JP3319073B2 - Immunological measurement method - Google Patents
Immunological measurement methodInfo
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- JP3319073B2 JP3319073B2 JP23086693A JP23086693A JP3319073B2 JP 3319073 B2 JP3319073 B2 JP 3319073B2 JP 23086693 A JP23086693 A JP 23086693A JP 23086693 A JP23086693 A JP 23086693A JP 3319073 B2 JP3319073 B2 JP 3319073B2
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- group
- antibody
- antigen
- compound
- measurement
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Description
【０００１】[0001]
【産業上の利用分野】本発明は、例えば血清、血漿、尿
等の体液中の微量成分を特異的に、迅速且つ正確に定量
し得る免疫学的測定方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological measurement method capable of specifically, quickly and accurately quantifying trace components in body fluids such as serum, plasma and urine.
【０００２】[0002]
【発明の背景】近年、多数検体、多項目同時分析が可能
な自動分析装置が普及し、抗原抗体反応を利用した微量
成分の免疫学的測定方法の自動分析装置への適用が試み
られている。BACKGROUND OF THE INVENTION In recent years, automatic analyzers capable of simultaneous analysis of many samples and multiple items have become widespread, and attempts have been made to apply a method for immunological measurement of trace components using an antigen-antibody reaction to an automatic analyzer. .
【０００３】自動分析装置に適用可能な方法としては、
抗原抗体反応に起因する濁度の変化を測定することによ
り目的の成分の測定を行う所謂免疫比濁法（ＴＩＡ）
や、抗原抗体反応に起因する散乱光強度の変化を測定す
ることにより目的の成分の測定を行う所謂免疫比ろう法
等が代表的なものとして挙げられる。[0003] Methods applicable to an automatic analyzer include:
The so-called immunoturbidimetry (TIA) for measuring a target component by measuring a change in turbidity caused by an antigen-antibody reaction.
A typical example is a so-called immunoratio method in which a target component is measured by measuring a change in scattered light intensity caused by an antigen-antibody reaction.
【０００４】しかしながら、これらの方法に於いては、
検体中の測定対象成分が少ない場合には（即ち、低濃度
域では）測定値が理論値より低くなるという現象が生
じ、測定対象成分を正確に測定することができないとい
う問題点がある。However, in these methods,
When the amount of the component to be measured in the sample is small (that is, in a low concentration range), a phenomenon that the measured value is lower than the theoretical value occurs, and there is a problem that the component to be measured cannot be measured accurately.
【０００５】このような問題点を克服するため、これら
の方法に於いては、抗原抗体反応促進剤であるポリエチ
レングリコール（ＰＥＧ）やコンドロイチン硫酸等を測
定系に多量に添加する方法や、検体の絶対量を増やす方
法等が検討されている。しかし、何れの方法に於いて
も、検体中の共存物質に起因する測定誤差や、非特異的
反応により生成する不溶物に起因する測定誤差等が生じ
るという問題点を有しており、更なる改良が望まれてい
る状況にある。[0005] In order to overcome such problems, in these methods, a method in which polyethylene glycol (PEG) or chondroitin sulfate as an antigen-antibody reaction accelerator is added in a large amount to a measurement system, Methods for increasing the absolute amount are being studied. However, any of the methods has a problem that a measurement error caused by a coexisting substance in a sample or a measurement error caused by an insoluble matter generated by a non-specific reaction occurs. There is a need for improvement.
【０００６】[0006]
【発明の目的】本発明は、上記した如き状況に鑑み成さ
れたもので、低濃度域においても測定対象成分を正確に
再現性よく、迅速且つ簡便に測定でき、自動分析装置へ
の適用が可能な免疫学的測定方法を提供することをその
目的とする。SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and enables accurate and reproducible measurement of a component to be measured, even in a low concentration range, quickly and easily, and is applicable to an automatic analyzer. The aim is to provide a possible immunological measurement method.
【０００７】[0007]
【発明の構成】本発明は、遊離の抗原と抗体との反応に
起因する濁度の変化又は散乱光強度の変化に基づいて測
定を行う微量成分の免疫学的測定方法に於いて、測定対
象である抗体（又は抗原）に対する抗原（又は抗体）と
して、ハプテンで予め修飾された抗原（又は抗体）を用
いることを特徴とする、該測定方法の発明である。SUMMARY OF THE INVENTION The present invention relates to a method for immunological measurement of a trace component, which is performed based on a change in turbidity or a change in scattered light intensity due to a reaction between a free antigen and an antibody. The invention of the measurement method, characterized in that an antigen (or antibody) previously modified with a hapten is used as an antigen (or antibody) for the antibody (or antigen).
【０００８】即ち、本発明者らは、共存物質の影響や非
特異的反応に起因する測定誤差が生じることなく、低濃
度域に於ける測定対象成分の測定感度を上げる方法を見
出すべく鋭意研究の結果、測定試薬中に添加する、測定
対象である抗体（又は抗原）に対する抗原（又は抗体）
を、例えばベンゼン環を有する化合物や複素環を有する
化合物等のハプテンで修飾することにより、低濃度域に
おいても反応感度が高く且つ直線性の良好な検量線が得
られることを見出し、本発明を完成するに至った。That is, the present inventors have conducted intensive studies to find a method for increasing the measurement sensitivity of a component to be measured in a low concentration range without causing measurement errors due to the effects of coexisting substances and nonspecific reactions. As a result, the antigen (or antibody) for the antibody (or antigen) to be measured added to the measurement reagent
Is modified with a hapten such as a compound having a benzene ring or a compound having a heterocyclic ring, thereby obtaining a calibration curve with high reaction sensitivity and good linearity even in a low concentration range. It was completed.
【０００９】本発明に於いて抗原（或は抗体）を修飾す
るために用いられるハプテンとしては、一般にハプテン
として知られているものであれば特に限定されることな
く挙げられるが、特にベンゼン環を有する化合物、複素
環を有する化合物等が好ましく挙げられる。The hapten used to modify the antigen (or antibody) in the present invention is not particularly limited as long as it is generally known as a hapten. And a compound having a heterocyclic ring.
【００１０】本発明に於いてハプテンとして用いられる
ベンゼン環を有する化合物としては、例えば置換又は無
置換の、フェニル基、トリル基、キシリル基、ナフチル
基等を有する化合物が挙げられ、置換基としては、例え
ばアルキル基、置換アルキル基（置換基としては、水酸
基、アルコキシ基、カルボキシル基、スルホ基、ハロゲ
ン原子等）、アルコキシ基、ニトロ基、アセチル基、カ
ルボキシル基、スルホ基、例えば塩素，臭素，沃素等の
ハロゲン原子等が挙げられる。Examples of the compound having a benzene ring used as a hapten in the present invention include a substituted or unsubstituted compound having a phenyl group, a tolyl group, a xylyl group, a naphthyl group and the like. For example, an alkyl group, a substituted alkyl group (as a substituent, a hydroxyl group, an alkoxy group, a carboxyl group, a sulfo group, a halogen atom, etc.), an alkoxy group, a nitro group, an acetyl group, a carboxyl group, a sulfo group such as chlorine, bromine, And halogen atoms such as iodine.
【００１１】本発明に係るベンゼン環を有する化合物の
具体例としては、例えばp-ニトロフェニル酢酸、4-メチ
ル安息香酸、3-(1-ナフチル)プロピオン酸等が好ましく
挙げられる。Specific examples of the compound having a benzene ring according to the present invention include, for example, p-nitrophenylacetic acid, 4-methylbenzoic acid, and 3- (1-naphthyl) propionic acid.
【００１２】本発明に於いてハプテンとして用いられる
複素環を有する化合物としては、例えばチアゾリル基、
チエニル基、フラニル基、ピラニル基、ピロリル基、イ
ミダゾリル基、ピラゾリル基、イソチアゾリル基、イソ
キサゾリル基、ピリミジニル基、ピリダジニル基、イン
ドリル基、プリニル基、キノリル基、イソキノリル基、
ピラゾリニル基、インドリニル基、モルホリノ基、ビオ
チニル基等の複素環基（何れも置換基を有していてもよ
い）を有する化合物が好ましく挙げられる。また、複素
環基の置換基としては、例えばアルキル基、置換アルキ
ル基（置換基としては、水酸基、アルコキシ基、カルボ
キシル基、スルホ基、ハロゲン原子等）、アルコキシ
基、ニトロ基、アセチル基、カルボキシル基、スルホ
基、例えば塩素，臭素，沃素等のハロゲン原子等が挙げ
られる。The compound having a heterocyclic ring used as a hapten in the present invention includes, for example, a thiazolyl group,
Thienyl group, furanyl group, pyranyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, isothiazolyl group, isoxazolyl group, pyrimidinyl group, pyridazinyl group, indolyl group, prenyl group, quinolyl group, isoquinolyl group,
Compounds having a heterocyclic group (any of which may have a substituent) such as a pyrazolinyl group, an indolinyl group, a morpholino group and a biotinyl group are preferred. Examples of the substituent of the heterocyclic group include, for example, an alkyl group, a substituted alkyl group (such as a hydroxyl group, an alkoxy group, a carboxyl group, a sulfo group, and a halogen atom), an alkoxy group, a nitro group, an acetyl group, and a carboxyl group. And sulfo groups, for example, halogen atoms such as chlorine, bromine and iodine.
【００１３】本発明に係る複素環を有する化合物の具体
例としては、例えばビオチン、2-チエニル酢酸、インド
ール酪酸、ピロール-2-カルボン酸等が好ましく挙げら
れる。Specific examples of the compound having a heterocycle according to the present invention include, for example, biotin, 2-thienylacetic acid, indolebutyric acid, pyrrole-2-carboxylic acid and the like.
【００１４】本発明に於いて、抗原又は抗体にベンゼン
環を有する化合物を修飾する方法としては、例えば以下
のような方法が挙げられる。即ち、例えばp-ニトロフェ
ニル酢酸を抗原又は抗体に修飾する場合には、p-ニトロ
フェニル酢酸に常法［例えばJ.Amer.Chem.Soc.,vol.85,
3039(1963);J.Amer.Chem.Soc.,vol.86, 1839(1964)
等］によりスクシンイミド基を導入し、得られた化合物
を常法［例えばZ.Anal.Chem.,vol.279, 143(1976);J.Cl
in.Endocrinol.Metab.,vol.44, 91(1977);Biochem.Biop
hys.Acta.,vol.403, 131(1975)等］により抗原又は抗体
と反応させることにより容易に行うことができる。尚、
p-ニトロフェニル酢酸にスクシンイミド基を導入した、
市販のＳＮＰＡ（N-Succinimidyl-p-nitrophenylacetat
e、株式会社同仁化学研究所製）試薬を用いればスクシ
ンイミド基の導入工程が省略できるので操作の簡略化が
図れる。In the present invention, examples of a method for modifying a compound having a benzene ring on an antigen or an antibody include the following methods. That is, for example, when p-nitrophenylacetic acid is modified into an antigen or an antibody, p-nitrophenylacetic acid can be modified by a conventional method [for example, J. Amer. Chem. Soc., Vol.
3039 (1963); J. Amer. Chem. Soc., Vol. 86, 1839 (1964)
And the like, and the resulting compound is subjected to a conventional method [for example, Z. Anal. Chem., Vol. 279, 143 (1976); J. Cl.
in.Endocrinol.Metab., vol. 44, 91 (1977); Biochem. Biop.
hyta. Acta., vol. 403, 131 (1975), etc.]. still,
A succinimide group was introduced into p-nitrophenylacetic acid,
Commercially available SNPA (N-Succinimidyl-p-nitrophenylacetat)
e) (manufactured by Dojindo Laboratories Co., Ltd.) If a reagent is used, the step of introducing a succinimide group can be omitted, thus simplifying the operation.
【００１５】その他の方法としては、ベンゼン環を有す
る化合物に常法によりマレイミド基を導入し［例えばJ.
Pharm.Dyn.,vol.4, 812-819(1981)等］、これを抗原や
抗体のチオール基と反応させる方法［例えばAnnals of
the New York Academy of Science,vol.254, 203(1975)
等］や、ベンゼン環を有する化合物に常法によりヒドラ
ジノ基を導入し［例えばJ.Biol.Chem.,vol.172, 71(194
8)等］、これと、アルデヒド化した抗原や抗体とを反応
させる方法［例えばBiotech.Appl.Biochem.,vol.9, 488
-496(1987)等］等が挙げられる。As another method, a maleimide group is introduced into a compound having a benzene ring by a conventional method [for example, J. Am.
Pharm. Dyn., Vol. 4, 812-819 (1981), etc.], and a method of reacting this with a thiol group of an antigen or an antibody [eg, Annals of
the New York Academy of Science, vol. 254, 203 (1975)
Hydrazino group is introduced into a compound having a benzene ring by a conventional method [for example, J. Biol. Chem., Vol.
8) etc.] and a method of reacting this with an aldehyde-converted antigen or antibody [eg Biotech. Appl. Biochem., Vol. 9, 488]
-496 (1987)].
【００１６】尚、ベンゼン環を有する化合物による抗原
又は抗体の修飾の程度としては、通常抗原又は抗体に対
してモル比で0.2〜10倍程度、好ましくは１〜５倍程度
が挙げられる。ベンゼン環を有する化合物による修飾量
が多い場合は抗原又は抗体の不溶性が高くなったり、抗
原抗体反応が妨げられる等問題が生じ、逆に修飾量が少
ない場合は感度が所期の目標に達しない等の問題が生じ
るため、注意が必要である。The degree of modification of the antigen or antibody with the compound having a benzene ring is usually about 0.2 to 10 times, preferably about 1 to 5 times the molar ratio of the antigen or antibody. When the amount of modification by the compound having a benzene ring is large, problems such as increased insolubility of the antigen or antibody or hindrance to the antigen-antibody reaction occur.On the contrary, when the amount of modification is small, the sensitivity does not reach the intended target. Care must be taken because problems such as these occur.
【００１７】次に、抗原又は抗体に例えばビオチン等の
複素環を有する化合物を結合させる方法としては、例え
ば市販のビオチン化試薬、例えばスクシンイミド基が導
入されたビオチン（例えば、ＮＨＳ-ビオチン）やN-ヒ
ドロキシコハク酸アミド（ＮＨＳ）とビオチンをスペー
サーを介して結合したもの等を抗体又は抗原蛋白のアミ
ノ基と反応させる方法、例えば市販の N-[6-(Biotinami
de)hexyl]-3'-(2'-pyridyldithio)propionamide（ビオ
チンーＨＰＤＰ）や N-Iodoacetyl-N-biotinylhexylened
iaminを抗原又は抗体のチオール基に反応させる方法
［例えばAnnals ofthe New York Academy of Science,v
ol.254, 203(1975)等］、ヒドラジノ基が導入されたビ
オチンをアルデヒド化された抗原又は抗体のアルデヒド
基に反応させる方法［例えばJ.Biol.Chem.,vol.172, 71
(1948);Biotech.Appl.Biochem.,vol.9, 488-496(1987)
等］等が好ましく挙げられる。Next, as a method for binding a compound having a heterocyclic ring such as biotin to an antigen or an antibody, for example, a commercially available biotinylation reagent such as biotin (for example, NHS-biotin) into which a succinimide group is introduced or N A method of reacting -hydroxysuccinamide (NHS) and biotin via a spacer with an amino group of an antibody or an antigen protein, for example, commercially available N- [6- (Biotinamido
de) hexyl] -3 '-(2'-pyridyldithio) propionamide (biotin-HPDP) and N-Iodoacetyl-N-biotinylhexylened
a method of reacting iamin with a thiol group of an antigen or an antibody [for example, Annals of the New York Academy of Science, v.
254, 203 (1975), etc.], and a method of reacting a biotin having a hydrazino group introduced thereto with an aldehyde group of an aldehyde-modified antigen or antibody [for example, J. Biol. Chem., vol.
(1948); Biotech.Appl.Biochem., Vol. 9, 488-496 (1987).
Etc.] are preferred.
【００１８】また、複素環を有する化合物による抗原又
は抗体の修飾の程度としては、抗原又は抗体に対してモ
ル比で0.2〜10倍程度、好ましくは１〜５倍程度が挙げ
られる。複素環を有する化合物による修飾量が多い場合
は抗原又は抗体の不溶性が高くなったり、抗原抗体反応
が妨げられる等問題が生じ、逆に修飾量が少ない場合は
感度が所期の目標に達しない等の問題が生じるため、注
意が必要である。The degree of modification of the antigen or antibody with the compound having a heterocyclic ring is about 0.2 to 10 times, preferably about 1 to 5 times, the molar ratio of the antigen or antibody. When the amount of modification with the compound having a heterocyclic ring is large, problems such as increased insolubility of the antigen or antibody and hindrance of the antigen-antibody reaction occur.On the contrary, when the amount of modification is small, the sensitivity does not reach the intended target. Care must be taken because problems such as these occur.
【００１９】本発明の方法に於いて、ハプテンにより修
飾されて用いられる抗原としては、例えばストレプトリ
ジンＯ（ＳＬＯ）、リウマチ因子（ＲＦ）、Ｂ型肝炎ウ
イルス表面抗原（ＨＢｓ）等が挙げられる。In the method of the present invention, examples of the antigen used after being modified with a hapten include streptolysin O (SLO), rheumatoid factor (RF), hepatitis B virus surface antigen (HBs) and the like.
【００２０】また、ハプテンにより修飾されて用いられ
る抗体はモノクロ−ナル抗体でもポリクロ−ナル抗体で
も何れにてもよく特に限定されない。また、その具体例
としては例えば抗Ｃ反応性蛋白質（抗ＣＲＰ）抗体、抗
免疫グロブリンＧ（抗ＩgＧ）抗体、抗免疫グロブリン
Ａ（抗ＩgＡ）抗体、抗免疫グロブリンＭ（抗ＩgＭ）抗
体、抗アルブミン抗体、抗Ｃ３抗体、抗Ｃ４抗体、抗α
-フェトプロテイン（抗ＡＦＰ）抗体等が挙げられる。The antibody used after being modified with the hapten may be either a monoclonal antibody or a polyclonal antibody, and is not particularly limited. Specific examples thereof include anti-C-reactive protein (anti-CRP) antibody, anti-immunoglobulin G (anti-IgG) antibody, anti-immunoglobulin A (anti-IgA) antibody, anti-immunoglobulin M (anti-IgM) antibody, Albumin antibody, anti-C3 antibody, anti-C4 antibody, anti-α
-Fetoprotein (anti-AFP) antibody and the like.
【００２１】本発明の測定方法を実施するに際して使用
するその他の試薬や、測定条件等（反応温度、反応時
間、測定波長、測定装置等）はすべて自体公知の免疫比
濁法や免疫比ろう法等に於けるそれらに準じて選択すれ
ば足りる。即ち、本発明の測定方法は、上記した如くし
てハプテンで修飾した抗原又は抗体を用いること以外は
自体公知の免疫比濁法や免疫比ろう法の測定操作法に準
じて実施すればよく、使用する自動分析装置、分光光度
計等も通常この分野で使用されているものは何れも例外
なく使用し得る。Other reagents and measurement conditions (reaction temperature, reaction time, measurement wavelength, measurement apparatus, etc.) used in carrying out the measurement method of the present invention are all known per se known immunoturbidimetry and immunoassay methods. It suffices to select according to those in etc. That is, the measurement method of the present invention may be performed according to a measurement operation method of a per se known immunoturbidimetry or immunoassay except that an antigen or antibody modified with a hapten is used as described above, Automatic analyzers, spectrophotometers, and the like to be used may be any of those usually used in this field without exception.
【００２２】本発明の測定法に於いて用いられる緩衝液
の具体例を挙げると、例えばトリス緩衝液、リン酸緩衝
液、ベロナール緩衝液、ホウ酸緩衝液、グッド緩衝液等
通常抗原抗体反応を利用した測定法に用いられている緩
衝液は全て挙げられ、そのpＨとしては抗原抗体反応を
抑制しない範囲であれば特に限定はされないが、通常５
〜９の範囲が好ましく用いられる。Specific examples of buffers used in the assay of the present invention include, for example, tris buffer, phosphate buffer, veronal buffer, borate buffer, good buffer and the like. All the buffers used in the assay method used can be mentioned, and the pH thereof is not particularly limited as long as it does not suppress the antigen-antibody reaction.
The range of ~ 9 is preferably used.
【００２３】本発明の方法によれば、測定対象成分が低
濃度の場合でも反応感度が高いため、低濃度域でも直線
性の良好な検量線が得られる。この理由は未だ定かでは
ないが、例えば反応に関与する抗原或は抗体をベンゼン
環を有する化合物や複素環を有する有する化合物等のハ
プテンで修飾したことにより、該抗原（又は抗体）の疎
水性が高まり、必然的に、反応の結果生じる抗原抗体複
合物の疎水性も高まるために、低濃度であっても目的の
抗原抗体複合物が不溶物として反応溶液から析出し易く
なるためではないかと考えられる。According to the method of the present invention, since the reaction sensitivity is high even when the concentration of the component to be measured is low, a calibration curve with good linearity can be obtained even in a low concentration range. Although the reason for this is not yet clear, for example, by modifying the antigen or antibody involved in the reaction with a hapten such as a compound having a benzene ring or a compound having a heterocyclic ring, the hydrophobicity of the antigen (or antibody) is reduced. It is thought that the target antigen-antibody complex is likely to precipitate out of the reaction solution as an insoluble substance even at a low concentration because the antigen-antibody complex resulting from the reaction also inevitably increases in hydrophobicity. Can be
【００２４】以下に実施例により、本発明を更に詳細に
述べるが、本発明はこれらにより何等限定されるもので
はない。Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
【００２５】[0025]
実施例１．抗ストレプトリジンＯ（ＡＳＯ）価の測定 (1)ストレプトリジンＯ（ＳＬＯ）の修飾 市販のＳＬＯ（和光純薬工業(株)製）を1.3 mg蛋白/ml
となるように100mM N-2-ヒドロキシエチルピペラジン-
N'-2-スルホン酸(HEPES)緩衝液(pH8.5) 10mlに溶解した
ものに、40mM N-Succinimidyl-4-nitrophenylacetate
（ＳＮＰＡ、株式会社同仁化学研究所製）を含むN,N-ジ
メチルホルムアミド溶液１mlを添加して、37℃で２時間
反応させた。反応終了後、反応液を0.9％NaCl溶液に対
して透析を行い、未反応のＳＮＰＡを除去して、修飾Ｓ
ＬＯ溶液を得た。 (2)抗ストレプトリジンＯ（ＡＳＯ）価の測定 （測定用試液の調製） 第１試液 3.5％ポリエチレングリコール6,000、0.9％NaCl及び0.1
％NaN3を含む50mM 3-(N-モルホリノ)プロパンスルホン
酸(MOPS)-NaOH緩衝液（pＨ7.4）を第１試液とした。 第２試液 上記(1)で得た修飾ＳＬＯを第１試薬に添加し、修飾Ｓ
ＬＯが150μg/mlの蛋白濃度となるように調整したもの
を第２試薬とした。 （測定機器）日立自動分析装置７０７０型を使用した。 （測定操作）ＡＳＯを所定濃度含む試料20μｌと第１試
液350μｌとを混合し、37℃で５分間インキュベートし
た後、測定主波長340nm副波長700nmに於ける吸光度を測
定した（検体盲検値）。次いでこれに第２試液50μｌを
添加し、37℃で５分間インキュベートした後、測定主波
長340nm副波長700nmに於ける吸光度を測定し、得られた
吸光度から、液量補正した検体盲検値を差引いた吸光度
を求めた（反応吸光度）。また、対照として、未修飾の
ＳＬＯを用いて同様にして調製した第２試液を用いた以
外は、同じ試料及び試液を用い、同様の操作を行って反
応吸光度を求めた。 （結果）得られた反応吸光度とＡＳＯ濃度との関係を示
す検量線を図１に示す。尚、図１に於いて、＋は修飾Ｓ
ＬＯを含む第２試液を用いて得られた結果を、また、□
は未修飾ＳＬＯを含む第２試液を用いて得られた結果を
夫々示す。図１から明らかな如く、修飾していないＳＬ
Ｏを用いた場合（従来法）では 200 U/ml 以下の領域で
は反応感度が低下して、測定が不可能となることが判
る。これに対し、修飾ＳＬＯを用いる本発明の方法によ
れば、100 U/ml以下の領域でも十分な反応感度があり、
測定が可能であることが判る。Embodiment 1 FIG. Measurement of anti-streptolysin O (ASO) value (1) Modification of streptolysin O (SLO) 1.3 mg protein / ml of commercially available SLO (manufactured by Wako Pure Chemical Industries, Ltd.)
100 mM N-2-hydroxyethylpiperazine-
N'-2-sulfonic acid (HEPES) buffer (pH 8.5) dissolved in 10 ml, 40 mM N-Succinimidyl-4-nitrophenylacetate
1 ml of a N, N-dimethylformamide solution containing (SNPA, manufactured by Dojindo Laboratories Inc.) was added and reacted at 37 ° C. for 2 hours. After completion of the reaction, the reaction solution was dialyzed against a 0.9% NaCl solution to remove unreacted SNPA,
An LO solution was obtained. (2) Measurement of anti-streptolysin O (ASO) value (Preparation of test solution) First test solution 3.5% polyethylene glycol 6,000, 0.9% NaCl and 0.1
A 50 mM 3- (N-morpholino) propanesulfonic acid (MOPS) -NaOH buffer (pH 7.4) containing% NaN3 was used as a first test solution. Second reagent solution The modified SLO obtained in the above (1) is added to the first reagent, and the modified SLO is added.
The second reagent was prepared so that LO was adjusted to a protein concentration of 150 μg / ml. (Measurement equipment) Hitachi automatic analyzer 7070 type was used. (Measurement procedure) After mixing 20 μl of a sample containing a predetermined concentration of ASO and 350 μl of the first test solution and incubating at 37 ° C. for 5 minutes, the absorbance was measured at a main wavelength of 340 nm and a secondary wavelength of 700 nm (sample blank value). . Next, 50 μl of the second test solution was added thereto, and the mixture was incubated at 37 ° C. for 5 minutes. Then, the absorbance at the main wavelength of 340 nm and the auxiliary wavelength of 700 nm was measured. The subtracted absorbance was determined (reaction absorbance). Further, as a control, the same operation was performed using the same sample and test solution except that the second test solution prepared similarly using unmodified SLO was used to determine the reaction absorbance. (Results) FIG. 1 shows a calibration curve showing the relationship between the obtained reaction absorbance and ASO concentration. In addition, in FIG.
The results obtained using the second reagent solution containing LO
Shows the results obtained using the second reagent containing unmodified SLO, respectively. As is clear from FIG. 1, the unmodified SL
In the case of using O (conventional method), the reaction sensitivity is lowered in the region of 200 U / ml or less, so that measurement becomes impossible. On the other hand, according to the method of the present invention using the modified SLO, there is sufficient reaction sensitivity even in the region of 100 U / ml or less,
It turns out that measurement is possible.
【００２６】実施例２．リウマチ因子（ＲＦ）の測定 (1)ビオチン修飾ヒトＩgＧの調製 市販のヒトＩgＧ 100mgを 50mM炭酸緩衝液(pH9)９mlに
溶解したものに、Biotinamidocaproate-N-Hydroxysucci
nimidoester（ＢＡＨＳ、PIERCE社製）９mgをN,N-ジメ
チルホルムアミド溶液１mlに溶解したものを添加し、５
℃で１昼夜反応させた。反応終了後、反応液を0.9％NaC
l溶液に対して透析し、未反応のＢＡＨＳを除去して、
ビオチン修飾ヒトＩgＧを得た。 (2)リウマチ因子（ＲＦ）の測定 （測定用試液の調製） 第１試液 実施例１の第１試液と同じ。 第２試液 生理食塩水(0.9%NaCl)にビオチン修飾ヒトＩgＧを１mg/
mlの蛋白濃度となるように添加したものを第２試液とし
た。 （測定機器）日立自動分析装置７０７０型を使用した。 （測定操作）ＲＦを所定濃度含む試料14μｌと第１試液
250μｌとを混合し、37℃で５分間インキュベートした
後、測定主波長340nm副波長700nmに於ける吸光度を測定
した（検体盲検値）。次いでこれに第２試液125μｌを
添加し、37℃で５分間インキュベートした後、測定主波
長340nm副波長700nmに於ける吸光度を測定し、得られた
吸光度から、液量補正した検体盲検値を差引いた吸光度
を求めた（反応吸光度）。また、対照として、ビオチン
で修飾していないヒトＩgＧを用いて同様にして調製し
た第２試液を用いた以外は、同じ試料及び試液を用い、
同様の操作を行って反応吸光度を求めた。 （結果）得られた反応吸光度とリウマチ因子（ＲＦ）濃
度との関係を示す検量線を図２に示す。尚、図２に於い
て、＋はビオチン修飾ヒトＩgＧを含む第２試液を用い
て得られた結果を、また、□は未修飾ヒトＩgＧを含む
第２試液を用いて得られた結果を夫々示す。図２から明
らかな如く、未修飾ヒトＩgＧを用いた場合（従来法）
ではＲＦを測定することは困難であることが判る。これ
に対し、ビオチン修飾ヒトＩgＧを用いる本発明の方法
によれば、ＲＦを感度良く測定することが可能となるこ
とが判る。Embodiment 2 FIG. Measurement of rheumatoid factor (RF) (1) Preparation of biotin-modified human IgG Biotinamidocaproate-N-Hydroxysucci was prepared by dissolving 100 mg of commercially available human IgG in 9 ml of 50 mM carbonate buffer (pH 9).
A solution obtained by dissolving 9 mg of nimidoester (BAHS, manufactured by PIERCE) in 1 ml of N, N-dimethylformamide solution is added, and 5
The reaction was carried out at ℃ for one day. After the reaction is completed, the reaction solution is 0.9% NaC
dialysis against the solution to remove unreacted BAHS,
Biotin-modified human IgG was obtained. (2) Measurement of rheumatoid factor (RF) (Preparation of reagent solution for measurement) First reagent solution Same as the first reagent solution of Example 1. Second test solution Biotin-modified human IgG was added to physiological saline (0.9% NaCl) at 1 mg /
A solution added to a protein concentration of ml was used as a second test solution. (Measurement equipment) Hitachi automatic analyzer 7070 type was used. (Measurement procedure) 14 μl of a sample containing a predetermined concentration of RF and the first reagent
After mixing with 250 µl and incubating at 37 ° C for 5 minutes, the absorbance was measured at a main wavelength of 340 nm and a subwavelength of 700 nm (sample blank value). Next, 125 μl of the second test solution was added thereto, and the mixture was incubated at 37 ° C. for 5 minutes. Then, the absorbance at the main wavelength of 340 nm and the auxiliary wavelength of 700 nm was measured. The subtracted absorbance was determined (reaction absorbance). As a control, the same sample and test solution were used except that a second test solution prepared similarly using human IgG not modified with biotin was used.
The same operation was performed to determine the reaction absorbance. (Results) FIG. 2 shows a calibration curve showing the relationship between the obtained reaction absorbance and the concentration of rheumatoid factor (RF). In FIG. 2, + represents the results obtained using the second reagent containing biotin-modified human IgG, and □ represents the results obtained using the second reagent containing unmodified human IgG. Show. As is clear from FIG. 2, when unmodified human IgG is used (conventional method)
Then, it turns out that it is difficult to measure RF. In contrast, according to the method of the present invention using biotin-modified human IgG, it can be seen that RF can be measured with high sensitivity.
【００２７】実施例３．抗ストレプトリジンＯ（ＡＳ
Ｏ）価の測定 (1)ビオチン修飾ストレプトリジンＯ（ＳＬＯ）の調製 市販のＳＬＯを1.3mg蛋白/mlとなるように30mMリン酸緩
衝液(pH7.5)に溶解したもの５mlに、N-Hydroxysuccinim
ido Biotin（ＨＳＢ、PIERCE社製）の30mM N,N-ジメチ
ルホルムアミド溶液0.5mlを添加し、37℃で１時間反応
させた。反応終了後、反応液を0.9％NaCl溶液に対して
透析し、未反応のＨＳＢを除去して、ビオチン修飾ＳＬ
Ｏを得た。 (2)ＡＳＯの測定 （測定用試液の調製） 第１試液 実施例１と同じものを用いた。 第２試液 上記(1)で得たビオチン修飾ＳＬＯを第１試薬に添加
し、ビオチン修飾ＳＬＯが150μg/mlの蛋白濃度となる
ように調整したものを第２試薬とした。 （測定機器）日立自動分析装置７０７０型を使用した。 （測定操作）ＡＳＯを所定濃度含む試料20μｌと第１試
液350μｌとを混合し、37℃で５分間インキュベートし
た後、測定主波長340nm副波長700nmに於ける吸光度を測
定した（検体盲検値）。次いでこれに第２試液50μｌを
添加し、37℃で５分間インキュベートした後、測定主波
長340nm副波長700nmに於ける吸光度を測定し、得られた
吸光度から、液量補正した検体盲検値を差引いた吸光度
を求めた（反応吸光度）。また、対照として、未修飾の
ＳＬＯを用いて同様にして調製した第２試液を用いた以
外は、同じ試料及び試液を用い、同様の操作を行って反
応吸光度を求めた。 （結果）得られた反応吸光度とＡＳＯ濃度との関係を示
す検量線を図３に示す。尚、図３に於いて、＋はビオチ
ン修飾ＳＬＯを含む第２試液を用いて得られた結果を、
また、□は未修飾ＳＬＯを含む第２試液を用いて得られ
た結果を夫々示す。図３から明らかな如く、修飾してい
ないＳＬＯを用いた場合（従来法）では 150 U/ml 以下
の領域では反応感度が低下して、測定が不可能となるこ
とが判る。これに対し、ビオチン修飾ＳＬＯを用いる本
発明の方法によれば、100 U/ml以下の領域でも十分な反
応感度があり、直線性の良好な検量線が得られることが
判る。Embodiment 3 FIG. Anti-streptolysin O (AS
O) Measurement of Value (1) Preparation of Biotin-Modified Streptolysin O (SLO) Commercially available SLO was dissolved in a 30 mM phosphate buffer (pH 7.5) to a concentration of 1.3 mg protein / ml. Hydroxysuccinim
0.5 ml of a 30 mM N, N-dimethylformamide solution of ido Biotin (HSB, manufactured by PIERCE) was added and reacted at 37 ° C. for 1 hour. After completion of the reaction, the reaction solution was dialyzed against a 0.9% NaCl solution to remove unreacted HSB, and the biotin-modified SL was removed.
O was obtained. (2) Measurement of ASO (Preparation of reagent solution for measurement) First reagent solution The same reagent as in Example 1 was used. Second reagent solution The biotin-modified SLO obtained in (1) was added to the first reagent, and the biotin-modified SLO was adjusted to have a protein concentration of 150 μg / ml. (Measurement equipment) Hitachi automatic analyzer 7070 type was used. (Measurement procedure) After mixing 20 μl of a sample containing a predetermined concentration of ASO and 350 μl of the first test solution and incubating at 37 ° C. for 5 minutes, the absorbance was measured at a main wavelength of 340 nm and a secondary wavelength of 700 nm (sample blank value). . Next, 50 μl of the second test solution was added thereto, and the mixture was incubated at 37 ° C. for 5 minutes. Then, the absorbance at the main wavelength of 340 nm and the auxiliary wavelength of 700 nm was measured. The subtracted absorbance was determined (reaction absorbance). Further, as a control, the same operation was performed using the same sample and test solution except that the second test solution prepared similarly using unmodified SLO was used to determine the reaction absorbance. (Results) FIG. 3 shows a calibration curve showing the relationship between the obtained reaction absorbance and ASO concentration. In FIG. 3, + represents the results obtained using the second test solution containing biotin-modified SLO,
In addition, □ shows the results obtained using the second reagent solution containing unmodified SLO, respectively. As is clear from FIG. 3, when unmodified SLO is used (conventional method), the reaction sensitivity is lowered in a region of 150 U / ml or less, and measurement becomes impossible. On the other hand, according to the method of the present invention using biotin-modified SLO, it can be seen that there is sufficient reaction sensitivity even in the region of 100 U / ml or less, and a calibration curve with good linearity can be obtained.
【００２８】[0028]
【発明の効果】以上述べた如く、本発明は、従来の方法
では測定不可能であった低濃度域に於いても測定対象成
分を正確に再現性よく、迅速且つ簡便に測定することが
できる方法を提供するものであり、斯業に貢献するとこ
ろ極めて大なる発明である。As described above, according to the present invention, the components to be measured can be measured accurately, with good reproducibility, quickly and easily even in a low concentration range which cannot be measured by the conventional method. It is a very large invention that provides a method and contributes to the industry.
【図面の簡単な説明】 図１は、実施例１で得られた検量線を示す。図２は、実
施例２で得られた検量線を示す。図３は、実施例３で得
られた検量線を示す。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a calibration curve obtained in Example 1. FIG. 2 shows the calibration curve obtained in Example 2. FIG. 3 shows the calibration curve obtained in Example 3.
図１に於いて、＋は修飾ストレプトリジンＯ（ＳＬＯ）
を含む第２試液を用いて得られた結果を、また、□は未
修飾ＳＬＯを含む第２試液を用いて得られた結果を夫々
示す。図２に於いて、＋はビオチン修飾ヒトＩgＧを含
む第２試液を用いて得られた結果を、また、□は未修飾
ヒトＩgＧを含む第２試液を用いて得られた結果を夫々
示す。図３に於いて、＋はビオチン修飾ＳＬＯを含む第
２試液を用いて得られた結果を、また、□は未修飾ＳＬ
Ｏを含む第２試液を用いて得られた結果を夫々示す。In FIG. 1, + represents modified streptolysin O (SLO)
, The results obtained using the second reagent containing unmodified SLO are shown, and the results obtained using the second reagent containing unmodified SLO are shown. In FIG. 2, + shows the results obtained using the second test solution containing biotin-modified human IgG, and □ shows the results obtained using the second test solution containing unmodified human IgG. In FIG. 3, + represents the results obtained using the second test solution containing biotin-modified SLO, and □ represents unmodified SL.
The results obtained using the second test solution containing O are shown respectively.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 平４−216465（ＪＰ，Ａ) 特開 平２−216055（ＪＰ，Ａ) 特開 昭63−231265（ＪＰ，Ａ) 特開 昭53−69825（ＪＰ，Ａ) 特開 平６−82449（ＪＰ，Ａ) 特開 平７−53597（ＪＰ，Ａ) 特開 平７−53405（ＪＰ，Ａ) 特表 平２−501410（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 33/536 G01N 33/53 ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-4-216465 (JP, A) JP-A-2-216055 (JP, A) JP-A-63-231265 (JP, A) JP-A-53-231 69825 (JP, A) JP-A-6-82449 (JP, A) JP-A-7-53597 (JP, A) JP-A-7-53405 (JP, A) JP-A-2-501410 (JP, A) (58) Field surveyed (Int. Cl. 7 , DB name) G01N 33/536 G01N 33/53
Claims (4)
の変化又は散乱光強度の変化に基づいて測定を行う微量
成分の免疫学的測定方法に於いて、測定対象である抗体
（又は抗原）に対する抗原（又は抗体）として、ハプテ
ンで予め修飾された抗原（又は抗体）を用いることを特
徴とする、該測定方法。In a method for immunological measurement of a trace component, the measurement is performed based on a change in turbidity or a change in scattered light intensity due to a reaction between a free antigen and an antibody. Or an antigen (or antibody) modified in advance with a hapten as the antigen (or antibody) to the antigen (or antibody).
は複素環を有する化合物である、請求項１に記載の測定
方法。2. The method according to claim 1, wherein the hapten is a compound having a benzene ring or a compound having a heterocyclic ring.
置換の、フェニル基、トリル基、キシリル基又はナフチ
ル基を有する化合物である、請求項２に記載の測定方
法。3. The method according to claim 2, wherein the compound having a benzene ring is a substituted or unsubstituted compound having a phenyl group, a tolyl group, a xylyl group or a naphthyl group.
の、チアゾリル基、チエニル基、フラニル基、ピラニル
基、ピロリル基、イミダゾリル基、ピラゾリル基、イソ
チアゾリル基、イソキサゾリル基、ピリミジニル基、ピ
リダジニル基、インドリル基、プリニル基、キノリル
基、イソキノリル基、ピラゾリニル基、インドリニル
基、モルホリノ基、ビオチニル基を有する化合物であ
る、請求項２に記載の測定方法。4. The compound having a heterocyclic ring is a substituted or unsubstituted thiazolyl group, thienyl group, furanyl group, pyranyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, isothiazolyl group, isoxazolyl group, pyrimidinyl group, pyridazinyl group. The method according to claim 2, wherein the compound has an indolyl group, a purinyl group, a quinolyl group, an isoquinolyl group, a pyrazolinyl group, an indolinyl group, a morpholino group, and a biotinyl group.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23086693A JP3319073B2 (en) | 1993-08-24 | 1993-08-24 | Immunological measurement method |
| EP94306064A EP0640836A3 (en) | 1993-08-24 | 1994-08-17 | Immunoassay method. |
| US08/291,895 US5728589A (en) | 1993-08-24 | 1994-08-17 | Immunoassay method |
| TW083107670A TW373073B (en) | 1993-08-24 | 1994-08-22 | Immunoassay method |
| KR1019940020832A KR100236864B1 (en) | 1993-08-24 | 1994-08-23 | Immunoassay method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23086693A JP3319073B2 (en) | 1993-08-24 | 1993-08-24 | Immunological measurement method |
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| Publication Number | Publication Date |
|---|---|
| JPH0763757A JPH0763757A (en) | 1995-03-10 |
| JP3319073B2 true JP3319073B2 (en) | 2002-08-26 |
Family
ID=16914538
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|---|---|---|---|
| JP23086693A Expired - Fee Related JP3319073B2 (en) | 1993-08-24 | 1993-08-24 | Immunological measurement method |
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| JP (1) | JP3319073B2 (en) |
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| WO2012068366A2 (en) | 2010-11-18 | 2012-05-24 | Yale University | Bifunctional molecules with antibody-recruiting and entry inhibitory activity against the human immunodeficiency virus |
| CN110114677B (en) * | 2016-12-15 | 2023-06-20 | 株式会社堀场制作所 | Method for determining suitability of concentration of test substance in concentration measurement using immune coagulation reaction, and sample analyzer having processing unit for the determination method |
-
1993
- 1993-08-24 JP JP23086693A patent/JP3319073B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0763757A (en) | 1995-03-10 |
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