JPH0315760A - Immunoassay - Google Patents
ImmunoassayInfo
- Publication number
- JPH0315760A JPH0315760A JP15135889A JP15135889A JPH0315760A JP H0315760 A JPH0315760 A JP H0315760A JP 15135889 A JP15135889 A JP 15135889A JP 15135889 A JP15135889 A JP 15135889A JP H0315760 A JPH0315760 A JP H0315760A
- Authority
- JP
- Japan
- Prior art keywords
- carrier
- antibody
- antigen
- measurement
- immobilized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 238000006243 chemical reaction Methods 0.000 abstract description 18
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- 230000035945 sensitivity Effects 0.000 abstract description 9
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- 238000002372 labelling Methods 0.000 description 14
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
【発明の詳細な説明】
く産業上の利用分野〉
本発明は免疫学的測定法に関する。更に詳細には、臨床
検査等の分野で使用され、抗原又は抗体が固定化された
不溶性担体を用いて検体中の微量成分を測定する免疫学
的測定法に関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an immunoassay method. More specifically, the present invention relates to an immunoassay method used in fields such as clinical testing, which measures trace components in a specimen using an insoluble carrier on which an antigen or antibody is immobilized.
く従来の技術及び発明が解決しようどする課題〉免疫学
的測定法は、抗原抗体反応の特異性を利用して、生体成
分（例えば、抗原、抗体、ハプテン、ホルモン等）、薬
剤等の微量成分を測定する方法であり、信頼性及び測定
精度が高いうえ、種々の抗原に対する抗体が容易に取得
し得ること等から、微量生体成分等の測定（定ｊｌ）法
として広く用いられている。このような免疫学的測定広
としては、使用される標識物質の差異により、放射免疫
測定法（Ｒ Ｉ Ａ法）、酵素免疫測定法（ＥＩＡ法）
、螢光免疫測定法（ＦＩＡ法）等が知られている。Problems to be Solved by Prior Art and the Invention> Immunological assays utilize the specificity of antigen-antibody reactions to detect trace amounts of biological components (e.g., antigens, antibodies, haptens, hormones, etc.), drugs, etc. It is a method for measuring components, and is widely used as a method for measuring trace biological components, etc., because it has high reliability and measurement accuracy, and antibodies against various antigens can be easily obtained. Broadly speaking, such immunoassays include radioimmunoassay (RIA method), enzyme immunoassay (EIA method), depending on the difference in the labeling substance used.
, fluorescence immunoassay (FIA method), and the like are known.
免疫学測定法には、その測定様式の差異により、抗原抗
体反応物と非反応物との分Ｍ操作（いわゆるＢ／Ｆ分離
）を必要とする方法と、Ｂ／Ｆ分離を必要としない方法
がある。Ｂ／Ｆ分離を必要としない方法は測定操作が簡
便ではあるが、検体中の測定対象物質が極微量の場合に
は測定精度が劣り、適切な方法とはいえない。かかる問
題から、Ｂ／Ｆ分離を行う方法が汎用されるが、この方
法の一種として、抗原又は抗体を固定化した不溶性担体
を用いてＢ／Ｆ分離を行う固相法が知られている。固相
法は、抗原抗体反応を固相上で行い、標識物質を含む抗
原抗体反応物を固相上に結合させる方法、抗原抗体反応
を液相で行い、標識物質を含む抗原抗体反応物を生成さ
せ、次いで該反応生成物を固相上に結合させる方法等に
より行われる。標識物質を含む抗原抗体反応物は固相上
に固定化されているので、反応終了後、担体を水洗する
方法、遠心分離法等により、容易にＢ／Ｆ分離を行うこ
とができ、操作が簡便であると共に測定精度の向上が図
れる。Due to differences in measurement formats, immunoassay methods include methods that require separation of antigen-antibody reactants and non-reactants (so-called B/F separation), and methods that do not require B/F separation. There is. Although a method that does not require B/F separation has a simple measurement operation, when the amount of the substance to be measured in the sample is extremely small, the measurement accuracy is poor and it cannot be said to be an appropriate method. Due to this problem, a method of B/F separation is widely used, and as one type of this method, a solid phase method is known in which B/F separation is performed using an insoluble carrier on which an antigen or antibody is immobilized. The solid-phase method is a method in which an antigen-antibody reaction is performed on a solid phase, and an antigen-antibody reaction product containing a labeling substance is bound to the solid phase. This is carried out by a method in which the reaction product is produced, and then the reaction product is bonded to a solid phase. Since the antigen-antibody reaction product containing the labeling substance is immobilized on the solid phase, B/F separation can be easily performed by washing the carrier with water, centrifugation, etc. after the reaction, and the operation is simple. It is simple and can improve measurement accuracy.
かかる固相法で用いられる不溶性担体としては、プラス
チック（例えば、ボリスチレン等）やガラス等の材質か
らなり、板状、粒状、チューブ状などの形状のものが汎
用され、これらの担体に抗原又は抗体を結合させること
が行われている。Insoluble carriers used in such solid-phase methods are commonly made of materials such as plastic (for example, boristyrene) and glass, and are in the form of plates, particles, tubes, etc., and these carriers are coated with antigens or antibodies. are being combined.
しかし、このような不溶性担体は、比表面積が小さく、
担体に結合できる抗原又は抗体の量や濃度が限られてお
り、十分量の抗原又は抗体を固定化するには多量の不溶
性担体を必要とする。その結果、検体と反応させる際に
、担体全体に検体が接触できるように検体を希釈して液
量を増やすことが必要となる。そのため抗原抗体反応が
緩慢に進行し、長時間の反応時間（通常、数時間〜１日
程度）を要するという問題がある。However, such insoluble carriers have a small specific surface area and
The amount and concentration of antigen or antibody that can be bound to a carrier is limited, and a large amount of insoluble carrier is required to immobilize a sufficient amount of antigen or antibody. As a result, when reacting with a sample, it is necessary to dilute the sample and increase the amount of liquid so that the sample can come into contact with the entire carrier. Therefore, there is a problem that the antigen-antibody reaction progresses slowly and requires a long reaction time (usually several hours to one day).
このような問題を解決するため、従来よりセファロース
、アガロース、セルロース、濾紙等を不溶性担体に用い
て、抗原又は抗体を比較的多量に固定化する方法が知ら
れている。しかしながら、この場合、担体上への非特異
的結合に起因するブランクの測定値の上昇、いわゆるバ
ックグランドの上昇が認められ、その結果、Ｓ／Ｎ比が
低くなり、測定感度が低下するという問題がある。In order to solve such problems, methods have been known in which a relatively large amount of antigen or antibody is immobilized using Sepharose, agarose, cellulose, filter paper, or the like as an insoluble carrier. However, in this case, an increase in the blank measurement value due to non-specific binding to the carrier, a so-called increase in background, is observed, resulting in a low S/N ratio and a decrease in measurement sensitivity. There is.
本発明は上記の従来技術の欠点を解消するために創案さ
れたもので、本発明者らが種々の検討を重ねた結果、不
溶性担体を改良することにより、反応時間を短縮し得る
と共にＢ／Ｆ分離を効率的に行うことができ、更にバッ
クグランドが低下し、測定感度を向上させ得ることを見
出して完成したもので、本発明の目的は測定精度及び感
度に優れると共に迅速測定を可能にする免疫学的測定法
を提供することにある。The present invention was devised to solve the above-mentioned drawbacks of the prior art, and as a result of various studies by the present inventors, it was possible to shorten the reaction time by improving the insoluble carrier, and to improve the B/ This invention was completed after discovering that F separation can be performed efficiently, the background can be further reduced, and measurement sensitivity can be improved.The purpose of the present invention is to achieve excellent measurement accuracy and sensitivity and to enable rapid measurement. The purpose of the present invention is to provide an immunoassay method for detecting
く課題を解決するための手段〉
上記の課題を解決すべくなされた、本発明の免疫学的測
定法は、抗原又は抗体（以下、便宜上、免疫反応性物質
という）が固定化された不溶性担体を用いる免疫学的測
定法であって、該免疫反応性物質が固定化された不溶性
担体がプロッキング剤で処理されていると共に不溶性担
体が多孔性プラスチックからなることを特徴とするもの
である。Means for Solving the Problems> The immunoassay method of the present invention, which was made to solve the above problems, uses an insoluble carrier on which an antigen or antibody (hereinafter referred to as an immunoreactive substance for convenience) is immobilized. This is an immunoassay method using an immunoassay, which is characterized in that an insoluble carrier on which the immunoreactive substance is immobilized is treated with a blocking agent, and the insoluble carrier is made of porous plastic.
上記構成からなる本発明の免疫学的測定法には、免疫反
応性物質が固定化された不溶性担体を用いる方法であれ
ばいずれの測定法（例えば、放射免疫測定法、酵素免疫
測定法、螢光免疫測定法等）も包含される。また、上記
各測定法において、測定様式として、例えば、競合法、
サンドイッチ法、固定化第二抗体を用いる方法などの様
々な方法が知られているが、これらのいずれの方法も包
含される。これらの免疫学的測定法及びその実施方法に
ついては、石川ら編「酵素免疫測定法」（１９７８年医
学書院刊行）、入江編「ラジオイムノアッセイＪ （
１９７４年講談社刊行）等に詳細に記載されている。不
溶性担体上に固定化される免疫反応性物質（すなわち、
抗原又は抗体）としては、検体中の測定対象物質が抗原
、ハブテン等の場合にはその抗体が、また測定対象物質
が抗体の場合にはその抗原が固定化される。The immunoassay method of the present invention having the above configuration may be any method using an insoluble carrier on which an immunoreactive substance is immobilized (e.g., radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, etc.). photoimmunoassay, etc.) are also included. In addition, in each of the above measurement methods, the measurement format includes, for example, competitive method,
Various methods are known, such as a sandwich method and a method using an immobilized second antibody, and any of these methods is included. These immunoassay methods and their implementation methods are described in "Enzyme immunoassay method" edited by Ishikawa et al. (published by Igaku Shoin in 1978), "Radioimmunoassay J" edited by Irie (
(published by Kodansha in 1974) etc. An immunoreactive substance immobilized on an insoluble carrier (i.e.
When the substance to be measured in the specimen is an antigen, habten, etc., the antibody is immobilized, and when the substance to be measured is an antibody, the antigen is immobilized.
また、本発明においては、不溶性担体として多孔性プラ
スチックが用いられており、使用される多孔性プラスチ
ックとしては、水性溶媒に対して不溶性且つ非膨潤性を
示す材質であればいずれのものも使用することができ、
孔径としては０．１〜２００μｍのものがよい。また、
多孔性プラスチックは、その表面にアミノ基、スルホ基
、カルボキシ基、チオール基等の官能基が導入されてい
てもよく、これらの官能基の導入は慣用の方法にて行う
ことができる。更に、多孔性プラスチックは相互に連絡
した空隙構造を有する通水性材料であることが好ましい
。かかる通水性の多孔性プラスチックを用いることによ
り、担体の洗浄をフロ一方式にて行うことができ、バッ
チ方式による担体の洗浄よりも、高効率且つ容易にＢ／
Ｆ分離を行え、測定精度の向上及び測定時間の短縮化に
寄与でき、また標識物質の種類によっては、標識活性の
測定もフロ一方式により行うことができるという利点を
有する。Furthermore, in the present invention, a porous plastic is used as an insoluble carrier, and any material that is insoluble and non-swellable in an aqueous solvent can be used as the porous plastic. It is possible,
The pore diameter is preferably 0.1 to 200 μm. Also,
A functional group such as an amino group, a sulfo group, a carboxy group, or a thiol group may be introduced onto the surface of the porous plastic, and these functional groups can be introduced by a conventional method. Furthermore, the porous plastic is preferably a water-permeable material having an interconnected pore structure. By using such a water-permeable porous plastic, the carrier can be washed in a single-flow system, which makes it possible to wash the carrier more efficiently and easily than in a batch system.
It has the advantage that F separation can be performed, which contributes to improving measurement accuracy and shortening measurement time, and depending on the type of labeling substance, labeling activity can also be measured using a single flow system.
上記の多孔性プラスチックからなる不溶性担体上への免
疫反応性物質の固定化は、物理化学的方法（例えば、吸
着法、イオン結合法等）、化学結合を介する方法等が挙
げられる。Immobilization of the immunoreactive substance onto the above-mentioned insoluble carrier made of porous plastic includes physicochemical methods (eg, adsorption method, ionic bonding method, etc.), methods via chemical bonding, and the like.
物理化学的方法による固定化は、０．０１〜ＩＭ程度の
適当な緩衝岐に免疫反応性物質を溶解し、ここに不溶性
担体を、室温又は冷却下、１〜１０時間程度浸漬し、次
いで水又は緩衝液で十分に洗浄することにより行われる
。Immobilization by physicochemical methods involves dissolving the immunoreactive substance in an appropriate buffer of about 0.01 to IM, immersing the insoluble carrier therein at room temperature or under cooling for about 1 to 10 hours, and then dipping it in water. Or by washing thoroughly with a buffer solution.
一方、化学結合を介する方法としては、結合剤を用いる
方法、結合性を有する物質対を用いる方法等が挙げられ
る。上記結合剤を用いる方法における結合剤としては、
例えば、グルタルアルデヒド、マロンジアルデヒド、ス
クシンジアルデヒド等のジアルデヒド類；ヘキサメチレ
ンジイソシアネート等のジイソシアネート類；Ｎ，Ｎ−
−ｏ−フェニレンジマレイミド、Ｎ，Ｎ−−ｍ−フェニ
レンジマレイミド等のジマレイミド類．Ｎ，Ｎ−一ジシ
クロへキシルカルボジイミド、Ｎ一エチル−Ｎ−− （
３−ジメチルアミノブロビル）カルボジイミド等のカル
ボジイミド類などが挙げられる。On the other hand, methods using chemical bonds include methods using a binder, methods using a pair of substances having binding properties, and the like. As the binder in the method using the above binder,
For example, dialdehydes such as glutaraldehyde, malondialdehyde, and succindialdehyde; diisocyanates such as hexamethylene diisocyanate; N,N-
Dimaleimides such as -o-phenylene dimaleimide and N,N--m-phenylene dimaleimide. N,N-1 dicyclohexylcarbodiimide, N-ethyl-N-- (
Examples include carbodiimides such as 3-dimethylaminobrovir) carbodiimide.
これらの結合剤を用いることにより、免疫反応性物質と
不溶性担体上の官能基とを反応させて結合させることが
できる。この反応は慣用の方法にて行うことができ、例
えば、千畑編「固定化酵素」（１９７５年講談社刊行）
等に記載されている。By using these binders, the immunoreactive substance and the functional group on the insoluble carrier can be reacted and bonded. This reaction can be carried out by a conventional method, such as "Immobilized Enzymes" edited by Chibata (published by Kodansha in 1975).
It is described in etc.
また、上記の結合性を有する物質対を用いる方法におけ
る物質対としては、例えば、アビジンービオチン対、コ
ンカナバリンＡ一糖類対等が挙げられ、これらは極めて
強固な非共有結合対を形成することが知られている。こ
の方法をアビジンービオチン対の例をもって説明すると
、まず、結合対の一方の物質（例えば、アビジン）を不
溶性担体に固定化（例えば、吸着法、上記の結合剤を用
いる方法等）し、一方、物質対の他方の物質（すなわち
ビオチン）を免疫反応性物質に固定化（例えば、上記の
結合剤を用いる方法等）しておく。In addition, examples of substance pairs in the method using substance pairs having the above-mentioned binding properties include avidin-biotin pairs and concanavalin A monosaccharide pairs, which are known to form extremely strong non-covalent bond pairs. It is being To explain this method using the example of an avidin-biotin pair, first, one substance of the binding pair (e.g. avidin) is immobilized on an insoluble carrier (e.g. adsorption method, method using the above-mentioned binding agent, etc.), and one , the other substance of the substance pair (ie, biotin) is immobilized on an immunoreactive substance (for example, by the method using the above-mentioned binding agent, etc.).
次いで、この両者を反応させると、アビジンとビオチン
が結合対を形成するので、該アビジンービオチン結合を
介して免疫反応性物質が不溶性担体上に固定化される。Next, when the two are reacted, avidin and biotin form a binding pair, and the immunoreactive substance is immobilized on the insoluble carrier via the avidin-biotin bond.
なお、不溶性担体上に固定化される免疫反応性物質の量
は特に限定されず、担体の空隙率、測定対象物質の種類
及び濃度、測定法の扛類等により適宜調整することがで
き、また固定化量の調整は、不溶性担体と反応させる際
の免疫反応性物質溶液の濃度、結合剤の使用量等を適宜
選択することにより行うことができる。Note that the amount of the immunoreactive substance immobilized on the insoluble carrier is not particularly limited, and can be adjusted as appropriate depending on the porosity of the carrier, the type and concentration of the substance to be measured, the characteristics of the measurement method, etc. The amount of immobilization can be adjusted by appropriately selecting the concentration of the immunoreactive substance solution, the amount of the binder used, etc. when reacting with the insoluble carrier.
かくして、免疫反応性物質が固定化された不溶性担体は
プロッキング剤で処理し、免疫反応性物質の非固定化部
をプロツキングする。ここで使用されるプロツキング剤
としては、目的とする免疫反応に関与しないものであれ
ばいずれのものも使用でき、例えば、脱脂粉乳、牛血清
アルブミン、ゼラチン及びその加水分解物、カゼイン及
びその加水分解物、デキストラン、ポリエチレングリコ
ール等が挙げられ、特に脱脂粉乳及び牛血清アルブミン
が好ましい。上記のブロッキング剤による不溶性担体の
処理は、例えば、０．５〜１０、好ましくは３〜５ （
Ｖ／Ｖ）％程度のブロッキング剤水溶液（又は分散液）
中に不溶性担体を浸漬し、ブロッキング剤を不溶性担体
上に吸着させ、次いで水洗することにより行われる。こ
れらのブロッキング剤を不溶性担体に吸着させ、免疫反
応性物質が固定化されていない箇所をブロッキングする
ことにより、免疫反応における非特異的反応を抑制する
ことができ、バックグランドを低下させることができる
。Thus, the insoluble carrier on which the immunoreactive substance is immobilized is treated with a blocking agent to block the non-immobilized portions of the immunoreactive substance. As the blocking agent used here, any agent that is not involved in the target immune reaction can be used, such as skim milk powder, bovine serum albumin, gelatin and its hydrolyzate, casein and its hydrolyzate. Examples include dextran, polyethylene glycol, etc., and skim milk powder and bovine serum albumin are particularly preferred. The treatment of the insoluble carrier with the above blocking agent is, for example, 0.5 to 10, preferably 3 to 5 (
V/V)% blocking agent aqueous solution (or dispersion)
This is carried out by immersing an insoluble carrier in the solution, adsorbing the blocking agent onto the insoluble carrier, and then washing with water. By adsorbing these blocking agents to an insoluble carrier and blocking areas where immunoreactive substances are not immobilized, non-specific reactions in immune reactions can be suppressed and background can be reduced. .
次に、不溶性担体として、上記の処理がなされた多孔性
プラスチックを用いる本発明の免疫学的測定法を、より
詳細に説明する。Next, the immunoassay method of the present invention using the above-treated porous plastic as an insoluble carrier will be explained in more detail.
その一例として、サンドイッチ法による免疫学的測定法
を用いて、測定対象物質としての抗原を測定（定ｆｌ）
する例をもって説明する。この方法においては、抗体が
固定化されると共にブロッキング剤で処理された多孔性
プラスチックからなる不溶性担体に、測定対象物質であ
る抗原を含有する検体を加え、検体中の抗原を不溶性担
体上の抗体で捕捉し、洗浄することによりＢ／Ｆ分離を
行う。次いで、担体に標識化抗体を加え、担体上に捕捉
された抗原をサンドイッチ状に挟み、過剰の標識化抗体
を洗浄により除去し、Ｂ／Ｆ分離を行う。かくして、標
識化抗体を含む抗原抗体反応物を担体上に固定化し、固
定化された標識化抗体の標識活性を測定する。一方、上
記の抗原含有検体の代わりに、濃度既知の抗原標準試料
を用いて、同様な方法により標識活性を測定し、抗原濃
度に対する標識活性の標準曲線（検量線）を作戊する。As an example, an antigen as a substance to be measured is measured using a sandwich immunoassay method (constant fl).
This will be explained using an example. In this method, a sample containing an antigen, which is the substance to be measured, is added to an insoluble carrier made of porous plastic on which antibodies are immobilized and treated with a blocking agent, and the antigen in the sample is transferred to the antibody on the insoluble carrier. B/F separation is performed by trapping and washing. Next, a labeled antibody is added to the carrier, the antigen captured on the carrier is sandwiched, excess labeled antibody is removed by washing, and B/F separation is performed. Thus, the antigen-antibody reaction product containing the labeled antibody is immobilized on the carrier, and the labeling activity of the immobilized labeled antibody is measured. On the other hand, instead of the antigen-containing sample described above, an antigen standard sample of known concentration is used to measure labeling activity in the same manner, and a standard curve (calibration curve) of labeling activity versus antigen concentration is created.
この標準曲線と抗原含有検体の標識活性とを対比するこ
とにより、抗原含有検体中の抗原量を定量することがで
きる（以下、便宜上、測定法Ｉという）。By comparing this standard curve with the labeling activity of the antigen-containing sample, the amount of antigen in the antigen-containing sample can be quantified (hereinafter referred to as measurement method I for convenience).
また、他の例として、第二抗体固体化担体を用いた競合
法に基いて、抗原（測定対象物質）の測定（定量）法を
説明する。この方法においては、測定対象物質である抗
原を含有する検体と一定量の標識化抗原とを第一抗体（
測定対象物質である抗原に対する抗体）に対して競合反
応させる。次いで第二抗体（第一抗体に対する抗体）が
固定化されると共にブロッキング剤で処理された多孔性
プラスチックからなる不溶性担体を加え、標識化抗原を
含む抗原抗体反応物を担体上に固定化し、水洗すること
により、Ｂ／Ｆ分離を行う。かくして固定化された標識
化抗原の標識活性を測定する。Furthermore, as another example, a method for measuring (quantifying) an antigen (substance to be measured) will be described based on a competitive method using a second antibody solidified carrier. In this method, a sample containing an antigen, which is the substance to be measured, and a certain amount of labeled antigen are combined with the first antibody (
A competitive reaction is made with the antibody against the antigen, which is the substance to be measured. Next, an insoluble carrier made of porous plastic on which the second antibody (antibody against the first antibody) is immobilized and treated with a blocking agent is added, and the antigen-antibody reaction product containing the labeled antigen is immobilized on the carrier, followed by washing with water. By doing so, B/F separation is performed. The labeling activity of the labeled antigen thus immobilized is measured.
一方、上記の抗原含有検体の代りに、濃度既知の抗原標
準試料を用いて、同様な方法により標識活性を測定し、
抗原濃度に対する標識活性の標準曲線を作成する。この
標準曲線と抗原含有検体の標識活性とを対比することに
より、抗原含有検体中の抗原量を定量することができる
（以下、便宜上、測定法■という）。On the other hand, instead of the above antigen-containing sample, an antigen standard sample of known concentration is used to measure the labeling activity by the same method,
Create a standard curve of label activity versus antigen concentration. By comparing this standard curve with the labeling activity of the antigen-containing sample, the amount of antigen in the antigen-containing sample can be quantified (hereinafter referred to as measurement method (for convenience)).
上記の測定法Ｉ及び■において、不溶性担体上に固体化
される抗体、標識化される抗体及び第二抗体は、ポリク
ローナル抗体（抗血清）及びモノクローナル抗体のいず
れであってもよく、またＦ（ａｂ−）２画分も使用する
ことができる。これらの抗体は従来から慣用の方法にて
得ることができる。なお、測定対象物質がハプテン等の
免疫原性のない物質の場合には、アルブミン、グロプリ
ン、ヘモグロビン等の慣用のキャリャーと結合させて免
疫抗原を調製した後、常法の抗体産生法により抗体を得
ることができる。In the above assay methods I and (2), the antibody solidified on the insoluble carrier, the labeled antibody, and the second antibody may be either a polyclonal antibody (antiserum) or a monoclonal antibody, and F ( ab-)2 fractions can also be used. These antibodies can be obtained by conventional methods. If the target substance to be measured is a non-immunogenic substance such as a hapten, the immunogenic antigen is prepared by binding it to a conventional carrier such as albumin, globulin, or hemoglobin, and then the antibody is generated using a conventional antibody production method. Obtainable.
また、標識化抗体及び標識化抗原は公知の方法にて調製
することができ、例えば、標識が酵素の場合には、前述
の結合剤を用いて酵素と抗体（又は抗原）とを反応させ
ることにより得られる。標１２５
識が放射性物質、例えば、 ■の場合には、常法の
クロラミンＴ法、ラクトバーオキシダーゼと過酸化水素
を用いる酵素法等により、抗体（又は抗原）を標識化す
ることができる。更に、標識が螢光物質の場合には、上
記の酵素標識と同様な方法にても行うことができるが、
螢光物質をマイクロカプセル中に封入し、このマイクロ
カプセルと抗体（又は抗原）とを吸着法、化学結合法等
により結合させて標識化する方法が好ましい。In addition, labeled antibodies and labeled antigens can be prepared by known methods. For example, when the label is an enzyme, the enzyme and antibody (or antigen) may be reacted using the above-mentioned binding agent. It is obtained by 125 When the marker is a radioactive substance, for example (2), the antibody (or antigen) can be labeled by a conventional chloramine T method, an enzymatic method using lactoveroxidase and hydrogen peroxide, or the like. Furthermore, if the label is a fluorescent substance, it can be carried out using the same method as the enzyme labeling described above.
A preferred method is to label a fluorescent substance by encapsulating it in microcapsules and bonding the microcapsules to an antibody (or antigen) by an adsorption method, a chemical bonding method, or the like.
標識活性の測定は、標識の種類に応じて適宜の方法が用
いられ、例えば、標識が放射性物質の場合にはその放射
能をカウントすることにより行われ、また標識が酵素の
場合には該酵素の基質を作用させ、常広に従って該基質
の減少量若しくは酵素反応生成物の増加量又はそれらの
変化速度を、吸光度測定等の方法で求めることにより行
われる。Label activity is measured using an appropriate method depending on the type of label. For example, if the label is a radioactive substance, it is performed by counting the radioactivity, or if the label is an enzyme, it is performed by counting the radioactivity. This is carried out by applying a substrate and determining the amount of decrease in the substrate, the amount of increase in the enzymatic reaction product, or the rate of change thereof using a method such as absorbance measurement according to Tsunehiro.
かかる標識酵素と基質の組み合わせの例としては、例え
ば、バーオキシダーゼと過酸化水素及び０−フエニレン
ジアミンとの組み合わせ、β−ガラクトシダーゼと０−
ニトロフエニルーβ一Ｄ−ガラクトシドとの組み合わせ
、アルカリホスファターゼとｐ−ニトロホスフエートと
の組み合わせ等が挙げられる。標識が螢光物質の場合に
は、螢光強度測定等により行われる。Examples of combinations of such labeling enzymes and substrates include peroxidase, hydrogen peroxide and 0-phenylenediamine, β-galactosidase and 0-phenylenediamine, and β-galactosidase and 0-phenylenediamine.
Examples include a combination of nitrophenyl-β-D-galactoside, a combination of alkaline phosphatase and p-nitrophosphate, and the like. If the label is a fluorescent substance, this is carried out by measuring the fluorescence intensity or the like.
なお、上記の測定法■及び■は、本発明の測定法のある
種の態様を示したものであり、これらの方法に限定され
るものではなく、測定対象物質等に応じて適宜変更する
ことができる。例えば、測定法■において、測定対象物
質として抗体を測定するには、抗原固定化担体及び標識
化抗原を用いればよく、また同様に、測定法Ｈにおいて
は、第−抗体の代りに抗原を用いると共に標識化抗体を
用いればよい。Note that the above measurement methods ■ and ■ indicate certain aspects of the measurement method of the present invention, and are not limited to these methods, and may be changed as appropriate depending on the substance to be measured, etc. I can do it. For example, in measurement method ①, to measure an antibody as the substance to be measured, an antigen-immobilized carrier and a labeled antigen may be used; similarly, in measurement method H, an antigen is used instead of the first antibody. A labeled antibody may be used together with the antibody.
次に、本発明の免疫学的測定法の一例を、サンドイッチ
法を用いた酵素免疫測定法により、測定対象物質として
の抗原を測定する例に基き、より具体的に説明する。Next, an example of the immunoassay method of the present invention will be described in more detail based on an example in which an antigen as a substance to be measured is measured by an enzyme immunoassay method using a sandwich method.
まず、抗体固定化担体を作製する。抗体固定化担体の作
製は、抗体又はそのＦ（ａｂ−）ｚ画分を１０〜２００
μｇ／１１程度となるように、適当な緩衝液（例えば、
０．１Ｍリン酸緩衝液、ｐＨ７程度）に溶解し、この溶
液に多孔性プラスチックを室温又は冷却下に０．５〜１
０時間、好ましくは１〜３時間程度浸漬した後、水又は
緩衝液で洗浄し、次いで、３〜５％（Ｗ／Ｖ）程度の濃
度のプロツキング剤溶液に室温又は冷却下に０．５〜５
時間、好ましくは１〜３時間程度浸漬した後、水又は緩
衝液で洗浄することにより行われる。First, an antibody-immobilized carrier is prepared. To prepare an antibody-immobilized carrier, the antibody or its F(ab-)z fraction is mixed with 10 to 200
A suitable buffer solution (for example,
0.1M phosphate buffer (about pH 7), and add porous plastic to this solution at room temperature or under cooling.
After being immersed for 0 hours, preferably 1 to 3 hours, it is washed with water or a buffer solution, and then soaked in a blocking agent solution with a concentration of 3 to 5% (W/V) at room temperature or under cooling for 0.5 to 3 hours. 5
This is carried out by immersing for a period of time, preferably about 1 to 3 hours, and then washing with water or a buffer solution.
次いで、かくして得られた抗体固定化担体を用いて抗原
を測定する。この測定操作としては、まず、測定対象物
質である抗原を含有する検体をそのまま又は適当な緩衝
液で濃度調整をした後、該担体に加え、検体中の抗原を
担体上の抗体で捕捉する。この反応は、冷却下〜加温下
、通常、室温程度で行われ、１〜１０分間程度、通常、
２〜５分間程度で終了する。次いで、該担体を適当な緩
衝岐で洗浄してＢ／Ｆ分離を行った後、適当な濃度の酵
素標識抗体溶液を添加し、担体上に固定化された抗原と
反応させることにより、酵素標識抗体を担体上に固定化
する。この反応は、冷却下〜加温下、通常、室温程度で
行われ、１〜１０分間程度、通常、２〜５分間程度で終
了する。かくして、酵素標識抗体を含む抗原抗体反応物
が固定化された担体は、緩衝液で洗浄することによりＢ
／Ｆ分離を行う。Ｂ／Ｆ分離された担体に、酵素の基質
溶液を添加し、所定時間（通常、１〜１０分間程度）反
応させた後、基質の減少量、酵素反応生成物の増加量等
を吸光度測定等の方法で測定し、担体上の標識酵素の酵
素活性を測定する。得られた測定値に基き、予め作成し
た標準曲線から、検体中の抗原量を求めることができる
。なお、標準曲線の作成は、上記の測定方法において、
検体の代りに濃度既知の抗原標準試料を用い、同様な操
作により酵素活性を求め、抗原濃度に対して酵素活性を
プロットすることにより得られる。Next, the antigen is measured using the antibody-immobilized carrier thus obtained. In this measurement operation, first, a sample containing an antigen, which is a substance to be measured, is added to the carrier either as is or after adjusting the concentration with an appropriate buffer solution, and the antigen in the sample is captured by an antibody on the carrier. This reaction is carried out under cooling to heating, usually at about room temperature, and usually for about 1 to 10 minutes.
It will finish in about 2 to 5 minutes. Next, after washing the carrier with an appropriate buffer to perform B/F separation, an enzyme-labeled antibody solution of an appropriate concentration is added and reacted with the antigen immobilized on the carrier, thereby removing the enzyme-labeled antibody. Immobilize the antibody on a carrier. This reaction is carried out under cooling to heating, usually at about room temperature, and is completed in about 1 to 10 minutes, usually about 2 to 5 minutes. In this way, the carrier on which the antigen-antibody reactant containing the enzyme-labeled antibody is immobilized can be washed with a buffer solution to obtain B.
/F Perform separation. After adding the enzyme substrate solution to the B/F separated carrier and reacting for a predetermined time (usually about 1 to 10 minutes), the amount of decrease in the substrate, the amount of increase in the enzyme reaction product, etc. is measured by absorbance measurement etc. The enzymatic activity of the labeled enzyme on the carrier is measured using the method described above. Based on the obtained measurement values, the amount of antigen in the sample can be determined from a standard curve prepared in advance. The standard curve is created using the above measurement method.
It can be obtained by using an antigen standard sample of known concentration instead of the specimen, determining the enzyme activity by the same procedure, and plotting the enzyme activity against the antigen concentration.
なお、上記の測定法において、Ｂ／Ｆ分離、酵素活性の
測定等は、不溶性担体として通水性材料を用いることに
より、フロ一方式により行うこともできる。また、使用
される緩衝液、反応時間、反応温度等は、測定対象物質
、標識酵素の種類等により、適宜選択することができる
。In addition, in the above measurement method, B/F separation, enzyme activity measurement, etc. can also be performed by a flow method by using a water-permeable material as an insoluble carrier. Further, the buffer solution, reaction time, reaction temperature, etc. to be used can be appropriately selected depending on the substance to be measured, the type of labeled enzyme, etc.
本発明の免疫学的測定法は種々の微量成分の測定（定量
）に用いることができ、測定対象物質としては、例えば
、α−フエトプロテイン、Ｃ反応性蛋白、ガン胎児性抗
原、ハブトグロビン、免疫グロブリン類等の血清蛋白質
、インスリン、甲状腺刺激ホルモン、成長ホルモン等の
ホルモン、ステロイドホルモン、ＨＢｓ抗原等のウイル
ス抗原、カナマイシン、テオフィリン等の薬剤などが挙
げられ、これらの物質を含む検体としては、例えば、血
清、血漿、尿、髄液、リンパ液等が挙げられる。The immunoassay method of the present invention can be used to measure (quantitate) various trace components, and examples of substances to be measured include α-fetoprotein, C-reactive protein, carcinoembryonic antigen, habtoglobin, Samples containing these substances include serum proteins such as immunoglobulins, hormones such as insulin, thyroid stimulating hormone, and growth hormone, steroid hormones, viral antigens such as HBs antigen, and drugs such as kanamycin and theophylline. Examples include serum, plasma, urine, cerebrospinal fluid, lymph fluid, and the like.
く発明の作用・効果〉
本発明の免疫学的測定法においては、不溶性担体として
多孔性プラスチックが用いられており、比表面積が大き
いので、担体上に免疫反応性物質を多量（高濃度）に固
定化できる。従って、検体を高度に希釈することなく、
抗原抗体反応を行うことができ、その結果、反応は短時
間に進行し迅速な測定を行うことができる。特に、多孔
性プラスチックは透水性に優れると共に耐圧性も良好な
ので、Ｂ／Ｆ分離を容易且つ高効率で行うことができる
。また、多孔性プラスチックは加工性に優れるので種々
の形態の担体とすることができる。Functions and Effects of the Invention In the immunoassay method of the present invention, porous plastic is used as an insoluble carrier, and since it has a large specific surface area, it is possible to place a large amount (high concentration) of an immunoreactive substance on the carrier. Can be fixed. Therefore, without highly diluting the sample,
An antigen-antibody reaction can be carried out, and as a result, the reaction proceeds in a short period of time, allowing rapid measurement. In particular, porous plastics have excellent water permeability and good pressure resistance, so B/F separation can be performed easily and with high efficiency. Furthermore, porous plastics have excellent processability and can be used as carriers in various forms.
更に、濾紙担体等を使用すると微細の剥離片がノイズと
なるが、多孔性プラスチックはかかる剥離片を生じない
ので、ノイズの発生を防止できる。Furthermore, when a filter paper carrier or the like is used, minute peeling pieces cause noise, but porous plastic does not produce such peeling pieces, so noise can be prevented from occurring.
また、本発明で用いられる多孔性プラスチック担体は、
ブロッキング剤により処理されているので、非特異的結
合が抑制され、測定精度の向上に寄与できると共にブラ
ンクの測定値の上昇を防ぎ、バックグランドを低下させ
ることができるので、Ｓ／Ｎ比が向上し、高感度で測定
を行うことができる。Furthermore, the porous plastic carrier used in the present invention is
Since it is treated with a blocking agent, non-specific binding is suppressed, which contributes to improving measurement accuracy, as well as preventing increases in blank measurement values and reducing background, improving the S/N ratio. It is possible to perform measurements with high sensitivity.
従って、本発明の免疫学的測定法によれば、短時間に測
定が終了し、測定精度及びハ１定感度を著しく向上させ
ることができるという効果を奏し、反応の迅速性、Ｂ／
Ｆ分離の容易性等から、自動分析化にも適した測定法で
ある。Therefore, according to the immunoassay method of the present invention, the measurement can be completed in a short time, and the measurement accuracy and constant sensitivity can be significantly improved.
This measurement method is also suitable for automatic analysis due to the ease of F separation.
く実施例〉
以下、実施例に基き本発明をより詳細に説明するが、本
発明はこれらの実施例に限定されるものではない。Examples> Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to these Examples.
実施例１
（１）抗体固定化担体の作製
多孔性担体として市販のプラスチック製多孔性担体（ボ
アーレックス社製、Ｘ−４８９７、厚さ１．６ｍｍ）を
約１５ＣＩｌｌＸ１０ＣＩ＋の大きさに裁断し、水洗し
た後、抗Ｃ反応性蛋白（ＣＲＰ）抗体のＦ（ａｂ）ｚ画
分を１００ｕｇ／ｘｉ含む０．１Ｍリン酸緩衝液（ｐＨ
７．０）中に浸漬させ、室温で１〜２時間放置した。次
いで、担体を取り出し、０，ＩＭリン酸緩衝液（ｐＨ７
．０）で洗浄し、更に脱脂粉乳を５％含むＯ．ＩＭホウ
酸緩衝液（ ｐ Ｈ ８ ． ５ ） ４ ０ １１
に１〜２時間浸漬して抗体固定化担体を作製した。Example 1 (1) Preparation of antibody-immobilized carrier A commercially available plastic porous carrier (manufactured by Boarex, X-4897, thickness 1.6 mm) was cut into a size of approximately 15CIllX10CI+, and washed with water. After that, 0.1M phosphate buffer (pH
7.0) and left at room temperature for 1 to 2 hours. Next, the carrier was taken out and added to 0.IM phosphate buffer (pH 7).
．． 0), and further added 5% skimmed milk powder. IM borate buffer (pH 8.5) 40 11
An antibody-immobilized carrier was prepared by immersing the carrier in water for 1 to 2 hours.
（２） Ｃ Ｒ Ｐの測定
上記（１）で作製した抗体固定化担体を直径５ＩＩＩ１
の円板状に裁断し、適当な容器に詰めて反応容器とした
。(2) Measurement of C R P The antibody-immobilized carrier prepared in (1) above was
The mixture was cut into disk shapes and packed into a suitable container to serve as a reaction container.
これに検体として、ＣＲＰを１００ｎ ｇ ／ １１含
む０．１ＭＩＪン酸緩衝液（ｐ Ｈ ７． ０， １
（Ｗ／Ｖ）％ウシ血清アルブミン含有、以下、測定緩
衝液という）を２０μｐ添加し、３分間放置して反応さ
せ、更に西洋ワサビバーオキシダーゼ標識抗ＣＲＰ抗体
を０．５単位／ ｘｌ含む測定緩衝液を５０μｇ添加し
、３分間反応させた。次いで、０．１Ｍリン酸緩衝液（
ｐＨ７．０）を１．５１１加えて洗浄した後、抗体固定
化担体を取り出した。この担体に２■／１！の０−フエ
ニレンジアミンを含む６ｍＭ過酸化水素溶液を０．５ｘ
Ｉ加えて３分間反応させた後、２Ｎ一硫酸を０．５ｖＩ
加えて反応を停止し、波長４９２ｎｍにおける吸光度を
測定した（３重測定）。To this, a 0.1 MIJ acid buffer (pH 7.0, 1
Add 20μp of (w/v)% bovine serum albumin (hereinafter referred to as measurement buffer), leave to react for 3 minutes, and add measurement buffer containing 0.5 units/xl of horseradish peroxidase-labeled anti-CRP antibody. 50 μg of the solution was added and reacted for 3 minutes. Then, 0.1M phosphate buffer (
After washing by adding 1.511 of pH 7.0), the antibody-immobilized carrier was taken out. 2■/1 for this carrier! 0.5x 6mM hydrogen peroxide solution containing 0-phenylenediamine
After adding 2N monosulfuric acid and reacting for 3 minutes, add 0.5vI of 2N monosulfuric acid.
In addition, the reaction was stopped, and the absorbance at a wavelength of 492 nm was measured (triplicate measurement).
一方、ブランクとして、検体の代わりに上記測定緩衝族
を用いて同様に測定した。On the other hand, as a blank, the same measurement was carried out using the above measurement buffer group instead of the specimen.
検体を３重測定した結果は、吸光度の平均値が１．８４
７、標準偏差が０．０３８、変動係数が２，０５％とな
り良好な測定再現性が得られた。As a result of triplicate measurement of the sample, the average value of absorbance was 1.84.
7. Standard deviation was 0.038, coefficient of variation was 2.05%, and good measurement reproducibility was obtained.
なお、ブランクの吸光度の測定値は０．０１９であった
。コノ結果、ＣＲＰ濃度１ ０ ０ ｎ ｇ ／ １１
における測定値のＳ／Ｎ比は９７．２となり、感度よく
判定できることが判った。Note that the measured value of the absorbance of the blank was 0.019. Results: CRP concentration 100 ng/11
The S/N ratio of the measured value was 97.2, indicating that the determination could be made with good sensitivity.
また、上記の測定操作に示されるように、本発明の測定
法は極めて短時間に反応が終了した。Further, as shown in the above measurement operation, the reaction was completed in an extremely short time in the measurement method of the present invention.
実施例２
実施例１で作製した抗体固定化担体を使用し、検体とし
て各種濃度のＣ Ｒ Ｐ ｉｌｌＪ定緩衝液溶液を５０
μｐ用いて、実施例１と同様の方法で吸光度を測定した
。Example 2 Using the antibody-immobilized carrier prepared in Example 1, 50% CRPillJ constant buffer solution of various concentrations was used as a specimen.
Absorbance was measured in the same manner as in Example 1 using μp.
その結果を第１図に示す。第１図から明らかなように、
ＣＲＰを短時間に広範囲に亘って測定できることが判っ
た。The results are shown in FIG. As is clear from Figure 1,
It has been found that CRP can be measured over a wide range in a short period of time.
実施例３
（１）抗体固定化担体の作製
多孔性担体として市販のプラスチック製多孔性担体（ボ
アーレックス社製、Ｘ−４８９７、厚サ１．６ｍｍ）を
約１ ５ ｃ＋ｎ Ｘ １ ０ ｃｒｎの大きさに裁断
し、水洗した後、抗α−フェトプロテイン（ＡＦＰ）抗
体のＦ（ａｂ−）２画分を５ ０ ｕ ｇ ／ ｘｌ含
む０，ＩＭリン酸緩衝液（ｐＨ７．０）中に浸漬させ、
室温で１〜２時間放置した。次いで、担体を取り出し、
０．１Ｍリン酸緩衝液（ｐＨ７．０）で洗浄し、更に脱
脂粉乳を５％含む０．１Ｍホウ酸緩衝液（ ｐ Ｈ ８
． ５ ） ４ ０ １１に１〜２時間浸漬させて
抗体固定化担体を作製した。Example 3 (1) Preparation of antibody-immobilized carrier A commercially available plastic porous carrier (manufactured by Boarex, X-4897, thickness 1.6 mm) was prepared using a porous carrier having a size of approximately 15 c+n x 10 crn. After cutting into pieces and washing with water, they were immersed in 0.IM phosphate buffer (pH 7.0) containing 50 μg/xl of the F(ab-)2 fraction of anti-α-fetoprotein (AFP) antibody. ,
It was left at room temperature for 1-2 hours. Next, take out the carrier,
Wash with 0.1M phosphate buffer (pH 7.0), and then wash with 0.1M borate buffer (pH 8) containing 5% skim milk powder.
．． 5) An antibody-immobilized carrier was prepared by immersing it in 4011 for 1 to 2 hours.
（２） Ａ Ｆ Ｐの測定
上記（１）で作製した抗体固定化担体を直径５ｆｆｉＩ
１の円板状に裁断し、適当な容器に詰めて反応容器とし
た。(2) Measurement of AFP The antibody-immobilized carrier prepared in (1) above was
1 was cut into a disk shape and packed into a suitable container to serve as a reaction container.
これに検体として、ＡＦＰを９ ２ ｎ ｇ ／ １１
含む測定緩衝液を２０μｐ添加し、３分間放置して反応
させ、更に西洋ワサビパーオキシダーゼ標識抗ＡＦＰ抗
体を１．０単位／１！含む測定緩衝液を５０μρ添加し
、３分間反応させた。次いで、０．１Ｍリン酸緩衝液（
ｐＨ７．０）を１．５ｆｆ，ｆ加えて洗浄した後、抗体
固定化担体を取り出した。To this, 92 ng/11 AFP was added as a sample.
Add 20μp of measurement buffer containing the solution, leave to react for 3 minutes, and add horseradish peroxidase-labeled anti-AFP antibody at 1.0 units/1! 50μρ of the measurement buffer containing the mixture was added and allowed to react for 3 minutes. Then, 0.1M phosphate buffer (
After washing by adding 1.5 ff, f of (pH 7.0), the antibody-immobilized carrier was taken out.
この担体に２ １Ｉｌｇ ／ １１の０−フエニレンジ
アミンを含む５ｍＭ過酸化水素溶液を０．５１１加えて
３分間反応させた後、２Ｎ一硫酸を０．５１１加えて反
応を停止し、波長４９２ｎｍにおける吸光度を測定した
（３重測定）。To this carrier was added 0.511 l of a 5 mM hydrogen peroxide solution containing 0-phenylenediamine of 2 lg/11 and allowed to react for 3 minutes, and then 0.51 l of 2N monosulfuric acid was added to stop the reaction. Absorbance was measured (triplicate measurements).
一方、ブランクとして、検体の代わりに前記測定緩衝液
を用いて同様に測定した。On the other hand, as a blank, the measurement was performed in the same manner using the measurement buffer instead of the specimen.
検体を３重測定した結果は、吸光度の平均値が０．７９
８、標準偏差が０．０１０、変動係数が１．２８％とな
り良好な測定再現性が得られた。As a result of triplicate measurement of the sample, the average value of absorbance was 0.79.
8. Standard deviation was 0.010, coefficient of variation was 1.28%, and good measurement reproducibility was obtained.
なお、ブランクの吸光度の測定値は０．０３７であった
。この結果、ＡＦＰ濃度９ ２ ｎ ｇ ／ １１にお
ける測定値のＳ／Ｎ比は２１．６となり、感度よく判定
できることが判った。Note that the measured value of the absorbance of the blank was 0.037. As a result, the S/N ratio of the measured value at an AFP concentration of 9 2 ng/11 was 21.6, indicating that determination could be made with good sensitivity.
また、上記の測定操作に示されるように、本発明の測定
法は極めて短時間に反応が終了した。Further, as shown in the above measurement operation, the reaction was completed in an extremely short time in the measurement method of the present invention.
実施例４
実施例３で作製した抗体固定化担体を使用し、検体とし
て各種濃度のＡ Ｆ Ｐ ＃Ｊ定緩衝液溶液を２０μｇ
用い、実施例３と同様の方法で吸光度を測定した。Example 4 Using the antibody-immobilized carrier prepared in Example 3, 20 μg of A F P #J constant buffer solution of various concentrations was added as a specimen.
The absorbance was measured using the same method as in Example 3.
その結果を第２図に示す。第２図から明らかなように、
ＡＦＰを短時間に広範囲に亘って測定できることが判っ
た。The results are shown in FIG. As is clear from Figure 2,
It has been found that AFP can be measured over a wide range in a short period of time.
第１図はＣＲＰを本発明の測定法により測定したときの
標準曲線、第２図はＡＦＰを本発明の測定法により測定
したときの標準曲線を示す図である。FIG. 1 is a standard curve when CRP is measured by the measuring method of the present invention, and FIG. 2 is a diagram showing a standard curve when AFP is measured by the measuring method of the present invention.
Claims (1)
疫学的測定法において、抗原又は抗体が固定化された不
溶性担体がブロッキング剤で処理されていると共に不溶
性担体が多孔性プラスチックからなることを特徴とする
免疫学的測定法。1. In an immunoassay using an insoluble carrier on which an antigen or antibody is immobilized, the insoluble carrier on which the antigen or antibody is immobilized is treated with a blocking agent, and the insoluble carrier is made of porous plastic. Characteristic immunoassay method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15135889A JPH0315760A (en) | 1989-06-13 | 1989-06-13 | Immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15135889A JPH0315760A (en) | 1989-06-13 | 1989-06-13 | Immunoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0315760A true JPH0315760A (en) | 1991-01-24 |
Family
ID=15516798
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15135889A Pending JPH0315760A (en) | 1989-06-13 | 1989-06-13 | Immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0315760A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59206761A (en) * | 1983-05-11 | 1984-11-22 | Igaku Seibutsugaku Kenkyusho:Kk | Enzyme immunomeasuring method |
| JPS60500384A (en) * | 1983-02-02 | 1985-03-22 | フア−マシア・ア−・ベ− | Method and apparatus for biological specific affinity reactions |
| JPS62261960A (en) * | 1986-05-08 | 1987-11-14 | Shinotesuto Kenkyusho:Kk | Labeled immune carrier and its using method |
| JPH01202662A (en) * | 1987-12-18 | 1989-08-15 | W R Grace & Co | Biological reagent bonded to porous support |
-
1989
- 1989-06-13 JP JP15135889A patent/JPH0315760A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60500384A (en) * | 1983-02-02 | 1985-03-22 | フア−マシア・ア−・ベ− | Method and apparatus for biological specific affinity reactions |
| JPS59206761A (en) * | 1983-05-11 | 1984-11-22 | Igaku Seibutsugaku Kenkyusho:Kk | Enzyme immunomeasuring method |
| JPS62261960A (en) * | 1986-05-08 | 1987-11-14 | Shinotesuto Kenkyusho:Kk | Labeled immune carrier and its using method |
| JPH01202662A (en) * | 1987-12-18 | 1989-08-15 | W R Grace & Co | Biological reagent bonded to porous support |
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