JPH02147956A - Enzyme immunological measurement method - Google Patents
Enzyme immunological measurement methodInfo
- Publication number
- JPH02147956A JPH02147956A JP30101688A JP30101688A JPH02147956A JP H02147956 A JPH02147956 A JP H02147956A JP 30101688 A JP30101688 A JP 30101688A JP 30101688 A JP30101688 A JP 30101688A JP H02147956 A JPH02147956 A JP H02147956A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- polyethylene glycol
- immunological measurement
- glycol derivative
- metabolic
- 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
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 34
- 238000000691 measurement method Methods 0.000 title claims abstract description 4
- 230000001900 immune effect Effects 0.000 title abstract 4
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 18
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 18
- 150000002334 glycols Chemical class 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 10
- 230000002503 metabolic effect Effects 0.000 claims abstract description 9
- 102000004157 Hydrolases Human genes 0.000 claims abstract description 5
- 108090000604 Hydrolases Proteins 0.000 claims abstract description 5
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims abstract description 4
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims abstract description 4
- 125000003118 aryl group Chemical group 0.000 claims abstract description 4
- 108090000471 glyoxylate dehydrogenase (acylating) Proteins 0.000 claims abstract description 4
- 108010093031 Galactosidases Proteins 0.000 claims abstract description 3
- 102000002464 Galactosidases Human genes 0.000 claims abstract description 3
- 108010056771 Glucosidases Proteins 0.000 claims abstract description 3
- 102000004366 Glucosidases Human genes 0.000 claims abstract description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 3
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims abstract 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims abstract 2
- 238000003018 immunoassay Methods 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 5
- 125000004448 alkyl carbonyl group Chemical group 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 3
- 108090000371 Esterases Proteins 0.000 claims description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 9
- -1 phosphate radical Chemical class 0.000 abstract description 6
- 229910019142 PO4 Inorganic materials 0.000 abstract description 3
- 239000010452 phosphate Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract 1
- 239000003550 marker Substances 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 23
- 239000000872 buffer Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 102000011923 Thyrotropin Human genes 0.000 description 6
- 108010061174 Thyrotropin Proteins 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000000370 acceptor Substances 0.000 description 3
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 2
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 2
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 2
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- FBWADIKARMIWNM-UHFFFAOYSA-N N-3,5-dichloro-4-hydroxyphenyl-1,4-benzoquinone imine Chemical compound C1=C(Cl)C(O)=C(Cl)C=C1N=C1C=CC(=O)C=C1 FBWADIKARMIWNM-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960003151 mercaptamine Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000276 potassium ferrocyanide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、酵素免疫測定法に関する。更に詳しくは、一
般式
％式％（１）
（式中、Ｒ１はリン酸基、アリール若しくはアルキルカ
ルボニル基又は糖残基、ＲはＲ１と同−又はアルキル基
であり、ｎは２以上である。）で表されるポリエチレン
グリコール誘導体及びポリエチレングリコール代謝酵素
を用い更に、前記ポリエチレングリコール誘導体を分解
する酵素を標識酵素として用いる酵素免疫測定法に関す
る。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an enzyme immunoassay. More specifically, the general formula % formula % (1) (wherein R1 is a phosphoric acid group, an aryl or alkyl carbonyl group, or a sugar residue, R is the same as R1 or an alkyl group, and n is 2 or more The present invention relates to an enzyme immunoassay method using a polyethylene glycol derivative represented by .
（従来の技術）
酵素免疫測定法（以下ＥＩＡと省略する）における比色
法は汎用されている分光光度計を用い測定できるなどの
簡単な測定法としての利点を有しているものの他の螢光
法、生物発光法、化学発光法に比べ感度が低いことが欠
点であった。この欠点を克服する方法として、酵素とし
てアルカリ性ホスファターゼ、基質としてＮＡＤＰを用
い生成するＮＡＤを酵素的サイクリングにより測定する
方法が開発された。 （Ａ、 Ｊｏｈａｎｎｓｓｏｎ
ら、Ｃｌ１ｎ。(Prior art) Although the colorimetric method in enzyme immunoassay (hereinafter abbreviated as EIA) has the advantage of being a simple measurement method such as being able to perform measurements using a commonly used spectrophotometer, other The disadvantage of this method is that it has lower sensitivity than the optical method, bioluminescence method, and chemiluminescence method. As a method to overcome this drawback, a method has been developed in which alkaline phosphatase is used as an enzyme and NADP is used as a substrate, and NAD produced is measured by enzymatic cycling. (A. Johansson
et al., Cl1n.
Ｃｈｅｍ、 Ａｃｔａ、 １４８． １１９−１２４
、 （１９８５））。Chem, Acta, 148. 119-124
, (1985)).
（発明の解決しようとする問題点）
しかしながら、前記方法は、蛍光法を上回る感度を得る
ことができるが、使用するＮＡＤ及びＮＡＤＰは、特に
アルカリ溶液中で不安定であり、容易に取り扱うことが
できない。また、両者は共に高価である。更に生体内、
特に血中に存在するＮＡＤまたはＮＡＤＨによりブラン
ク値の上昇がみとめられ正確な測定を行うことができな
いという欠点を有していた。(Problems to be Solved by the Invention) However, although the above method can obtain higher sensitivity than the fluorescence method, the NAD and NADP used are unstable, especially in alkaline solutions, and cannot be easily handled. Can not. Moreover, both are expensive. Furthermore, in vivo,
In particular, it has been found that the blank value increases due to NAD or NADH present in the blood, making it impossible to perform accurate measurements.
（問題点を解決するための手段）
これら問題点を解決するため、本発明者等は、前記一般
式（１）で表されるポリエチレングリコール誘導体及び
ポリエチレングリコール代謝酵素を用い、更に前記ポリ
エチレングリコール誘導体を分解する酵素を標識酵素と
して用いるＥＩＡを見出し、本発明を完成させた。本発
明は、前記一般式（１）で表されるポリエチングリコー
ル誘導体を基質として用い酵素免疫測定を行うものであ
る。(Means for Solving the Problems) In order to solve these problems, the present inventors used a polyethylene glycol derivative represented by the general formula (1) and a polyethylene glycol metabolic enzyme, and further The present invention was completed by discovering EIA using an enzyme that decomposes as a labeling enzyme. The present invention is to perform enzyme immunoassay using the polyethine glycol derivative represented by the general formula (1) as a substrate.
前記一般式（１）で表されるポリエチレングリコール誘
導体のＲ′として表される基としてはリン酸基、アリー
ル若しくはアルキルカルボニル基又は糖残基、ＲはＲ’
と同−又はアルキル基であり、ｎとしては２以上の重
合度のものを使用することができる。より具体的には
ＣＨ３０（ＣＨ２−Ｃ１１２０）、１−ＣＨｚＣＨｚ−
ＯｐＨ＋”ＣＨ：＋０（ＣＨｚ−ＣＨｚＯ） 、 −Ｃ
ｔｌｚＣｌｌｚ−ＯＧａｌＣＨａＯ（ＣＨｚ−ＣＨｚＯ
）、１−ＣＨｚＣＨｚ−ＯＧＩＵＣＨｉＯ（ＣＨｚ−Ｃ
ＨｚＯ）ｎ−ＣＨｚＣＨｚ−ＯＡｃＣＨｘＯ（Ｃｆｂ−
ＣＨｚＯ）、１−ＣＨｚＣＨｚ−ＯＢＺ等を挙げること
ができる。（式中、Ｇａｌ はガラクトース基、Ｇｌｕ
はグルコース基、八Ｃはアセチル基であり、Ｂｚはベン
ゾイル基である。ｎは２〜１０００である。）
前記一般式（Ｉ）で表されるポリエチレングリコール誘
導体は、抗原抗体反応径測定系に存在する標識酵素によ
り分解されポリエチレングリコールに誘導される。この
際使用される標識酵素は前記ポリエチレングリコールｍ
８体のＲ又はＲ１で表される基に依存し選択される。そ
の酵素としては。The group represented by R' of the polyethylene glycol derivative represented by the general formula (1) is a phosphoric acid group, an aryl or alkyl carbonyl group, or a sugar residue, and R is R'
It is the same as or an alkyl group, and n can be used having a degree of polymerization of 2 or more. More specifically, CH30 (CH2-C1120), 1-CHzCHz-
OpH+”CH:+0(CHz-CHzO), -C
tlzCllz-OGalCHaO(CHz-CHzO
), 1-CHzCHz-OGIUCHiO(CHz-C
HzO)n-CHzCHz-OAcCHxO(Cfb-
CHzO), 1-CHzCHz-OBZ, and the like. (In the formula, Gal is a galactose group, Glu
is a glucose group, 8C is an acetyl group, and Bz is a benzoyl group. n is 2-1000. ) The polyethylene glycol derivative represented by the general formula (I) is decomposed by a labeled enzyme present in the antigen-antibody reaction diameter measurement system and is induced to polyethylene glycol. The labeling enzyme used at this time is the polyethylene glycol m
The selection depends on the group represented by 8 R or R1. As for the enzyme.
例えば、アルカリ性若しくは酸性ホスファターゼ、ガラ
クトシダーゼ、グルコシダーゼ、エステラーゼなどを使
用することができる。For example, alkaline or acid phosphatase, galactosidase, glucosidase, esterase, etc. can be used.
標識酵素により誘導されたポリエチレングリコールは、
ポリエチレングリコール代謝酵素により以下例示にある
様に分解を受ける。Polyethylene glycol induced by labeled enzyme is
It is degraded by polyethylene glycol metabolic enzymes as illustrated below.
たとえば、前記一般式（１）で表されるポリエチレング
リコール誘導体として、下記一般式（Ｉ゛）で表される
化合物を用い、標識酵素としてアルカリ性ホスファター
ゼを用いた場合の分解反応は下記の如くである。For example, when a compound represented by the following general formula (I゛) is used as the polyethylene glycol derivative represented by the above general formula (1) and alkaline phosphatase is used as the labeling enzyme, the decomposition reaction is as follows. .
Ｃ）１：＋０（Ｃ１１ｚ−ＣＨｚＯ）ｌｌ−ＣＩｌｚＣ
）ｔｚ−ＯＰＯｚ”（ビ）
＝−）ＣＨ：＋０（ＣＨｉ−ＣＩ（ア０）ｆｉ−ＣＨ２
ＣＨ２−ＯＨＬ−−−ＣＨｚＯ（ＣＨｚ−ＣＨｚＯ）ｎ
−Ｈ＋
Ｃ）ＩＯｃＯＯＨ
（ＣＯＯＨ）ｚ
（１）アルコールデヒドロゲナーゼ
（２）アルデヒドデヒドロゲナーゼ
（３）エーテルポンドハイドロゲナーゼ（４）グリオキ
シレートデヒドロゲナーゼ本発明におけるポリエチレン
グリコール代謝酵素は前記（１）〜（４）に例示した酵
素全体を示すものである。これらの酵素の（１）に関し
ては特願昭６２６２３５６号に、（２）に関しては特願
昭６２−６２３５７に、（３）に関しては昭和６３年度
日本農芸化学会大会講演要旨集１２８頁（１９８８）に
、（４）に関して特願昭６１−２６２５７４３号に開示
されているものを好適に使用することができる。C) 1:+0(C11z-CHzO)ll-CIlzC
)tz-OPOz"(Bi) =-)CH:+0(CHi-CI(A0)fi-CH2
CH2-OHL---CHzO(CHz-CHzO)n
-H+ C) IOcOOH (COOH)z (1) Alcohol dehydrogenase (2) Aldehyde dehydrogenase (3) Etherpond hydrogenase (4) Glyoxylate dehydrogenase The polyethylene glycol metabolic enzymes in the present invention are the aforementioned (1) to (4). This shows the entire enzyme exemplified in . Regarding these enzymes, (1) can be found in Japanese Patent Application No. 6262356, (2) can be found in Japanese Patent Application No. 62-62357, and (3) can be found in 1988 Japanese Society of Agricultural Chemistry Conference Abstracts, page 128 (1988). Regarding (4), what is disclosed in Japanese Patent Application No. 61-2625743 can be suitably used.
本発明は、前記反応式に示した如く、（１）〜（４）の
酵素の存在する反応においてそれぞれ１電子ずつの供給
を受けその電子が電子受容体に捕捉され、その電子受容
体の電子受容量の状態の変化を測定することよにり達成
される。In the present invention, as shown in the reaction formula, one electron is supplied to each of the enzymes (1) to (4) in the reaction in which the enzyme is present, and the electron is captured by an electron acceptor. This is accomplished by measuring changes in the state of receptivity.
本発明で使用することのできる電子受容体は、たとえば
、フェリシアン化カリウム、ツェナチアジンサルフェー
ト（ＰＭＳ） 、ジクロロフェノールインドフェノール
（ＰＣＰＩＦ）、ニトロテトラヅリウムブル−（ＮＴＢ
）等である。Electron acceptors that can be used in the present invention include, for example, potassium ferricyanide, zenatiazine sulfate (PMS), dichlorophenolindophenol (PCPIF), nitrotetradurium blue (NTB
) etc.
電子受容体の電子受容量の状態の変化は、例えば、フェ
リシアン化カリウムの場合にはフェロシアン化カリウム
への変化量をその吸光度より求めることにより行うこと
ができる。For example, in the case of potassium ferricyanide, the state of the electron acceptance amount of the electron acceptor can be changed by determining the amount of change to potassium ferrocyanide from its absorbance.
本発明を実施するにあたっては、通常緩衝液中で行うも
のであり、トリス塩酸緩衝液を好適に使用することがで
きる。The present invention is usually carried out in a buffer, and a Tris-HCl buffer can be suitably used.
本発明における酵素反応は２０〜４０°Ｃの範囲を選び
行うことができる。The enzyme reaction in the present invention can be carried out at a temperature in the range of 20 to 40°C.
本発明の測定対象物は、α−フェトプロティン（ＡＦＰ
）、癌胎児性抗原（ＣＥＡ）等の癌関連抗原、ＨＢｓ等
の抗原、甲状腺刺激ホルモン（ＴＳ Ｈ）などのホルモ
ン、ＩｇＧ、、ＩｇＭ等の抗体を挙げることができる。The measurement target of the present invention is α-fetoprotein (AFP).
), cancer-related antigens such as carcinoembryonic antigen (CEA), antigens such as HBs, hormones such as thyroid stimulating hormone (TSH), and antibodies such as IgG, IgM, etc.
（作用）
本発明は、ＥＩＡにおいてポリエチレングリコール誘導
体およびその代謝酵素を用い増幅作用を行い高感度の免
疫測定を行うものである。(Function) The present invention performs highly sensitive immunoassay by performing an amplification effect using a polyethylene glycol derivative and its metabolic enzyme in EIA.
（実施例） 以下、実施例により本発明をさらに詳しく説明する。(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例１ 甲状腺刺激ホルモン（ＴＳＨ）の測定１材料
の調整）
抗Ｔ Ｓ Ｈ抗体感作用の１７８インチビーズを、マウ
スモノクローナル抗ＴＳＨ−β抗体で感作した。Example 1 Measurement of Thyroid Stimulating Hormone (TSH) 1 Preparation of Materials 178-inch beads for anti-TSH antibody sensitization were sensitized with a mouse monoclonal anti-TSH-β antibody.
一方マウスモノクローナル抗ＴＳＨ抗体は、ペプシンに
よりＦ（ａｂ’）ｚとし２−メルカプトエチルアミン（
０，１Ｍ溶液）で還元しＧＭＢ （Ｎ Ｃｒ−ｍａｌｅ
ｊｍｊｄｏｂｕｔｙｒｏｘｙ））化したアルカリ性ホス
ファターゼと反応させ抗体酵素コンジュゲートを作製し
た。On the other hand, mouse monoclonal anti-TSH antibody was converted to F(ab')z by pepsin and 2-mercaptoethylamine (
GMB (N Cr-male
An antibody-enzyme conjugate was prepared by reacting with alkaline phosphatase.
被検液および抗体酵素コンジュゲートに用いた緩衝液は
Ｏ，ＬＭ）リス塩酸、１ｍＭＭｇｃ１ｇおよび１％ＢＳ
Ａ（ｐＨ７，５）である。基質緩衝液は０．１ ｍＭ
ＭｇＣ１ｚ （ｐＨ９，５）および基質としてモノメ
トキシポリエチレングリコールリン酸エステル（ｎ＝
１００ （１００ｍｇ／ｍｌ）を用いた。The buffers used for the test solution and antibody-enzyme conjugate were O, LM) Lis-HCl, 1mM Mgc1g, and 1%BS.
A (pH 7.5). Substrate buffer is 0.1mM
MgC1z (pH 9,5) and monomethoxypolyethylene glycol phosphate ester (n=
100 (100mg/ml) was used.
（ＴＳＨの測定）
ＴＳＨ含有被検溶液（１５μｌ ）（０，４，８゜１６
．３２μＵ／ｍｌ）を抗体酵素コンジュゲート溶液１３
５μｌと混合し、この溶液の上記のように感作した１ｚ
４インチビーズ１個を加え、室温にて２時間インキュベ
ートした。 その後、ビーズを０．１Ｍ）リス緩衝液（
ｐ Ｈ７，５）で４回洗い、反応試験管へ移した。上記
の基質を含む緩衝液 ２００μｌを試験管に加えた。さ
らに１時間後、Ｏ，１Ｍトリス塩酸緩衝液（ｐＨ８，０
） １ｍｌ中、フェリシアン化カリウム（１０ｌｌｍ
ｏｌ）、アルコールデヒドロゲナーゼ（ＩＯＵ）、アル
デヒドデヒドロゲナーゼ（ＩＯＵ）およびエーテルボン
ドハイドラーゼ（ＩＯＵ）、グリオキシレートデヒドロ
ゲナーゼ（１００）よりなる反応液中で３７°Ｃ１１時
間反応を行い、０． ５ ｍｌ フエリックヂュバノー
ル液（リン酸９５ ｍｌ 、ＳＤ３ ３ｇ。(Measurement of TSH) TSH-containing test solution (15 μl) (0,4,8°16
．． 32 μU/ml) as antibody enzyme conjugate solution 13
1z sensitized as above of this solution.
One 4 inch bead was added and incubated for 2 hours at room temperature. Then, add the beads to 0.1M) Lys buffer (
The mixture was washed four times with pH 7.5) and transferred to a reaction tube. 200 μl of buffer containing the above substrate was added to the test tube. After another hour, O, 1M Tris-HCl buffer (pH 8,0
) Potassium ferricyanide (10llm) in 1ml
ol), alcohol dehydrogenase (IOU), aldehyde dehydrogenase (IOU), ether bond hydrolase (IOU), and glyoxylate dehydrogenase (100) at 37°C for 11 hours. 5 ml Ferric Dubanol solution (phosphoric acid 95 ml, SD3 3 g.
Ｆｅ（ＳＯ４）３・ｎＨｚｏ ５ ｇに水を加え１１
とした溶液）を加えて反応を停止させる。さらに３．５
ｍｌの水を加え、２０分間放置し、色素が安定してがら
６６０ｎｍの吸光度を測定した。Add water to 5 g of Fe(SO4)3・nHzo and add 11
solution) to stop the reaction. Another 3.5
ml of water was added and left to stand for 20 minutes, and while the dye was stable, the absorbance at 660 nm was measured.
比較として、上記抗原抗体反応後洗浄したビーズに１０
ｍＭｐ−二トロフェニルリン酸、０． １Ｍトリス塩酸
緩衝液、１ ｍＭＭ ｇ Ｃ１ｔを含む基質液４００μ
ｍを加え、３７°Ｃ１１時間反応させ、０．２Ｎ Ｎ
ａＯＨ６００μｌを加え反応を停止させ、波長４５０ｎ
ｍの吸光度を測定した。その結果を表１に示した。（Ｐ
ＮＰ法）
（発明の効果）
本発明は、ＥＩＡにおいて高感度の測定ができるように
なった。For comparison, beads washed after the above antigen-antibody reaction were treated with 10
mM p-ditrophenyl phosphate, 0. 400μ of substrate solution containing 1M Tris-HCl buffer, 1mM gClt
m, reacted at 37°C for 11 hours, and added 0.2N N
Add 600μl of aOH to stop the reaction, and change the wavelength to 450n.
The absorbance of m was measured. The results are shown in Table 1. (P
NP method) (Effects of the invention) The present invention enables highly sensitive measurement in EIA.
Claims (3)
−ＯＲ＾１で表されるポリエチレングリコール誘導体及
びポリエチレングリコール代謝酵素を用い、更に、前記
ポリエチレングリコール誘導体を分解する酵素を標識酵
素として用いる酵素免疫測定法（式中、Ｒ＾１はリン酸
基、アリール若しくはアルキルカルボニル基又は糖残基
、ＲはＲ＾１と同一又はアルキル基であり、ｎは２以上
である。）。(1) General formula RO(CH_2-CH_2O)_n-CH_2CH_2
Enzyme immunoassay using a polyethylene glycol derivative represented by -OR^1 and a polyethylene glycol metabolic enzyme, and further using an enzyme that decomposes the polyethylene glycol derivative as a labeling enzyme (wherein R^1 is a phosphate group, aryl or alkylcarbonyl group or sugar residue, R is the same as R^1 or an alkyl group, and n is 2 or more).
ゼ、グルコシダーゼ又はエステラーゼを用いる請求項１
の測定法。(2) Claim 1 in which phosphatase, galactosidase, glucosidase or esterase is used as the labeling enzyme.
measurement method.
ルデヒドハイドロラーゼ、エーテルボンドハイドラーゼ
及びグリオキシレートデヒドロゲナーゼを用いる請求項
１の測定法。(3) The measuring method according to claim 1, wherein alcohol dehydrogenase, aldehyde hydrolase, ether bond hydrolase, and glyoxylate dehydrogenase are used as metabolic enzymes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30101688A JPH02147956A (en) | 1988-11-30 | 1988-11-30 | Enzyme immunological measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30101688A JPH02147956A (en) | 1988-11-30 | 1988-11-30 | Enzyme immunological measurement method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02147956A true JPH02147956A (en) | 1990-06-06 |
Family
ID=17891826
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP30101688A Pending JPH02147956A (en) | 1988-11-30 | 1988-11-30 | Enzyme immunological measurement method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02147956A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5434051A (en) * | 1991-07-26 | 1995-07-18 | E. I. Du Pont De Nemours And Company | Assay with signal detection in the presence of a suspended solid support |
| WO2012099904A1 (en) | 2011-01-18 | 2012-07-26 | General Atomics | Hydrolase enzyme substrates and uses thereof |
-
1988
- 1988-11-30 JP JP30101688A patent/JPH02147956A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5434051A (en) * | 1991-07-26 | 1995-07-18 | E. I. Du Pont De Nemours And Company | Assay with signal detection in the presence of a suspended solid support |
| US5654159A (en) * | 1991-07-26 | 1997-08-05 | Dade International Inc. | Assay with signal detection in the presence of a suspended solid support |
| WO2012099904A1 (en) | 2011-01-18 | 2012-07-26 | General Atomics | Hydrolase enzyme substrates and uses thereof |
| US8795979B2 (en) | 2011-01-18 | 2014-08-05 | General Atomics | Hydrolase enzyme substrates and uses thereof |
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