JPH0153422B2 - - Google Patents
Info
- Publication number
- JPH0153422B2 JPH0153422B2 JP56185646A JP18564681A JPH0153422B2 JP H0153422 B2 JPH0153422 B2 JP H0153422B2 JP 56185646 A JP56185646 A JP 56185646A JP 18564681 A JP18564681 A JP 18564681A JP H0153422 B2 JPH0153422 B2 JP H0153422B2
- Authority
- JP
- Japan
- Prior art keywords
- ans
- thyroxine
- luminescence
- hydrogen peroxide
- oxalate
- 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
Links
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 229940088598 enzyme Drugs 0.000 claims description 11
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 claims description 9
- 238000003018 immunoassay Methods 0.000 claims description 8
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- 229940034208 thyroxine Drugs 0.000 claims description 7
- 239000004366 Glucose oxidase Substances 0.000 claims description 5
- 229940116332 glucose oxidase Drugs 0.000 claims description 5
- 238000002372 labelling Methods 0.000 claims description 4
- 238000002796 luminescence method Methods 0.000 claims description 4
- 108010015776 Glucose oxidase Proteins 0.000 claims description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- ADRVRLIZHFZKFE-UHFFFAOYSA-N ethanediperoxoic acid Chemical compound OOC(=O)C(=O)OO ADRVRLIZHFZKFE-UHFFFAOYSA-N 0.000 claims description 3
- 235000019420 glucose oxidase Nutrition 0.000 claims description 3
- GEVPIWPYWJZSPR-UHFFFAOYSA-N tcpo Chemical compound ClC1=CC(Cl)=CC(Cl)=C1OC(=O)C(=O)OC1=C(Cl)C=C(Cl)C=C1Cl GEVPIWPYWJZSPR-UHFFFAOYSA-N 0.000 claims description 2
- FWEOQOXTVHGIFQ-UHFFFAOYSA-N 8-anilinonaphthalene-1-sulfonic acid Chemical compound C=12C(S(=O)(=O)O)=CC=CC2=CC=CC=1NC1=CC=CC=C1 FWEOQOXTVHGIFQ-UHFFFAOYSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 5
- 238000003127 radioimmunoassay Methods 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- VFEXYZINKMLLAK-UHFFFAOYSA-N 2-(trichloromethyl)oxirane Chemical compound ClC(Cl)(Cl)C1CO1 VFEXYZINKMLLAK-UHFFFAOYSA-N 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000007982 barbital buffer Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- PROQIPRRNZUXQM-UHFFFAOYSA-N (16alpha,17betaOH)-Estra-1,3,5(10)-triene-3,16,17-triol Natural products OC1=CC=C2C3CCC(C)(C(C(O)C4)O)C4C3CCC2=C1 PROQIPRRNZUXQM-UHFFFAOYSA-N 0.000 description 1
- XFOOPZIJVVDYHI-BQBZGAKWSA-N (2s)-2-amino-6-[[(2r)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-6-oxohexanoic acid Chemical compound OC(=O)[C@@H](N)CCCC(=O)N[C@@H](CS)C(=O)NCC(O)=O XFOOPZIJVVDYHI-BQBZGAKWSA-N 0.000 description 1
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 108010015133 Galactose oxidase Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 229940125717 barbiturate Drugs 0.000 description 1
- HNYOPLTXPVRDBG-UHFFFAOYSA-N barbituric acid Chemical compound O=C1CC(=O)NC(=O)N1 HNYOPLTXPVRDBG-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- PROQIPRRNZUXQM-ZXXIGWHRSA-N estriol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H]([C@H](O)C4)O)[C@@H]4[C@@H]3CCC2=C1 PROQIPRRNZUXQM-ZXXIGWHRSA-N 0.000 description 1
- 229960001348 estriol Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- YAUHDTOEJHVKJO-UHFFFAOYSA-N n-heptylformamide Chemical compound CCCCCCCNC=O YAUHDTOEJHVKJO-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 229940035722 triiodothyronine Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明は、酵素免疫測定法における新規な定量
方法に関し、更に詳しくは新規な発光反応による
定量方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel quantitative method in enzyme immunoassay, and more particularly to a novel quantitative method using a luminescence reaction.
抗原抗体反応を利用して、生体内の微量の物質
を定量する方法としては、螢光免疫測定法
（FIA）、放射免疫測定法（RIA）、酵素免疫測定
法（EIA）など種々の方法が使用されていた。な
かでも高感度で定量できるRIAが広く使用されて
いた。しかしながら、同位元素標識化合物に不安
定なものが多いこと、人体に対する危険性および
環境汚染の立場からその取り扱いが厳しく廃棄が
困難であることなど、問題点が多い。そこで最近
になり、安全性に問題がなく実験室の制約もな
く、また測定に使用される物質も比較的安定で保
存でき、操作も比較的容易であるEIAが注目され
た。とくに、標識として使用される酵素の活性の
測定において、RIAに匹敵する高感度の測定法、
即ち螢光反応や発光反応を利用する方法が開発さ
れるに到り、更に重要視されてきた。その中で
も、発光反応を利用した方法がより感度が高い。 There are various methods to quantify minute amounts of substances in living organisms using antigen-antibody reactions, such as fluorescence immunoassay (FIA), radioimmunoassay (RIA), and enzyme immunoassay (EIA). It was used. Among these, RIA, which allows for highly sensitive and quantitative determination, was widely used. However, there are many problems, such as the fact that many of the isotope-labeled compounds are unstable, and that they are difficult to handle and difficult to dispose of due to the danger to the human body and environmental pollution. Therefore, recently, EIA has attracted attention because it has no safety issues, there are no laboratory restrictions, the substances used for measurement are relatively stable and can be stored, and it is relatively easy to operate. In particular, it is a highly sensitive measurement method comparable to RIA in measuring the activity of enzymes used as labels.
That is, with the development of methods that utilize fluorescent reactions and luminescent reactions, even more importance has been placed on them. Among these, methods using luminescent reactions have higher sensitivity.
従来、発光反応を利用したEIAには、発光物質
としてルミノール、イソルミノールなどが利用さ
れている。一方、生体成分は通常他の蛋白質と結
合している場合が多く、８−アニリノ−１−ナフ
タレンスルフオン（ANS）によつてこの結合を
切つた後に測定が行なわれることが多い。例えば
サイロキシンT4のEIAにおいて、TBG（サイロイ
ド・バインデイング・グロブリン）ブロツカーと
してANSが用いられるが、洗滌を繰返しても
ANSが残り、この微量に残存するANSのために
酵素活性測定が妨害される。 Conventionally, EIA using luminescence reactions has used luminol, isoluminol, etc. as luminescent substances. On the other hand, biological components are often bound to other proteins, and measurements are often performed after this bond is broken with 8-anilino-1-naphthalenesulfon (ANS). For example, in the EIA of thyroxine T 4 , ANS is used as a TBG (thyroid binding globulin) blocker, but even after repeated washing,
ANS remains, and this small amount of ANS interferes with enzyme activity measurements.
本発明者は、このようなANSの影響をカバー
するような発光物質について種々研究したとこ
ろ、上記ANS自身がパーオキシオギザレート発
光法［アナリテイカ・キミカ・アクタ97巻21頁
（1978年）］の発光物質としても優れた結果を与え
ることを見い出して本発明をなした。 The present inventor conducted various studies on luminescent substances that could cover the effects of such ANS, and found that the above-mentioned ANS itself is based on the peroxyogyzalate luminescence method [Analytica Chimica Acta Vol. 97, p. 21 (1978)] The present invention was made based on the discovery that it also provides excellent results as a luminescent material.
本発明に係る酵素免疫測定法の方法は、標識に
使用された酵素によつて発生した過酸化水素をパ
ーオキシオギザレート発光法によつて測定する方
法において、発光物質としてANSを使用するこ
とを特徴とするものである。 The enzyme immunoassay method according to the present invention uses ANS as a luminescent substance in a method of measuring hydrogen peroxide generated by an enzyme used for labeling by peroxyoxalate luminescence method. It is characterized by:
本発明に係る測定方法は、酵素反応によつて生
じた過酸化水素を含む溶液に、ANSおよびオギ
ザレートの夫々の溶液を加え、反応混合液よりの
光量をを測定することによつて容易に実施され
る。 The measurement method according to the present invention can be easily carried out by adding respective solutions of ANS and oxalate to a solution containing hydrogen peroxide produced by an enzymatic reaction and measuring the amount of light emitted from the reaction mixture. be done.
上記の方法において使用される過酸化水素を含
む溶液とは、標識酵素として過酸化水素を生成す
る各種のオキシダーゼ例えばグルコースオキシダ
ーゼ、ガラクトースオキシダーゼ、コレステロー
ルオキシダーゼなどをを使用した通常の酵素免疫
法の測定システムによつて得られる過酸化水素を
含む反応液である。ANSは中性または塩基性の
緩衝液、特にPH８〜9.5程度の緩衝液例えばバル
ビツール緩衝液の溶液として使用され、通常血清
アルブミンが添加される。オギザレートとしては
好ましくはビス（２，４，６−トリクロロフエニ
ル）オギザレート（TCPO）が使用され、それら
は有機溶媒例えば酢酸エチル溶液として使用され
る。 The solution containing hydrogen peroxide used in the above method is a measurement system for normal enzyme immunoassay using various oxidases that produce hydrogen peroxide as labeling enzymes, such as glucose oxidase, galactose oxidase, and cholesterol oxidase. This is a reaction solution containing hydrogen peroxide obtained by. ANS is used as a solution in a neutral or basic buffer, especially a buffer with a pH of about 8 to 9.5, such as a barbiturate buffer, and serum albumin is usually added. Bis(2,4,6-trichlorophenyl)oxalate (TCPO) is preferably used as oxalate, which is used in solution in an organic solvent, for example in ethyl acetate.
本発明に係る方法は、通常EIAの対象とされる
物質に対して適用することができる。例えばサイ
ロキシンT4、トリヨードサイロニンT3、エスト
リオール、プロゲステロン、コルチゾール、イン
シユリン、HPL、HCG、TSH、α−フエトプロ
テイン、カルチノエンブリオ抗原（CEA）、フエ
リチン、β2−ミクログロブリン、HBsAg、IgG、
IgM、IgA、IgEなどの定量にも使用することが
できる。 The method according to the present invention can be applied to substances that are normally subject to EIA. For example, thyroxine T4 , triiodothyronine T3 , estriol, progesterone, cortisol, insulin, HPL, HCG, TSH, α-fetoprotein, carcinoembryoantigen (CEA), ferritin, β2 -microglobulin, HBsAg. , IgG,
It can also be used to quantify IgM, IgA, IgE, etc.
次に、二抗体固相法によるT4の測定に適用し
た実施例をあげて本発明の方法を具体的に説明す
るが、本発明の方法はこれによつて限定されるも
のではない。 Next, the method of the present invention will be specifically explained with reference to an example applied to the measurement of T 4 by the two-antibody solid phase method, but the method of the present invention is not limited thereto.
参考例 １
抗サイロキシン血清の調製
カルボジイミド法により、サイロキシンを牛血
清アルブミンBSAに結合し、常法によりウサギ
への免疫により抗体を産生させた（クリニカル・
ケミストリー20巻10号1353頁1974年）。Reference Example 1 Preparation of anti-thyroxine serum Thyroxine was bound to bovine serum albumin BSA using the carbodiimide method, and antibodies were produced by immunizing rabbits using a conventional method (clinical).
Chemistry Vol. 20, No. 10, p. 1353, 1974).
参考例 ２
サイロキシン−グルコースオキシダーゼ結合物
の調製
10ml用ナス型コルベンにサイロキシン遊離酸３
mgを95％ジメチルホルムアミド１mlに溶解し、グ
ルコースオキシダーゼ10mgを含む水溶液１mlを加
え、撹拌し、１％グルタルアルデヒド溶液0.21ml
を加え、室温で２時間反応する。得られた結合物
は0.05Mリン酸緩衝液（PH8.0）に透析し、
Tyopalカラムクロマトグラフイーにより精製し
た。Reference Example 2 Preparation of thyroxine-glucose oxidase conjugate Add thyroxine free acid 3 to a 10 ml eggplant-shaped kolben.
Dissolve mg in 1 ml of 95% dimethylformamide, add 1 ml of an aqueous solution containing 10 mg of glucose oxidase, stir, and 0.21 ml of 1% glutaraldehyde solution.
and react at room temperature for 2 hours. The obtained conjugate was dialyzed against 0.05M phosphate buffer (PH8.0),
Purified by Tyopal column chromatography.
実施例
サイロキシンの酵素免疫測定法
ａ イムノアツセイ
ポリスチレンチユーブ（10×75mm）に0.12M
バルビタール緩衝液（PH8.6、0.2％BSA含有）
で希釈した抗サイロキシン血清（×400）溶液
0.1ml、試料（サイロキシン：0.05〜50ng／0.1
ml）水溶液0.1ml、サイロキシン−グルコース
オキシダーゼ結合物（×1000）溶液0.1mlを加
え、次いで精製された家兎のIgGを結合させた
セフアローズゲルを使用したアフイニテイクロ
マトグラフイーにより精製した抗家兎IgG山羊
IgGをコーテイングしたビーズ（ポリスチレン
製：6φ×６mm歯車形）１個を加え、よく撹拌
し、４℃で一夜放置して反応させる。反応液を
吸引除去後、生理食塩水２mlを加えて洗浄し、
吸引除去をを行なう。この操作をさらに２回繰
返した後、酢酸緩衝液（0.01M、PH5.0）に溶
解した0.5Mグルコース溶液0.3mlを加え、室温
で３時間反応させる。Example Enzyme immunoassay for thyroxine a Immunoassay 0.12M in polystyrene tube (10 x 75 mm)
Barbital buffer (PH8.6, containing 0.2% BSA)
Antithyroxine serum (x400) solution diluted with
0.1ml, sample (thyroxine: 0.05-50ng/0.1
ml) Add 0.1 ml of the aqueous solution and 0.1 ml of thyroxine-glucose oxidase conjugate (x1000) solution, then add the antibody purified by Affinity chromatography using Sepharose gel conjugated with purified rabbit IgG. rabbit IgG goat
Add one IgG-coated bead (made of polystyrene: 6φ x 6mm gear shape), stir well, and leave to react overnight at 4°C. After removing the reaction solution by suction, wash by adding 2 ml of physiological saline.
Perform suction removal. After repeating this operation two more times, 0.3 ml of a 0.5M glucose solution dissolved in acetate buffer (0.01M, PH5.0) is added, and the mixture is allowed to react at room temperature for 3 hours.
生成した過酸化水素はANS−TCPO系によ
る発光反応により測定する。 The generated hydrogen peroxide is measured by a luminescent reaction using the ANS-TCPO system.
ｂ 化学発光法
ガラスチユーブ（10φ×75mm）に反応溶液
（生成した過酸化水素）100μ、0.2Mバルビタ
ール緩衝液（PH9.0）に0.02％ANS、0.1％BSA
を含むANS溶液100μ、酢酸エチルに溶解し
たTCPO5mM溶液200μを加え、Photon
Counter（浜松テレビ社製HTV−C767）により
測定した。測定結果が第１図に示されている。b Chemiluminescence method 100μ of reaction solution (produced hydrogen peroxide) in a glass tube (10φ x 75mm), 0.02% ANS, 0.1% BSA in 0.2M barbital buffer (PH9.0)
Add 100μ of ANS solution containing 200μ of 5mM TCPO solution in ethyl acetate, and add Photon
Measurement was performed using a Counter (HTV-C767 manufactured by Hamamatsu Television Co., Ltd.). The measurement results are shown in FIG.
第１図はT4の検量線であり、横軸は試料T4の
量、縦軸はT4の添加に伴なう発光強度の相対値
を示す。B0はT4無添加のときの発光強度であり、
ＢはT4を添加したときの発光強度である。
FIG. 1 is a calibration curve for T 4 , where the horizontal axis shows the amount of sample T 4 and the vertical axis shows the relative value of the luminescence intensity associated with the addition of T 4 . B 0 is the luminescence intensity when T 4 is not added,
B is the luminescence intensity when T4 is added.
Claims (1)
化水素をパーオキシオギザレート発光法によつて
測定する方法において、発光物質として８−アニ
リノ−１−ナフタレンスルフオン酸を使用するこ
とを特徴とする酵素免疫測定法。 ２ パーオキシオギザレート発光法においてオギ
ザレートとしてビス（２，４，６−トリクロロフ
エニル）オギザレートを使用する特許請求の範囲
第１項記載の方法。 ３ 標識に使用される酵素がグルコースオキシダ
ーゼである特許請求の範囲第１項乃至第２項記載
の方法。 ４ 測定される対象が生体内に存在する低分子化
合物および高分子化合物の中より選択された一種
である特許請求の範囲第１項乃至第３項のいずれ
か一項に記載の方法。 ５ 測定される対象がサイロキシンである特許請
求の範囲第１項乃至第３項のいずれか１項に記載
の方法。[Scope of Claims] 1. A method for measuring hydrogen peroxide generated by an enzyme used for labeling by peroxyoxalate luminescence method, in which 8-anilino-1-naphthalenesulfonic acid is used as a luminescent substance. An enzyme immunoassay method characterized by using. 2. The method according to claim 1, wherein bis(2,4,6-trichlorophenyl)oxalate is used as the oxalate in the peroxyoxalate luminescence method. 3. The method according to claims 1 and 2, wherein the enzyme used for labeling is glucose oxidase. 4. The method according to any one of claims 1 to 3, wherein the target to be measured is one selected from low-molecular compounds and high-molecular compounds existing in living organisms. 5. The method according to any one of claims 1 to 3, wherein the target to be measured is thyroxine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18564681A JPS5886458A (en) | 1981-11-19 | 1981-11-19 | Measuring method for enzyme immunity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18564681A JPS5886458A (en) | 1981-11-19 | 1981-11-19 | Measuring method for enzyme immunity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5886458A JPS5886458A (en) | 1983-05-24 |
| JPH0153422B2 true JPH0153422B2 (en) | 1989-11-14 |
Family
ID=16174407
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18564681A Granted JPS5886458A (en) | 1981-11-19 | 1981-11-19 | Measuring method for enzyme immunity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5886458A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5737261A (en) * | 1980-08-15 | 1982-03-01 | Otsuka Pharmaceut Co Ltd | Measuring method of physiological active substance |
-
1981
- 1981-11-19 JP JP18564681A patent/JPS5886458A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5737261A (en) * | 1980-08-15 | 1982-03-01 | Otsuka Pharmaceut Co Ltd | Measuring method of physiological active substance |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5886458A (en) | 1983-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4842997A (en) | Enhanced luminescent and luminometric assay | |
| US4478817A (en) | Detecting or quantifying substances using labelling techniques | |
| JP3611579B2 (en) | Chemiluminescent immunoassay for antibody detection | |
| JP2651060B2 (en) | Equipment for simultaneous calibration heterogeneous immunoassays | |
| EP0247796A1 (en) | Solid phase immunoassay method | |
| US5188939A (en) | Displacement immunoassay utilizing an oligavalent labelled antibody | |
| JPH0716438B2 (en) | Use of the oxidase enzyme system in chemiluminescence analysis | |
| EP0522677B1 (en) | Method of measuring concentration of immunoreactant using electrochemiluminescence | |
| JP2000508075A (en) | Luminescence-specific binding assay | |
| JPH0648996B2 (en) | Amplified emission analysis | |
| US4637985A (en) | Assay processes and materials therefor | |
| JP2603422B2 (en) | Analyte measuring method and measuring means | |
| JPH06501559A (en) | Amplified heterogeneous chemiluminescence immunoassay | |
| JP3395673B2 (en) | Specific binding pair measurement method using micro oxygen electrode | |
| JPH0153422B2 (en) | ||
| EP0682254B1 (en) | Enzyme linked chemiluminescent assay | |
| AU703940B2 (en) | Regenerable solid phase for carrying out specific binding reactions | |
| JP2672151B2 (en) | Enzyme immunoassay using magnetic substance | |
| CA2053120A1 (en) | Quantitation by chemiluminescence using photosensitive substance | |
| JPH04203968A (en) | Method for measuring chemical emission immunity | |
| JP2657672B2 (en) | Ultrasensitive specific antibody assay | |
| JP3694044B2 (en) | Luminescence method of acridinium derivative and detection method of test substance using the method | |
| JPH035539B2 (en) | ||
| JP3792899B2 (en) | Chemiluminescent enzyme immunoassay method | |
| JPH0333231B2 (en) |