JPH08262024A - Kit for immunoassay of in vivo substance and immunoassay method - Google Patents
Kit for immunoassay of in vivo substance and immunoassay methodInfo
- Publication number
- JPH08262024A JPH08262024A JP1081196A JP1081196A JPH08262024A JP H08262024 A JPH08262024 A JP H08262024A JP 1081196 A JP1081196 A JP 1081196A JP 1081196 A JP1081196 A JP 1081196A JP H08262024 A JPH08262024 A JP H08262024A
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- substance
- particles
- magnetic particles
- measured
- magnetic
- Prior art date
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、磁性を有する粒子
と磁性を有しない粒子とを用いる抗原−抗体反応を利用
して生体内物質を測定する方法において、体液などの検
体を用いる測定が可能で、上記体液中の測定妨害物質の
影響を受けることなく、またＢ／Ｆ分離（bound/free分
離、すなわち、抗原と抗体により形成される免疫複合体
／未反応の遊離体を分離すること）などの複雑な操作を
必要とせずに、測定対象物質の濃度上昇を吸光度値の増
加で検出し、高感度で信頼性の高い測定結果が得られる
免疫測定用キットおよび免疫測定方法に関する。TECHNICAL FIELD The present invention relates to a method for measuring an in-vivo substance utilizing an antigen-antibody reaction using magnetic particles and non-magnetic particles, which enables measurement using a specimen such as a body fluid. And B / F separation (bound / free separation, that is, separation of immune complex / unreacted educt formed by antigen and antibody) without being affected by the measurement-interfering substances in the body fluid. The present invention relates to an immunoassay kit and an immunoassay method that can detect a concentration increase of a substance to be measured by an increase in absorbance value and can obtain a highly sensitive and reliable measurement result without requiring a complicated operation such as.
【０００２】[0002]
【従来の技術】従来、血清、血漿、尿などの体液中の抗
原または抗体を測定対象物質として、抗体または抗原感
作磁性粒子を用いる免疫学的な測定が行われてきた。こ
のような測定方法は、特開昭55−156866号公報、特開昭
61−128168号公報、特開平１−193647号公報、特開平３
−59459 号公報、および特開平３−128462号公報などで
開示されている。2. Description of the Related Art Conventionally, immunological measurements have been carried out using antibodies or antigen-sensitized magnetic particles with antigens or antibodies in body fluids such as serum, plasma and urine as the substances to be measured. Such measuring methods are disclosed in JP-A-55-156866 and JP-A-
61-128168, JP-A-1-193647, JP-A-3
-59459 and Japanese Patent Laid-Open No. 3-128462.
【０００３】特開昭55−156866号公報には、抗原または
抗体の担持粒子である第一の磁性粒子を用いて検体中の
測定対象物質との複合体を形成させ、この複合体とは結
合するが遊離の第一の粒子（測定対象物質と結合してい
ない）とは結合しない第二の粒子と上記複合体とを結合
させて凝集させ、凝集していない第一または第二の粒子
を標識により選択的に検定することによって検体中に含
まれる抗体または抗原の量を測定する方法が開示されて
いる。そして、上記粒子は磁性体を含んでもよく、凝集
体の分離は遠心または集磁によって行うことが開示され
ている。In Japanese Patent Laid-Open No. 55-156866, a first magnetic particle, which is a particle carrying an antigen or an antibody, is used to form a complex with a substance to be measured in a sample, and the complex is bound to this complex. However, the first particles or the second particles that are not aggregated are aggregated by binding the second particles that do not bind to the free first particles (which are not bound to the substance to be measured) and the complex. A method of measuring the amount of antibody or antigen contained in a sample by selectively assaying with a label is disclosed. It is disclosed that the particles may include a magnetic substance, and the aggregates are separated by centrifugation or magnetism collection.
【０００４】また、特開平１−193647号公報には、抗体
または抗原を担持させた磁性粒子と同じく抗体または抗
原を担持させた非磁性粒子とを抗原または抗体と反応さ
せて凝集体を形成させ、これらを反応液から分離した
後、反応液中に残存する非磁性粒子の量を吸光度または
散乱光で測定することにより、定性的または定量的に測
定する方法が開示されている。特開平３−59459 号公報
には、磁性粒子と非磁性粒子により凝集体を形成させ、
これらを反応液から分離した後、反応液中に残存する非
磁性粒子を濁度測定することにより、定性的または定量
的に測定する方法が開示されている。Further, in JP-A-1-193647, magnetic particles carrying an antibody or an antigen and non-magnetic particles carrying an antibody or an antigen are reacted with the antigen or the antibody to form an aggregate. There is disclosed a method of qualitatively or quantitatively measuring the amount of non-magnetic particles remaining in the reaction solution by measuring the absorbance or scattered light after separating them from the reaction solution. JP-A-3-59459 discloses that an aggregate is formed by magnetic particles and non-magnetic particles,
A method of qualitatively or quantitatively measuring the nonmagnetic particles remaining in the reaction solution after separating them from the reaction solution is disclosed.
【０００５】特開平３−128462号公報には、抗体または
抗原と反応した、あるいは未反応の磁性粒子および磁性
粒子と非磁性粒子との凝集物を反応液から分離した後、
反応液中に残存する粒子（非磁性）量を目視または光学
的に測定することにより検体中の抗体量を定量し、これ
らの抗体のＩｇのクラスを判別する方法が開示されてい
る。さらに、特開昭61−128168号公報には、抗体または
抗原を担持させた磁化可能粒子である不溶性担体（磁性
粒子）と磁化可能粒子でない標識粒子（非磁性粒子）と
を用いて反応を行い、反応混合物に磁場を付与すること
によって、この反応混合物中の磁化粒子または磁化粒子
を含む凝集粒子を集磁により分離し、この凝集粒子中の
標識粒子を標識強度で測定する免疫学的分析方法が開示
されている。In Japanese Patent Laid-Open No. 3-128462, after separating magnetic particles that have reacted with antibodies or antigens or unreacted and aggregates of magnetic particles and non-magnetic particles from a reaction solution,
A method is disclosed in which the amount of particles (non-magnetic) remaining in the reaction solution is visually or optically measured to quantify the amount of antibody in the sample and to determine the Ig class of these antibodies. Further, in JP-A-61-128168, a reaction is carried out using an insoluble carrier (magnetic particle) which is a magnetizable particle carrying an antibody or an antigen and a labeled particle (non-magnetic particle) which is not a magnetizable particle. An immunological analysis method in which magnetized particles or agglomerated particles containing magnetized particles in the reaction mixture are separated by magnetic attraction by applying a magnetic field to the reaction mixture, and the labeled particles in the agglomerated particles are measured by the labeling intensity. Is disclosed.
【０００６】[0006]
【発明が解決しようとする課題】これらの方法において
は、抗原濃度の上昇につれて吸光度が上昇する検量線を
得るために、以下の手順が取られている。すなわち、上
記抗体または抗原担持磁性粒子と標識粒子（非磁性粒
子）とを反応させた後、これら２つの粒子によって形成
された免疫複合体と集磁されずに残った非磁性粒子とを
Ｂ／Ｆ分離し、洗浄する工程を経た後に、上記免疫複合
体中の非磁性粒子の量をその標識によって検出するとい
うものである。したがって、上述の方法においては、Ｂ
／Ｆ分離とその後の洗浄工程が不可欠であった。In these methods, the following procedure is taken in order to obtain a calibration curve whose absorbance increases as the antigen concentration increases. That is, after reacting the above-mentioned antibody- or antigen-bearing magnetic particles with labeled particles (non-magnetic particles), the immune complex formed by these two particles and the non-magnetic particles remaining without being magnetized are After the steps of F separation and washing, the amount of nonmagnetic particles in the immune complex is detected by the label. Therefore, in the above method, B
/ F separation and subsequent washing steps were essential.
【０００７】そして、これらの公報で開示された方法で
得られる検量線は、一般的なイムノアッセイやＥＩＡ法
などで得られる右上がりのものであるが、ラテックス凝
集法、免疫比濁法等で過剰の抗原を用いた場合は、図１
に示したように抗原−抗体の複合体が形成されずに抗原
濃度が高いところで右上がりの曲線が急激に下がるとい
うプロゾーン現象が生じる場合があった。図１中、プロ
ゾーン現象をおこした範囲を矢印で示す。さらに、この
ような測定に用いる検体は、測定対象物質以外にも多く
の生体内物質を含み、これらのうちのいくつかは測定妨
害物質として作用するため、測定前に検体からこのよう
な測定妨害物質を除去しておかなければ、測定精度の低
下が生じるなどといった問題点があり、信頼性に欠ける
場合があった。The calibration curves obtained by the methods disclosed in these publications are upward-sloping curves obtained by general immunoassays, EIA methods, etc., but are excessive by latex agglutination method, immunoturbidimetric method, etc. When using the antigen of
In some cases, the prozone phenomenon in which the upward-sloping curve sharply drops at a high antigen concentration without formation of the antigen-antibody complex as shown in FIG. In FIG. 1, the range in which the prozone phenomenon occurs is indicated by an arrow. Furthermore, the sample used for such measurement contains many in-vivo substances in addition to the substance to be measured, and some of them act as measurement interfering substances. If the substance is not removed, there is a problem that the measurement accuracy may be deteriorated and the reliability may be insufficient.
【０００８】本発明は、以上のような問題点を解決する
ためになされたものであり、洗浄工程が不要で、プロゾ
ーン現象を起こさない右上がりの検量線を得ることがで
き、測定精度および信頼性の高い、生体内物質の免疫測
定用キットおよび免疫測定方法を提供することにある。The present invention has been made in order to solve the above-mentioned problems, does not require a washing step, and can obtain a calibration curve that rises to the right without causing the prozone phenomenon. An object of the present invention is to provide a highly reliable immunoassay kit for an in-vivo substance and an immunoassay method.
【０００９】[0009]
【課題を解決するための手段】すなわち、本発明は、抗
原−抗体反応を利用して生体内物質を測定する免疫測定
用キットであって、少なくとも、測定対象物質と免疫学
的に結合する物質を担持させた不溶性磁性粒子、および
測定対象物質を担持させた特定波長吸収領域を有する不
溶性非磁性粒子を含むことを特徴とする生体内物質の免
疫測定用キットである。また、本発明は、検体中の測定
妨害物質と免疫学的に結合する物質を担持させた不溶性
磁性粒子をさらに含むことを特徴とする。That is, the present invention provides an immunoassay kit for measuring an in-vivo substance using an antigen-antibody reaction, which is a substance that immunologically binds to at least a substance to be measured. An in-vivo substance immunoassay kit characterized by comprising insoluble magnetic particles carrying R and insoluble non-magnetic particles having a specific wavelength absorption region carrying a substance to be measured. Further, the present invention is characterized in that it further comprises insoluble magnetic particles carrying a substance that immunologically binds to the measurement-interfering substance in the sample.
【００１０】さらに、本発明は、抗原−抗体反応を利用
して生体内物質を測定する免疫測定方法であって、
（１）測定対象物質と免疫学的に結合する物質を担持さ
せた不溶性磁性粒子を含む溶液、および測定対象物質を
担持させた特定波長吸収領域を有する不溶性非磁性粒子
を含む溶液を、任意の順序で検体と混合して混合液と
し、（２）上記混合液に磁場を付与して、上記混合液中
で生成した上記磁性粒子と測定対象物質とが形成した免
疫複合体、および未結合の磁性粒子を集磁し、（３）残
留する上記非磁性粒子の濃度を吸光度によって測定する
生体内物質を測定する免疫測定方法である。Further, the present invention is an immunoassay method for measuring an in-vivo substance using an antigen-antibody reaction,
(1) A solution containing insoluble magnetic particles carrying a substance that immunologically binds to a measurement target substance and a solution containing insoluble non-magnetic particles having a specific wavelength absorption region supporting a measurement target substance (2) A magnetic field is applied to the mixed solution to mix with the sample in order to form a mixed solution, and the immune complex formed by the magnetic particles and the substance to be measured formed in the mixed solution, and unbound An immunoassay method in which magnetic particles are collected and (3) the concentration of the remaining non-magnetic particles is measured by absorbance to measure an in-vivo substance.
【００１１】本発明はまた、検体中の測定妨害物質と免
疫学的に結合する物質を担持させた不溶性磁性粒子をさ
らに加え、上記磁性粒子と測定妨害物質とが形成した免
疫複合体をさらに集磁することを特徴とする。In addition, the present invention further comprises insoluble magnetic particles carrying a substance that immunologically binds to a measurement-interfering substance in a sample, and further collects an immune complex formed by the magnetic particles and the measurement-interfering substance. It is characterized by being magnetized.
【００１２】以下に本発明を詳細に説明する。本発明の
抗原−抗体反応による生体内物質の免疫測定用キット
は、少なくとも、測定対象物質と免疫学的に結合する物
質を担持させた不溶性磁性粒子（以後、磁性粒子（Ａ）
と記す）と、測定対象物質を担持させた特定波長吸収領
域を持つ不溶性非磁性粒子（以下、非磁性粒子（Ｂ）と
記す）とを含むことを特徴とするものである。これらの
粒子は、溶液中に含まれていてもよい。The present invention will be described in detail below. The kit for immunoassay of an in-vivo substance by the antigen-antibody reaction of the present invention comprises at least insoluble magnetic particles (hereinafter, magnetic particles (A)) carrying a substance that immunologically binds to a substance to be measured.
And) insoluble non-magnetic particles (hereinafter, referred to as non-magnetic particles (B)) having a specific wavelength absorption region supporting a substance to be measured. These particles may be contained in the solution.
【００１３】本発明においては、抗原−抗体反応におけ
る測定対象物質を含む検体として、ヒト、動物の体液、
具体的には、全血、血清、血漿、尿、リンパ液、脊髄
液、関節液、唾液、汗、乳汁、胃液、膵液、腸液、胆
汁、涙液などを用いることができる。そのなかでも、従
来前処理を必要とした有色の検体、例えば、全血、乳
汁、胆汁等に用いることが好ましく、特に、微少の検体
量で済むことから全血の測定上好適である。In the present invention, as the sample containing the substance to be measured in the antigen-antibody reaction, human and animal body fluids,
Specifically, whole blood, serum, plasma, urine, lymph, spinal fluid, synovial fluid, saliva, sweat, milk, gastric juice, pancreatic juice, intestinal juice, bile, tear fluid and the like can be used. Among them, it is preferable to use it for a colored sample that has conventionally required pretreatment, for example, whole blood, milk, bile, etc., and it is particularly suitable for measurement of whole blood because it requires a small amount of sample.
【００１４】測定対象物質は、検体中に含まれる抗原、
抗体のいずれであってもよく、抗原の場合、抗体産生能
を有するものであればよい。また、測定対象物質と免疫
学的に結合する物質は、上記抗原または抗体と特異的に
結合する抗原、抗体であればよく、抗体の中には抗−抗
体も含まれる。抗体は、一般に、ポリクローナル抗体、
モノクローナル抗体に大別され、さらに、これらの抗体
は、ＩｇＡ、ＩｇＭ、ＩｇＧ、ＩｇＥおよびＩｇＤの５
つのクラスに分けられるが、同時にこれらの抗体はタン
パクであるため、免疫原性と抗原性とを有し、それ自身
が抗原となり得る。The substance to be measured is an antigen contained in the sample,
It may be any antibody, and in the case of an antigen, any antigen may be used as long as it has antibody-producing ability. The substance that immunologically binds to the substance to be measured may be an antigen or an antibody that specifically binds to the above-mentioned antigen or antibody, and the antibody also includes anti-antibody. Antibodies are generally polyclonal antibodies,
It is roughly classified into monoclonal antibodies, and these antibodies are further classified into IgA, IgM, IgG, IgE and IgD 5
Although these antibodies are divided into two classes, at the same time, since these antibodies are proteins, they have immunogenicity and antigenicity, and can be antigens themselves.
【００１５】本発明は、抗原として上述の抗体、あるい
は上述の抗体をパパインやペプシンで分解して得られる
Ｆab、Ｆ(ab')2フラグメント、またはγ−グロブリンな
どの他、種々の物質を使用することができる。具体的に
は、アルブミン；ＨＣＧ（絨毛性性腺刺激ホルモン）、
ＡＦＰ（α−フェトプロテイン）、ＣＥＡ（癌胎児性抗
原）、ＣＲＰ（Ｃ反応性タンパク）、カルジオリピン抗
原、ＨＢｓ（Ｂ型肝炎ウイルスの表面抗原）、ヒト成長
ホルモン、ヘモグロビン、Ｌｐ（ａ）（リポプロテイン
（ａ））などのタンパク；Ａｐｏ−ＡＩ、Ａｐｏ−ＡI
I、Ａｐｏ−ＣII、Ａｐｏ−ＣIII 、Ａｐｏ−Ｂ、Ａｐ
ｏ−Ｅなどのアポリポタンパク；Ａ型、Ｂ型、Ｏ型、Le
a 型、Leb 型抗原などの糖鎖抗原である血液型物質；ス
テロイドホルモン、コンカナバリンＡ、各種プロスタグ
ランジンなどのハプテンと担体の結合した物質；などを
挙げることができる。In the present invention, various substances other than the above-mentioned antibody or Fab, F (ab ') 2 fragment obtained by degrading the above-mentioned antibody with papain or pepsin, or γ-globulin are used as an antigen. can do. Specifically, albumin; HCG (chorionic gonadotropin),
AFP (α-fetoprotein), CEA (carcinoembryonic antigen), CRP (C-reactive protein), cardiolipin antigen, HBs (hepatitis B virus surface antigen), human growth hormone, hemoglobin, Lp (a) (lipoprotein) (A)) and other proteins; Apo-AI, Apo-AI
I, Apo-CII, Apo-CIII, Apo-B, Ap
apolipoprotein such as o-E; A type, B type, O type, Le
Examples thereof include blood group substances that are sugar chain antigens such as a- type and Le b- type antigens; substances in which haptens such as steroid hormones, concanavalin A, and various prostaglandins are bound to carriers.
【００１６】本発明で用いる抗体としては、抗アルブミ
ン抗体、抗Ｌｐ（ａ）抗体、抗ＣＲＰ抗体、抗ヒト成長
ホルモン抗体、抗ＨＣＧ抗体、抗ＡＦＰ抗体、抗ＣＥＡ
抗体、抗ヒト凝固ファクター抗体、抗ＨＢｓ抗体などの
タンパクに対する抗体；抗ＩｇＡ抗体、抗ＩｇＭ抗体、
抗ＩｇＧ抗体、抗ＩｇＥ抗体、抗ＩｇＤ抗体および抗γ
−グロブリン抗体などの免疫グロブリンからなる抗体；
抗ステロイドホルモン抗体、抗ＤＮＡ抗体、抗プロスタ
グランジン抗体などのハプテンに対する抗体といったも
のを挙げることができる。The antibodies used in the present invention include anti-albumin antibodies, anti-Lp (a) antibodies, anti-CRP antibodies, anti-human growth hormone antibodies, anti-HCG antibodies, anti-AFP antibodies, anti-CEA.
Antibodies against proteins such as antibodies, anti-human coagulation factor antibodies, anti-HBs antibodies; anti-IgA antibodies, anti-IgM antibodies,
Anti-IgG antibody, anti-IgE antibody, anti-IgD antibody and anti-γ
An antibody consisting of an immunoglobulin such as a globulin antibody;
Examples thereof include antibodies to haptens such as anti-steroid hormone antibody, anti-DNA antibody, anti-prostaglandin antibody.
【００１７】本発明においては、上述の抗原、抗体など
の測定対象物質を非磁性粒子に担持させ、この粒子自
体、または水溶液中にこの粒子を分散して後述の磁性体
粒子（Ａ）と組み合わせて生体内物質の免疫測定用キッ
トとし、また、このキットを免疫測定に用いる。本発明
で使用する非磁性粒子の素材としては、有機高分子物
質、無機物質のいずれでもよい。有機高分子物質の例と
しては、ゼラチン粒子、ポリスチレン粒子、スチレン−
ブタジエン共重合体粒子、またはスチレン−（メタ）ア
クリル酸エステル共重合体粒子があり、これらは乳化重
合によりラテックスとして得ることができる。In the present invention, the substances to be measured such as the above-mentioned antigens and antibodies are carried on non-magnetic particles, and the particles themselves or dispersed in an aqueous solution and combined with magnetic particles (A) described later. To be used as an immunoassay kit for in-vivo substances, and this kit is used for immunoassay. The material of the non-magnetic particles used in the present invention may be either an organic polymer substance or an inorganic substance. Examples of organic polymer substances include gelatin particles, polystyrene particles, styrene-
There are butadiene copolymer particles or styrene- (meth) acrylic acid ester copolymer particles, which can be obtained as a latex by emulsion polymerization.
【００１８】上記（メタ）アクリル酸エステル共重合体
粒子の形成に用いるモノマーとしては、例えば（メタ）
アクリル酸−２−ヒドロキシエチル、（メタ）アクリル
酸−２−ヒドロキシプロピル、（メタ）アクリル酸−１
−メチル−２−ヒドロキシエチル、モノメタクリル酸グ
リセロール、２−アクリルアミド−２−メチルプロパン
スルホン酸、メタクリル酸−２−スルホエチル、メタク
リル酸アシッドホスホキシエチル、メタクリル酸−３−
クロロ−２−アシッドホスホキシプロピル、メタクリル
酸アシッドホスホキシプロピル、（メタ）アクリル酸エ
チル、（メタ）アクリル酸−ｎ−ブチル、（メタ）アク
リル酸−ｉ−ブチル、（メタ）アクリル酸−２−エチル
ヘキシル、メタクリル酸ラウリル、メタクリル酸シクロ
ヘキシル、（メタ）アクリル酸アミド、Ｎ−メチロール
アクリルアミド、Ｎ−ブトキシメチルアクリルアミド、
（メタ）アクリル酸グリシジル、および（メタ）アクリ
ル酸メチルグリシジルなどがある。Examples of the monomer used for forming the (meth) acrylic acid ester copolymer particles include (meth)
2-Hydroxyethyl acrylate, 2-Hydroxypropyl (meth) acrylate, (Meth) acrylic acid-1
-Methyl-2-hydroxyethyl, glycerol monomethacrylate, 2-acrylamido-2-methylpropanesulfonic acid, 2-sulfoethyl methacrylate, acid phosphoxyethyl methacrylate, methacrylic acid-3-
Chloro-2-acid phosphoroxypropyl, acid phosphoroxypropyl methacrylate, ethyl (meth) acrylate, n-butyl (meth) acrylate, i-butyl (meth) acrylate, (meth) acrylic acid-2 -Ethylhexyl, lauryl methacrylate, cyclohexyl methacrylate, (meth) acrylic acid amide, N-methylolacrylamide, N-butoxymethylacrylamide,
Examples thereof include glycidyl (meth) acrylate and methylglycidyl (meth) acrylate.
【００１９】無機物質の例としては、シリカ、アルミ
ナ、酸化チタン等を挙げることができる。Examples of inorganic materials include silica, alumina, titanium oxide and the like.
【００２０】非磁性粒子は、特定波長吸収領域を有すれ
ばよく、好ましくは着色剤を公知の方法で含有させて着
色粒子とする。特定波長吸収領域とは１９０〜１，００
０ｎｍの波長領域をいい、この範囲に最大吸収波長を有
し、吸光度が測定できる着色剤を用いることが好まし
い。特に、３５０〜７００ｎｍで吸光度が測定できる着
色剤を用いることが検体中のタンパクが有する紫外線吸
収を感知しないことから好ましい。上記非磁性粒子の着
色は特に限定されず、半透明であってもよい。The non-magnetic particles have only to have a specific wavelength absorption region, and preferably, a coloring agent is added by a known method to obtain colored particles. The specific wavelength absorption region is 190 to 1,000
It means a wavelength region of 0 nm, and it is preferable to use a colorant having a maximum absorption wavelength in this range and capable of measuring absorbance. In particular, it is preferable to use a coloring agent whose absorbance can be measured at 350 to 700 nm because it does not detect the ultraviolet absorption of the protein in the sample. The coloring of the nonmagnetic particles is not particularly limited and may be semitransparent.
【００２１】非磁性粒子の粒径は特に限定されないが、
０．０１〜１００μｍが好ましく、さらに０．１〜３０
μｍが好ましい。粒径が０．０１μｍ未満では、抗体感
作時に粒子同士の凝集が起きやすくなり、また遠心分離
が困難となるからであり、１００μｍを超えると水溶液
中で短時間に沈降し、抗原−抗体反応に好ましい浮遊性
の良い粒子が得られないためである。The particle size of the non-magnetic particles is not particularly limited,
0.01 to 100 μm is preferable, and further 0.1 to 30
μm is preferred. This is because if the particle size is less than 0.01 μm, aggregation of particles is likely to occur during antibody sensitization, and centrifugation is difficult, and if it exceeds 100 μm, the particles precipitate in an aqueous solution in a short time and the antigen-antibody reaction occurs. This is because it is not possible to obtain particles having good floating properties.
【００２２】本発明で使用する磁性粒子は、上述の有機
高分子物質、無機物質粒子に、公知の方法で、鉄、磁性
酸化鉄などの磁性体を含有させて磁性粒子とするか、ま
たは有機高分子物質や無機物質の粒子を核としてフェラ
イト被覆を行いフェライト被覆粒子を形成する。一方、
磁性粒子の粒径は、０．０１μｍ未満では粒子の製造が
困難であり、たとえ製造できても集磁性不良であるこ
と、また、平均粒径が１００μｍを超えると表面積が大
きくなって分散不良となり測定感度が低下することか
ら、０．０１〜１００μｍのものが好ましく、より好ま
しくは０．１〜３０μｍのものである。The magnetic particles used in the present invention may be magnetic particles obtained by incorporating a magnetic material such as iron or magnetic iron oxide into the above-mentioned organic polymer particles or inorganic material particles by a known method. Ferrite coating is performed by using particles of a polymer substance or an inorganic substance as a core to form ferrite-coated particles. on the other hand,
If the particle size of the magnetic particles is less than 0.01 μm, it is difficult to manufacture the particles, and even if it can be manufactured, the magnetism is poor, and if the average particle size exceeds 100 μm, the surface area becomes large and the dispersion becomes poor. Since the measurement sensitivity decreases, it is preferably from 0.01 to 100 μm, more preferably from 0.1 to 30 μm.
【００２３】本発明の磁性粒子は上述のようにして得ら
れる磁性粒子を更に高分子処理により粒子表面を被覆し
て用いてもよい。高分子処理に用いる高分子化合物のう
ち、合成高分子としては、例えば、シラン、ナイロン又
はポリスチレン等を挙げることができる。また、天然高
分子としてはゼラチン、キチン等の蛋白質や天然ゴム等
を挙げることができる。更に、これら合成高分子、天然
高分子の共重合体あるいは混合物も使用することができ
る。The magnetic particles of the present invention may be obtained by coating the surface of the magnetic particles obtained as described above with a polymer treatment. Among the polymer compounds used for polymer processing, examples of synthetic polymers include silane, nylon, polystyrene and the like. Examples of natural polymers include proteins such as gelatin and chitin, natural rubber and the like. Furthermore, copolymers or mixtures of these synthetic polymers and natural polymers can also be used.
【００２４】本発明においては、非磁性粒子には、上述
した検体中の測定対象物質を、また磁性粒子には測定対
象物質と免疫学的に結合する物質をそれぞれ担持させ
て、磁性粒子（Ａ）および非磁性粒子（Ｂ）を形成す
る。ところで、検体中に赤血球、ヘモグロビン、ビリル
ビンなどの生体内物質、あるいはフェノバルビタール、
フェニトイン、ジゴキシン、イミプラミン、テオフィリ
ン、ペニシリンなどの薬物が存在する場合は、これらが
測定妨害物質となって測定感度や測定精度に悪影響を及
ぼす。本発明においては、上述の磁性粒子（Ａ）および
非磁性粒子（Ｂ）に加えて、さらに測定妨害物質と免疫
学的に結合する物質を担持させた磁性粒子（以後、磁性
粒子（Ｃ）と記す）を調製・添加し、これらの測定妨害
物質を除去することができる。磁性粒子（Ｃ）の原料と
なる磁性粒子は、上述した磁性粒子（Ａ）の原料と同様
のものを使用して製造することができる。磁性粒子
（Ｃ）は、２種以上を使用してもよい。In the present invention, the non-magnetic particles are loaded with the substance to be measured in the sample described above, and the magnetic particles are loaded with the substance that immunologically binds to the substance to be measured. ) And non-magnetic particles (B) are formed. By the way, in-vivo substances such as red blood cells, hemoglobin, bilirubin, or phenobarbital in the sample,
When drugs such as phenytoin, digoxin, imipramine, theophylline, and penicillin are present, they act as measurement interfering substances and adversely affect measurement sensitivity and measurement accuracy. In the present invention, in addition to the above-mentioned magnetic particles (A) and non-magnetic particles (B), magnetic particles (hereinafter referred to as magnetic particles (C)) carrying a substance that immunologically binds to a measurement-interfering substance. (Note) can be prepared and added to remove these measurement-interfering substances. The magnetic particles that are the raw material of the magnetic particles (C) can be manufactured using the same materials as the raw materials of the magnetic particles (A) described above. Two or more kinds of magnetic particles (C) may be used.
【００２５】上記の測定対象物質または測定妨害物質と
免疫学的に結合するとは、これらの物質と特異的に結合
することを意味し、測定対象物質または測定妨害物質が
上述したような抗原である場合にはこれらに対する抗体
と、測定対象物質または測定妨害物質が上述したような
抗体である場合には、これらに対する抗原または抗抗体
との間に形成される結合をいう。Immunologically binding to the substance to be measured or the substance that interferes with the measurement means to specifically bind to these substances, and the substance to be measured or the substance to be measured is an antigen as described above. In this case, it refers to the bond formed between the antibody against them and the antigen or anti-antibody against them when the substance to be measured or the substance that interferes with the measurement is an antibody as described above.
【００２６】上述の粒子に、測定対象物質またはこれら
に免疫学的に結合する物質を担持させる方法は、磁性粒
子または非磁性粒子に共通である。すなわち、上述した
各粒子に、測定対象物質または測定対象物質と免疫学的
に結合する物質を物理的に吸着させる方法、または、化
学的に担持させる方法がある。The above-mentioned method of supporting the substance to be measured or the substance which is immunologically bound to these substances is common to magnetic particles and non-magnetic particles. That is, there is a method of physically adsorbing a substance to be measured or a substance that immunologically binds to the substance to be measured, or a method of chemically supporting the substance on each particle described above.
【００２７】具体的には、上述の抗体または抗原などを
適当な緩衝液中で前記粒子と反応させることにより、物
理的に吸着させることができる。この反応に使用する緩
衝溶液としては、リン酸緩衝液、トリス−塩酸緩衝液、
炭酸緩衝液などを挙げることができ、吸着反応の際のｐ
Ｈに応じて、適切な緩衝作用を発揮し得る緩衝液を適宜
選択する。反応は、上述の粒子と測定対象物質または測
定対象物質と免疫学的に結合する物質とを室温にて混合
することにより容易に進行し、目的とする物質を担持し
た粒子を得ることができる。また、化学的に担持させる
方法では、所謂ペプチド結合法におけるカルボジイミド
法やグルタルアルデヒド法等を採用して、目的とする物
質を担持した粒子を得ることができる。Specifically, the above-mentioned antibody or antigen can be physically adsorbed by reacting with the particles in an appropriate buffer. The buffer solution used in this reaction includes a phosphate buffer solution, a Tris-hydrochloric acid buffer solution,
Carbonic acid buffer etc. can be mentioned, and p at the time of adsorption reaction
Depending on H, a buffer solution capable of exhibiting an appropriate buffer action is appropriately selected. The reaction easily proceeds by mixing the above-mentioned particles and the substance to be measured or the substance to be immunologically bound to the substance to be measured at room temperature, and particles carrying the target substance can be obtained. Further, in the method of chemically supporting, a so-called peptide binding method such as a carbodiimide method or a glutaraldehyde method can be adopted to obtain particles supporting a target substance.
【００２８】担持させる抗体としては、上述のポリクロ
ーナル抗体またはモノクローナル抗体が使用でき、例え
ば、γ−グロブリン、ＩｇＧ、ＩｇＭなどのクラスに属
する免疫グロブリン、Ｆ(ab')2、Ｆabなどのフラグメン
トのいずれの抗体も使用することができる。また、担持
させる抗原としては、細胞片、ハプテン−担体の結合
体、タンパク、免疫複合体、天然または合成高分子など
が用いられる。ハプテンと化学的に結合させる担体とし
ては、アルブミンなどの異種タンパクが汎用される。As the antibody to be carried, the above-mentioned polyclonal antibody or monoclonal antibody can be used, and examples thereof include immunoglobulins belonging to the classes such as γ-globulin, IgG, and IgM, and fragments such as F (ab ') 2 and Fab. Antibodies of can also be used. Further, as the antigen to be carried, cell debris, hapten-carrier conjugate, protein, immune complex, natural or synthetic polymer, etc. are used. A heterologous protein such as albumin is generally used as a carrier to be chemically bound to the hapten.
【００２９】一般に、粒子に担持させる抗原または抗体
の量は、粒子の種類などによって大きな幅があるが、
０．００１mg/ml 〜２０mg/ml 、好ましくは０．００５
mg/ml〜５mg/ml の範囲である。免疫学的に結合する物
質を担持した各粒子を、それぞれ、ＢＳＡ（ウシ胎児血
清アルブミン）などの血清を含んでもよい上述のリン酸
緩衝液、トリス塩酸衝液等の水性溶媒中に、０．０１〜
１０重量％となるように分散し、懸濁液であるラテック
スとして、本発明の免疫測定用キットの一部とする。Generally, the amount of the antigen or antibody to be carried on the particles has a wide range depending on the kind of the particles,
0.001 mg / ml to 20 mg / ml, preferably 0.005
It is in the range of mg / ml to 5 mg / ml. Each of the particles carrying the substance to be immunologically bound is subjected to 0.01% in an aqueous solvent such as the above-mentioned phosphate buffer solution or Tris-hydrochloric acid buffer solution which may contain serum such as BSA (fetal bovine serum albumin). ~
Dispersion is performed so as to be 10% by weight, and a latex as a suspension is used as a part of the immunoassay kit of the present invention.
【００３０】本発明の免疫測定用キットは、上述のよう
に、少なくとも測定対象物質と免疫学的に結合する物質
を担持した磁性粒子（Ａ）と測定対象物質を担持した１
種以上の非磁性粒子（Ｂ）を含むものである。上記免疫
測定用キットは、さらに検体中の測定妨害物質と免疫学
的に結合する物質を担持させた１種以上の磁性を有する
磁性粒子（Ｃ）を含んでもよい。また、これらの粒子が
溶液中に含まれている場合、必要に応じてＮａＮ3 など
の防腐剤を添加してもよい。As described above, the immunoassay kit of the present invention comprises a magnetic particle (A) carrying at least a substance that immunologically binds to the substance to be measured and a substance to be measured.
It contains one or more kinds of non-magnetic particles (B). The immunoassay kit may further include one or more types of magnetic particles (C) having magnetism, which carry a substance that immunologically binds to a measurement-interfering substance in a sample. Further, when these particles are contained in the solution, a preservative such as NaN 3 may be added if necessary.
【００３１】上記免疫測定用キットは、これらの他に、
検量線を作成するための標準試料とそれを希釈するため
の水溶液、検体希釈用の水溶液などを含んでもよい。こ
のような水溶液としては、例えば、水、生理食塩水、上
述したリン酸緩衝液、トリス−塩酸緩衝液などを挙げる
ことができる。上述した磁性粒子および非磁性粒子が含
まれてもよい水溶性溶媒、検量線作成用標準試料および
その希釈用緩衝液、および検体希釈用緩衝液は、それぞ
れ個別に、表面処理を行ったガラス製容器、またはポリ
プロピレンなどのプラスチック製容器などに入れて保存
する。The above-mentioned immunoassay kit includes, in addition to these,
It may contain a standard sample for preparing a calibration curve, an aqueous solution for diluting the standard sample, an aqueous solution for diluting a sample, and the like. Examples of such an aqueous solution include water, physiological saline, the above-mentioned phosphate buffer solution, Tris-hydrochloric acid buffer solution, and the like. The above-mentioned water-soluble solvent that may contain magnetic particles and non-magnetic particles, the standard sample for preparing the calibration curve and its dilution buffer, and the sample dilution buffer are each made of glass that has been surface treated. Store in a container or a plastic container such as polypropylene.
【００３２】本発明の測定方法は、以下に説明する
（１）〜（３）の工程を含む。本発明の免疫測定用キッ
トを用いる場合には、まず、第（１）工程として、上述
の（Ａ）、（Ｂ）の粒子と検体とを混合する。上述の各
粒子の使用量は、測定対象物質を担持させた非磁性粒子
（Ｂ）１容量に対して、測定対象物質と免疫学的に結合
する物質を担持させた磁性粒子（Ａ）を１／４〜４容量
倍の範囲で用いることが、非磁性粒子を効率よく磁性粒
子と反応させるために好ましい。より好ましくは、１〜
４容量倍である。粒子（Ａ）、（Ｂ）と測定対象物質と
の混合順序は特に限定されないが、磁性粒子（Ａ）をま
ず測定対象物質と混合し、次に非磁性粒子（Ｂ）を加え
るか、あるいは非磁性粒子（Ｂ）をまず測定対象物質と
混合し、次に磁性粒子（Ａ）を加えるのが好ましい。ま
た、必要により、測定妨害物質と免疫学的に結合する物
質を担持した磁性粒子（Ｃ）を上記（Ａ）、（Ｂ）の粒
子と混合してもよい。この場合、上記の（Ｃ）粒子は、
上記非磁性粒子（Ｂ）１容量に対し、１／１０〜１０容
量倍の範囲で用いることができる。特に好ましくは、１
／２〜２容量倍である。The measuring method of the present invention includes the steps (1) to (3) described below. When the immunoassay kit of the present invention is used, first, in the step (1), the particles (A) and (B) described above are mixed with the sample. The amount of each of the above-mentioned particles to be used is such that 1 volume of the non-magnetic particles (B) carrying the substance to be measured carries 1 volume of the magnetic particles (A) carrying the substance immunologically bound to the substance to be measured. It is preferable to use it in the range of / 4 to 4 times the volume in order to efficiently react the non-magnetic particles with the magnetic particles. More preferably 1 to
4 times the capacity. The mixing order of the particles (A) and (B) and the substance to be measured is not particularly limited, but the magnetic particles (A) are first mixed with the substance to be measured, and then the non-magnetic particles (B) are added, or It is preferable to first mix the magnetic particles (B) with the substance to be measured, and then add the magnetic particles (A). If necessary, magnetic particles (C) carrying a substance that immunologically binds to the measurement-interfering substance may be mixed with the particles (A) and (B). In this case, the above-mentioned (C) particles are
The non-magnetic particles (B) can be used in a range of 1/10 to 10 times the volume of the non-magnetic particles (B). Particularly preferably, 1
/ 2 to 2 times the capacity.
【００３３】上述の粒子（Ａ）、（Ｂ）、および（Ｃ）
の混合順序は特に限定されないが、磁性粒子（Ｃ）、磁
性粒子（Ａ）および検体をまず混合し、十分に反応させ
た後に非磁性粒子（Ｂ）を混合する方法が、測定感度お
よび再現性を良くする点で好ましい。さらに好ましく
は、上記磁性粒子（Ｃ）を検体とまず混合し、その後上
記磁性粒子（Ａ）を混合し十分に反応させた後、さらに
非磁性粒子（Ｂ）を混合する方法である。また、粒子
（Ａ）、（Ｂ）、および（Ｃ）を検体と一括して混合す
る方法も、簡易かつ迅速な測定が可能となる点で好まし
い。Particles (A), (B), and (C) described above
The order of mixing is not particularly limited, but the method of first mixing the magnetic particles (C), the magnetic particles (A) and the sample and allowing them to react sufficiently, and then mixing the non-magnetic particles (B) is not suitable for measurement sensitivity and reproducibility. Is preferable in terms of improving More preferably, the magnetic particles (C) are first mixed with a sample, and then the magnetic particles (A) are mixed and sufficiently reacted, and then the non-magnetic particles (B) are mixed. In addition, a method of mixing particles (A), (B), and (C) together with a sample is also preferable in that simple and quick measurement is possible.
【００３４】ここで混合する検体の量は、磁性粒子
（Ａ）１容量に対して１／６０〜１／３容量であればよ
く、必要により緩衝液などで適宜希釈して用いてもよ
い。The amount of the sample mixed here may be 1/60 to 1/3 volume with respect to 1 volume of the magnetic particles (A), and may be appropriately diluted with a buffer solution or the like if necessary.
【００３５】これら粒子と検体との混合は、通常、室温
（２０〜３０℃）で行うが、各粒子に担持された上記各
物質と対象物質との間で生じる免疫学的な結合反応の速
度を上げるため、３０〜４０℃に加温してもよく、ま
た、試薬の安定性を維持するため４〜２０℃に冷却して
行ってもよい。これら粒子と測定対象物質との反応時間
は特に限定されないが、１〜６０分とすれば、免疫反応
がほぼ終了し、測定感度および再現性ともに良好であ
る。The mixing of these particles and the specimen is usually carried out at room temperature (20 to 30 ° C.), but the rate of the immunological binding reaction that occurs between each of the substances supported on each particle and the target substance. In order to raise the temperature, it may be heated to 30 to 40 ° C, or in order to maintain the stability of the reagent, it may be cooled to 4 to 20 ° C. The reaction time between these particles and the substance to be measured is not particularly limited, but if it is 1 to 60 minutes, the immune reaction is almost completed, and the measurement sensitivity and reproducibility are good.
【００３６】本発明の測定方法の第（２）工程は、第
（１）工程で得られる上述の各粒子と検体との混合物に
磁場を付与して集磁する工程である。磁場の強さは特に
限定されないが、上記混合物が全量２００μｌの場合、
１，０００〜１０，０００ガウスが好適である。磁場を
付与するに際しては、市販の集磁装置、例えば、日本ペ
イント社製のギャザリン（GATHERIN）などを使用するこ
とができる。The step (2) of the measuring method of the present invention is a step of applying a magnetic field to the mixture of the above-mentioned particles and the sample obtained in the step (1) to collect the magnetic field. The strength of the magnetic field is not particularly limited, but when the total amount of the mixture is 200 μl,
1,000 to 10,000 gauss is preferred. When applying the magnetic field, a commercially available magnetizer, such as GATHERIN manufactured by Nippon Paint Co., Ltd., can be used.
【００３７】この第（２）工程においては、未反応の磁
性粒子（Ａ）、（Ｃ）並びに磁性粒子（Ａ）と免疫学的
に結合した非磁性粒子（Ｂ）、磁性粒子（Ａ）と測定対
象物質とが形成した複合体、および磁性粒子（Ｃ）と反
応妨害物質とが形成した複合体を集磁する。第（２）工
程の集磁時間は、特に限定されないが、１秒〜１０分間
行うのが好ましい。特に好ましくは、１０秒〜５分程度
である。In the second step (2), unreacted magnetic particles (A), (C) and non-magnetic particles (B) immunologically bound to the magnetic particles (A) and magnetic particles (A) are used. The complex formed by the substance to be measured and the complex formed by the magnetic particles (C) and the reaction interfering substance are magnetized. The magnetizing time in the step (2) is not particularly limited, but it is preferably 1 second to 10 minutes. Particularly preferably, it is about 10 seconds to 5 minutes.
【００３８】第（３）工程では、集磁されなかった非磁
性粒子（Ｂ）の量を、１９０〜１，０００ｎｍの範囲、
好ましくは３５０〜７００ｎｍの波長を用いて吸光度を
測定する。より好ましくは、これら非磁性粒子に上述し
た色素が含まれている場合、それらの色素が吸収を示す
範囲の波長を用いる。In the third step (3), the amount of non-magnetic particles (B) that has not been magnetized is set in the range of 190 to 1,000 nm
The absorbance is preferably measured using a wavelength of 350 to 700 nm. More preferably, when these non-magnetic particles contain the above-mentioned dye, a wavelength in the range in which the dye absorbs is used.
【００３９】本発明の免疫測定方法によれば、検体中の
測定対象物質を定性的あるいは定量的に測定することが
できる。定量的に測定する場合には、目的とする測定対
象物を既知の濃度含む標準検体を調製し、上述した波長
を用いて吸光度を測定して検量線を作成した後、この検
量線を用いて、未知濃度の検体の吸光度の測定値から定
量する。定性的に測定する場合には、一定量の測定対象
物質と免疫学的に結合する物質を担持した粒子と検体と
を混合し、集磁後に、目視で測定対象物質の有無を判定
する。According to the immunoassay method of the present invention, the substance to be measured in the sample can be qualitatively or quantitatively measured. In the case of quantitative measurement, prepare a standard sample containing a known concentration of the target measurement object, create a calibration curve by measuring the absorbance using the wavelength described above, and then use this calibration curve , Quantify from the measured absorbance value of a sample of unknown concentration. In the case of qualitative measurement, a certain amount of a substance to be measured and particles carrying a substance that binds immunologically are mixed with a sample, and after magnetism collection, the presence or absence of the substance to be measured is visually determined.
【００４０】本発明は、例えば図２に示すように、磁性
粒子（Ａ）上の抗体に、測定対象物質である抗原と非磁
性粒子（Ｂ）上の抗原とが競合しながら結合した免疫複
合体を反応容器側壁に集磁し、残余の非磁性粒子（Ｂ）
の量を吸光度測定で検知するものであり、粒子の凝集の
有無を検知するものではない。したがって、前述したよ
うなプロゾーン現象は生じない。また、抗原量増加に伴
って磁性粒子（Ａ）と検体中の抗原からなる免疫複合体
が多くなり、残余の非磁性粒子（Ｂ）の量も増加するた
め、右上がりの検量線が得られる。The present invention is, for example, as shown in FIG. 2, an immune complex in which an antibody on a magnetic particle (A) is bound by an antigen as a substance to be measured and an antigen on a non-magnetic particle (B) while competing with each other. The body is magnetized on the side wall of the reaction vessel, and the remaining non-magnetic particles (B)
Is detected by measuring the absorbance, and the presence or absence of particle aggregation is not detected. Therefore, the prozone phenomenon as described above does not occur. Further, as the amount of the antigen increases, the amount of the immune complex composed of the magnetic particles (A) and the antigen in the sample increases, and the amount of the remaining non-magnetic particles (B) also increases, so that a calibration curve rising to the right can be obtained. .
【００４１】本発明の原理によれば、検体中に遊離して
存在する抗原の方が非磁性粒子（Ｂ）の抗原よりも粒子
による立体障害を受けないため、磁性粒子（Ａ）の抗体
と優先的に結合する。したがって、検体中に存在する遊
離の抗原量の多少にかかわらず、最初に遊離の抗原が結
合し、残りの抗原結合部位に非磁性粒子（Ｂ）の抗原が
結合する。According to the principle of the present invention, the free antigens present in the sample are less sterically hindered by the particles than the non-magnetic particles (B), so that the antigens of the magnetic particles (A) are used. Join preferentially. Therefore, regardless of the amount of free antigen present in the sample, the free antigen first binds and the antigen of the non-magnetic particles (B) binds to the remaining antigen binding site.
【００４２】すなわち、図２ｂ1 に示すように、検体中
に存在する抗原量が少ないときは、磁性粒子（Ａ）の抗
体と非磁性粒子（Ｂ）の抗原とが結合した複合体が多く
なり、反応容器中に残存する非磁性粒子（Ｂ）の量が少
なくなる。したがって、吸光度を測定すると測定値は小
さくなる（図２ｃ1 ）。逆に、検体中に存在する抗原量
が多い場合には（図２ｂ2 ）、磁性粒子（Ａ）の抗体と
非磁性粒子（Ｂ）の抗原とが結合した複合体が少なくな
り、反応容器中に残存する非磁性粒子（Ｂ）の量が多く
なる（図２ｃ2 ）。したがって、吸光度を測定すると測
定値は高くなる。That is, as shown in FIG. 2b 1 , when the amount of the antigen present in the sample is small, the number of complexes in which the antibody of the magnetic particle (A) and the antigen of the non-magnetic particle (B) are bound increases. The amount of the non-magnetic particles (B) remaining in the reaction container is reduced. Therefore, the measured value becomes smaller when the absorbance is measured (FIG. 2c 1 ). On the contrary, when the amount of the antigen present in the sample is large (FIG. 2b 2 ), the complex in which the antibody of the magnetic particle (A) and the antigen of the non-magnetic particle (B) are bound to each other is reduced, and thus the reaction container is in the reaction vessel. The amount of the non-magnetic particles (B) remaining in the film increases (FIG. 2c 2 ). Therefore, the measured value becomes high when the absorbance is measured.
【００４３】以上のように、反応容器中に残存する非磁
性粒子（Ｂ）の量は、検体中に存在する抗原の量が多く
なれば増加するという正の相関関係を有する。また、本
発明においては、形成された免疫複合体はその大きさに
かかわらず集磁され残存した非磁性粒子（Ｂ）を測定す
るため、従来の凝集によるプロゾーン現象は生じない。As described above, the amount of the non-magnetic particles (B) remaining in the reaction container has a positive correlation that it increases as the amount of the antigen present in the sample increases. Further, in the present invention, since the formed immune complex measures the non-magnetic particles (B) remaining after being magnetized regardless of its size, the conventional prozone phenomenon due to aggregation does not occur.
【００４４】本発明の方法を用いて全血中のＣＲＰ（Ｃ
反応性タンパク質）量を測定する例を、具体的に説明す
る。ＣＲＰは、組織破壊を伴うような炎症性疾患等の時
に増量する血清中のβグロブリンである。Using the method of the present invention, CRP (C
An example of measuring the amount of reactive protein will be specifically described. CRP is β-globulin in serum, which is increased during inflammatory diseases involving tissue destruction.
【００４５】検体として全血を用いて測定を行う場合に
は、測定妨害物質となる赤血球やヘモグロビンなどがＣ
ＲＰとともに存在する。この場合は、検体中の赤血球と
特異的に結合する抗赤血球抗体などを担持させた磁性粒
子（Ｃ）、検体中のＣＲＰと特異的に結合する抗ＣＲＰ
抗体を担持させた磁性粒子（Ａ）、およびＣＲＰを担持
させた非磁性粒子（Ｂ）を混合して用いる。血清または
血漿を用いる場合には、通常上記磁性粒子（Ｃ）は不用
であるが、溶血している場合には抗ヘモグロビン抗体を
担持させた磁性粒子（Ｃ）を用いるとよい。When the measurement is carried out using whole blood as a sample, red blood cells and hemoglobin which are interfering substances for measurement are C
Exists with RP. In this case, magnetic particles (C) carrying an anti-erythrocyte antibody or the like that specifically binds to red blood cells in the sample, anti-CRP that specifically binds to CRP in the sample
The magnetic particles carrying the antibody (A) and the non-magnetic particles carrying the CRP (B) are mixed and used. When serum or plasma is used, the above-mentioned magnetic particles (C) are usually unnecessary, but when hemolyzed, it is preferable to use the magnetic particles (C) carrying an anti-hemoglobin antibody.
【００４６】これら３種類の粒子を室温で混合した後に
この混合粒子を検体と混合すると、ＣＲＰはＣＲＰを担
持させた磁性粒子（Ｂ）と競合しながら、抗ＣＲＰ抗体
を担持させた磁性粒子（Ａ）と結合する。抗ＣＲＰ抗体
を担持させた磁性粒子（Ａ）は（Ｂ）に担持されたＣＲ
Ｐよりも検体中に含まれるＣＲＰと優先的に結合し、免
疫複合体を形成する。結合反応は、約２０分程度行わせ
る。上述の検体以外に、検量線作成のために、ＣＲＰ標
準溶液から一定の濃度範囲のＣＲＰ含有試料を調製し、
上述の検体と同様に各粒子と混合して免疫複合体を形成
させる。その後、上述した量の磁場をかけて上述の未反
応の磁性粒子および磁性粒子を含む免疫複合体を集磁す
る。When these three kinds of particles are mixed at room temperature and then the mixed particles are mixed with a sample, CRP competes with CRP-carrying magnetic particles (B), while anti-CRP antibody-carrying magnetic particles ( Combine with A). Magnetic particles (A) loaded with anti-CRP antibody are CR loaded on (B)
It preferentially binds to CRP contained in the sample rather than P to form an immune complex. The binding reaction is performed for about 20 minutes. In addition to the above-mentioned samples, in order to prepare a calibration curve, prepare a CRP-containing sample in a certain concentration range from a CRP standard solution,
As in the case of the above-mentioned specimen, the particles are mixed with each other to form an immune complex. Then, the above-mentioned unreacted magnetic particles and the immune complex containing the magnetic particles are attracted by applying the above-mentioned magnetic field.
【００４７】したがって、検体中に赤血球などの測定妨
害物質が存在しない場合、磁性粒子（Ａ）とＣＲＰによ
って形成された免疫複合体、未反応の磁性粒子（Ａ）、
および磁性粒子（Ａ）と非磁性粒子（Ｂ）によって形成
された免疫複合体が集磁される。検体中に測定妨害物質
が存在する場合には、上記の粒子、免疫複合体に加え
て、未反応の磁性粒子（Ｃ）、および磁性粒子（Ｃ）と
赤血球などの測定妨害物質との免疫複合体が集磁され
る。集磁されずに反応容器中に残る粒子は、いずれの場
合も、ＣＲＰを担持させた非磁性粒子（Ｂ）のみであ
る。Therefore, when there is no measurement interfering substance such as red blood cells in the sample, the immune complex formed by the magnetic particles (A) and CRP, unreacted magnetic particles (A),
And the immune complex formed by the magnetic particles (A) and the non-magnetic particles (B) is magnetized. When a measurement-interfering substance is present in the sample, in addition to the above particles and the immune complex, unreacted magnetic particles (C), and an immune complex of the magnetic particle (C) and a measurement-interfering substance such as red blood cells. The body is magnetized. In any case, the particles remaining in the reaction container without being magnetized are only the non-magnetic particles (B) supporting CRP.
【００４８】このように磁性粒子および磁性粒子を含む
免疫複合体を集磁し、検量線作成用試料および各検体中
に集磁されずに残った非磁性粒子（Ｂ）の量を、１９０
〜１, ０００ｎｍの範囲の適切な波長で吸光度測定す
る。検量線作成用試料の吸光度の測定値をプロットし
て、検量線を作成する。各検体の吸光度の測定値を用い
て、上記の検量線からそれらの検体中のＣＲＰ濃度を読
み取りＣＲＰ濃度を決定する。なお、ＣＲＰ以外にも、
動脈硬化性疾患の発症時に増加するタンパクであるＬｐ
（ａ）などを用いて同様に測定することができる。In this way, the magnetic particles and the immune complex containing the magnetic particles were magnetized, and the amount of the non-magnetic particles (B) remaining without being magnetized in the sample for preparing the calibration curve and each specimen was set to 190.
Measure absorbance at an appropriate wavelength in the range of up to 1,000 nm. A calibration curve is created by plotting the measured absorbance values of the sample for creating a calibration curve. Using the measured absorbance of each sample, the CRP concentration in each sample is read from the above calibration curve to determine the CRP concentration. In addition to CRP,
Lp, a protein that increases at the onset of arteriosclerosis
The same measurement can be performed using (a) and the like.
【００４９】[0049]
【実施例】以下に実施例を示して、具体的に本発明を説
明するが、本発明はこれらに何等限定されるものではな
い。EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.
【００５０】（実施例１） （１）ＣＲＰ担持非磁性粒子の調製法 平均粒子径０．１７μｍの非磁性青色粒子を、固形分１
％（ｗ／ｗ）となるように、１ｍｌの０．０２％界面活
性剤含有２０ｍＭリン酸緩衝液（ｐＨ７．４、以下ＰＢ
と略す）中に分散させた。この分散溶液（１ｍｌ）にＣ
ＲＰ濃度２４．６ｍｇ／ｄｌのヒト血清（ＡＴＡＢ社
製）１００μｌを加えて攪拌した後、冷蔵庫中にて３日
間感作させた。(Example 1) (1) Method for preparing CRP-supporting non-magnetic particles Non-magnetic blue particles having an average particle diameter of 0.17 μm were mixed with a solid content of 1
% (W / w) of 1 ml of 20 mM phosphate buffer solution (pH 7.4, hereinafter PB containing 0.02% surfactant).
Abbreviated). Add C to this dispersion solution (1 ml)
100 μl of human serum (ATAB Co., Ltd.) having an RP concentration of 24.6 mg / dl was added, stirred, and then sensitized in a refrigerator for 3 days.
【００５１】感作の終了した上記分散溶液を、１２，０
００ｒｐｍで２０分間遠心分離して上澄み液を取り除
き、沈殿物である青色粒子を１ｍｌの１％ＢＳＡ含有Ｐ
Ｂに再度分散させた。この操作を３回繰り返し、最終的
に上記の１％ＢＳＡ含有ＰＢに分散させ、粒子量０．２
％（ｗ／ｗ）のＣＲＰ担持非磁性粒子（Ｂ−１）溶液と
した。After the sensitization, the above dispersion solution was added to 12,0
The supernatant was removed by centrifuging at 00 rpm for 20 minutes, and the blue particles as a precipitate were dissolved in 1 ml of P containing 1% BSA.
Redispersed in B. This operation was repeated 3 times, and finally dispersed in the above PB containing 1% BSA to give a particle amount of 0.2.
% (W / w) of CRP-supported non-magnetic particle (B-1) solution.
【００５２】（２）抗ＣＲＰヤギ抗体担持磁性粒子の調
製法 平均粒子径１．３５μｍのスチレン−メタクリル酸共重
合ポリマー被覆磁性粒子を、固形分１％（ｗ／ｗ）とな
るように、１ｍｌのＰＢ中に分散させた。この分散溶液
（１ｍｌ）に、抗ヒトＣＲＰヤギ抗体（ＡＴＡＢ社製）
０．２５ｍｇを加えて攪拌した後、冷蔵庫中にて３日間
感作させた。(2) Method for Preparing Anti-CRP Goat Antibody-Supporting Magnetic Particles Magnetic particles coated with a styrene-methacrylic acid copolymer having an average particle diameter of 1.35 μm were added to 1 ml so that the solid content was 1% (w / w). Dispersed in PB. An anti-human CRP goat antibody (manufactured by ATAB) was added to this dispersion solution (1 ml).
After adding 0.25 mg and stirring, it was sensitized in a refrigerator for 3 days.
【００５３】感作の終了した分散溶液を１２，０００ｒ
ｐｍで２０分間遠心分離して上澄み液を取り除き、沈殿
物である磁性粒子を１ｍｌの１％ＢＳＡ含有ＰＢに再度
分散させた。この操作を３回繰り返し、最終的に上記の
１％ＢＳＡ含有ＰＢに分散させた。この溶液を上記の緩
衝液で粒子量０．８％（ｗ／ｗ）の抗ＣＲＰヤギ抗体担
持磁性粒子（Ａ−１）溶液とした。After the sensitization, 12,000 r of the dispersed solution was added.
The supernatant was removed by centrifugation at pm for 20 minutes, and the magnetic particles as a precipitate were dispersed again in 1 ml of PB containing 1% BSA. This operation was repeated 3 times and finally dispersed in the above PB containing 1% BSA. This solution was used as a solution of anti-CRP goat antibody-supported magnetic particles (A-1) having a particle amount of 0.8% (w / w) with the above buffer solution.
【００５４】（３）抗ヒト赤血球抗体担持磁性粒子の調
製法 平均粒子径１．３５μｍのスチレン−メタクリル酸共重
合ポリマー被覆磁性粒子を、固形分１％（ｗ／ｗ）とな
るように、１ｍｌのＰＢ中に分散させた。この分散溶液
（１ｍｌ）に抗ヒト赤血球ウサギ抗体（オルガノンテク
ニカ（ORGNON TEKNIKA) 社製、総タンパク23.5mg/ml ）
０．２５ｍｇを加えて攪拌後、冷蔵庫中にて３日間感作
させた。(3) Method for Preparing Magnetic Particles Bearing Anti-Human Red Blood Cell Antibodies Magnetic particles coated with a styrene-methacrylic acid copolymer having an average particle diameter of 1.35 μm were added to 1 ml so as to have a solid content of 1% (w / w). Dispersed in PB. An anti-human red blood cell rabbit antibody (ORGNON TEKNIKA, total protein 23.5 mg / ml) was added to this dispersion solution (1 ml).
After adding 0.25 mg and stirring, it was sensitized in a refrigerator for 3 days.
【００５５】この溶液を１２，０００ｒｐｍで２０分間
遠心分離して上澄み液を取り除き、沈殿である磁性粒子
を１ｍｌの１％ＢＳＡ含有ＰＢに分散させた。この操作
を３回繰り返し、最終的に上記の１％ＢＳＡ含有ＰＢに
分散させた。この溶液を上記緩衝液で粒子量０．４％
（ｗ／ｗ）の抗ヒト赤血球抗体担持磁性粒子（Ｃ−１）
溶液とした。This solution was centrifuged at 12,000 rpm for 20 minutes to remove the supernatant, and the magnetic particles as a precipitate were dispersed in 1 ml of PB containing 1% BSA. This operation was repeated 3 times and finally dispersed in the above PB containing 1% BSA. This solution was added to the above buffer solution to give a particle amount of 0.4%.
(W / w) anti-human red blood cell antibody-supporting magnetic particles (C-1)
It was a solution.
【００５６】（４）キットの作製 （１）〜（３）で調製したＣＲＰ担持非磁性青色粒子
（Ｂ−１）、抗ＣＲＰヤギ抗体担持磁性粒子（Ａ−
１）、抗ヒト赤血球ウサギ抗体担持磁性粒子（Ｃ−１）
をそれぞれ含む分散溶液、検量線用ＣＲＰ標準液（ＡＴ
ＡＢ社製造）、および標準液希釈用緩衝液（３０ｍＭリ
ン酸緩衝液）によりＣＲＰ測定用キットを構成した。(4) Preparation of kit CRP-supporting non-magnetic blue particles (B-1) prepared in (1) to (3), anti-CRP goat antibody-supporting magnetic particles (A-)
1), magnetic particles (C-1) carrying an anti-human red blood cell rabbit antibody
Dispersion solution containing CRP standard solution for calibration curve (AT
A CRP measurement kit was constructed with a buffer solution (30 mM phosphate buffer solution) for diluting a standard solution) (manufactured by AB).
【００５７】（５）操作方法 １）５０μｌの磁性粒子（Ａ−１）溶液と、５０μｌの
磁性粒子（Ｃ−１）溶液と、検体である１０μｌの血液
を、９６ウェルマイクロプレートの各ウェルに入れ、２
０分間室温にて反応させた。検量線作成用に、上記標準
液を用いて濃度既知の試料を調製し、上記検体同様に１
０μｌを（Ｂ−１）溶液および（Ｃ−１）溶液とともに
各ウェルに入れ、２０分間室温にて反応させた。 ２）上記１）の各ウェル内の反応混合液に、１００μｌ
の非磁性青色粒子（Ｂ−１）溶液をさらに加え、２０分
間室温にて反応させた。(5) Operation method 1) 50 μl of the magnetic particle (A-1) solution, 50 μl of the magnetic particle (C-1) solution, and 10 μl of blood as a sample were put in each well of a 96-well microplate. Put 2
The reaction was carried out for 0 minutes at room temperature. To prepare a calibration curve, prepare a sample of known concentration using the standard solution described above, and
0 μl was placed in each well together with the (B-1) solution and the (C-1) solution, and the mixture was reacted for 20 minutes at room temperature. 2) Add 100 μl to the reaction mixture in each well of 1) above.
The non-magnetic blue particle (B-1) solution of was further added and reacted at room temperature for 20 minutes.
【００５８】３）次に、集磁装置ギャザリン（日本ペイ
ント社製）をマイクロタイタープレートの下方に５分間
はめ込み、室温で静置した。 ４）集磁されなかった非磁性青色粒子（Ｂ−１）をマイ
クロプレートリーダー（東ソー社製）により、５７０ｎ
ｍでその吸光度を測定した。検量線作成用標準試料の吸
光度測定値を用いて検量線を作成した結果を図３に示
す。図３に示した検量線の作成範囲から明らかなよう
に、全血中のＣＲＰの量を微量の検体を用いて高感度で
測定することが可能であった。3) Next, a magnetizer Gatherin (manufactured by Nippon Paint Co., Ltd.) was fitted under the microtiter plate for 5 minutes and allowed to stand at room temperature. 4) 570n of non-magnetic blue particles (B-1) which were not collected by a microplate reader (manufactured by Tosoh Corporation)
The absorbance was measured in m. FIG. 3 shows the result of creating a calibration curve using the absorbance measurement values of the standard sample for creating the calibration curve. As is clear from the range of preparation of the calibration curve shown in FIG. 3, it was possible to measure the amount of CRP in whole blood with high sensitivity using a small amount of sample.
【００５９】（実施例２） （１）Ｌｐ（ａ）担持非磁性粒子の調製法 平均粒子径０．１７μｍの非磁性青色粒子を、固形分１
％（ｗ／ｗ）となるように、１ｍｌの０．０２％界面活
性剤含有２０ｍＭリン酸緩衝液（ｐＨ７．４、以下ＰＢ
と略す）中に分散させる。この分散溶液（１ｍｌ）にヒ
トＬｐ（ａ）血清（ＩＩＣ社製造、１００ｍｇ／ｄｌ）
０．２５ｍｇを加えて攪拌後、冷蔵庫中にて３日間感作
させた。Example 2 (1) Method for Preparing Lp (a) -Supporting Non-Magnetic Particles Non-magnetic blue particles having an average particle diameter of 0.17 μm were mixed with solid content 1
% (W / w) of 1 ml of 20 mM phosphate buffer solution (pH 7.4, hereinafter PB containing 0.02% surfactant).
Abbreviated). Human Lp (a) serum (manufactured by IIC, 100 mg / dl) was added to this dispersion solution (1 ml).
After adding 0.25 mg and stirring, it was sensitized in a refrigerator for 3 days.
【００６０】感作の終了した分散溶液を、１２，０００
ｒｐｍで２０分間遠心分離して上澄み液を取り除き、分
離された非磁性青色粒子を１ｍｌの１％ＢＳＡ（ウシ胎
児血清アルブミン）含有ＰＢに分散させた。この操作を
３回繰り返し、最終的に上記の１％ＢＳＡ含有ＰＢに再
分散させた。上記緩衝液で粒子量１％（ｗ／ｗ）のＬｐ
（ａ）担持非磁性青色粒子（Ｂ−２）溶液とした。After the sensitization, the dispersed solution was added to 12,000.
The supernatant was removed by centrifugation at rpm for 20 minutes, and the separated nonmagnetic blue particles were dispersed in 1 ml of PB containing 1% BSA (fetal bovine serum albumin). This operation was repeated 3 times, and finally redispersed in the above PB containing 1% BSA. Lp with a particle amount of 1% (w / w) in the above buffer solution
(A) A supported non-magnetic blue particle (B-2) solution was prepared.
【００６１】（２）抗Ｌｐ（ａ）ヤギ抗体担持磁性粒子
の調製法 平均粒子径１．３５μｍのスチレン−メタクリル酸共重
合ポリマー被覆磁性粒子を、固形分１％（ｗ／ｗ）とな
るように、１ｍｌのＰＢに分散させた。この分散溶液
（１ｍｌ）に抗ヒトＬｐ（ａ）ヤギ抗体（インターナシ
ョナル エンザイムス(International Enzymes) 社製
造、20.3mg/ml(ベッカー法(Becker Method)による) ）
０．２５ｍｇを加えて攪拌した後、冷蔵庫中にて３日間
感作させた。(2) Method for Preparing Anti-Lp (a) Goat Antibody-Supporting Magnetic Particles Styrene-methacrylic acid copolymerized polymer-coated magnetic particles having an average particle size of 1.35 μm have a solid content of 1% (w / w). Then, it was dispersed in 1 ml of PB. Anti-human Lp (a) goat antibody (20.3 mg / ml (by Becker Method) manufactured by International Enzymes) was added to this dispersion solution (1 ml).
After adding 0.25 mg and stirring, it was sensitized in a refrigerator for 3 days.
【００６２】感作の終了した分散溶液から、磁気分離装
置を用いて反応容器中の液を取り除き、分離された磁性
粒子を１ｍｌの１％ＢＳＡ含有ＰＢに再分散させた。こ
の操作を３回繰り返し、最終的に上記の１％ＢＳＡ含有
ＰＢに分散させた。上記緩衝液で粒子量１％（ｗ／ｗ）
の抗Ｌｐ（ａ）ヤギ抗体担持磁性粒子（Ａ−２）溶液と
した。The liquid in the reaction vessel was removed from the dispersion solution after the sensitization by using a magnetic separator, and the separated magnetic particles were redispersed in 1 ml of PB containing 1% BSA. This operation was repeated 3 times and finally dispersed in the above PB containing 1% BSA. Particle amount 1% (w / w) with the above buffer
The anti-Lp (a) goat antibody-supporting magnetic particle (A-2) solution was prepared.
【００６３】（３）抗ヒト赤血球抗体担持磁性粒子の調
製法 平均粒子径１．３５μｍのスチレン−メタクリル酸共重
合被覆ポリマー磁性粒子を、固形分１％（ｗ／ｗ）とな
るように、１ｍｌのＰＢに分散させた。この分散溶液
（１ｍｌ）に抗ヒト赤血球ウサギ抗体０．２５ｍｇを加
えて攪拌後、冷蔵庫中にて３日間感作させた。(3) Method for preparing magnetic particles carrying anti-human erythrocyte antibody 1 mg of styrene-methacrylic acid copolymer-coated polymer magnetic particles having an average particle diameter of 1.35 μm were added so that the solid content was 1% (w / w). Dispersed in PB. To this dispersion solution (1 ml), 0.25 mg of anti-human red blood cell rabbit antibody was added, stirred and sensitized in a refrigerator for 3 days.
【００６４】その後、磁気分離装置を用いて上澄み液を
取り除き、分離された磁性粒子を１ｍｌの１％ＢＳＡ含
有ＰＢに再分散させた。この操作を３回繰り返し、最終
的に上記の１％ＢＳＡ含有ＰＢに分散させた。上記緩衝
液で粒子量１％（ｗ／ｗ）の抗ヒト赤血球ウサギ抗体担
持磁性粒子（Ｃ−２）とした。After that, the supernatant was removed using a magnetic separator, and the separated magnetic particles were redispersed in 1 ml of PB containing 1% BSA. This operation was repeated 3 times and finally dispersed in the above PB containing 1% BSA. The above-mentioned buffer solution was used to prepare anti-human red blood cell rabbit antibody-supporting magnetic particles (C-2) having a particle amount of 1% (w / w).
【００６５】（４）キットの作製 （１）〜（３）で調製された（Ａ−２）、（Ｂ−２）、
および（Ｃ−２）の各粒子を含む溶液、検量線作成用Ｌ
ｐ（ａ）標準液、標準液希釈用緩衝液（３０ｍＭリン酸
緩衝液）により、免疫測定用キットを構成した。(4) Preparation of kit (A-2), (B-2) prepared in (1) to (3),
And a solution containing each particle of (C-2), L for preparing a calibration curve
The p (a) standard solution and the buffer solution for diluting the standard solution (30 mM phosphate buffer solution) constituted an immunoassay kit.
【００６６】（５）測定波長の選択 非磁性青色粒子（Ｂ−２）の吸収波長プロファイルを、
紫外領域である１９０ｎｍから可視領域である１，００
０ｎｍの範囲でスキャンすることにより検討し、５７０
ｎｍを測定波長とした。(5) Selection of measurement wavelength The absorption wavelength profile of the non-magnetic blue particles (B-2) is
190 nm, which is the ultraviolet region, to 1,00 which is the visible region
Examined by scanning in the range of 0 nm, 570
nm was used as the measurement wavelength.
【００６７】（６）測定方法 １）Ｌｐ（ａ）濃度が、１、１０、５０、１００ｍｇ／
ｄｌとなるようにＬｐ（ａ）標準液（１００ｍｇ／ｄ
ｌ）を血液に混合し、検体とした。 ２）上記検体１０μｌと１％ＢＳＡ含有ＰＢ１００μｌ
を９６ウェルマイクロプレートの各ウェルに入れて混合
した。検量線作成用に、上記Ｌｐ（ａ）標準液を同様に
上記リン酸緩衝液とともに別のウェルに入れて混合し
た。(6) Measuring method 1) Lp (a) concentration is 1, 10, 50, 100 mg /
Lp (a) standard solution (100 mg / d)
l) was mixed with blood to obtain a sample. 2) 10 μl of the above sample and 100 μl of PB containing 1% BSA
Were placed in each well of a 96-well microplate and mixed. For preparation of the calibration curve, the Lp (a) standard solution was similarly put in another well together with the phosphate buffer solution and mixed.
【００６８】３）上記各ウェルに、磁性粒子（Ａ−２）
６０μｌおよび磁性粒子（Ｃ−２）２０μｌを加え、１
０分間室温にて反応させた。 ４）上記各ウェルに、非磁性青色粒子（Ｂ−２）２０μ
ｌを加え、１０分間室温にて反応させた。3) The magnetic particles (A-2) were added to each of the wells.
Add 60 μl and 20 μl of magnetic particles (C-2) and
The reaction was carried out for 0 minutes at room temperature. 4) 20 μ of non-magnetic blue particles (B-2) in each well
1 was added and reacted at room temperature for 10 minutes.
【００６９】５）次に、集磁装置ギャザリンをマイクロ
タイタープレートの下方に５分間はめ込み、非磁性青色
粒子（Ｂ−２）のＬｐ（ａ）抗原と磁性粒子（Ａ−２）
の抗体とが形成した免疫複合体、赤血球と磁性粒子
（Ｃ）の抗ヒト赤血球とが結合した免疫複合体、および
未反応の磁性粒子をウェル側壁に集磁した。 ６）各ウェルの反応液中に残存する非磁性青色粒子（Ｂ
−２）濃度を、マイクロプレートリーダーを用いて５７
０ｎｍで吸光度を測定し、図４に示す検量線を０〜１０
０ｍｇ／ｄｌの範囲で作成した。5) Next, the magnetizer Gatherin was fitted under the microtiter plate for 5 minutes, and the Lp (a) antigen of the non-magnetic blue particles (B-2) and the magnetic particles (A-2).
The immune complex formed by the antibody of 1), the immune complex formed by binding the red blood cells and the anti-human red blood cells of the magnetic particles (C), and the unreacted magnetic particles were collected on the side wall of the well. 6) Non-magnetic blue particles (B) remaining in the reaction solution in each well
-2) Adjust the concentration to 57 using a microplate reader.
The absorbance was measured at 0 nm, and the calibration curve shown in FIG.
It was prepared in the range of 0 mg / dl.
【００７０】７）上述の検量線より全血検体の測定値を
読み取った。表１に、Ｌｐ（ａ）検量線および全血検体
の実測値を示す（ｎ＝５）。 7) The measured value of the whole blood sample was read from the above calibration curve. Table 1 shows the Lp (a) calibration curve and the actually measured values of whole blood samples (n = 5).
【００７１】さらに、図４に示したように、Ｌｐ（ａ）
を用いても右上がりの検量線が得られ、ＣＲＰの場合と
同様にプロゾーン現象は見られず、高感度での測定が可
能であった。さらに、全血中のＬｐ（ａ）を測定する場
合、血清分離などの前処理を必要とせずに低濃度から高
濃度までの定量が可能であった。以上より本発明の免疫
測定方法は、酵素免疫測定法や、ラジオアイソトープ免
疫測定法のように、Ｂ／Ｆ分離を必要とせず、簡易で迅
速に高感度の測定を行えることが示された。Further, as shown in FIG. 4, Lp (a)
A calibration curve rising to the right was obtained by using, and the prozone phenomenon was not observed as in the case of CRP, and measurement with high sensitivity was possible. Furthermore, when measuring Lp (a) in whole blood, quantification from low concentration to high concentration was possible without requiring pretreatment such as serum separation. From the above, it was demonstrated that the immunoassay method of the present invention does not require B / F separation, unlike the enzyme immunoassay method and the radioisotope immunoassay method, and can perform simple and rapid highly sensitive measurement.
【００７２】[0072]
【発明の効果】本発明によれば、検体中に含まれる測定
対象物質、および測定対象物質と免疫学的に結合する物
質との磁性粒子複合体を集磁により除去できるので、残
された非磁性粒子の量を吸光度測定することにより、右
上がりかつプロゾーン現象のない検量線を得ることがで
き、市販のニーズに適合したキットおよびそれを利用す
る測定方法を提供することができる。According to the present invention, the substance to be measured contained in the sample and the magnetic particle complex of the substance to be immunologically bound to the substance to be measured can be removed by magnetic attraction, so that the residual non-remaining substances can be removed. By measuring the amount of magnetic particles by absorbance, it is possible to obtain a calibration curve that does not rise to the right and does not have the prozone phenomenon, and it is possible to provide a kit that meets commercial needs and a measurement method using the kit.
【００７３】また、本発明の免疫測定方法により得られ
る検量線は、凝集の有無を検知するのではなく、集磁後
の反応液中に残存する非磁性粒子の量を測定することに
基づくため、凝集法で見られるようなプロゾーン現象を
起こすことがない。さらに、非磁性粒子の量を吸光度測
定により直接検出するために、酵素免疫測定法やラジオ
アイソトープ免疫測定法に必要なＢ／Ｆ分離およびその
後の洗浄工程などの煩雑な操作を必要とせず、本発明の
免疫測定用キットを用いることにより、簡易かつ迅速に
所望の測定対象物質の濃度を測定することができる。加
えて、本発明の免疫測定方法では、使用する検体の量が
極めて少量であり、しかも全血を用いた高感度の測定が
可能であるため、新生児など、血液が少量しか採取でき
ない場合に特に有用である。Since the calibration curve obtained by the immunoassay method of the present invention is based on measuring the amount of non-magnetic particles remaining in the reaction solution after magnetism collection, rather than detecting the presence or absence of aggregation. , The prozone phenomenon as seen in the agglutination method does not occur. Furthermore, in order to directly detect the amount of non-magnetic particles by absorbance measurement, complicated operations such as B / F separation and subsequent washing step necessary for enzyme immunoassay and radioisotope immunoassay are not required, and By using the immunoassay kit of the invention, the concentration of a desired substance to be measured can be measured simply and quickly. In addition, in the immunoassay method of the present invention, the amount of the sample to be used is extremely small, and since highly sensitive measurement using whole blood is possible, it is particularly useful when a small amount of blood can be collected, such as in a newborn baby. It is useful.
【図１】 従来法により生ずるプロゾーン現象を示す検
量線の図である。FIG. 1 is a diagram of a calibration curve showing a prozone phenomenon generated by a conventional method.
【図２】 検体中に存在する抗原と抗原担持非磁性粒子
を競合的に反応させた場合の、抗原量の多少による反応
容器中に残存する非磁性粒子量の相違を示す図である。FIG. 2 is a diagram showing a difference in the amount of nonmagnetic particles remaining in a reaction container depending on the amount of antigen when the antigen present in a sample and the antigen-bearing nonmagnetic particles are reacted competitively.
【図３】 既知濃度のＣＲＰを含む標準試料を波長５７
０ｎｍの吸光度で測定して得られた検量線を示す図であ
る。FIG. 3 shows a standard sample containing a known concentration of CRP at a wavelength of 57.
It is a figure which shows the calibration curve obtained by measuring by the light absorbency of 0 nm.
【図４】 既知濃度のＬｐ（ａ）を含む標準試料を波長
５７０ｎｍの吸光度で測定して得られた検量線を示す図
である。FIG. 4 is a diagram showing a calibration curve obtained by measuring a standard sample containing a known concentration of Lp (a) by absorbance at a wavelength of 570 nm.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 33/543 ５４１ Ｇ０１Ｎ 33/543 ５４１Ａ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display location G01N 33/543 541 G01N 33/543 541A
Claims (4)
定する免疫測定用キットであって、少なくとも、測定対
象物質と免疫学的に結合する物質を担持させた不溶性磁
性粒子、および測定対象物質を担持させた特定波長吸収
領域を有する不溶性非磁性粒子を含むことを特徴とする
生体内物質の免疫測定用キット。1. An immunoassay kit for measuring an in-vivo substance using an antigen-antibody reaction, which comprises at least insoluble magnetic particles carrying a substance that immunologically binds to a substance to be measured, and measurement. An in-vivo substance immunoassay kit comprising insoluble non-magnetic particles having a specific wavelength absorption region supporting a target substance.
る物質を担持させた不溶性磁性粒子をさらに含むことを
特徴とする請求項１記載の生体内物質の免疫測定用キッ
ト。2. The kit for immunoassay of an in-vivo substance according to claim 1, further comprising insoluble magnetic particles carrying a substance that immunologically binds to a measurement-interfering substance in a sample.
る免疫測定方法であって、（１）測定対象物質と免疫学
的に結合する物質を担持させた不溶性磁性粒子を含む溶
液、および測定対象物質を担持させた特定波長吸収領域
を有する不溶性非磁性粒子を含む溶液を、任意の順序で
検体と混合して混合液とし、（２）前記混合液に磁場を
付与して、前記混合液中で生成した前記磁性粒子と測定
対象物質とが形成した免疫複合体、および未結合の磁性
粒子を集磁し、（３）残留する前記非磁性粒子の濃度を
吸光度によって測定することを特徴とする生体内物質を
測定する免疫測定方法。3. An immunoassay method for measuring an in-vivo substance by an antigen-antibody reaction, which comprises (1) a solution containing insoluble magnetic particles carrying a substance that immunologically binds to a substance to be measured, and measurement. A solution containing insoluble non-magnetic particles having a specific wavelength absorption region supporting a target substance is mixed with a sample in an arbitrary order to form a mixed solution, and (2) a magnetic field is applied to the mixed solution to form the mixed solution. The immunocomplex formed by the magnetic particles and the substance to be measured formed inside and the unbound magnetic particles are magnetized, and (3) the concentration of the remaining nonmagnetic particles is measured by absorbance. Immunoassay method for measuring a substance in a living body.
疫学的に結合する物質を担持させた不溶性磁性粒子をさ
らに加え、前記磁性粒子と測定妨害物質とが形成した免
疫複合体をさらに集磁することを特徴とする請求項３記
載の生体内物質の免疫測定方法。4. An insoluble magnetic particle carrying a substance that immunologically binds to a measurement-interfering substance in a sample is added to the mixed solution to form an immune complex formed by the magnetic particle and the measurement-interfering substance. The method for immunoassay of a substance in a living body according to claim 3, further comprising collecting magnetism.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1081196A JPH08262024A (en) | 1995-01-26 | 1996-01-25 | Kit for immunoassay of in vivo substance and immunoassay method |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7-10627 | 1995-01-26 | ||
| JP1062795 | 1995-01-26 | ||
| JP1081196A JPH08262024A (en) | 1995-01-26 | 1996-01-25 | Kit for immunoassay of in vivo substance and immunoassay method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08262024A true JPH08262024A (en) | 1996-10-11 |
Family
ID=26345927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1081196A Withdrawn JPH08262024A (en) | 1995-01-26 | 1996-01-25 | Kit for immunoassay of in vivo substance and immunoassay method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08262024A (en) |
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| JP2003512625A (en) * | 1999-10-15 | 2003-04-02 | ウェーブセンス エルエルシー | Systems and methods for performing magnetic chromatography measurements |
| JP2004331953A (en) * | 2003-04-16 | 2004-11-25 | Sekisui Chem Co Ltd | Magnetic material encapsulating particle, immunity measuring particle and immunity measuring method |
| WO2010074265A1 (en) * | 2008-12-25 | 2010-07-01 | ユニバーサル・バイオ・リサーチ株式会社 | Method for pretreating specimen and method for assaying biological substance |
| WO2011125877A1 (en) * | 2010-03-31 | 2011-10-13 | 積水メディカル株式会社 | Measurement method using immunochromatography, test strip for immunochromatography, and measurement reagent kit for immunochromatography |
| WO2012077737A1 (en) * | 2010-12-08 | 2012-06-14 | 株式会社クレハ | Anti-psk polyclonal antibody, psk immunological analysis method using same, and psk immunological analysis kit |
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-
1996
- 1996-01-25 JP JP1081196A patent/JPH08262024A/en not_active Withdrawn
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