JPH01232263A - Immunoassay of triiodothyronine ingestion rate - Google Patents
Immunoassay of triiodothyronine ingestion rateInfo
- Publication number
- JPH01232263A JPH01232263A JP5823288A JP5823288A JPH01232263A JP H01232263 A JPH01232263 A JP H01232263A JP 5823288 A JP5823288 A JP 5823288A JP 5823288 A JP5823288 A JP 5823288A JP H01232263 A JPH01232263 A JP H01232263A
- Authority
- JP
- Japan
- Prior art keywords
- triiodothyronine
- antibody
- binding globulin
- thyroxine
- immunoassay
- 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
- 229940035722 triiodothyronine Drugs 0.000 title claims abstract description 41
- 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 title claims abstract description 39
- 238000003018 immunoassay Methods 0.000 title claims abstract description 8
- 230000037406 food intake Effects 0.000 title abstract 3
- 102000006395 Globulins Human genes 0.000 claims abstract description 12
- 108010044091 Globulins Proteins 0.000 claims abstract description 12
- 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 28
- 238000000034 method Methods 0.000 claims description 15
- 102000002248 Thyroxine-Binding Globulin Human genes 0.000 claims description 13
- 108010000259 Thyroxine-Binding Globulin Proteins 0.000 claims description 13
- 125000006850 spacer group Chemical group 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 101000837639 Homo sapiens Thyroxine-binding globulin Proteins 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 5
- 238000002372 labelling Methods 0.000 abstract description 5
- 102100028709 Thyroxine-binding globulin Human genes 0.000 abstract 4
- 230000001268 conjugating effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 8
- 239000012491 analyte Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 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 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 229940034208 thyroxine Drugs 0.000 description 5
- 238000000926 separation method Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 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 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102000007584 Prealbumin Human genes 0.000 description 1
- 108010071690 Prealbumin Proteins 0.000 description 1
- 102000009488 Thyroxine-Binding Proteins Human genes 0.000 description 1
- 108010048889 Thyroxine-Binding Proteins Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 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
- 238000000265 homogenisation Methods 0.000 description 1
- 102000048799 human SERPINA7 Human genes 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は血中のトリヨードサイロニン摂取率免疫測定法
に関するものである。本発明のトリヨードサイロニン摂
取率免疫測定法は、抗サイロキシン結合グロブリン抗体
を用いることにより、これまで報告されたどの方法より
も簡単明瞭な分析系で安全かつ容易にトリヨードサイロ
ニン摂取率を測定する方法である。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an immunoassay method for triiodothyronine uptake in blood. The triiodothyronine uptake rate immunoassay method of the present invention uses an antithyroxine-binding globulin antibody to safely and easily measure the triiodothyronine uptake rate with a simpler and clearer analysis system than any previously reported method. It is a method of measurement.
［従来の技術］
従来の酵素免疫測定法におけるトリヨードサイロニン摂
取率の測定は、抗トリヨードサイロニン抗体に対して、
既知の外因性トリヨードサイロニン及びサイロキシン結
合グロブリンに反応しない酵素標識されたトリヨードサ
イロニン並びに被検体を反応させるもので、まず外因性
トリヨードサイロニンがサイロキシン結合グロブリンの
不飽和部分に摂取される。残った外因性トリヨードサイ
ロニンと酵素標識されたトリヨードサイロニンが抗トリ
ヨードサイロニン抗体に対して競争的に反応が進行しサ
イロキシン結合グロブリンの不飽和度が測定できるとい
うもので非常に複雑な反応系である。[Prior art] In the conventional enzyme immunoassay method, the triiodothyronine uptake rate is measured using anti-triiodothyronine antibodies.
This method involves reacting with a known exogenous triiodothyronine and an enzyme-labeled triiodothyronine that does not react with thyroxine-binding globulin, as well as the analyte. Ru. The remaining exogenous triiodothyronine and enzyme-labeled triiodothyronine undergo a competitive reaction against the anti-triiodothyronine antibody, allowing the degree of unsaturation of thyroxine-binding globulin to be measured, which is extremely complex. It is a reaction system.
［発明が解決しようとする課題〕
本発明の目的は、このような従来の方法よりも簡便な分
析系でトリヨードサイロニン摂取率をｎ１定できる方法
を提供することにある。[Problems to be Solved by the Invention] An object of the present invention is to provide a method for determining the triiodothyronine intake rate n1 using a simpler analysis system than such conventional methods.
［課題を解決するための手段］
本発明者らは、上記問題点に関し鋭意検討を重ねた結果
、本発明に到達した。[Means for Solving the Problems] The present inventors have made extensive studies regarding the above-mentioned problems, and as a result, have arrived at the present invention.
即ち本発明は、抗サイロキシン結合グロブリン抗体、サ
イロキシン結合グロブリンと反応しうる。That is, the present invention can react with anti-thyroxine-binding globulin antibodies and thyroxine-binding globulin.
トリヨードサイロニン標識結合体を用いるトリヨードサ
イロニン摂取率免疫測定法である。This is a triiodothyronine uptake immunoassay using a triiodothyronine-labeled conjugate.
本発明では抗サイロキシン結合グロブリン抗体を用いる
。この抗体は、ポリクローナル抗体であってもよいが、
モノクローナル抗体であることが好ましい。モノクロー
ナル抗体を用いる場合、そのサイロキシン結合グロブリ
ン認識部位は、サイロキシン結合グロブリンのトリヨー
ドサイロニン結合部位及びその近傍に持たないことが必
要である。本発明では、サイロキシン結合グロブリンを
抗サイロキシン結合グロブリン抗体とトリヨードサイロ
ニンとではさむことによって、トリヨードサイロニンの
摂取率を測定するものだからである。In the present invention, an anti-thyroxine-binding globulin antibody is used. This antibody may be a polyclonal antibody, but
Preferably, it is a monoclonal antibody. When using a monoclonal antibody, it is necessary that its thyroxine-binding globulin recognition site does not exist in or near the triiodothyronine-binding site of thyroxine-binding globulin. This is because the present invention measures the uptake rate of triiodothyronine by sandwiching thyroxine-binding globulin between an anti-thyroxine-binding globulin antibody and triiodothyronine.
該抗体は１３／Ｐ分離を容易にするため、固相化担体に
固相化する。固相化担体としては、通常、抗体を固相化
できるものならいずれを用いてもよく、例えば、プラス
チック、ガラス製のビーズ１ プレートなどが使用でき
る。固相化の方法には特に限定はなく、例えば、物理的
吸着法、化学結合法などを用いればよい。The antibody is immobilized on an immobilized carrier to facilitate 13/P separation. As the immobilized carrier, any carrier that can immobilize the antibody may be used, such as a plastic or glass bead plate. There is no particular limitation on the method of solid phase formation, and for example, a physical adsorption method, a chemical bonding method, etc. may be used.
一方、トリヨードサイロニンに標識を施したものを用い
る。標識としては、酵素、放射性物質。On the other hand, labeled triiodothyronine is used. Labels include enzymes and radioactive substances.
蛍光物質などを使用する。例えば、酵素の場合は、アル
カリ性ホスファターゼ、パーオキシダーゼなどが使用で
き、各酵素に応じた基質も用いられる。Use fluorescent substances, etc. For example, in the case of enzymes, alkaline phosphatase, peroxidase, etc. can be used, and substrates suitable for each enzyme can also be used.
ｔｉ ２はトリヨードサイロニンにつける。この際、ト
リヨードサイロニンに限定されるのは、サイロキシンは
、トリヨードサイロニンよりサイロキシン結合グロブリ
ンへの結合能が約２０倍大きいため、すでに結合してい
るトリヨードサイロニンを追い出して、結合してしまう
ことが考えられるからである。さらに、サイロキシンは
、トリヨードサイロニンに比べ、サイロキシン結合プレ
アルブミンの影響をうけることが一般に知られているか
らである。Ti 2 is attached to triiodothyronine. In this case, thyroxine is limited to triiodothyronine because its ability to bind to thyroxine-binding globulin is approximately 20 times greater than that of triiodothyronine, so thyroxine is able to expel the already bound triiodothyronine and bind to thyroxine. This is because you may end up doing something like this. Furthermore, it is generally known that thyroxine is more affected by thyroxine-binding prealbumin than triiodothyronine.
本発明では、サイロキシン結合グロブリンと反応しうる
トリヨードサイロニンの標識結合体（以下、コンジュゲ
ートという）を用いる。トリヨードサイロニンは直接標
識化すると、サイロキシン結合グロブリンと反応しなく
なると考えられている。これはコンジュゲートの立体障
害によるものと考えられる。したがって、トリヨードサ
イロニンと標識との間にスペーサーを導入してコンジュ
ゲートとする。In the present invention, a labeled conjugate of triiodothyronine (hereinafter referred to as a conjugate) that can react with thyroxine-binding globulin is used. Direct labeling of triiodothyronine is thought to prevent it from reacting with thyroxine-binding globulin. This is thought to be due to steric hindrance of the conjugate. Therefore, a spacer is introduced between triiodothyronine and the label to form a conjugate.
スペーサーとしては、←ＣＨ２−トーｔを用いるのが好
ましい。ｎは２〜５がスペーサーとして適当である。ス
ペーサーと標識及びトリヨードサイロニンとを結合する
方法には、特に限定はなく、種々の方法を採用できる。As the spacer, it is preferable to use ←CH2-t. A suitable value for n as a spacer is 2 to 5. There are no particular limitations on the method of binding the spacer, label, and triiodothyronine, and various methods can be employed.
例えば、スペーサーの末端にカルボニルを導入し、それ
とトリヨードサイロニン又は標識のアミノ基とを結合さ
せるなどによってスペーサーを導入する。For example, the spacer is introduced by introducing a carbonyl at the end of the spacer and bonding it to triiodothyronine or an amino group of a label.
以上のようにして製造したコンジュゲートと固相化抗体
を用いて、サイロキシン結合グロブリンのトリヨードサ
イロニン摂取率を測定する。測定方法は、被検体に固相
化抗体及びコンジュゲートを反応せしめ、固相に検体を
介して結合したコンジュゲートの、又は遊離のコンジュ
ゲートの標識の程度を測定すればよい。Using the conjugate and immobilized antibody produced as described above, the triiodothyronine uptake rate of thyroxine-binding globulin is measured. The measurement method is to react the immobilized antibody and conjugate with the analyte, and measure the degree of labeling of the conjugate bound to the solid phase via the analyte or the free conjugate.
固相化抗体、被検体、コンジュゲートの添加順序には特
に限定はなく、被検体を固相化抗体とコンジュゲートの
どちらと先に反応させるか、若しくは同時に反応させる
か、また途中でのＢ／Ｐ分離の有無など、必要に応じて
行えばよい。ただし、固相化抗体と被検体を反応せしめ
、Ｂ／Ｐ分離後、固相とコンジュゲートを反応させる２
段階法を用いた方が、標識を感度よ＜ ａ’ｔ＋＋定す
ることができるので、好ましい方法である。しかし、標
識の性能がよければ、コンジュゲート、被検体、固相化
抗体を反応させた後に１回のＢ／Ｐ分離だけで感度よく
測定することができる。There is no particular limitation on the order of addition of the immobilized antibody, the analyte, and the conjugate, and whether the analyte is reacted with the immobilized antibody or the conjugate first, or at the same time, and whether B. /P separation may be performed as necessary. However, if the immobilized antibody is reacted with the analyte, and after B/P separation, the solid phase is reacted with the conjugate.
Using a stepwise method is the preferred method because it allows the sensitivity of the label to be determined. However, if the performance of the label is good, sensitive measurements can be performed with just one B/P separation after reacting the conjugate, analyte, and immobilized antibody.
［発明の効果コ
本発明によって非標識のトリヨードサイロニン、サイロ
キシンやそれらの抗体を用いることなく、抗サイロキシ
ン結合グロブリン抗体を用いて、これまで報告された方
法よりも簡単、明瞭な分析系で安全かつ容易にトリヨー
ドサイロニン摂取率を測定できる。[Effects of the Invention] The present invention uses an anti-thyroxine-binding globulin antibody without using unlabeled triiodothyronine, thyroxine or their antibodies, and with a simpler and clearer analysis system than previously reported methods. Triiodothyronine intake rate can be measured safely and easily.
［実施例コ
以下の実施例において本発明を更に詳細に説明する。な
お本発明は下記実施例に限定されるものではない。また
％は重量％を示す。トリヨードサイロニンは以下Ｔ３と
記す。[Example] The present invention will be explained in further detail in the following example. Note that the present invention is not limited to the following examples. Moreover, % indicates weight %. Triiodothyronine is hereinafter referred to as T3.
・Ｔ３−アルカリ性ホスファターゼコンジュゲートの作
製
Ｔ３を塩化チオニル存在下でメタノールと反応させ、メ
チル化Ｔ３塩酸塩を生成させる。これをピリジン存在下
のジオキサン中で無水グルタル酸と反応させ、アミノ基
にヘミグルタレート基を導入した。このうちのカルボキ
シル基をジシクロへキシルカルボジイミド存在下でＮ−
ハイドロキシサクシイミドと反応させ、活性化エステル
を合成し、これをアルカリ性ホスファターゼとコンシュ
ゲイジョンしてＴ３−アルカリ性ホスファターゼ溶液と
して用いた。- Preparation of T3-alkaline phosphatase conjugate T3 is reacted with methanol in the presence of thionyl chloride to produce methylated T3 hydrochloride. This was reacted with glutaric anhydride in dioxane in the presence of pyridine to introduce hemiglutarate groups into the amino groups. Among these, the carboxyl group was converted to N- in the presence of dicyclohexylcarbodiimide.
An activated ester was synthesized by reacting with hydroxysuccinimide, and this was consugated with alkaline phosphatase and used as a T3-alkaline phosphatase solution.
・抗サイロキシン結合グロブリン抗体固相化担体の調製
吸着法でポリスチレンビーズ（φ１．５−１．８ ｍｍ
）に対して、常法によって作製した抗サイロキシン結合
グロブリンモノクローナル抗体１．０−１０．０μｇを
結合させ、これを抗体同相化担体として用いた。・Preparation of antithyroxine-binding globulin antibody immobilized carrier using polystyrene beads (φ1.5-1.8 mm) by adsorption method.
) was bound with 1.0-10.0 μg of an anti-thyroxine-binding globulin monoclonal antibody prepared by a conventional method, and used as an antibody homogenization carrier.
ここで用いる抗サイロキシン結合グロブリンモノクロー
ナル抗体は、サイロキシン結合グロブリン認識部位を、
Ｔ３結合部位の近傍に持たないものであった。The anti-thyroxine-binding globulin monoclonal antibody used here has a thyroxine-binding globulin recognition site,
It did not have it near the T3 binding site.
・Ｔ３−摂取率標準液の調製
ヒトサイロキシン結合グロブリン（ＲＩＡ社製）溶液を
ＰＢＳ緩衝液に順次添加することにより、濃度０ 、５
、１０．２０．４０．５０ｕ ｇ／ｍｌの溶液を調製
して各々Ｔ３−摂取率０．１０．２０．４０．８０．１
００％の溶液とした。これらをＴ３−摂取率標準液とし
て用いた。・Preparation of T3-uptake rate standard solution By sequentially adding human thyroxine binding globulin (manufactured by RIA) solution to PBS buffer, concentrations of 0, 5
, 10.20.40.50 u g/ml solutions were prepared and the T3-uptake rate was 0.10.20.40.80.1 respectively.
00% solution. These were used as T3-uptake standard solutions.
・Ｔ３−摂取率測定方法
１２個の抗体固相化ビーズを試験管にとった。次に各試
験管にＴ３−摂取率標準液及び血清検体の各１５０μｍ
を加えて３７℃で１時間反応させた。次いで０．２％の
Ｔｖｅｅｎ２０を含むＰＢＳ緩衝液で反応を止め、これ
で３回洗浄を行なった。次いで１０％ゼラチンを含む１
５０ＩＩＩＭトリスー塩酸緩衝液（ｐＨ８，ｆｌｉ）で
希釈した０、０１％コンジュゲートを各試験管に１００
μｍを加えて３７℃で１時間反応させた。次いで０．２
％のＴｖｅｅｎ２０を含むＰＢＳｗｉ街液で反応を止め
、これで３回洗浄を行い洗浄液を吸引した。次いで１ｍ
Ｍ４−メチルウンベリフェリルホスフェート１００μｌ
を加え３７℃で１０分間酵素反応を行なった。- T3-uptake rate measurement method Twelve antibody-immobilized beads were placed in a test tube. Next, add 150 μm each of T3-uptake standard solution and serum sample to each test tube.
was added and reacted at 37°C for 1 hour. Next, the reaction was stopped with a PBS buffer containing 0.2% Tveen20, and the plate was washed three times with this buffer. Then 1 containing 10% gelatin
0.01% conjugate diluted in 50IIIM Tris-HCl buffer (pH 8, fli) was added to each test tube.
μm was added and reacted at 37° C. for 1 hour. then 0.2
The reaction was stopped with PBSwi street solution containing % Tveen20, and washing was performed three times with this solution, and the washing solution was aspirated. then 1m
M4-methylumbelliferyl phosphate 100μl
was added and the enzymatic reaction was carried out at 37°C for 10 minutes.
停止液としてＯ，ＬＭ Ｅ Ｄ Ｔ Ａを含むＯＡ４
Ｍリン酸水索二カリウムを５００μｌ加えこの反応液を
停止液で３０倍に希釈して生成した４−メチルウンベリ
フェロンの蛍光強度をΔＩＩＩ定した。図１にその結果
（検量線）を示した。また表１に、本実施例と同様の方
法でｌｐｊ定した、甲状腺機能亢進症患者、甲状腺機能
低下症患者及び正常者のＴ３−摂取率（％）を示した。OA4 containing O, LMEDTA as stop liquid
500 μl of dipotassium M phosphate was added and the reaction solution was diluted 30 times with a stop solution, and the fluorescence intensity of the produced 4-methylumbelliferone was determined by ΔIII. The results (calibration curve) are shown in FIG. Table 1 also shows the T3-intake rate (%) of hyperthyroid patients, hypothyroid patients, and normal subjects, determined by lpj using the same method as in this example.
表１Table 1
図１は、実施例で測定したＴ３摂取率と蛍光強度の関係
を表した検量線を示す図である。FIG. 1 is a diagram showing a calibration curve showing the relationship between T3 uptake rate and fluorescence intensity measured in Examples.
Claims (3)
ン結合グロブリンと反応しうるトリヨードサイロニン標
識結合体を用いるトリヨードサイロニン摂取率免疫測定
法。(1) Triiodothyronine uptake immunoassay using an anti-thyroxine-binding globulin antibody and a triiodothyronine-labeled conjugate that can react with thyroxine-binding globulin.
（ I ）に示すものを用いる特許請求の範囲第１項記載
の測定法。 ▲数式、化学式、表等があります▼（ I ） （ｎ＝２〜５）(2) The measuring method according to claim 1, in which a spacer represented by formula (I) is used as a spacer when synthesizing the labeled conjugate. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (n=2-5)
ロキシン結合グロブリン認識部位をトリヨードサイロニ
ン結合部位の近傍に持たないモノクローナル抗体である
特許請求の範囲第１項記載の測定法。(3) The assay method according to claim 1, wherein the anti-thyroxine-binding globulin antibody is a monoclonal antibody that does not have a thyroxine-binding globulin recognition site in the vicinity of the triiodothyronine-binding site.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5823288A JPH01232263A (en) | 1988-03-14 | 1988-03-14 | Immunoassay of triiodothyronine ingestion rate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5823288A JPH01232263A (en) | 1988-03-14 | 1988-03-14 | Immunoassay of triiodothyronine ingestion rate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01232263A true JPH01232263A (en) | 1989-09-18 |
Family
ID=13078344
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5823288A Pending JPH01232263A (en) | 1988-03-14 | 1988-03-14 | Immunoassay of triiodothyronine ingestion rate |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01232263A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01305358A (en) * | 1988-04-15 | 1989-12-08 | Boehringer Mannheim Gmbh | Method of thyroxine bonding capacity and reference solution ideal therefor |
| JPH01305359A (en) * | 1988-04-15 | 1989-12-08 | Boehringer Mannheim Gmbh | Method and reference liquid for measuring thyroxine or triiodothyronine in serum |
-
1988
- 1988-03-14 JP JP5823288A patent/JPH01232263A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01305358A (en) * | 1988-04-15 | 1989-12-08 | Boehringer Mannheim Gmbh | Method of thyroxine bonding capacity and reference solution ideal therefor |
| JPH01305359A (en) * | 1988-04-15 | 1989-12-08 | Boehringer Mannheim Gmbh | Method and reference liquid for measuring thyroxine or triiodothyronine in serum |
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