JP3147992B2 - Labeling reagent for immunoassay - Google Patents
Labeling reagent for immunoassayInfo
- Publication number
- JP3147992B2 JP3147992B2 JP13891192A JP13891192A JP3147992B2 JP 3147992 B2 JP3147992 B2 JP 3147992B2 JP 13891192 A JP13891192 A JP 13891192A JP 13891192 A JP13891192 A JP 13891192A JP 3147992 B2 JP3147992 B2 JP 3147992B2
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- JP
- Japan
- Prior art keywords
- labeling reagent
- aminoglycan
- modified
- chitosan
- solution
- 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.)
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Description
【0001】[0001]
【産業上の利用分野】本発明は、免疫測定法による生理
活性物質の測定に使用される標識試薬に関し、アミノ基
の20〜80%が飽和脂肪酸でアシル化されているアミ
ノグリカンを用いることにより、標識試薬の凍結保存が
可能となった標識試薬に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a labeling reagent used for the measurement of a physiologically active substance by an immunoassay, and uses an aminoglycan in which 20 to 80% of amino groups are acylated with a saturated fatty acid. The present invention relates to a labeling reagent capable of cryopreservation of the labeling reagent.
【0002】[0002]
【従来の技術】従来、免疫測定法に用いる標識試薬の高
感度化を図る手段としては、複数の反応活性基を有し、
その大部分の反応活性基が多数の標識物質(酵素、色
素、発光物質等)で修飾された化合物を用いることによ
って、免疫物質1分子当りの標識量を増加させるため
に、本発明者は特願平2−506243号において複数
のアミノ基を有するアミノグリカンを提案した。2. Description of the Related Art Conventionally, as a means for increasing the sensitivity of a labeling reagent used in an immunoassay, a method having a plurality of reactive groups,
By using a compound in which most of the reactive groups are modified with a large number of labeling substances (enzymes, dyes, luminescent substances, etc.), the present inventor has specially attempted to increase the amount of labeling per molecule of immunological substance. No. 2,506,243 proposed an aminoglycan having a plurality of amino groups.
【0003】[0003]
【発明が解決しようとする課題】アミノグリカンは、入
手の容易さからキトサンが一般に用いられるが、市販の
キトサンは甲殻類から抽出したキチンを酸により処理し
て製造され、そのアミノ基の10%未満がアセチル化さ
れている。このようなキトサンは水に対する溶解度が低
いため、これを用いた標識試薬を凍結あるいは凍結乾燥
して保存し、使用時に解凍・溶解した場合、その一部が
不溶のまま残存し、免疫測定時の感度や再現性が低下す
るという問題点があった。As aminoglycan, chitosan is generally used because of its easy availability. Commercially available chitosan is produced by treating chitin extracted from crustaceans with an acid, and 10% of the amino group is obtained. Are less than acetylated. Since such chitosan has a low solubility in water, the labeling reagent using the chitosan is frozen or freeze-dried and stored, and when thawed and dissolved at the time of use, a part of the chitosan remains insoluble and remains undissolved during immunoassay. There is a problem that sensitivity and reproducibility are reduced.
【0004】[0004]
【課題を解決するための手段】本発明者は、キトサンな
どのアミノグリカンのアミノ基の20〜80%をアシル
化し、これを用いて製造された標識試薬は、凍結保存や
凍結乾燥保存ができることを見い出した。本発明は、免
疫物質と結合し、標識体で修飾されたアミノグリカンが
アミノ基の20〜80%が飽和脂肪酸でアシル化されて
いるアミノグリカンである免疫測定用標識試薬である。Means for Solving the Problems The present inventors acylate 20 to 80% of the amino groups of aminoglycans such as chitosan and the like, and the labeling reagent produced using the acylates can be stored frozen or lyophilized. I found The present invention is a labeling reagent for immunoassay, wherein the aminoglycan modified with a label and bound to an immunological substance is an aminoglycan in which 20 to 80% of amino groups are acylated with a saturated fatty acid.
【0005】以下、本発明の標識試薬の製造法を説明す
る。 分子量が104 〜5×106 、好ましくは105 〜1
06 であるキトサン、ポリガラクトサミンのようなアミ
ノグリカンを、炭素数1〜6の飽和脂肪酸の水溶液に溶
解し、縮合剤を加えて反応させ、アミノ基の20〜80
%をアシル化し、これを透析又はゲル濾過等により精製
する。該アシル化度は、ケイ光分光光度計により励起波
長225nmにおける460nmの蛍光を測定することによ
り、分子量及び濃度既知のアミノグリカンを標準とし
て、概算して求めることができる。また、該脂肪酸と縮
合剤の組合わせの代わりに、該脂肪酸の無水物を用いて
もよい。Hereinafter, a method for producing the labeling reagent of the present invention will be described. The molecular weight is 10 4 to 5 × 10 6 , preferably 10 5 to 1
0 6 chitosan is, the aminoglycan such as polygalactosamine, dissolved in an aqueous solution of saturated fatty acids having 1 to 6 carbon atoms, is reacted by addition of a condensing agent, 20 to 80 of the amino group
%, Which is purified by dialysis or gel filtration. The degree of acylation can be roughly determined by measuring fluorescence at 460 nm at an excitation wavelength of 225 nm with a fluorescence spectrophotometer, using aminoglycans of known molecular weight and concentration as standards. Further, instead of the combination of the fatty acid and the condensing agent, an anhydride of the fatty acid may be used.
【0006】上記によりアシル化されたアミノグリ
カンに、色素、酵素等の標識体を混合し、縮合剤を加え
て反応させ、アシル化されていない残余のアミノ基に標
識体を反応させることにより、標識体で修飾されたアミ
ノグリカンを得る。標識体の色素としては、フルオレセ
イン、ローダミン類、クマリン系色素及びシアニン色素
(例えば日本感光色素研究所製NK1160)等が用い
られる。酵素としては、ペルオキシダーゼ、ルシフェラ
ーゼ等の酸化還元酵素、又はホスファターゼ、グルコシ
ダーゼもしくはエステラーゼ等の加水分解酵素等が用い
られる。酵素の基質としては、酵素反応により発色又は
蛍光を発する物質の他、酵素反応により発光する物質、
例えばペルオキシダーゼに対してはルミノール、ルシフ
ェラーゼに対してはルシフェリン及びホスファターゼに
対してはAPPDMが用いられる。[0006] By mixing a labeled substance such as a dye or an enzyme with the aminoglycan acylated as described above, adding a condensing agent and reacting the mixture, and reacting the labeled substance with the remaining amino group which is not acylated, An aminoglycan modified with a label is obtained. Fluorescein, rhodamines, coumarin-based dyes and cyanine dyes (for example, NK1160 manufactured by Japan Photographic Dye Laboratories) and the like are used as the dyes for the label. As the enzyme, an oxidoreductase such as peroxidase or luciferase, or a hydrolase such as phosphatase, glucosidase or esterase is used. As a substrate for the enzyme, other than a substance that emits color or fluorescence by the enzyme reaction, a substance that emits light by the enzyme reaction,
For example, luminol is used for peroxidase, luciferin for luciferase, and APPDM for phosphatase.
【0007】標識体で修飾されたアミノグリカンに、
免疫物質(抗原又は抗体)を縮合剤と共に加えて反応さ
せて標識試薬を得る。免疫物質は、標識もしくはアシル
化されていないアミノ基に結合させる。上記、、
で用いられる縮合剤としては、カルボジイミド類、N−
ブロモスクシイミド等を用いることができる。また、標
識体で修飾されたアミノグリカンが、ビオチンとアビジ
ンを介して複数の標識体で修飾されたものを用いれば、
免疫物質1分子当りの標識量を増加させることができ、
好ましい。そのような標識試薬は、前記の工程におい
て、標識体を反応させる代わりにビオチンを反応させ
て、残余のアミノ基にビオチンを結合させ、前記の工
程後、標識体で修飾されたアビジンを混合して、例えば
式(I)のような標識試薬を得る。[0007] The aminoglycan modified with a label,
An immunological substance (antigen or antibody) is added together with a condensing agent and reacted to obtain a labeling reagent. The immunizing agent is attached to a non-labeled or non-acylated amino group. the above,,
As the condensing agent used in the above, carbodiimides, N-
Bromosuccinimide or the like can be used. In addition, if the aminoglycan modified with a label is modified with a plurality of labels via biotin and avidin,
It is possible to increase the amount of label per molecule of the immune substance,
preferable. Such a labeling reagent is obtained by reacting biotin instead of reacting the label in the above-mentioned step to bind biotin to the remaining amino group, and mixing avidin modified with the label after the above-mentioned step. Thus, for example, a labeling reagent of the formula (I) is obtained.
【0008】[0008]
【化1】 Embedded image
【0009】さらに、ビオチン+アミノグリカン+免疫
物質と、標識体で修飾されたアビジンとは結合していな
くてもよい。このような場合、ビオチン+アミノグリカ
ン+免疫物質(抗原等)と光ファイバー上の免疫物質
(抗体)とを免疫反応させ、さらに標識体で修飾された
アビジンを反応させる。このように後から標識アビジン
を反応させることにより、免疫反応中に標識体が酸化さ
れたり加水分解されることを防止することができる。Furthermore, biotin + aminoglycan + immune substance and avidin modified with a label may not be bound. In such a case, biotin + aminoglycan + immune substance (antigen or the like) is immunoreacted with an immunological substance (antibody) on an optical fiber, and further, avidin modified with a label is reacted. Thus, by reacting the labeled avidin later, it is possible to prevent the labeled body from being oxidized or hydrolyzed during the immune reaction.
【0010】[0010]
【作用】アミノグリカン中の遊離のアミノ基は、水酸基
と水素結合して分子間で強固に結合している。そのた
め、アミノ基がプロトン化されにくいので、水に対して
難溶性となる。しかし、一部のアミノ基がアシル化され
ると、その箇所の水素結合が妨げられるため、プロトン
化されやすくなり結果的に水に溶解しやすくなる。一
方、アシル化が進み過ぎると遊離のアミノ基数が少なく
なり、再び水に対して難溶性となる。従って、アミノ基
の20〜80%、好ましくは25〜30%がアシル化さ
れたアミノグリカンを用いれば、凍結や凍結乾燥後も水
に可溶な標識試薬を作成するこができる。The free amino group in the aminoglycan forms a hydrogen bond with the hydroxyl group and is tightly bound between the molecules. Therefore, since the amino group is not easily protonated, it becomes sparingly soluble in water. However, when a part of the amino group is acylated, hydrogen bonding at the site is hindered, so that the amino group is easily protonated and consequently easily dissolved in water. On the other hand, if the acylation proceeds too much, the number of free amino groups decreases, and the compound becomes sparingly soluble in water again. Therefore, by using an aminoglycan in which 20 to 80%, preferably 25 to 30% of amino groups are acylated, a water-soluble labeling reagent can be prepared even after freezing or freeze-drying.
【0011】[0011]
【発明の効果】アミノ基の20〜80%をアシル化した
アミノグリカンを用いて製造された標識試薬は、凍結保
存又は凍結乾燥保存したものを再溶解したとき、沈殿を
生ずることがなく、このような標識試薬を用いることに
より、標識試薬の感度や信頼性が向上した。The labeling reagent produced by using aminoglycan in which 20 to 80% of the amino groups are acylated does not cause precipitation when the frozen or lyophilized one is redissolved. By using such a labeling reagent, the sensitivity and reliability of the labeling reagent were improved.
【0012】[0012]
実施例1 カルシトニンの測定:サンドイッチ法 (1)アセチル化度5%、分子量約106 のキトサン
を、10%酢酸に3mg/mlとなるように溶解した。次い
でこのキトサン溶液に水溶性カルボジイミドを60mg/
mlとなるように添加し、室温で6時間反応させた。この
反応混合物を蒸留水で透析し、アセチル化度約30%の
キトサン原液を得た。 (2)水100mlに炭酸ナトリウム4mgとビオチン10
mgを溶解した。このビオチン溶液に前記(1)のキトサ
ン原液2mlを混合し、さらに水溶性カルボジイミド10
0mgを添加して、一晩反応させた。次いで、これに0.
2 g/ml炭酸ナトリウムと0.1 g/mlの塩化ナトリウ
ムの混合液4mlを加えて、ビオチン化キトサンを沈殿さ
せた。遠心分離により沈殿を回収した後、この沈殿を
0.1 g/ml炭酸ナトリウムと0.3 g/ml塩化ナトリ
ウムの混合液で2回洗浄した。この沈殿を10mMリン酸
緩衝液(pH7)2mlに懸濁し、同緩衝液中で4℃、一晩
透析し、透析物をビオチン化キトサン懸濁液とした。Example 1 Measurement of Calcitonin: Sandwich Method (1) Chitosan having a degree of acetylation of 5% and a molecular weight of about 10 6 was dissolved in 10% acetic acid to a concentration of 3 mg / ml. Next, 60 mg / water-soluble carbodiimide was added to this chitosan solution.
ml, and reacted at room temperature for 6 hours. This reaction mixture was dialyzed against distilled water to obtain a chitosan stock solution having an acetylation degree of about 30%. (2) 4 mg of sodium carbonate and 10 of biotin in 100 ml of water
mg was dissolved. To this biotin solution, 2 ml of the chitosan stock solution of the above (1) was mixed, and then water-soluble carbodiimide 10
0 mg was added and reacted overnight. Then, add 0.
Biotinylated chitosan was precipitated by adding 4 ml of a mixture of 2 g / ml sodium carbonate and 0.1 g / ml sodium chloride. After collecting the precipitate by centrifugation, the precipitate was washed twice with a mixed solution of 0.1 g / ml sodium carbonate and 0.3 g / ml sodium chloride. This precipitate was suspended in 2 ml of 10 mM phosphate buffer (pH 7) and dialyzed in the same buffer at 4 ° C. overnight to give a dialyzed product as a biotinylated chitosan suspension.
【0013】(3)前記(2)のビオチン化キトサン懸
濁液に、ウサギ由来ヒトカルシトニン抗体100μg を
加え、さらに水溶性カルボジイミド10mgを添加して、
4℃で6時間反応させた。この反応混合物を、10mMリ
ン酸カリウム緩衝液(pH7.0)で一晩透析し、次いで
陰イオン交換カラムを用いて未反応物質を除去し、ヒト
カルシトニン抗体が結合したビオチン化キトサンを得
た。 (4)アビジン1mg及びトリエチルアミン0.2mlをエ
タノール1mlに溶解した溶液にシアニン色素NK116
0 2mgを加えて充分に溶解し、さらにジシクロヘキシ
ルカルボジイミド0.3mlを加えて、室温で一晩反応さ
せた。遠心分離により沈殿を回収した後、この沈殿をエ
タノールで2回洗浄し、さらに遠心分離により沈殿を回
収し、沈殿中に残っているエタノールを減圧除去した。
この残留物を20mM酢酸緩衝液(pH6.5)2mlに溶解
し、NK1160で修飾されたアビジンを得た。(3) 100 μg of rabbit-derived human calcitonin antibody is added to the biotinylated chitosan suspension of (2), and 10 mg of water-soluble carbodiimide is further added.
The reaction was performed at 4 ° C. for 6 hours. The reaction mixture was dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.0), and then unreacted substances were removed using an anion exchange column to obtain biotinylated chitosan to which a human calcitonin antibody was bound. (4) Cyanine dye NK116 was added to a solution of 1 mg of avidin and 0.2 ml of triethylamine in 1 ml of ethanol.
0 mg was added and dissolved sufficiently. Further, 0.3 ml of dicyclohexylcarbodiimide was added and reacted at room temperature overnight. After recovering the precipitate by centrifugation, the precipitate was washed twice with ethanol, and the precipitate was recovered by centrifugation, and the ethanol remaining in the precipitate was removed under reduced pressure.
This residue was dissolved in 2 ml of 20 mM acetate buffer (pH 6.5) to obtain NK1160-modified avidin.
【0014】(5)水0.5mlに硫酸ニッケル10mgを
溶解し、さらにエタノール2.5mlを加えた。この際生
じた沈殿を遠心分離により除去し、採取した上清をNi
−エタノール溶液とした。次に20mM水酸化カリウムの
エタノール溶液0.4mlに、Ni−エタノール溶液0.
1mlと50%グルタルアルデヒド50μl を添加し、反
応溶液とした。 (6)ポリメタクリル酸メチル樹脂製光ファイバー(三
菱レイヨン製)を3cmに切り、両端面をポリシングフィ
ルムで研磨した。この光ファイバーの片面を、前記
(5)の反応溶液に50℃で10分間浸漬した後、20
mM塩酸、次いでリン酸緩衝生理食塩水(PBS)で洗浄
した。次にこの光ファイバーを2mg/mlのヤギ由来カル
シトニン抗体溶液に浸漬し、4℃で一晩放置した。光フ
ァイバーを溶液から取り出し、1%NaBH4 水溶液に
15分間浸漬した後、PBSで洗浄して、ヤギ由来ヒト
カルシトニン抗体固定化センサーを作成し、これを検出
部とした。(5) 10 mg of nickel sulfate was dissolved in 0.5 ml of water, and 2.5 ml of ethanol was further added. The precipitate formed at this time was removed by centrifugation, and the collected supernatant was washed with Ni
-Ethanol solution. Next, Ni-ethanol solution was added to 0.4 ml of 20 mM potassium hydroxide in ethanol solution.
1 ml and 50 μl of 50% glutaraldehyde were added to prepare a reaction solution. (6) An optical fiber made of polymethyl methacrylate resin (manufactured by Mitsubishi Rayon) was cut into 3 cm, and both end faces were polished with a polishing film. After immersing one side of this optical fiber in the reaction solution of the above (5) at 50 ° C. for 10 minutes,
Washed with mM hydrochloric acid followed by phosphate buffered saline (PBS). Next, this optical fiber was immersed in a 2 mg / ml goat-derived calcitonin antibody solution and left at 4 ° C. overnight. Removed fiber from the solution, was dipped for 15 minutes in 1% NaBH 4 solution, washed with PBS, and creates a goat human calcitonin antibody immobilized sensor was a detector this.
【0015】(7)前記(3)のウサギ由来ヒトカルシ
トニン抗体が結合したビオチン化キトサンと、前記
(4)のNK1160で修飾されたアビジンを混合して
標識試薬を得た。これを凍結乾燥して、−20℃で2週
間保存した (8)各濃度のカルシトニン溶液10μl に前記(6)
の検出部を浸漬し、室温に30分放置後、前記(7)の
凍結乾燥粉末を蒸留水に懸濁した溶液に30分浸漬し
た。検出部を0.2%トゥイーン20含有1M KSCN
水溶液で洗浄し、図1に示す装置にて蛍光強度を測定し
たところ、0.3ng/mlまで測定できた。これは、前記
(7)の標識試薬を凍結乾燥保存せずに、調製後直ちに
用いた場合と同じ感度であった。(7) The labeled reagent was obtained by mixing the biotinylated chitosan to which the rabbit-derived human calcitonin antibody of (3) was bound and the avidin modified with NK1160 of (4). This was freeze-dried and stored at −20 ° C. for 2 weeks. (8) 10 μl of each concentration of calcitonin solution was added to the above (6).
Was immersed in a solution obtained by suspending the freeze-dried powder of the above (7) in distilled water for 30 minutes. 1M KSCN containing 0.2% Tween 20
After washing with an aqueous solution and measuring the fluorescence intensity with the apparatus shown in FIG. 1, it was possible to measure up to 0.3 ng / ml. This was the same sensitivity as when the labeling reagent of (7) was used immediately after preparation without lyophilization and storage.
【0016】比較例1 (1)アセチル化処理を行わなかったキトサン(アセチ
ル化度5%)を用いた以外は、実施例1と同様の方法で
標識試薬を調製し、これを凍結乾燥して、−20℃で2
週間保存した。 (2)前記(1)の凍結乾燥標品を蒸留水に懸濁しても
溶解しないため、測定不能であった。Comparative Example 1 (1) A labeling reagent was prepared in the same manner as in Example 1 except that chitosan (acetylation degree: 5%) not subjected to acetylation treatment was used, and this was lyophilized. At -20 ° C
Saved for a week. (2) Even if the freeze-dried sample of (1) was suspended in distilled water, it did not dissolve, so that measurement was impossible.
【0017】実施例2 カルシトニンの測定:サンドイ
ッチ法 (1)アセチル化度1%、分子量約105 のポリガラク
トサミンを実施例1の(1)と同様の方法でアセチル化
した。生成物のアセチル化度は約40%であった。 (2)キトサンの代わりに、前記(1)のポリガラクト
サミンを用いた以外は、実施例1の(2)と同様の方法
で、ヒトカルシトニン抗体が結合したビオチン化ポリガ
ラクトサミンを得た。 (3)実施例1の(3)〜(7)と同様の方法を用い
て、NK1160で修飾された標識試薬及びヤギ由来ヒ
トカルシトニン抗体固定化センサーを作成した。 (4)実施例1の(8)と同様の方法で、ヒトカルシト
ニンを測定したところ、0.4ng/mlまで測定できた。Example 2 Measurement of Calcitonin: Sandwich Method (1) Polygalactosamine having a degree of acetylation of 1% and a molecular weight of about 10 5 was acetylated in the same manner as in Example 1 (1). The degree of acetylation of the product was about 40%. (2) A biotinylated polygalactosamine to which a human calcitonin antibody was bound was obtained in the same manner as in (2) of Example 1, except that the polygalactosamine of (1) was used instead of chitosan. (3) A labeling reagent modified with NK1160 and a goat-derived human calcitonin antibody-immobilized sensor were prepared using the same method as in (3) to (7) of Example 1. (4) When human calcitonin was measured in the same manner as in (8) of Example 1, it could be measured up to 0.4 ng / ml.
【0018】比較例2 (1)アセチル化処理を行わなかったポリガラクトサミ
ンキトサン(アセチル化度1%)を用いた以外は、実施
例2と同様の方法で標識試薬を調製し、これを凍結乾燥
して、−20℃で2週間保存した。 (2)前記(1)の凍結乾燥標品を蒸留水に懸濁しても
溶解しないため、測定不能であった。Comparative Example 2 (1) A labeling reagent was prepared in the same manner as in Example 2 except that polygalactosamine chitosan (acetylation degree: 1%) not subjected to acetylation treatment was used, and this was lyophilized. And stored at −20 ° C. for 2 weeks. (2) Even if the freeze-dried sample of (1) was suspended in distilled water, it did not dissolve, so that measurement was impossible.
【0019】実施例3 ヒト絨毛性性腺刺激ホルモン
(hCG)の測定:競合法 (1)アセチル化度5%、分子量約106 のキトサン
を、5%プロピオン酸に3mg/mlとなるように溶解し、
次いでこの溶液に水溶性カルボジイミド(CHMC)を
50mg/mlとなるように添加して、室温で6時間反応さ
せた。生成物のアシル化度は約25%であった。 (2)実施例1の(2)と同様の方法で、ビオチン化キ
トサン懸濁液を得た。 (3)ウサギ由来ヒトカルシトニン抗体100μg の代
わりに、ヒト絨毛性性腺刺激ホルモン(hCG)100
μg を加えた以外は、実施例1の(3)と同様の方法で
hCGが結合したビオチン化キトサンを得た。 (4)アビジン1mg及びトリエチルアミン0.2mlをエ
タノール1mlに溶解した溶液に、フルオレセインイソチ
オシアネート(フルオレセイン)1.8mgを溶解し、遮
光下、室温で6時間反応させた。遠心分離により沈殿を
回収した後、この沈殿をエタノールで2回洗浄し、さら
に遠心分離により沈殿を回収し、沈殿中のエタノールを
減圧除去した。この残留物を50mM酢酸緩衝液(pH5.
5)1mlに懸濁し、フルオレセインで修飾されたアビジ
ンを得た。Example 3 Measurement of Human Chorionic Gonadotropin (hCG): Competition Method (1) Chitosan having a degree of acetylation of 5% and a molecular weight of about 10 6 was dissolved in 5% propionic acid to a concentration of 3 mg / ml. And
Next, water-soluble carbodiimide (CHMC) was added to the solution at a concentration of 50 mg / ml and reacted at room temperature for 6 hours. The degree of acylation of the product was about 25%. (2) A biotinylated chitosan suspension was obtained in the same manner as in Example 1, (2). (3) Instead of rabbit calcitonin antibody 100 μg, human chorionic gonadotropin (hCG) 100
Except for adding μg, a biotinylated chitosan to which hCG was bound was obtained in the same manner as in Example 1, (3). (4) 1.8 mg of fluorescein isothiocyanate (fluorescein) was dissolved in a solution of 1 mg of avidin and 0.2 ml of triethylamine in 1 ml of ethanol, and reacted at room temperature for 6 hours under light shielding. After recovering the precipitate by centrifugation, the precipitate was washed twice with ethanol, and the precipitate was recovered by centrifugation, and the ethanol in the precipitate was removed under reduced pressure. This residue was treated with 50 mM acetate buffer (pH 5.
5) The suspension was suspended in 1 ml to obtain avidin modified with fluorescein.
【0020】(5)カルシトニン抗体の代わりにhCG
抗体を用いた以外は、実施例1の(5)及び(6)と同
様の方法でhCG抗体固定化センサーを作成し、これを
検出部とした。 (6)前記(3)のhCGが結合したビオチン化キトサ
ンと、前記(4)のフルオレセインで修飾されたアビジ
ンを混合し標識試薬を得た。これを−20℃で1週間凍
結保存した。 (7)各濃度のhCG溶液10μlに、前記(6)の凍
結保存品を解凍して一定量添加し、さらに前記(5)の
検出部を浸漬して室温に30分放置した。この検出部を
0.2%トゥイーン20含有PBSで洗浄し、次いで1
%炭酸ナトリウム溶液に浸漬し、図2に示す装置を用い
て780nmと1360nmの半導体レーザを照射して51
8nmの蛍光を測定したところ、0.4ng/mlまで測定で
きた。(5) hCG instead of calcitonin antibody
An hCG antibody-immobilized sensor was prepared in the same manner as in (5) and (6) of Example 1 except that an antibody was used, and this was used as a detection unit. (6) The biotinylated chitosan bound to hCG of (3) and avidin modified with fluorescein of (4) were mixed to obtain a labeling reagent. This was stored frozen at -20 ° C for one week. (7) The cryopreserved product of (6) was thawed and added in a fixed amount to 10 μl of the hCG solution at each concentration, and the detection section of (5) was immersed and left at room temperature for 30 minutes. The detection part was washed with PBS containing 0.2% Tween 20, and then washed with PBS.
% Sodium carbonate solution, and irradiated with a semiconductor laser of 780 nm and 1360 nm using the apparatus shown in FIG.
When the fluorescence at 8 nm was measured, it could be measured up to 0.4 ng / ml.
【0021】比較例3 (1)アシル化処理を行わなかったキトサン(アセチル
化度5%)を用いた以外は、実施例3と同様の方法で標
識試薬を調製し、これを−20℃で1週間凍結保存し
た。 (2)前記(1)の凍結保存標品を室温に20分放置し
て解凍したところ、一部不溶性の沈殿が生じたため、測
定値にばらつきが生じ、50ng/mlまでしか検出できな
かった。Comparative Example 3 (1) A labeling reagent was prepared in the same manner as in Example 3 except that chitosan (acetylation degree: 5%) not subjected to an acylation treatment was used. Stored frozen for one week. (2) When the cryopreserved sample of (1) was left at room temperature for 20 minutes and thawed, insoluble precipitates were partially formed, and the measured values fluctuated, and only up to 50 ng / ml could be detected.
【0022】実施例4 ヒト絨毛性性腺刺激ホルモン
(hCG)の測定:競合法 (1)実施例3の(1)〜(4)と同様の方法で、hC
Gが結合したビオチン化キトサン及びフルオレセインで
修飾されたアビジンを得た。 (2)前記(1)のhCGが結合したビオチン化キトサ
ン及びフルオレセインで修飾されたアビジンを、それぞ
れ別々に凍結乾燥して、−20℃で1カ月凍結保存し
た。 (3)実施例3の(5)と同様の方法でhCG抗体固定
化センサーを作成し、これを検出部とした。 (4)各濃度のhCG溶液10μl に、前記(2)のh
CGが結合したビオチン化キトサンの凍結乾燥標品を蒸
留水に溶解した溶液を一定量添加し、さらに前記(3)
の検出部を浸漬して室温に30分放置した。次いで、こ
の検出部を0.2%トゥイーン20含有PBSで洗浄
し、前記(2)のフルオレセインで修飾されたアビジン
の凍結乾燥標品を蒸留水に溶解した溶液に浸漬して、室
温に10分放置した。この検出部を0.2%トゥイーン
20含有PBSで洗浄し、次いで1%炭酸ナトリウム溶
液に浸漬し、図2に示す装置を用いて780nmと136
0nmの半導体レーザを照射して518nmの蛍光を測定し
たところ、0.4ng/mlまで測定できた。Example 4 Measurement of Human Chorionic Gonadotropin (hCG): Competition Method (1) In the same manner as in (3) to (4) of Example 3, hC
G-linked biotinylated chitosan and avidin modified with fluorescein were obtained. (2) The biotinylated chitosan bound to hCG and the avidin modified with fluorescein of (1) above were separately lyophilized and cryopreserved at -20 ° C for one month. (3) An hCG antibody-immobilized sensor was prepared in the same manner as in (3) of Example 3 and used as a detection unit. (4) Add 10 μl of the hCG solution of each concentration to the hCG of (2).
A fixed amount of a solution obtained by dissolving a freeze-dried preparation of biotinylated chitosan to which CG is bound in distilled water is added, and then the above (3)
Was immersed and left at room temperature for 30 minutes. Next, the detection portion was washed with PBS containing 0.2% Tween 20, and immersed in a solution of the freeze-dried sample of avidin modified with fluorescein (2) in distilled water, and allowed to stand at room temperature for 10 minutes. I left it. The detection part was washed with PBS containing 0.2% Tween 20, then immersed in 1% sodium carbonate solution, and 780 nm and 136 nm were measured using the apparatus shown in FIG.
Irradiation with a semiconductor laser of 0 nm and measurement of fluorescence at 518 nm showed that the measurement was possible up to 0.4 ng / ml.
【0023】比較例4 (1)アセチル化度5%、分子量約106 のキトサン
を、30%酢酸に3mg/mlとなるように溶解し、次いで
このキトサン溶液に水溶性カルボジイミドを20mg/ml
となるように添加し、さらに2時間おきに20mg/mlず
つ3回添加し、最終的に80mg/mlとして、室温で16
時間反応させた。この反応混合物を蒸留水に透析し、ア
セチル化度約85%のキトサン原液を得た。 (2)実施例1の(2)〜(7)と同様の方法で、NK
1160で修飾された標識試薬及びヤギ由来ヒトカルシ
トニン抗体固定化センサーを作成した。 (3)各濃度のカルシトニン溶液10μl に前記(2)
の検出部を浸漬し、室温に30分放置後、前記(2)の
凍結乾燥粉末を蒸留水に懸濁した溶液に30分浸漬し
た。このとき浸漬を開始してから10分ぐらいするとわ
ずかに沈殿が生じた。検出部を0.2%トゥイーン20
含有1M KSCN水溶液で洗浄し、図1に示す装置にて
蛍光強度を測定したところ、10mg/mlまでしか測定で
きなかった。Comparative Example 4 (1) Chitosan having a degree of acetylation of 5% and a molecular weight of about 10 6 was dissolved in 30% acetic acid so as to have a concentration of 3 mg / ml. Then, 20 mg / ml of a water-soluble carbodiimide was added to the chitosan solution.
, And 3 times 20 mg / ml every 2 hours to a final concentration of 80 mg / ml.
Allowed to react for hours. The reaction mixture was dialyzed against distilled water to obtain a chitosan stock solution having a degree of acetylation of about 85%. (2) In the same manner as in (2) to (7) of Example 1, NK
A labeling reagent modified with 1160 and a goat-derived human calcitonin antibody-immobilized sensor were prepared. (3) Add 10 μl of each concentration of calcitonin solution to (2)
Was immersed in a solution in which the freeze-dried powder of the above (2) was suspended in distilled water for 30 minutes. At this time, about 10 minutes after the start of immersion, a slight precipitation occurred. 0.2% Tween 20 detector
After washing with a 1M aqueous KSCN solution and measuring the fluorescence intensity with the apparatus shown in FIG. 1, only up to 10 mg / ml could be measured.
【図1】1個のレーザを使用する蛍光測定系FIG. 1 Fluorescence measurement system using one laser
【図2】2個のレーザを使用する蛍光測定系FIG. 2 Fluorescence measurement system using two lasers
【符合の説明】 1 光ファイバー 2 レーザ 3 光軸合わせのためのガイドレール 4 検出部 5 フィルター 6 蛍光検出器 7 ハーフミラー[Description of References] 1 Optical fiber 2 Laser 3 Guide rail for optical axis alignment 4 Detector 5 Filter 6 Fluorescence detector 7 Half mirror
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭63−258579(JP,A) 国際公開90/13029(WO,A1) (58)調査した分野(Int.Cl.7,DB名) G01N 33/48 - 33/98 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-63-258579 (JP, A) WO 90/13029 (WO, A1) (58) Fields investigated (Int. Cl. 7 , DB name) G01N 33/48-33/98
Claims (3)
アミノグリカンが、アミノ基の20〜80%が飽和脂肪
酸でアシル化されているアミノグリカンであり、凍結保
存又は凍結乾燥保存されることを特徴とする免疫測定用
標識試薬。1. An aminoglycan which binds to an immune substance and is modified with a label is an aminoglycan in which 20 to 80% of amino groups are acylated with a saturated fatty acid, and is stored frozen or freeze-dried. A labeling reagent for immunoassay, comprising:
が、ビオチンとアビジンを介して複数の標識体で修飾さ
れている請求項1記載の免疫測定用標識試薬。2. The labeling reagent for immunoassay according to claim 1, wherein the aminoglycan modified with the label is modified with a plurality of labels via biotin and avidin.
アミノグリカンと、標識体で修飾されたアビジンの組み
合わせからなる免疫測定用標識試薬であって、前記アミ
ノグリカンのアミノ基の20〜80%が飽和脂肪酸でア
シル化されており、凍結保存又は凍結乾燥保存されるこ
とを特徴とする免疫測定用標識試薬。3. An immunoassay labeling reagent comprising a combination of an aminoglycan modified with biotin, which binds to an immune substance, and avidin modified with a label, wherein the labeling reagent is 20 to 80% of the amino group of the aminoglycan. Is acylated with a saturated fatty acid, and is stored frozen or freeze-dried.
Priority Applications (1)
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JP13891192A JP3147992B2 (en) | 1992-05-29 | 1992-05-29 | Labeling reagent for immunoassay |
Applications Claiming Priority (1)
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JP13891192A JP3147992B2 (en) | 1992-05-29 | 1992-05-29 | Labeling reagent for immunoassay |
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JPH05333026A JPH05333026A (en) | 1993-12-17 |
JP3147992B2 true JP3147992B2 (en) | 2001-03-19 |
Family
ID=15233028
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JP13891192A Expired - Lifetime JP3147992B2 (en) | 1992-05-29 | 1992-05-29 | Labeling reagent for immunoassay |
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US6217848B1 (en) * | 1999-05-20 | 2001-04-17 | Mallinckrodt Inc. | Cyanine and indocyanine dye bioconjugates for biomedical applications |
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1992
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