ
、１３ｂ・・・検査容器１３中のウェル、１４・・・電
磁石（傾斜磁界発生装置）、１５・・・レーザ光源（入
射光学系）、１６・・・偏向器（入射光学系）、１７ａ
、１７ｂ・・・集光レンズ（受光光学系）、１８ａ、１
８ｂ・・・スリット（受光光学系）、１９ａ、１９ｂ・
・・光電子増倍管（受光光学系）、２０・・・検査容器
の水平移動装置、２１・・・電子回路部（識別機構）、
３１・・・雰囲気槽、３２・・・超常磁性体超微粒子原
料、３３・・・レーザ、３４・・・レーザビーム、３５
・・・ミラー、３６・・・真空ポンプ、３７・・・蒸発
粒子の捕集管、３８・・・超常磁性体超微粒子の回収槽
、３９・・・真空ポンプ、４０・・・分散溶液、４１・
・・磁気フィルター、４２・・・雰囲気ガス、４３・・
・リークバルブ、４４・・・液体窒素容器、４５・・・
液体窒素、４６・・・バルブ、７′Ｉ７・・バルジ。 ５ＣFigure 1 and Figure 4 are for explaining an example of the C1F magnetic immunoassay method according to the present invention, and Figure 1 is a schematic diagram showing a superparamagnetic label dispersed in a dispersion solvent. ,
Figure 2 is a schematic diagram showing an antibody immobilized on the surface of a non-magnetic microsphere, and Figure 3 is a schematic diagram showing a superparamagnetic material-labeled specimen complex whose magnetization direction is easily aligned by a magnetic field during antigen-antibody reaction. , FIG. 4 is a schematic diagram showing the process of concentrating and detecting the complex shown in FIG. 3. FIG. 5 is a schematic configuration diagram without showing an example of the laser magnetic immunoassay device according to the present invention, and FIG. 6 and FIG. 7 show measurement results obtained using the measuring device shown in FIG. 6
The figure is a graph showing the time dependence of the amount of scattered light in the presence of a specimen, Figure 7 is a graph showing the time dependence of scattered light 1 in the absence of a specimen, and Figure 8 is a superparamagnetic label according to the present invention. FIG. 9 is a process diagram showing an example of the method for manufacturing the superparamagnetic material W! according to the present invention. FIG. 1 is a schematic cross-sectional view showing an example of a manufacturing apparatus suitably used in carrying out a method for manufacturing a medical body. 1... Superparamagnetic ultrafine particles, 2... Antibodies, 3...
Arrow indicating the direction of easy magnetization of superparamagnetic ultrafine particles, 4...
・Antibody, 5... Nonmagnetic microspheres, 6... Virus (antigen), 7... Superparamagnetic label, 8... Nonmagnetic antibody complex, 9... Superparamagnetic label Specimen complex, 10.
... Laser incident beam, 11... Output beam, 11a, 1
1b...Scattered light flux, 12.12a, 12b...Magnetic pole piece (gradient magnetic field generator), 13...Test container, 13a
, 13b...well in test container 13, 14...electromagnet (gradient magnetic field generator), 15...laser light source (incident optical system), 16...deflector (incident optical system), 17a
, 17b... converging lens (light receiving optical system), 18a, 1
8b...Slit (light receiving optical system), 19a, 19b.
... Photomultiplier tube (light receiving optical system), 20 ... Horizontal movement device for inspection container, 21 ... Electronic circuit section (identification mechanism),
31... Atmosphere tank, 32... Superparamagnetic ultrafine particle raw material, 33... Laser, 34... Laser beam, 35
... Mirror, 36 ... Vacuum pump, 37 ... Collection tube for evaporated particles, 38 ... Collection tank for superparamagnetic ultrafine particles, 39 ... Vacuum pump, 40 ... Dispersion solution, 41・
...Magnetic filter, 42...Atmosphere gas, 43...
・Leak valve, 44...Liquid nitrogen container, 45...
Liquid nitrogen, 46... valve, 7'I7... bulge. 5C

Claims (5)

【特許請求の範囲】[Claims] （１）超常磁性体超微粒子に１つの抗原あるいは抗体を
付加した超常磁性体標識体と、検体たる抗体あるいは抗
原とを抗原抗体反応させる第１工程と、該第１工程後の
超常磁性体標識体と検体との複合体である超常磁性体標
識検体複合体を含む溶液に磁界を作用させて該超常磁性
体標識検体複合体を定められた位置に誘導・濃縮させる
第２工程と、未反応の該超常磁性体標識体と抗原抗体反
応後の該超常磁性体標識検体複合体とを濃縮位置への到
達時間差により識別する工程を少なくとも含むことを特
徴とするレーザ磁気免疫測定方法。(1) A first step of causing an antigen-antibody reaction between a superparamagnetic label in which one antigen or antibody is added to superparamagnetic ultrafine particles and an antibody or antigen as a specimen, and a superparamagnetic label after the first step. a second step in which a magnetic field is applied to a solution containing a superparamagnetic-labeled specimen complex, which is a complex of a body and a specimen, to guide and concentrate the superparamagnetic-labeled specimen complex to a predetermined position; A laser magnetic immunoassay method comprising at least the step of identifying the superparamagnetic labeled substance and the superparamagnetic labeled specimen complex after antigen-antibody reaction based on the difference in arrival time to a concentration position. （２）請求項１に記載の第１工程において、抗原抗体反
応を磁界中で行ない、抗原抗体反応後の超常磁性体標識
検体複合体の磁化方向を揃える処理を施すことを特徴と
するレーザ磁気免疫測定方法。(2) In the first step according to claim 1, the antigen-antibody reaction is performed in a magnetic field, and a process is performed to align the magnetization direction of the superparamagnetic material-labeled specimen complex after the antigen-antibody reaction. Immunoassay method. （３）超常磁性体超微粒子に１つの抗原あるいは抗体を
付加した超常磁性体標識体と、該超常磁性体標識体と検
体たる抗体あるいは抗原とを抗原抗体反応させた超常磁
性体標識検体複合体とを含む溶液を収容する検査容器と
、該超常磁性体標識検体複合体を検査容器の１点に誘導
・濃縮する傾斜磁界発生装置と、レーザ光を検査容器の
超常磁性体標識検体複合体の濃縮位置へ導く入射光学系
と、超常磁性体標識検体複合体からの出射光及び超常磁
性体標識検体複合体を含まない溶液からの出射光をそれ
ぞれ受光する光学系とを少なくとも含むレーザ磁気免疫
測定装置であって、上記超常磁性体標識検体複合体と未
反応の超常磁性体標識体とを識別する機構を具備したこ
とを特徴とするレーザ磁気免疫測定装置。(3) A superparamagnetic labeled substance in which one antigen or antibody is added to superparamagnetic ultrafine particles, and a superparamagnetic labeled specimen complex in which the superparamagnetic labeled substance is subjected to an antigen-antibody reaction with the antibody or antigen serving as the specimen. a gradient magnetic field generator that guides and concentrates the superparamagnetically labeled specimen complex to one point in the test container; A laser magnetic immunoassay that includes at least an input optical system that guides to a concentration position and an optical system that receives emitted light from a superparamagnetically labeled analyte complex and emitted light from a solution that does not contain the superparamagnetically labeled analyte complex, respectively. 1. A laser magnetic immunoassay device, comprising a mechanism for distinguishing between the superparamagnetic label sample complex and unreacted superparamagnetic label. （４）請求項１または２に記載のレーザ磁気免疫測定方
法に用いる超常磁性体標識体であつて、超常磁牲体から
なる超微粒子の表面が生物的に活性な物質で被覆され、
該被覆層に抗原あるいは抗体が固定化されてなることを
特徴とするレーザ磁気免疫測定用超常磁性体標識体。(4) A superparamagnetic label used in the laser magnetic immunoassay method according to claim 1 or 2, wherein the surface of the ultrafine particles made of a superparamagnetic substance is coated with a biologically active substance;
A superparamagnetic label for laser magnetic immunoassay, characterized in that an antigen or antibody is immobilized on the coating layer. （５）超常磁性体超微粒子の表面に活性な物質からなる
被覆層を形成する工程と、被覆層が形成された磁性体超
微粒子のうち超常性体のみを分離・回収する工程と、超
常磁性体超微粒子の被覆層に抗原あるいは抗体を固定化
する工程とを少なくとも含むことを特徴とするレーザ磁
気免疫測定用超常磁性体標識体の製造方法。(5) A step of forming a coating layer made of an active substance on the surface of the superparamagnetic ultrafine particles, a step of separating and collecting only the superparamagnetic substance from the magnetic ultrafine particles on which the coating layer has been formed, and 1. A method for producing a superparamagnetic label for laser magnetic immunoassay, the method comprising at least the step of immobilizing an antigen or antibody on a coating layer of ultrafine particles.