JPH0333231B2 - - Google Patents
Info
- Publication number
- JPH0333231B2 JPH0333231B2 JP6803683A JP6803683A JPH0333231B2 JP H0333231 B2 JPH0333231 B2 JP H0333231B2 JP 6803683 A JP6803683 A JP 6803683A JP 6803683 A JP6803683 A JP 6803683A JP H0333231 B2 JPH0333231 B2 JP H0333231B2
- Authority
- JP
- Japan
- Prior art keywords
- thyroxine
- enzyme
- god
- glucose oxidase
- prepared
- 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
- 238000000034 method Methods 0.000 claims description 11
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- 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
- 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
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 229940088598 enzyme Drugs 0.000 claims description 7
- 229940034208 thyroxine Drugs 0.000 claims description 7
- 239000004366 Glucose oxidase Substances 0.000 claims description 6
- 229940116332 glucose oxidase Drugs 0.000 claims description 6
- 238000003018 immunoassay Methods 0.000 claims description 6
- 238000004020 luminiscence type Methods 0.000 claims description 5
- 238000002965 ELISA Methods 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 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 description 2
- 150000001718 carbodiimides Chemical class 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 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 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 239000007982 barbital buffer Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 238000002796 luminescence method Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- VFEXYZINKMLLAK-UHFFFAOYSA-N 2-(trichloromethyl)oxirane Chemical compound ClC(Cl)(Cl)C1CO1 VFEXYZINKMLLAK-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- -1 diethylaminopropyl Chemical group 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 description 1
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- ZTSGDCMQRKBJKC-ZDUSSCGKSA-N methyl (2s)-2-amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoate Chemical compound IC1=CC(C[C@H](N)C(=O)OC)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 ZTSGDCMQRKBJKC-ZDUSSCGKSA-N 0.000 description 1
- MLBBKTFJGPTWFM-ZOWNYOTGSA-N methyl (2s)-2-amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoate;hydrochloride Chemical compound Cl.IC1=CC(C[C@H](N)C(=O)OC)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 MLBBKTFJGPTWFM-ZOWNYOTGSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 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 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Endocrinology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明はサイロキシン（以下「T4」と略す）
の定量法に関し、更に詳しくは酵素免疫測定法に
よつて感度よくT4を測定するための改良に関す
るものである。[Detailed Description of the Invention] The present invention relates to thyroxine (hereinafter abbreviated as "T 4 ")
The present invention relates to a method for quantifying T4, and more specifically, to an improvement for measuring T4 with high sensitivity by enzyme immunoassay.
本発明者等は、生体微量物質測定の手段とし
て、従来、人体への危険性や環境汚染に若干問題
を残しながらも、その高感度性の故に広く使用さ
れている放射免疫測定法に代えて、安全性、操作
性に全く問題のない酵素免疫測定法の実用化を試
み、その高感度化について研究を重ね、既にパー
オキシオギザレート発光法を使用する酵素免疫測
定法の改良について発明した〔特願昭56−
185646；日本薬学会第102年会（昭和57年４月）〕
が、更に個々の生体成分について、より優れた感
度で測定する方法について鋭意研究を重ねていた
ところ、特定のT4誘導体の組合せを使用するこ
とにより、非常に高感度でT4が定量しうること
を見出して本発明を完成した。 The present inventors have proposed an alternative method for measuring biological trace substances, which has traditionally been widely used due to its high sensitivity, although it poses some problems regarding danger to the human body and environmental pollution. , tried to put the enzyme immunoassay method to practical use without any problems in terms of safety and operability, conducted research on how to increase its sensitivity, and has already invented an improved enzyme immunoassay method that uses peroxyogyzalate luminescence method. [Special application 1984-
185646; 102nd Annual Meeting of the Pharmaceutical Society of Japan (April 1982)]
However, as we continued our intensive research into methods for measuring individual biological components with even greater sensitivity, we discovered that by using a combination of specific T4 derivatives, T4 could be quantified with extremely high sensitivity. They discovered this and completed the present invention.
即ち、本発明は、８−アニリノー１−ナフタレ
ンスルフオン酸（以下「ANS」と略す）を発光
物質として使用し、標識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
て測定する酵素免疫測定法において、サイロキシ
ン−ヘミグルタルアミド−担体蛋白結合により調
製した抗サイロキシン抗血清（以下「抗T4抗血
清」と略す）とカルボジイミド法により調製した
サイロキシン−グルコースオキシダーゼ−結合物
（以下「T4−GOD」と略す）若しくはグルタルア
ルデヒド法により調製したサイロキシン−グルコ
ースオキシダーゼ結合物（以下「T4−DOD（G.
A.）」と略す）とよりなる糸を使用してサイロキ
シンの定量をすることを特徴とする酵素免疫測定
法である。 That is, the present invention uses 8-anilino-1-naphthalenesulfonic acid (hereinafter abbreviated as "ANS") as a luminescent substance, and generates hydrogen peroxide by a labeled enzyme using a peroxyogyzalate luminescence method. In the enzyme-linked immunosorbent assay, anti-thyroxine antiserum prepared by thyroxine-hemiglutaramide-carrier protein conjugation (hereinafter referred to as "anti- T4 antiserum") and thyroxine-glucose oxidase conjugate prepared by the carbodiimide method are used. (hereinafter abbreviated as “T 4 -GOD”) or a thyroxine-glucose oxidase conjugate prepared by the glutaraldehyde method (hereinafter “T 4 -DOD (G.
This is an enzyme-linked immunosorbent assay characterized by the use of strands of thread (A.) to quantify thyroxine.
本発明の方法は、前記公知の方法によつて容易
に実施される。即ち、抗T4抗血清、T4標準液お
よびT4−GOD若しくはT4−GOD（G.A.）よりな
る混合物に、第２抗体を吸着させた不溶体（例え
ばビーズ）を添加して反応させ、反応後洗滌した
不溶体とグルコースとを反応させ、生じた過酸化
水素を含む溶液にANSおよびオギザレートを添
加し、発光光量を測定することによつて行なわれ
る。 The method of the present invention is easily carried out by the above-mentioned known methods. That is, to a mixture consisting of anti -T4 antiserum, T4 standard solution, and T4 -GOD or T4 -GOD (GA), an insoluble body (e.g., beads) adsorbed with the second antibody is added and reacted. This is carried out by reacting the insoluble material washed after the reaction with glucose, adding ANS and oxalate to the resulting solution containing hydrogen peroxide, and measuring the amount of emitted light.
反応に使用されるオギザレートとしては、通常
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（以下「TCPO」と略す）が
あげられる。また抗T4抗血清およびT4−GOD
（G.A.）に使用されるサイロキシンなる語は遊離
の酸或いは低級アルキルエステルの両者を含むも
ので、溶解性のよいエステルが誘導体の製造につ
いては好ましい。 The oxalate used in the reaction is usually preferably bis(2,4,6-trichlorophenyl) oxalate (hereinafter abbreviated as "TCPO"). Also anti -T4 antiserum and T4 -GOD
The term thyroxine used in (GA) includes both free acids and lower alkyl esters, and esters with good solubility are preferred for the production of derivatives.
次に実施例をあげて本発明を更に具体的に説明
するが本発明はこれによつて限定されるものでは
ない。 Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.
参考例 １
抗T4抗血清の調製
アメリカ特許第4040907号の方法に準じて、サ
イロキシンメチルエステル、グルタル酸無水物お
よび牛血清アルブミン（BSA）より、サイロキ
シン−ヘミグルタ−ルアミド−BSA結合物をつ
くり、常法によりウサギへの免疫により抗体（以
下「抗T4−ｇ−BSA抗血清と略す）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- T4 antiserum A thyroxine-hemiglutaramide-BSA conjugate was prepared from thyroxine methyl ester, glutaric anhydride and bovine serum albumin (BSA) according to the method of US Patent No. 4,040,907. An antibody (hereinafter referred to as "anti- T4 -g-BSA antiserum") was produced by immunizing rabbits using a conventional method (Clinical Chemistry Vol. 20, No. 10, p. 1353).
(1974).
参考例 ２
T4−GODの調製
サイロキシン 4.2mgを95％ジメチルホルムア
ミド0.6mlに溶かし、室温で30分撹拌した後、グ
ルコースオキシダーゼ10.7mgを水１mlに溶かして
添加して30分撹拌する。１−エチル−３−（３−
ジエチルアミノプロピル）カルボジイミド塩酸塩
（以下「ECDI」と略す）2.6mgを粉末のまま添加
し、更に20分後、３時間後および４時間後ECDI2
mgを水0.1mlに溶かした溶液を追加する。反応混
合物を４℃に放置した後、PH7.0の0.05Mリン酸
緩衝液に透析した後、ToyopalHW50（商品名）
カラムクロマトグラフイーにより精製した。Reference Example 2 Preparation of T 4 -GOD 4.2 mg of thyroxine is dissolved in 0.6 ml of 95% dimethylformamide and stirred at room temperature for 30 minutes, then 10.7 mg of glucose oxidase dissolved in 1 ml of water is added and stirred for 30 minutes. 1-ethyl-3-(3-
2.6 mg of diethylaminopropyl) carbodiimide hydrochloride (hereinafter abbreviated as "ECDI") was added as a powder, and after another 20 minutes, 3 hours, and 4 hours, ECDI2
Add a solution of mg dissolved in 0.1 ml of water. After the reaction mixture was left at 4°C, it was dialyzed against 0.05M phosphate buffer of PH7.0 and then purified using Toyopal HW50 (trade name).
Purified by column chromatography.
参考例 ３
T4−GOD（G.A.）の調製
10ml用ナス型コルベンにサイロキシンメチルエ
ステル塩酸塩５mgを95％ジメチルホルムアミド１
ml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）
に透孔し、ToyopalHW50カラムクロマトグラフ
イーにより調製した。Reference Example 3 Preparation of T 4 -GOD (GA) 5 mg of thyroxine methyl ester hydrochloride was added to 10 ml of eggplant-shaped Kolben in 95% dimethylformamide.
ml, add 1 ml of an aqueous solution containing 10 mg of glucose oxidase, stir, add 0.21 ml of 1% glutaraldehyde solution, and react at room temperature for 2 hours.
The obtained conjugate was added to 0.05M phosphate buffer (PH8.0)
and prepared by Toyopal HW50 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および
400μｇ／mlANS含有）で希釈した抗T4−ｇ−
BSA抗血清（×105）溶液200μ、試料（サイ
ロキシン：4.6〜320pg／デイスク、デイスクは
径３mmの紙）、T4−GOD（G.A.）（×2000）
溶液100μ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、37℃で３時間反応させる。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、
４℃で１夜放置して反応させる。Example Enzyme immunoassay for thyroxine a Immunoassay 0.12M in polystyrene tube (10 x 75 mm)
Barbital buffer (PH8.6, 0.2% BSA and
Anti-T 4 -g- diluted with 400 μg/ml ANS)
BSA antiserum (×10 5 ) solution 200 μ, sample (thyroxine: 4.6 to 320 pg/disk, disk is paper with a diameter of 3 mm), T 4 -GOD (GA) (× 2000)
Add 100μ of the solution and then add purified rabbit
Add one bead (made of polystyrene: 6φ x 6mm gear shape) coated with anti-rabbit IgG goat IgG purified by Affinity chromatography using Sepharose gel bound with IgG, stir well, and incubate at 37°C. Let it react for 3 hours. After removing the reaction solution by suction, add 2 ml of physiological saline for washing, and remove by suction. Repeat this operation two more times.
After repeating twice, acetate buffer (0.01M, PH5.0)
Add 0.3ml of 0.5M glucose solution dissolved in
Leave to react overnight at 4°C.
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および0.1％
BSAを含むANS溶液100μ、酢酸エチルに溶
解した5mMTCPO溶液200μを加え、Photon
Counter（アロカ社製ルミネツセンスリーダー）
により測定した。測定結果より求めた検量線が
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 and 0.1% in 0.2M barbital buffer (PH9.0)
Add 100μ of ANS solution containing BSA, 200μ of 5mMTCPO solution dissolved in ethyl acetate, and Photon
Counter (Luminescence sense reader manufactured by Aloka)
It was measured by A calibration curve obtained from the measurement results is shown in FIG.
上記実施例において、T4−GOD（G.A.）の代
りにT4−GODを使用することによつて、T4−ｇ
−BSA・T4−GOD系によるT4の検量線が得られ
る。 In the above example, by using T 4 -GOD instead of T 4 -GOD(GA), T 4 -g
A calibration curve for T 4 is obtained using the −BSA・T 4 −GOD system.
第１図はT4の検量線であり、線ＡおよびＢは、
夫々、T4−GOD（G.A.）およびT4−GODを使用
した場合を示す。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。
Figure 1 is the calibration curve for T 4 , and curves A and B are
The cases using T 4 -GOD (GA) and T 4 -GOD are shown, respectively. The horizontal axis is the amount of sample T 4 , and the vertical axis is T 4
The relative values of luminescence intensity associated with the addition of are shown. B 0 is
B is the luminescence intensity when T 4 is not added, and B is the luminescence intensity when T 4 is added.
Claims (1)
を発光物質として使用し、標識酵素によつて発生
した過酸化水素をパーオキシオギザレート発光法
によつて測定する酵素免疫測定法において、サイ
ロキシン−ヘミグルタルアミド−担体蛋白結合に
より調製した抗サイロキシン抗血清とカルボジイ
ミド法により調製したサイロキシン−グルコース
オキシダーゼ−結合物若しくはグルタルアルデヒ
ド法により調製したサイロキシン−グルコースオ
キシダーゼ結合物とよりなる系を使用してサイロ
キシンの定量をすることを特徴とする酵素免疫測
定法。1 In an enzyme-linked immunosorbent assay that uses 8-anilino-1-naphthalenesulfonic acid as a luminescent substance and measures hydrogen peroxide generated by a labeled enzyme by peroxyogyzalate luminescence, thyroxine-hemiglutar Thyroxine is quantified using a system consisting of an antithyroxine antiserum prepared by amide-carrier protein binding and a thyroxine-glucose oxidase conjugate prepared by the carbodiimide method or a thyroxine-glucose oxidase conjugate prepared by the glutaraldehyde method. An enzyme immunoassay method characterized by:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6803683A JPS59193357A (en) | 1983-04-18 | 1983-04-18 | Measuring method of enzyme immunity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6803683A JPS59193357A (en) | 1983-04-18 | 1983-04-18 | Measuring method of enzyme immunity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS59193357A JPS59193357A (en) | 1984-11-01 |
| JPH0333231B2 true JPH0333231B2 (en) | 1991-05-16 |
Family
ID=13362164
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6803683A Granted JPS59193357A (en) | 1983-04-18 | 1983-04-18 | Measuring method of enzyme immunity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS59193357A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06105252B2 (en) * | 1986-02-05 | 1994-12-21 | 三共株式会社 | Enzyme-linked immunosorbent assay for free thyroxine |
-
1983
- 1983-04-18 JP JP6803683A patent/JPS59193357A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS59193357A (en) | 1984-11-01 |
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