JPS5945103B2 - Method for producing anti-ursodeoxycholic acid antibody and detection reagent comprising the same - Google Patents
Method for producing anti-ursodeoxycholic acid antibody and detection reagent comprising the sameInfo
- Publication number
- JPS5945103B2 JPS5945103B2 JP3846877A JP3846877A JPS5945103B2 JP S5945103 B2 JPS5945103 B2 JP S5945103B2 JP 3846877 A JP3846877 A JP 3846877A JP 3846877 A JP3846877 A JP 3846877A JP S5945103 B2 JPS5945103 B2 JP S5945103B2
- Authority
- JP
- Japan
- Prior art keywords
- ursodeoxycholic acid
- acid
- ursodeoxycholic
- acid antibody
- antibody
- 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
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
【発明の詳細な説明】
本発明は抗ウルソデオキシコール酸抗体の製造方法およ
びそれからなるウルソデオキシコール酸一の検出用試薬
に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an anti-ursodeoxycholic acid antibody and a reagent for detecting ursodeoxycholic acid comprising the same.
最近、肝機能の診断方法として、グリココール酸、ケノ
デオキシコール酸などを負荷して一定時間後、血中のそ
の胆汁酸濃度を測定する方法が報告されている。Recently, as a method for diagnosing liver function, a method has been reported in which glycocholic acid, chenodeoxycholic acid, etc. are loaded and the bile acid concentration in the blood is measured after a certain period of time.
つまり肝機能が正常であると負荷した胆汁酸が肝臓で処
理される結果、血中濃度の上昇は少ないかまたはすみや
かに低下するが、肝機能が低下していると上昇の程度が
強く持続する現象を利用するものである。しかし、グリ
ココール酸、ケノデオキシコール酸の場合、その抗体が
生体内に存在する異種胆汁酸と反応する事と、その胆汁
酸自体が生体内にかなり存在するため、抗体を用いて血
中の対応する胆汁酸濃度を測定した結果は、負荷した胆
汁酸がそのまま血中濃度へ反映したものとはいえない。
したがつて肝機能診断の精度の点で問題がある。そこで
、本発明者らは、生体内にほとんど存在しない胆汁酸の
ウルソデオキシコール酸を選び、特異性の高いその抗体
の製造を試みた。In other words, when liver function is normal, the loaded bile acids are processed by the liver, resulting in a small increase in blood concentration or a rapid decrease, but when liver function is impaired, the increase is strong and persists. It utilizes phenomena. However, in the case of glycocholic acid and chenodeoxycholic acid, the antibodies react with different types of bile acids present in the body, and since the bile acids themselves are present in large quantities in the body, antibodies are used to detect the corresponding substances in the blood. The results of measuring bile acid concentrations cannot be said to reflect the loaded bile acids directly in the blood concentration.
Therefore, there is a problem with the accuracy of liver function diagnosis. Therefore, the present inventors selected ursodeoxycholic acid, a bile acid that hardly exists in living organisms, and attempted to produce highly specific antibodies for the bile acid.
本発明は、ウルソデオキシコール酸とウシ血清アルブミ
ンの結合物で人以外の哺乳動物を免疫し、抗血清を採取
することにより抗ウルソデオキシコール酸抗体を製造す
る方法である。The present invention is a method for producing anti-ursodeoxycholic acid antibodies by immunizing a non-human mammal with a conjugate of ursodeoxycholic acid and bovine serum albumin and collecting antiserum.
ウルソデオキコール酸とウシ血清アルブミンの結合物は
、例えば、トリーｎ−ブチルアミンおよびイソブチルク
ロロホルメートの存在下、両者を混合する事により得ら
れる。A conjugate of ursodeoxycholic acid and bovine serum albumin can be obtained, for example, by mixing the two in the presence of tri-n-butylamine and isobutyl chloroformate.
免疫動物としては、ウサギ、モルモツト、山羊など人以
外の哺乳動物が用いられる。As the immunized animal, mammals other than humans such as rabbits, guinea pigs, and goats are used.
免疫する際には、フロインド完全アジバントを抗原と混
合したもので免疫する事、および免疫は１回だけでなく
、数回追加免疫する事が抗体価の高い抗血清を得る上で
好ましい。本発明で得られた抗ウルソデオキシコール酸
抗体は特異性が高く、ウルソデオキシコール酸、および
これが生体内で変換される可能性のあるグリコウルソデ
オキシコール酸タウロウルソデオキシコール酸とは１０
０％反応するが、生体内に存在する異種胆汁酸とはほと
んど反応しない。When immunizing, it is preferable to immunize with Freund's complete adjuvant mixed with the antigen, and to immunize not only once but several times in order to obtain an antiserum with a high antibody titer. The anti-ursodeoxycholic acid antibody obtained in the present invention has high specificity, and is highly specific for ursodeoxycholic acid and glycoursodeoxycholic acid and tauroursodeoxycholic acid, which can be converted in vivo.
0% reaction, but almost no reaction with foreign bile acids present in the body.
この特異性は、グリココール酸、ケノデオキシコール酸
に対する抗血清の特異性とは比較にならない程高いもの
である。この事は生体が７α−ＯＨと７β−ＯＨを区別
して認識している事を示している。したがつて、ウルソ
デオキシコール酸を負荷し、一・定時間後の血中濃度を
、ＲＩＡ法、ＥＬＩＳＡ法などの免疫学的検出方法を採
用し、本発明の抗ウルソデオキシコール酸抗体を試薬と
して用いて測定すれば、肝機能の診断をより正確に行な
う事ができる。また、本発明の抗ウルソデオキシコール
酸抗体からなる試薬は、上記の肝機能診断以外にも、種
々のサンプル中のウルソデオキシコール酸を免疫学的検
出方法を用いて検出する場合にも用いる事ができる。次
に実施例を示し、本発明をさらに詳しく説明する。This specificity is incomparably higher than that of antisera against glycocholic acid and chenodeoxycholic acid. This indicates that living organisms recognize 7α-OH and 7β-OH separately. Therefore, ursodeoxycholic acid is loaded and the blood concentration is measured after a certain period of time using an immunological detection method such as RIA method or ELISA method, and the anti-ursodeoxycholic acid antibody of the present invention is used as a reagent. If used as a measurement method, liver function can be diagnosed more accurately. In addition, the reagent comprising the anti-ursodeoxycholic acid antibody of the present invention can be used not only for the above liver function diagnosis but also for detecting ursodeoxycholic acid in various samples using immunological detection methods. Can be done. EXAMPLES Next, the present invention will be explained in more detail with reference to Examples.
実施例 １
ウルソデオキシコール酸（３９２Ｔ１１ｆ）のジオキサ
ン溶液２０ｄを４℃に冷やし、この溶液にトリ一ｎ−ブ
チルアミン２０３ｍｇおよびイソブチルクロロホルメー
ト１８０ηを加えて３０分間撹拌した。Example 1 20d of a dioxane solution of ursodeoxycholic acid (392T11f) was cooled to 4°C, and 203mg of tri-n-butylamine and 180η of isobutyl chloroformate were added to this solution and stirred for 30 minutes.
この反応液にウシ血清アルブミン１．４９を溶かしたジ
オキサン−水（１：１）の混合液（６０ｄ）を滴下、つ
いで１Ｎ一水酸化ナトリウム溶液２Ｔ！１ｔを加えて撹
拌、さらに１時間後上記水酸化ナ[■■た。減圧下にジ
オキサンを留去後、水１００ｄを加え、得られた懸濁液
をセルロースチユーブを用いて２昼夜流水中で透析を行
なつた。透析後、熱２２０ｍ１，をそのまま真空凍結乾
燥し、ウルソデオ ニキシコール酸とウシ血清アルブミ
ンの結合物を白色粉末として１．１ｆ！得た。得られた
結合物をリン酸緩衝液に溶解して０．８Ｗｉ／ｄとし、
これにフロインド完全アジバントを等量混合した。A mixed solution (60d) of dioxane and water (1:1) in which 1.49% of bovine serum albumin was dissolved was added dropwise to this reaction solution, and then 2T of 1N sodium monohydroxide solution was added! After 1 hour, the above sodium hydroxide was added. After dioxane was distilled off under reduced pressure, 100 d of water was added, and the resulting suspension was dialyzed in running water for 2 days and nights using a cellulose tube. After dialysis, 220ml of heat was directly vacuum freeze-dried to obtain 1.1f white powder of a combination of ursodeonyxicolic acid and bovine serum albumin. Obtained. The obtained conjugate was dissolved in phosphate buffer to give a concentration of 0.8 Wi/d,
To this was mixed an equal amount of Freund's complete adjuvant.
この液を０．２ｍ１ずつウサギの背中、４手、足の各２
ケ所、計６ケ所に注射して免疫した。さらに２週間毎に
同様の方法で追加免疫した。免疫開始後４ケ月目に採血
して抗血清を分離し、抗ウルソデオキシコール酸抗体を
得た。得られた抗体の特異性は極めて高く、ウルソデオ
キシコール酸、グリコウルソデオキシコール酸タウロウ
ルソデオキシコール酸とは１００％反応するが、リトコ
ール酸とは０．２％、グリコケノデオキシコール酸、タ
ウロケンデオキシコール酸およびケノデオキシコール酸
とは０．１％以下の極めてわずかな交叉反応しか認めら
れなかつた。Apply 0.2ml of this solution to the rabbit's back, 4 hands, and 2 feet each.
Immunization was achieved by injecting at six locations in total. Furthermore, booster immunizations were given in the same manner every two weeks. Four months after the start of immunization, blood was collected, antiserum was separated, and anti-ursodeoxycholic acid antibody was obtained. The specificity of the obtained antibody is extremely high; it reacts 100% with ursodeoxycholic acid, glycoursodeoxycholic acid, and tauroursodeoxycholic acid, but only reacts with lithocholic acid at 0.2%, glycochenodeoxycholic acid, and taurochendeoxycholic acid. Only very slight cross-reactivity of 0.1% or less with cholic acid and chenodeoxycholic acid was observed.
また、その他の生体内に存在する胆汁酸とは、４μ９に
至るまで全く反応しなかつた。本発明の抗ウルソデオキ
シコール酸抗体を試薬として用いてＲＩＡを行なつた。Furthermore, it did not react at all with other bile acids present in living organisms up to 4μ9. RIA was performed using the anti-ursodeoxycholic acid antibody of the present invention as a reagent.
アツセイ系は、被検血清、標識抗原〔１１，１２−３田
一ウルソデオキシコール酸、抗ウルソデオキシコール酸
抗体（１６０倍希釈）、胆汁酸を含まない血清各０．１
Ｔ１１１と０．０１Ｍリン酸緩衝液（ＰＨ７．４）０．
６ｄからなり、４２℃、６０分間インキユベーシヨンし
た後、遊離型と結合型はポリエチレングリコール法で分
離し、上清（遊離型）を液体シンチレーシヨンカウンタ
一にて測定し、遊離型％を求めさらに結合型％を算出し
た。図１に示すように、ウルソデオキシコール酸１０〜
２００ピコモルで極めて良好な標準曲線が得られた。本
方法により、血中のウルソデオキシコール酸の濃度を測
定した。ウルソデオキシコール酸２５Ｗ９を経口投与し
て３０分後の血中ウルソデオキシコール酸濃度の上昇は
、正常人で約２００〜１，４００Ｐモル／Ｍｌｌ慢性肝
炎患者で約８００〜４，６００Ｐモル／Ｍｌｌ肝硬変患
者で約１，７００〜４，６００Ｐモル／ｄであつた。し
たがつて肝機能の診断にこの方法を用いる事ができる。The assay system consists of test serum, labeled antigen [11, 12-3 Taichi ursodeoxycholic acid, anti-ursodeoxycholic acid antibody (160-fold dilution), and bile acid-free serum at 0.1% each.
T111 and 0.01M phosphate buffer (PH7.4) 0.
After incubation at 42°C for 60 minutes, the free form and bound form were separated using the polyethylene glycol method, and the supernatant (free form) was measured using a liquid scintillation counter to determine the percentage of free form. Furthermore, the binding type % was calculated. As shown in Figure 1, ursodeoxycholic acid 10~
A very good standard curve was obtained at 200 pmol. By this method, the concentration of ursodeoxycholic acid in blood was measured. Thirty minutes after oral administration of ursodeoxycholic acid 25W9, the increase in blood ursodeoxycholic acid concentration is approximately 200 to 1,400 Pmol/Mll in normal people and approximately 800 to 4,600 Pmol/Mll in chronic hepatitis patients. In patients with liver cirrhosis, it was approximately 1,700 to 4,600 Pmol/d. Therefore, this method can be used to diagnose liver function.
図１はＲＩＡ法における、ウルソデオキシコール酸と〔
１１，１２−３Ｈ〕−ウルソデオキシコール酸結合型％
との標準曲線を示す。Figure 1 shows ursodeoxycholic acid and [
11,12-3H]-ursodeoxycholic acid bond type%
The standard curve is shown.
Claims (1)
合物で人以外の哺乳動物を免疫し、抗血清を採取する事
を特徴とする抗ウルソデオキシコール酸抗体の製造方法
。 ２ 哺乳動物がウサギである特許請求の範囲第１項記載
の抗ウルソデオキシコール酸抗体の製造方法。 ３ 抗ウルソデオキシコール酸抗体からなるウルソデオ
キシコール酸の検出用試薬。[Scope of Claims] 1. A method for producing an anti-ursodeoxycholic acid antibody, which comprises immunizing a mammal other than a human with a conjugate of ursodeoxycholic acid and bovine serum albumin, and collecting antiserum. 2. The method for producing an anti-ursodeoxycholic acid antibody according to claim 1, wherein the mammal is a rabbit. 3. A reagent for detecting ursodeoxycholic acid comprising an anti-ursodeoxycholic acid antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3846877A JPS5945103B2 (en) | 1977-04-06 | 1977-04-06 | Method for producing anti-ursodeoxycholic acid antibody and detection reagent comprising the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3846877A JPS5945103B2 (en) | 1977-04-06 | 1977-04-06 | Method for producing anti-ursodeoxycholic acid antibody and detection reagent comprising the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS53124613A JPS53124613A (en) | 1978-10-31 |
| JPS5945103B2 true JPS5945103B2 (en) | 1984-11-02 |
Family
ID=12526066
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3846877A Expired JPS5945103B2 (en) | 1977-04-06 | 1977-04-06 | Method for producing anti-ursodeoxycholic acid antibody and detection reagent comprising the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5945103B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61175005U (en) * | 1985-04-22 | 1986-10-31 |
-
1977
- 1977-04-06 JP JP3846877A patent/JPS5945103B2/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61175005U (en) * | 1985-04-22 | 1986-10-31 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS53124613A (en) | 1978-10-31 |
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