JPH0765988B2 - Fractional measurement method for trace components - Google Patents
Fractional measurement method for trace componentsInfo
- Publication number
- JPH0765988B2 JPH0765988B2 JP2002170A JP217090A JPH0765988B2 JP H0765988 B2 JPH0765988 B2 JP H0765988B2 JP 2002170 A JP2002170 A JP 2002170A JP 217090 A JP217090 A JP 217090A JP H0765988 B2 JPH0765988 B2 JP H0765988B2
- Authority
- JP
- Japan
- Prior art keywords
- substance
- measured
- lectin
- measurement
- complex
- 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.)
- Expired - Lifetime
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 ［発明の利用分野］ 本発明は、例えば血清，血液，血漿，尿等の生体体液、
リンパ球、血球、各種細胞類等の生体由来の試料中に存
在する、同一の作用を有し、且つ同一の検出可能な化学
特性を有する２以上の測定対象物質を、その化学的又は
／及び物理的な性質に応じて、迅速に、容易に且つ精度
良く分別測定する方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Use of the Invention] The present invention relates to biological fluids such as serum, blood, plasma and urine,
Two or more substances to be measured, which have the same action and the same detectable chemical properties, present in a sample derived from a living body such as lymphocytes, blood cells, and various cells are chemically or / and The present invention relates to a method for performing a quick and easy and accurate fractional measurement according to physical properties.
［発明の背景］ 生体試料中に含まれる微量成分の中には、同一の作用を
有するが化学的又は／及び物理的に異なる性質を有する
ものが存在する。例えば、蛋白質部分又は／及び糖鎖部
分の構造が異なる酵素（アイソザイム）や、糖鎖構造の
異なるホルモン等の生理活性物質がそれに相当する。こ
れらの試料中の量を、種々の性質に応じて分別測定する
ことができれば、臨床上有効な指標が得られることは良
く知られている。BACKGROUND OF THE INVENTION Among trace components contained in a biological sample, there are substances that have the same action but have chemically or / and physically different properties. For example, an enzyme (isozyme) having a different protein or / and sugar chain structure, or a physiologically active substance such as a hormone having a different sugar chain structure corresponds thereto. It is well known that a clinically effective index can be obtained if the amounts in these samples can be separately measured according to various properties.
これらを分別測定する一般的な方法としては、例えば電
気泳動法、イムノアッセイ法、抗体やインヒビターを用
いた酵素活性の阻害を利用した方法等が挙げられる。し
かしながら、これらの方法には、測定に時間を要する、
定量性が低い等の問題点があり、必ずしも実用的な方法
とはいい難い。As a general method for separately measuring these, for example, an electrophoresis method, an immunoassay method, a method utilizing inhibition of enzyme activity using an antibody or an inhibitor, and the like can be mentioned. However, these methods require time for measurement,
There are problems such as low quantification, and it is not always a practical method.
一方、このような問題点を解決する方法として、例え
ば、イオン交換クロマトグラフィ用充填剤を充填したカ
ラムを用いた高速液体クロマトグラフィ（HPLC）によ
り、乳酸脱水素酵素のアイソザイムを分別測定する方法
が提案されている（J.Chromatogr.,374,45〜50頁,198
6、J.Chromatogr.,378,456〜461頁,1986）。しかしなが
ら、この方法に於いても、分別測定の対象となり得るも
のはある程度限られており、しかも分別のための測定条
件は測定対象物質に応じて設定する必要がある等の問題
点を有しているので、必ずしも良い方法であるとは言い
難く、更なる改良が望まれていた。On the other hand, as a method of solving such a problem, for example, a method of separately measuring lactate dehydrogenase isozymes by high performance liquid chromatography (HPLC) using a column packed with a packing material for ion exchange chromatography has been proposed. (J. Chromatogr., 374 , 45-50 pages, 198
6, J. Chromatogr., 378 , 456-461, 1986). However, even in this method, there are some limits to what can be the target of separation measurement, and there is a problem that the measurement conditions for separation must be set according to the substance to be measured. Therefore, it is hard to say that this is a good method, and further improvements have been desired.
［発明の目的］ 本発明は、上記した如き状況に鑑みなされたもので、生
体由来の試料中に存在する、同一の作用を有し、且つ同
一の検出可能な化学特性を有する２以上の測定対象物質
を、その化学的又は／及び物理的な性質に応じて、迅速
に、容易に且つ精度良く分別測定し得る方法を提供する
ことを目的とする。[Object of the Invention] The present invention has been made in view of the above situation, and two or more measurements that are present in a sample derived from a living body and have the same action and the same detectable chemical property. It is an object of the present invention to provide a method capable of rapidly and easily and accurately performing a fractional measurement of a target substance according to its chemical or / and physical properties.
［発明の構成］ 本発明は、同一の作用を有し、且つ同一の検出可能な化
学特性を有する２以上の測定対象物質（以下、単に、測
定対象物質と略記する。）を含む試料を、測定対象物質
の少なくとも１つに対しては結合能を有するが、それら
の少なくとも１つとは結合しない物質（以下、単に、結
合能物質と略記する。）と混合して反応させた後、測定
対象物質と結合能物質との複合体（以下、単に、複合体
と略記する。）と、遊離の測定対象物質とを高速液体ク
ロマトグラフィにより分離し、複合体中の測定対象物質
の量又は／及び遊離の測定対象物質の量を測定すること
により試料中の測定対象物質の何れかの量を測定するこ
とを特徴とする分別測定方法の発明である。[Structure of the Invention] The present invention provides a sample containing two or more substances to be measured (hereinafter, simply referred to as a substance to be measured) that have the same action and the same detectable chemical property. The substance to be measured is mixed with a substance that has a binding ability to at least one of the substances to be measured but does not bind to at least one of them (hereinafter, simply referred to as a substance having a binding property) and then reacted. A complex of a substance and a binding substance (hereinafter simply referred to as a complex) and a free substance to be measured are separated by high-performance liquid chromatography, and the amount of the substance to be measured in the complex and / or release of the substance. The invention of the fractional measurement method is characterized in that the amount of any of the measurement target substances in the sample is measured by measuring the amount of the measurement target substance.
本発明の分別測定方法を実施するには、例えば以下のよ
うにして行えばよい。To carry out the fractional measurement method of the present invention, for example, the following may be performed.
即ち、先ず測定対象物質を含む生体由来の試料と結合能
物質とを、要すれば適当な緩衝液中に添加、混合して反
応させ、複合体を形成させた後、該複合体と遊離の測定
対象物質とを所定の充填剤を充填したカラムを装着した
HPLCにより分離する。次いで、分離された複合体に含ま
れる測定対象物質の量又は／及び遊離の測定対象物質の
量を、測定対象物質の性質に応じた測定方法により求め
れば、試料中の測定対象物質の何れかの量が求められ
る。That is, first, a sample of biological origin containing a substance to be measured and a binding substance are added, if necessary, in an appropriate buffer and mixed to react to form a complex, which is then separated from the complex. A column filled with a predetermined packing material was attached to the substance to be measured.
Separate by HPLC. Then, if the amount of the measurement target substance contained in the separated complex or / and the amount of the free measurement target substance is obtained by the measurement method according to the property of the measurement target substance, any of the measurement target substances in the sample Is required.
本発明の分別測定方法により測定可能な測定対象物質と
しては、それ自身が何らかの方法により測定（検出）可
能であって、且つ、結合能物質、即ち測定対象物質の少
なくとも１つとは互いに強い相互作用（affinity;親和
力或は親和性）を及ぼしあい、強固な複合体を形成する
が、それらの少なくとも１つとは結合しない性質を有す
る物質が存在するものであれば、特に限定することなく
挙げられるが、例えば血清，血液，血漿，尿等の生体体
液、リンパ液、血球、各種細胞類等の生体由来の試料中
に含まれる酵素等が代表的なものとして挙げられる。更
に具体的には、例えばアミラーゼ，アルカリホスファタ
ーゼ，酸性ホスファターゼ，γ−グルタミルトランスフ
ェラーゼ（γ−GTP），リパーゼ，クレアチンキナーゼ
（CK），乳酸脱水素酵素（LDH），グルタミン酸オキザ
ロ酢酸トランスアミナーゼ（GOT），グルタミン酸ピル
ビン酸トランスアミナーゼ（GPT），レニン，プロテイ
ンキナーゼ（PK），チロシンキナーゼ等の酵素等が挙げ
られる。The measurement target substance that can be measured by the differential measurement method of the present invention is itself a substance that can be measured (detected) by any method, and has a strong interaction with a binding substance, that is, at least one of the measurement target substances. (Affinity; affinity or affinity) to form a strong complex, but there is a substance having a property of not binding to at least one of them, without any particular limitation. Representative examples include enzymes contained in biological body fluids such as serum, blood, plasma, urine, etc., lymph fluids, blood cells, and samples derived from living bodies such as various cells. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate. Examples include enzymes such as pyruvate transaminase (GPT), renin, protein kinase (PK), and tyrosine kinase.
本発明に係る測定対象物質に対する結合能物質として
は、測定対象物質の少なくとも１つとは互いに強い相互
作用（affinity;親和力或は親和性）を及ぼしあい、強
固な複合体を形成するが、測定対象物質の少なくとも１
つとは結合しない性質であれば特に限定することなく挙
げられるが、例えば抗原性を有する物質（ハプテンを含
む。）の特定の部分構造或は抗原決定部位に対する抗
体、特定構造の糖鎖に対して結合能を有する例えばコン
カナバリンA,レンズマメレクチン，インゲンマメレクチ
ン，ダツラレクチン，ヒイロチャワンタケレクチン，ヒ
ママメレクチン，ピーナッツレクチン，小麦胚芽レクチ
ン等のレクチン類、例えばアミラーゼ，クレアチンキナ
ーゼ（CK），グルタミン酸オキザロ酢酸トランスアミナ
ーゼ（GOT）等の酵素に対するインヒビター等が挙げら
れる。The substance capable of binding to the substance to be measured according to the present invention has a strong interaction (affinity; affinity or affinity) with at least one of the substances to be measured to form a strong complex. At least one of the substances
There is no particular limitation as long as it is a property that does not bind to one. For example, an antibody against a specific partial structure or antigenic determinant site of a substance having an antigenicity (including hapten) Lectins that have binding ability, such as concanavalin A, lentil lectin, kidney bean lectin, duck lalectin, chrysanthemum lectin, lima bean lectin, peanut lectin, wheat germ lectin, such as amylase, creatine kinase (CK), glutamate oxaloacetate transaminase ( Examples include inhibitors for enzymes such as GOT).
本発明に係る測定対象物質が有する検出可能な化学特性
としては、例えば酵素活性、蛍光性、発光性或は紫外部
に吸収を有する性質等が挙げられる。The detectable chemical properties of the substance to be measured according to the present invention include, for example, enzyme activity, fluorescence, luminescence, and a property of absorbing in the ultraviolet.
本発明の分別測定方法に於いて、測定対象物質と結合能
物質とを反応させて、複合体を形成させる際の反応条件
としては、測定対象物質や結合能物質を変質させてしま
う様な条件でさえなければ特に限定されないが、例えば
酵素免疫測定法（EIA），ラジオイムノアッセイ（RI
A），蛍光免疫測定法（FIA），アフィニティクロマトグ
ラフィ等の自体公知の方法に於いて採用されている複合
体等を形成させる際の反応条件に準じて行われるのが一
般的である。例えば、反応時に緩衝液を用いる場合に
は、使用される緩衝剤やその他の試薬はこれら自体公知
の方法に於いて用いられるものを適宜選択して用いれば
よい。In the differential measurement method of the present invention, the reaction conditions for reacting the substance to be measured and the binding substance to form a complex are conditions such that the substance to be measured or the binding substance is altered. There is no particular limitation as long as it does not include, for example, enzyme immunoassay (EIA), radioimmunoassay (RI
A), fluorescent immunoassay (FIA), affinity chromatography and the like are generally used according to the reaction conditions for forming the complex and the like which are employed in known methods. For example, when a buffer solution is used in the reaction, the buffer agent and other reagents to be used may be appropriately selected from those used in the methods known per se.
本発明の分別測定方法に於いて、複合体を形成させる際
の結合能物質の使用濃度は、測定対象物質の検量限界や
測定感度をどの程度に設定するかによって適宜設定すれ
ばよく、特に限定されない。尚、結合能物質は通常１種
を用いれば足りるが、要すれば２種以上組み合わせて用
いてもよい。この場合、任意の測定対象物質上の異なる
部位に各々結合する性質を有する２種類以上の結合能物
質を用いれば、結果的に該測定対象物質から成る複合体
の分子量が大きくなり、また、場合によっては等電点も
変動すること等から、複合体と遊離の測定対象物質との
分離がより容易となり、測定精度の向上を計ることがで
きる。また、測定対象物質が、例えばＡ、Ｂ及びＣの混
合物である場合に、例えばＡに対する結合能物質とＢに
対する結合能物質とを併せて用いれば、Ａ、Ｂ及びＣを
各々同時に分別測定することも可能となる。In the fractional measurement method of the present invention, the concentration of the binding substance used in forming the complex may be appropriately set depending on how much the calibration limit or measurement sensitivity of the substance to be measured is set, and is not particularly limited. Not done. It should be noted that one type of binding substance is usually used, but if necessary, two or more types may be used in combination. In this case, if two or more kinds of binding substances having the property of respectively binding to different sites on an arbitrary substance to be measured are used, as a result, the molecular weight of the complex consisting of the substance to be measured becomes large. In some cases, the isoelectric point also changes, so that the complex and the free substance to be measured become easier to separate, and the measurement accuracy can be improved. When the substance to be measured is, for example, a mixture of A, B, and C, if, for example, a substance capable of binding to A and a substance capable of binding to B are used together, A, B, and C are separately and separately measured. It is also possible.
本発明の分別測定方法に於いて、反応時のpHとしては、
複合体が形成されるのを妨げない範囲であれば特に限定
されるものではないが、通常２〜10、好ましくは５〜９
の範囲が挙げられる。反応時の温度も、複合体が形成さ
れるのを妨げない範囲であれば特に限定されるものでは
ないが、通常０〜50℃、好ましくは20〜40℃の範囲が好
ましく挙げられる。反応時間は、複合体が形成されるの
に要する時間が測定対象物質と結合能物質との性質によ
り異なるので、各々の性質に応じて数秒間乃至数時間適
宜反応させればよい。In the fractionation measuring method of the present invention, as the pH during the reaction,
It is not particularly limited as long as it does not prevent the formation of the complex, but is usually 2 to 10, preferably 5 to 9
The range of is mentioned. The temperature during the reaction is also not particularly limited as long as it does not prevent the formation of the complex, but it is usually 0 to 50 ° C., preferably 20 to 40 ° C. The reaction time depends on the properties of the substance to be measured and the binding substance because the time required for forming the complex depends on the properties of the substance to be measured and the binding substance.
本発明の分別測定方法に於いて、複合体と遊離の測定対
象物質の分離に用いられるHPLCとしては、装置自身は通
常分析の分野に於いて用いられているもので定流速のも
のであれば特に問題なく用いることができるが、分離用
カラムに使用する充填剤は、複合体と、遊離の測定対象
物質との間にどのような性質の差があるかにより種々の
ものが適宜選択されて使用されなければならないことは
言うまでもない。即ち、例えば複合体の分子量が遊離の
測定対象物質の分子量の約1.2倍以上、好ましくは1.5倍
以上、更に好ましくは２倍以上ある場合にはゲル濾過
（ゲルクロマトグラフィ）用の充填剤が適しており、例
えば複合体の等電点と遊離型の測定対象物質の等電点と
の差がpHで0.05以上、好ましくは0.2以上ある場合には
イオン交換クロマトグラフィ用或は等電点クロマトグラ
フィ用の充填剤が適しており、例えば複合体と遊離型の
測定対象物質の疎水性がかなり異なっている場合には疎
水クロマトグラフィ用充填剤、逆相クロマトグラフィ用
充填剤或はハイドロキシアパタイトが適している。In the fractional measurement method of the present invention, as the HPLC used for separating the complex and the free substance to be measured, the device itself is one that is usually used in the field of analysis and has a constant flow rate. Although it can be used without any particular problem, the packing material used for the separation column is appropriately selected from various ones depending on the difference in properties between the complex and the free substance to be measured. It goes without saying that it must be used. That is, for example, when the molecular weight of the complex is about 1.2 times or more, preferably 1.5 times or more, and more preferably 2 times or more the molecular weight of the substance to be measured, a packing material for gel filtration (gel chromatography) is suitable. For example, when the difference between the isoelectric point of the complex and the isoelectric point of the free substance to be measured is 0.05 or more at pH, preferably 0.2 or more, packing for ion exchange chromatography or isoelectric focusing chromatography is performed. Suitable agents are hydrophobic packing materials for reversed phase chromatography, packing materials for reverse phase chromatography, or hydroxyapatite, for example, when the hydrophobicity of the substance to be measured is significantly different from that of the complex.
HPLCにより複合体と遊離の測定対象物質との分離を行う
際に用いられる溶媒（溶離液）としては、形成された複
合体が再び測定対象物質と結合能物質とに分離されるよ
うなことがなく、且つ複合体に含まれる測定対象物質の
検出可能な化学特性を失わしめるようなものでなければ
特に限定されることなく挙げられるが、通常は例えばEI
A,RIA,FIA,アフィニティクロマトグラフィ等の自体公知
の方法に於いて緩衝液として用いられているようなもの
が好ましく用いられる。具体例としては、例えばリン酸
塩，酢酸塩，クエン酸塩，グッド（Good）の緩衝剤，ト
リス（ヒドロキシエチル）アミノメタン等の緩衝剤、例
えば塩化ナトリウム，塩化カリウム，硫酸アンモニウム
等の塩類、例えばメタノール，エタノール，イソプロピ
ルアルコール，アセトニトリル，テトラヒドロフラン等
の極性有機溶媒類及び界面活性剤等を、複合体と遊離の
測定対象物質の性質に応じて適宜選択し、添加、混合し
て調製された、pH2〜10の緩衝液が好ましく用いられ
る。As the solvent (eluent) used when separating the complex and the free substance to be measured by HPLC, it is possible that the formed complex is again separated into the substance to be measured and the binding substance. There is no particular limitation as long as it does not impair the detectable chemical properties of the substance to be measured contained in the complex.
Those used as buffers in methods known per se such as A, RIA, FIA and affinity chromatography are preferably used. Specific examples include, for example, phosphates, acetates, citrates, Good's buffers, buffers such as tris (hydroxyethyl) aminomethane, salts such as sodium chloride, potassium chloride, ammonium sulfate, etc. PH2 prepared by appropriately selecting, adding, and mixing polar organic solvents such as methanol, ethanol, isopropyl alcohol, acetonitrile, and tetrahydrofuran, and surfactants, etc., according to the properties of the complex and the free substance to be measured. -10 buffers are preferably used.
本発明の分別測定方法に於いて、HPLCにより分離された
複合体中に含まれる測定対象物質或は遊離の測定対象物
質の測定は、測定対象物質の有する検出可能な化学特性
の種類に応じて夫々所定の方法に従って実施される。例
えば、測定対象物質が酵素の場合にはEIAの常法、例え
ば「酵素免疫測定法、蛋白質 核酸 酵素 別冊 No.3
1、北川常廣・南原利夫・辻章夫・石川榮治編集、51〜6
3頁、共立出版（株）、1987年９月10日発行」等に記載
された方法に準じて測定を行えばよく、測定対象物質が
蛍光性物質の場合には蛍光光度計等の測定機器を用いる
FIAの常法、例えば「図説蛍光抗体、川生明著、第１
版、（株）ソフトサイエンス社、1983」等に記載された
方法に準じて測定を行えばよく、測定対象物質が発光性
物質の場合にはフォトンカウンター等の測定機器を用い
る常法、例えば「酵素免疫測定法、蛋白質 核酸 酵素
別冊 No.31、北川常廣・南原利夫・辻章夫・石川榮
治編集、252〜263頁、共立出版（株）、1987年９月10日
発行」等に記載された方法に準じて測定を行えばよい。
更に、測定対象物質が紫外部に吸収を有する物質の場合
には分光光度計等の測定機器を用いる常法によって測定
を行えばよい。In the fractional measurement method of the present invention, the measurement of the measurement target substance or the free measurement target substance contained in the complex separated by HPLC depends on the type of detectable chemical property of the measurement target substance. Each is performed according to a predetermined method. For example, when the substance to be measured is an enzyme, a conventional EIA method such as “enzyme immunoassay, protein / nucleic acid / enzyme separate volume No. 3” is used.
1, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51-6
Page 3, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 "and the like. If the substance to be measured is a fluorescent substance, a measuring instrument such as a fluorometer Use
FIA standard method, for example, "Illustrated fluorescent antibody, Akira Kawao, No. 1"
Edition, Soft Science Co., Ltd., 1983 ”and the like, and the measurement may be performed according to the method described therein. When the measurement target substance is a luminescent substance, a conventional method using a measurement device such as a photon counter, for example,“ Enzyme-linked immunosorbent assay, protein / nucleic acid enzyme separate volume No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 252-263, Kyoritsu Shuppan Co., Ltd., published September 10, 1987. The measurement may be performed according to the above method.
Further, when the substance to be measured is a substance having absorption in the ultraviolet, the measurement may be carried out by a conventional method using a measuring instrument such as a spectrophotometer.
本発明の分別測定方法に於いて、HPLCによる分離後の測
定方式としては、例えば「最新液体クロマトグラフィ、
原昭二・辻章夫編、第１版、92〜104頁、南山堂、1978
年２月１日発行」等に記載されているような、HPLCのカ
ラムからの流出液をそのまま検出部に導き、流出液中の
複合体中に含まれる測定対象物質の量或は遊離の測定対
象物質の量を直接測定する方式が、測定が迅速に行える
のでより好ましい。この場合に、測定対象物質が例えば
酵素であれば、HPLCのカラムと検出部との間に、酵素活
性測定用の試薬を添加し流出液と反応させる、所謂ポス
トカラム法の反応部を設ける必要があることは言うまで
もない。測定対象物質が酵素である場合に該反応部に於
いて用いられる酵素活性測定用の試薬は、常法、例えば
「酵素免疫測定法、蛋白質 核酸 酵素 別冊 No.3
1、北川常廣・南原利夫・辻章夫・石川榮治編集、51〜6
3頁、共立出版（株）、1987年９月10日発行」等に記載
された方法に準じて調製したものを用いてもよいし、市
販されている臨床検査用キットの試薬を適宜選択して利
用してもよい。また、測定対象物質が酵素以外の場合に
於いても、検出感度を増加させる目的で所定の試薬を添
加、反応させるために、HPLCのカラムと検出部との間に
適当な反応部を設けることは任意である。In the fractionation measuring method of the present invention, as the measuring method after separation by HPLC, for example, "latest liquid chromatography,
Edited by Shoji Hara and Akio Tsuji, first edition, pages 92-104, Nanzandou, 1978.
Directly flow the effluent from the HPLC column to the detection unit as described in "Published on February 1, 2000" and measure the amount or release of the substance to be measured contained in the complex in the effluent. The method of directly measuring the amount of the target substance is more preferable because the measurement can be performed quickly. In this case, if the substance to be measured is, for example, an enzyme, it is necessary to provide a so-called post-column method reaction unit between the HPLC column and the detection unit to add a reagent for measuring enzyme activity and react with the effluent. It goes without saying that there is. When the substance to be measured is an enzyme, the reagent for measuring the enzyme activity used in the reaction part is a conventional method, for example, "enzyme immunoassay, protein nucleic acid enzyme separate volume No. 3".
1, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51-6
3 page, Kyoritsu Shuppan Co., Ltd., published September 10, 1987 "may be used, or the reagents of commercially available clinical test kits may be appropriately selected. You may use it. Even when the substance to be measured is other than an enzyme, an appropriate reaction part should be provided between the HPLC column and the detection part in order to add and react a predetermined reagent for the purpose of increasing the detection sensitivity. Is optional.
本発明の分別測定方法に於いて、結合能物質として抗体
を用いる場合には、目的に応じて使用する抗体を適宜ペ
プシン，パパイン等の酵素を用いて消化してＦ（ab′）
2、Fab′或はFabとして使用することが望ましい。即
ち、通常の抗血清中に抗体として含まれる免疫グロブリ
ンは通常IgGクラスであるが、このIgGの分子量は約15万
であり、これと測定対象物質が反応した結果得られる複
合体と遊離の測定対象物とを例えばゲルクロマトグラフ
ィの原理で分離しようとすれば、測定対象物質の分子量
が約５万以上なければ分離が難しくなり、必然的に測定
対象物質の種類が限られてくる。従って、使用する抗体
を酵素により消化してＦ（ab′）2（分子量約10万）、F
ab′（分子量約５万）或はFab（分子量約５万）として
用いれば、分子量が１万程度以上のものが測定対象物質
と成り得るので好ましい。また、抗体として１つの抗原
認識部位のみと結合する性質を備えたモノクローナル抗
体を用いた場合には、測定対象物質１個当りに１個（測
定対象物質が２量体や３量体等になっている場合には単
量体あたりに１個）の結合能物質が結合するため、複合
体がHPLCにより溶出されて来る時間がほぼ一定となるの
でより好ましい。この場合に、これを消化してFab′或
はFabとして用いれば、前記した如き利点が生じるので
更に好ましいことは言うまでもない。In the differential measurement method of the present invention, when an antibody is used as the binding substance, the antibody used according to the purpose is appropriately digested with an enzyme such as pepsin or papain to obtain F (ab ').
2 , preferably used as Fab 'or Fab. That is, immunoglobulin contained as an antibody in ordinary antisera is usually of IgG class, but the molecular weight of this IgG is about 150,000, and the complex and free measurement obtained as a result of reaction of this IgG with the substance to be measured. If an object is to be separated by, for example, the principle of gel chromatography, separation will be difficult unless the molecular weight of the measurement target substance is about 50,000 or more, and the types of measurement target substances are inevitably limited. Therefore, the antibody to be used is digested with an enzyme to produce F (ab ') 2 (molecular weight of about 100,000).
When it is used as ab '(molecular weight of about 50,000) or Fab (molecular weight of about 50,000), a substance having a molecular weight of about 10,000 or more can be a substance to be measured, which is preferable. When a monoclonal antibody having the property of binding to only one antigen recognition site is used as the antibody, one per measurement target substance (the measurement target substance is a dimer or trimer, etc.). In this case, the binding ability substance (1 per monomer) is bound, so that the time when the complex is eluted by HPLC becomes almost constant, which is more preferable. In this case, it is needless to say that it is more preferable to digest this and use it as Fab 'or Fab because the above-mentioned advantages will be produced.
本発明に於いて用いられる結合能物質としての抗体は、
常法、例えば「免疫学実験入門、第２刷、松橋直ら、
（株）学会出版センター、1981」等に記載の方法に準じ
て、馬、牛、羊、兎、山羊、ラット、マウス等の動物に
測定対象物質を免疫して作製されるポリクローナル抗体
でも、或はまた常法、即ちケラーとミルスタイン（G.K
hler and C.Milstein;Nature,256,495,1975）により
確立された細胞融合法に従い、マウスの腫瘍ラインから
の細胞と、測定対象物質で予め免疫されたマウスの脾細
胞とを融合させて得られるハイブリドーマが産生する単
クローン性抗体でも何れにてもよく、これらを単独で或
はこれらを適宜組み合わせて用いる等は任意である。The antibody as the binding substance used in the present invention is
Conventional methods, such as "Introduction to immunological experiments, 2nd edition, Nao Matsuhashi,
Polyclonal antibody prepared by immunizing animals such as horses, cows, sheep, rabbits, goats, rats, and mice with the substance to be measured according to the method described in "Society Press Center, 1981", or the like, or Is also conventional, namely Keller and Milstein (GK
hler and C. Milstein; Nature, 256, 495, 1975), and a hybridoma obtained by fusing cells from a mouse tumor line with splenocytes of a mouse previously immunized with a substance to be measured according to the cell fusion method established by Any of the monoclonal antibodies produced by the above may be used, and these may be used alone or in an appropriate combination thereof.
本発明の分別測定方法によれば、測定に要する時間は数
十分から数時間程度であり、必要な測定操作自体は、測
定対象物質を含む試料と結合能物質を混合した後、HPLC
により複合体と遊離の測定対象物質とを分離、検出する
のみである。これらのことから明らかなように、本願発
明の分別測定方法は、従来の同様な目的の分別測定方法
に比べて、簡便に且つ迅速に目的の測定を行うことがで
きる。According to the fractional measurement method of the present invention, the time required for measurement is several tens of minutes to several hours, and the necessary measurement operation itself is HPLC after mixing the sample containing the substance to be measured and the binding ability substance.
It only separates and detects the complex and the free substance to be measured. As is clear from these, the fractional measurement method of the present invention can perform the desired measurement more easily and rapidly than the conventional fractional measurement method for the same purpose.
以下に実施例を挙げて、本発明を更に具体的に説明する
が、本発明はこれらにより何ら限定されるものではな
い。Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.
［実施例］ 実施例1.α−アミラーゼアイソザイムの分別測定 （溶離液） 20mM NaCl及び2mM CaCl2を含む75mM N,N−ビス（２−ヒ
ドロキシエチル）−２−アミノエタンスルホン酸（BE
S）・NaOH緩衝液（pH7.6）を溶離液とした。[Examples] Example 1. Fractional measurement of α-amylase isozyme (eluent) 75 mM N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BE containing 20 mM NaCl and 2 mM CaCl 2
S) -NaOH buffer (pH 7.6) was used as the eluent.
（基質液） ｐ−ニトロフェニル ベンジル−α−マルトペンタオシ
ドを14mg/ml、α−グルコシダーゼを150U/ml及びグルコ
アミラーゼを300U/mlとなるように、上記溶離液に溶解
したものを基質液とした。(Substrate solution) p-nitrophenyl benzyl-α-maltopentaoside 14 mg / ml, α-glucosidase 150 U / ml and glucoamylase 300 U / ml dissolved in the above eluent to obtain a substrate solution And
（抗体液） 抗ヒト唾液型α−アミラーゼ（マウス）モノクローナル
抗体（和光純薬工業（株）製）を、上記溶離液に0.5mg/
mlの蛋白濃度となるように添加して抗体液とした。(Antibody solution) Anti-human saliva type α-amylase (mouse) monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the above eluent at 0.5 mg /
The antibody solution was added so that the protein concentration would be ml.
（試料） ヒト血清を生理食塩水で適宜希釈した溶液を試料とし
た。(Sample) A solution prepared by appropriately diluting human serum with physiological saline was used as a sample.
（HPLCの使用条件） システムの概略を第２図に示す。(Usage conditions for HPLC) Fig. 2 shows the outline of the system.
・カラム:0.75φ×30cm（東ソー（株）社製、 TSKgel G2000SW）。-Column: 0.75φ x 30 cm (TSKgel G2000SW manufactured by Tosoh Corporation).
・流速：溶離液;1.0ml/min.、基質液;0.1ml/ min.。Flow rate: eluent: 1.0 ml / min., Substrate solution: 0.1 ml / min.
・反応部:0.04φ×900cm（37℃保温）。・ Reaction part: 0.04φ x 900 cm (37 ° C heat retention).
・検出:405nmの吸光度を測定した。-Detection: Absorbance at 405 nm was measured.
（測定操作） 抗体液300μｌと試料30μｌとを混合し、37℃で30分間
反応させた後、混合液の50μｌをHPLCにより分析した。(Measurement procedure) 300 μl of the antibody solution and 30 μl of the sample were mixed and reacted at 37 ° C. for 30 minutes, and then 50 μl of the mixed solution was analyzed by HPLC.
（結果） HPLCによる分析の結果、α−アミラーゼのピークが２つ
の観察され、唾液型α−アミラーゼと膵臓型α−アミラ
ーゼが分離できることが判った。(Results) As a result of analysis by HPLC, two peaks of α-amylase were observed, and it was found that salivary α-amylase and pancreatic α-amylase can be separated.
また、膵臓型α−アミラーゼを生理食塩水中に適宜添加
したものを試料として、同様の操作を行い、得られるピ
ーク面積値と膵臓型α−アミラーゼの活性値との関係を
示す検量線を作成した。In addition, using a sample prepared by appropriately adding pancreatic α-amylase to physiological saline, the same operation was performed to prepare a calibration curve showing the relationship between the obtained peak area value and the activity value of pancreatic α-amylase. .
結果を第１図に示す。尚、第１図は横軸の各膵臓型α−
アミラーゼの活性値（U/l）に対して得られたピーク面
積値を縦軸に沿ってプロットした点を結んだものであ
る。The results are shown in Fig. 1. Incidentally, FIG. 1 shows each pancreatic type α- on the horizontal axis.
It is the result of connecting the points obtained by plotting the peak area values obtained with respect to the activity value (U / l) of amylase along the vertical axis.
第１図から明らかな如く、原点を通り且つ直線性の良好
な検量線が得られた。As is clear from FIG. 1, a calibration curve that passed through the origin and had good linearity was obtained.
実施例2.γ−グルタミルトランスフェラーゼ（γ−GT
P）アイソザイムの分別測定 （溶離液） 70mMグリシルグリシン、0.15M NaCl及び2mM EDTAを含む
0.1Mトリス（ヒドロキシエチル）アミノメタン−HCl緩
衝液（pH8.1）を溶離液とした。Example 2.γ-glutamyl transferase (γ-GT
P) Fractional measurement of isozyme (eluent) Contains 70 mM glycylglycine, 0.15 M NaCl and 2 mM EDTA
The eluent was 0.1 M tris (hydroxyethyl) aminomethane-HCl buffer (pH 8.1).
（レクチン溶液） 50mM 2−（Ｎ−モルホリノ）エタンスルホン酸（MES）
緩衝液（pH7.0）にレンズマメレクチン（LCA-A）を3mg/
mlとなるように溶解したものをレクチン溶液とした。(Lectin solution) 50 mM 2- (N-morpholino) ethanesulfonic acid (MES)
Lentil bean lectin (LCA-A) 3 mg / in buffer solution (pH 7.0)
A lectin solution was prepared by dissolving the solution so that the amount became ml.
（基質液） γ−グルタミル−７−アミド−４−メチルクマリンを0.
4mMとなるように、上記溶離液に溶解したものを基質液
とした。(Substrate solution) γ-glutamyl-7-amido-4-methylcoumarin was added to 0.2%.
What was dissolved in the above eluent so as to be 4 mM was used as a substrate solution.
（試料） 新鮮なヒト血清を試料とした。(Sample) Fresh human serum was used as a sample.
（HPLCの使用条件） システムの概略は第２図に同じ。(HPLC usage conditions) The outline of the system is the same as in Fig. 2.
・カラム:0.94φ×25cm（デュポン社製、 Zorbax GF-250）。-Column: 0.94φ x 25 cm (Zorbax GF-250 manufactured by DuPont).
・流速：溶離液;1.0ml/min.、基質液;0.5ml/ min.。Flow rate: eluent: 1.0 ml / min., Substrate solution: 0.5 ml / min.
・反応部:0.04φ×900cm（40℃保温）。・ Reaction part: 0.04φ x 900 cm (40 ℃ insulation).
・検出：励起波長395nm、蛍光波長480nmで蛍 光を測定した。-Detection: Fluorescence was measured at an excitation wavelength of 395 nm and a fluorescence wavelength of 480 nm.
（測定操作） レクチン溶液20μｌと試料20μｌとを混合し、37℃で30
分間反応させた後、混合液の15μｌをHPLCにより分析し
た。(Measurement procedure) Mix 20 μl of the lectin solution with 20 μl of the sample, and mix at 37 ° C for 30
After reacting for a minute, 15 μl of the mixture was analyzed by HPLC.
（結果） HPLCによる分析の結果、フコースの付いた糖鎖を有する
γ−GTPとLCA-Aとの複合体は5.8分後に、フコースの付
いた糖鎖を有さないγ−GTPは11.3分後に溶出してくる
ことが判った。(Results) As a result of analysis by HPLC, a complex of γ-GTP having a sugar chain with fucose and LCA-A was after 5.8 minutes, and γ-GTP without a sugar chain with fucose was after 11.3 minutes. It was found to be eluting.
実施例3.乳酸脱水素酵素（LDH）アイソザイムの分別測
定 （溶離液） 0.15M NaClを含む10mMトリス（ヒドロキシエチル）アミ
ノメタン−HCl緩衝液（pH8.7）を溶離液とした。Example 3. Fractional measurement of lactate dehydrogenase (LDH) isozyme (eluent) 10 mM tris (hydroxyethyl) aminomethane-HCl buffer (pH 8.7) containing 0.15 M NaCl was used as an eluent.
（抗体液） 市販の抗ヒトLDH（H-subunit）モノクローナル抗体液
（ICNバイオメディカルズ社製）を溶離液で10倍希釈し
たものを抗体液とした。(Antibody solution) A commercially available anti-human LDH (H-subunit) monoclonal antibody solution (manufactured by ICN Biomedicals) was diluted 10-fold with an eluent to obtain an antibody solution.
（基質液） 385mM L−乳酸及び22mM β−NAD+を含む500mMトリス
（ヒドロキシエチル）アミノメタン−HCl緩衝液（pH8.
7）を基質液とした。(Substrate solution) 500 mM tris (hydroxyethyl) aminomethane-HCl buffer solution containing 385 mM L-lactic acid and 22 mM β-NAD + (pH 8.
7) was used as the substrate solution.
（試料） 新鮮なヒト血清を試料とした。(Sample) Fresh human serum was used as a sample.
（HPLCの使用条件） システムの概略は第２図に同じ。(HPLC usage conditions) The outline of the system is the same as in Fig. 2.
・カラム:0.8φ×30cm（山村化学研究所（株） 社製、YMC−パック Diol-300）。-Column: 0.8φ x 30 cm (YMC-Pack Diol-300, manufactured by Yamamura Chemical Laboratory Co., Ltd.).
・流速：溶離液;1.0ml/min.、基質液;0.1ml/ min.。Flow rate: eluent: 1.0 ml / min., Substrate solution: 0.1 ml / min.
・反応部:0.04φ×900cm（37℃保温）。・ Reaction part: 0.04φ x 900 cm (37 ° C heat retention).
・検出：励起波長370nm、蛍光波長465nmで蛍 光を測定した。-Detection: Fluorescence was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 465 nm.
（測定操作） 抗体液120μｌと試料30μｌとを混合し、30℃で60分間
反応させた後、混合液の100μｌをHPLCにより分析し
た。(Measurement procedure) After 120 μl of the antibody solution and 30 μl of the sample were mixed and reacted at 30 ° C. for 60 minutes, 100 μl of the mixed solution was analyzed by HPLC.
（結果） HPLCによる分析の結果、LDH1は11.1分後、LDH2は10.1分
後、LDH3は9.3分後、LDH4は8.9分後、LDH5は8.3分後に
夫々溶出された。(Results) As a result of HPLC analysis, LDH 1 was eluted after 11.1 minutes, LDH 2 after 10.1 minutes, LDH 3 after 9.3 minutes, LDH 4 after 8.9 minutes, and LDH 5 after 8.3 minutes.
［発明の効果］ 以上述べた如く、本発明は、生体由来の試料中の測定対
象物質を、その化学的又は／及び物理的な性質に応じ
て、迅速に、容易に且つ精度良く分別測定し得る方法を
提供するものである。本発明の方法によれば、測定に要
する時間は数十分から数時間程度であり、必要な測定操
作自体は、測定対象物質を含む試料と結合能物質を混合
した後、HPLCにより複合体と遊離の測定対象物質とを分
離、検出するのであるので、従来の同様な目的の分別測
定方法に比べて、簡便に且つ迅速に目的の測定を行うこ
とができる点に顕著な効果を有する発明であり、斯業に
貢献するところ大なる発明である。[Effects of the Invention] As described above, the present invention provides rapid, easy, and accurate differential measurement of a substance to be measured in a biological sample according to its chemical or / and physical properties. It provides a method of obtaining. According to the method of the present invention, the time required for measurement is about several tens of minutes to several hours, and the necessary measurement operation itself is to prepare a complex by HPLC after mixing the sample containing the substance to be measured and the binding ability substance. Since it separates and detects a free substance to be measured, it is an invention that has a remarkable effect in that the desired measurement can be carried out easily and rapidly, as compared with the conventional fractionation measurement method for similar purposes. Yes, it is a great invention that contributes to this industry.
第１図は、実施例１に於いて得られた検量線を示す。 第２図は、実施例１、２及び３で使用したHPLCのシステ
ムの概略図を示したものである。FIG. 1 shows the calibration curve obtained in Example 1. FIG. 2 is a schematic view of the HPLC system used in Examples 1, 2 and 3.
Claims (6)
化学特性を有する２以上の測定対象物質（以下、単に、
測定対象物質と略記する。）を含む試料を、測定対象物
質の少なくとも１つに対しては結合能を有するが、それ
らの少なくとも１つとは結合しない物質（以下、単に、
結合能物質と略記する。）と混合して反応させた後、測
定対象物質と結合能物質との複合体（以下、単に、複合
体と略記する。）と、遊離の測定対象物質とを高速液体
クロマトグラフイにより分離し、複合体中の測定対象物
質の量又は／及び遊離の測定対象物質の量を測定するこ
とにより試料中の測定対象物質の何れかの量を測定する
ことを特徴とする分別測定方法。1. Two or more substances to be measured having the same action and the same detectable chemical property (hereinafter, simply,
Abbreviated as the measurement target substance. ) Containing a sample, which has a binding ability to at least one of the substances to be measured, but does not bind to at least one of them (hereinafter, simply,
Abbreviated as a binding substance. ) Is mixed and reacted, and then the complex of the substance to be measured and the binding substance (hereinafter simply referred to as the complex) and the free substance to be measured are separated by high performance liquid chromatography. A method for fractionation measurement, which comprises measuring the amount of a measurement target substance in a complex or / and the amount of a free measurement target substance to measure any amount of the measurement target substance in a sample.
の分別測定方法。2. The differential measurement method according to claim 1, wherein the substance to be measured is an enzyme.
１つに対して結合能を有するが、少なくとも１つは結合
しない抗体又はレクチンである請求項１に記載の分別測
定方法。3. The fractional measurement method according to claim 1, wherein the binding substance is an antibody or a lectin that has a binding ability to at least one of the substances to be measured, but at least one does not.
に記載の分別測定方法。4. The antibody according to claim 3, which is a monoclonal antibody.
Separation measurement method described in.
メレクチン、インゲンマメレクチン、ダツラレクチン、
ヒイロチャワンタケレクチン、ヒママメレクチン、ピー
ナッツレクチン又は小麦胚芽レクチンである請求項３に
記載の分別測定方法。5. The lectin is concanavalin A, lentil lectin, kidney bean lectin, datura lectin,
The fractionation measuring method according to claim 3, wherein the method is a Hiirochawantake lectin, a chickpea lectin, a peanut lectin, or a wheat germ lectin.
を、ゲル濾過（ゲルクロマトグラフィ）用充填剤、イオ
ン交換クロマトグラフィ用充填剤、疎水クロマトグラフ
ィ用充填剤、等電点クロマトグラフィ用充填剤、逆相ク
ロマトグラフィ用充填剤又はハイドロキシアパタイトを
充填したカラムを装着した高速液体クロマトグラフィに
より行う請求項１〜５の何れかに記載の分別測定方法。6. Separation of the complex and the free substance to be measured is performed by gel packing (gel chromatography) packing, ion exchange chromatography packing, hydrophobic chromatography packing, isoelectric focusing packing packing, The fractional measurement method according to any one of claims 1 to 5, which is carried out by high performance liquid chromatography equipped with a column packed with a packing material for reverse phase chromatography or hydroxyapatite.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002170A JPH0765988B2 (en) | 1990-01-09 | 1990-01-09 | Fractional measurement method for trace components |
| DE69115518T DE69115518T2 (en) | 1990-01-09 | 1991-01-02 | Process for the separation and measurement of track components |
| AT91300008T ATE131933T1 (en) | 1990-01-09 | 1991-01-02 | METHOD FOR SEPARATING AND MEASURING TRACK COMPONENTS |
| ES91300008T ES2080886T3 (en) | 1990-01-09 | 1991-01-02 | PROCEDURE FOR SEPARATING AND MEASURING TRACE COMPONENTS. |
| EP91300008A EP0441470B1 (en) | 1990-01-09 | 1991-01-02 | Process for separating and measuring trace components |
| DK91300008.9T DK0441470T3 (en) | 1990-01-09 | 1991-01-02 | Method for separation and measurement of trace components |
| US08/488,009 US5780247A (en) | 1990-01-09 | 1995-06-07 | Process for separating and measuring trace components |
| GR950403705T GR3018568T3 (en) | 1990-01-09 | 1995-12-29 | Process for separating and measuring trace components |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002170A JPH0765988B2 (en) | 1990-01-09 | 1990-01-09 | Fractional measurement method for trace components |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03206964A JPH03206964A (en) | 1991-09-10 |
| JPH0765988B2 true JPH0765988B2 (en) | 1995-07-19 |
Family
ID=11521890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2002170A Expired - Lifetime JPH0765988B2 (en) | 1990-01-09 | 1990-01-09 | Fractional measurement method for trace components |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0765988B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2181109A1 (en) | 1995-07-18 | 1997-01-19 | Nobuko Imajo | Polypeptide and process for measuring living body components using the same |
| JP6157862B2 (en) * | 2013-01-29 | 2017-07-05 | テルモ株式会社 | Method for separating target substance-receptor complex and free receptor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61110059A (en) * | 1984-11-05 | 1986-05-28 | Olympus Optical Co Ltd | Immunological analysis |
| JPH01257266A (en) * | 1988-04-06 | 1989-10-13 | Unitika Ltd | Reagent for measuring activity of creatine kinase and measuring method using said agent |
| JPH01316660A (en) * | 1988-03-07 | 1989-12-21 | Progen Biotechnik Gmbh | Immunological detection of material and composition and test kit used therefor |
| JPH02176563A (en) * | 1988-12-28 | 1990-07-09 | Tosoh Corp | Method for separating b/f and method for measuring antigen quantity by using this method |
-
1990
- 1990-01-09 JP JP2002170A patent/JPH0765988B2/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61110059A (en) * | 1984-11-05 | 1986-05-28 | Olympus Optical Co Ltd | Immunological analysis |
| JPH01316660A (en) * | 1988-03-07 | 1989-12-21 | Progen Biotechnik Gmbh | Immunological detection of material and composition and test kit used therefor |
| JPH01257266A (en) * | 1988-04-06 | 1989-10-13 | Unitika Ltd | Reagent for measuring activity of creatine kinase and measuring method using said agent |
| JPH02176563A (en) * | 1988-12-28 | 1990-07-09 | Tosoh Corp | Method for separating b/f and method for measuring antigen quantity by using this method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03206964A (en) | 1991-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Maurer et al. | Validated electrospray liquid chromatographic–mass spectrometric assay for the determination of the mushroom toxins α-and β-amanitin in urine after immunoaffinity extraction | |
| AU7744098A (en) | Device and method for obtaining clinically significant analyte ratios | |
| AU615823B2 (en) | Enzyme-linked immunoassay for measurement of cyclosporin a levels in whole blood samples | |
| US4912033A (en) | Creatine kinase MB determination method | |
| EP0487301A1 (en) | Method of stabilizing enzyme conjugates | |
| EP3165928B1 (en) | A method for quantifying anti-tnf antibodies | |
| EP1207394A2 (en) | Immunoassay for HIV protease inhibitors | |
| KR100231628B1 (en) | Process for separating and measuring glycoprotein | |
| EP0968422B1 (en) | Improving performance of binding assays by use of more than one label | |
| US4624916A (en) | Process and composition for the rapid quantitation of small levels of creative kinase-MB isoenzyme | |
| JP3208668B2 (en) | Method and kit for measuring tyrosine phosphorylation and dephosphorylation activity | |
| US5919642A (en) | Competitive binding assays having improved linearity | |
| EP3264085A1 (en) | Immunoassay method and assay reagent used in said method | |
| JP3070418B2 (en) | Differential measurement of glycoproteins | |
| JPH0765988B2 (en) | Fractional measurement method for trace components | |
| JP2596574B2 (en) | Creatine kinase MB assay | |
| Vacik et al. | Separation and measurement of isoenzymes and other proteins by high-performance liquid chromatography | |
| US5780247A (en) | Process for separating and measuring trace components | |
| JPH0421819B2 (en) | ||
| JP3102611B2 (en) | Complex separation method | |
| US8021849B2 (en) | Methods and kits for the determination of sirolimus in a sample | |
| Tijssen et al. | Enzyme-linked immunosorbent assays and developments in techniques using latex beads | |
| JPH0731202B2 (en) | New method for fractional analysis of trace components | |
| JPH0566222A (en) | Material analyzing method | |
| JP3376955B2 (en) | Complex separation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080719 Year of fee payment: 13 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100719 Year of fee payment: 15 |
|
| EXPY | Cancellation because of completion of term | ||
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100719 Year of fee payment: 15 |