JPH04182499A - Ecgonine-protein conjugate and derivative thereof - Google Patents
Ecgonine-protein conjugate and derivative thereofInfo
- Publication number
- JPH04182499A JPH04182499A JP30977490A JP30977490A JPH04182499A JP H04182499 A JPH04182499 A JP H04182499A JP 30977490 A JP30977490 A JP 30977490A JP 30977490 A JP30977490 A JP 30977490A JP H04182499 A JPH04182499 A JP H04182499A
- Authority
- JP
- Japan
- Prior art keywords
- ecgonine
- protein
- group
- globulin
- cocaine
- 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
- PHMBVCPLDPDESM-YWIQKCBGSA-N Ecgonine Natural products C1[C@H](O)[C@@H](C(O)=O)[C@H]2CC[C@@H]1N2C PHMBVCPLDPDESM-YWIQKCBGSA-N 0.000 claims abstract description 30
- PHMBVCPLDPDESM-UHFFFAOYSA-N d-Pseudoekgonin Natural products C1C(O)C(C(O)=O)C2CCC1N2C PHMBVCPLDPDESM-UHFFFAOYSA-N 0.000 claims abstract description 30
- PHMBVCPLDPDESM-FKSUSPILSA-N ecgonine Chemical compound C1[C@H](O)[C@H](C(O)=O)[C@H]2CC[C@@H]1N2C PHMBVCPLDPDESM-FKSUSPILSA-N 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 125000003277 amino group Chemical group 0.000 claims abstract description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 10
- 241000287828 Gallus gallus Species 0.000 claims abstract description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 7
- 108010074605 gamma-Globulins Proteins 0.000 claims abstract description 7
- 108060003552 hemocyanin Proteins 0.000 claims abstract description 5
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims 1
- 229960003920 cocaine Drugs 0.000 abstract description 10
- ZPUCINDJVBIVPJ-LJISPDSOSA-N cocaine Chemical compound O([C@H]1C[C@@H]2CC[C@@H](N2C)[C@H]1C(=O)OC)C(=O)C1=CC=CC=C1 ZPUCINDJVBIVPJ-LJISPDSOSA-N 0.000 abstract description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 abstract description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 abstract description 3
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 230000002163 immunogen Effects 0.000 abstract description 2
- 229960003771 cocaine hydrochloride Drugs 0.000 abstract 2
- PIQVDUKEQYOJNR-VZXSFKIWSA-N cocaine hydrochloride Chemical compound [Cl-].O([C@H]1C[C@@H]2CC[C@@H]([NH+]2C)[C@H]1C(=O)OC)C(=O)C1=CC=CC=C1 PIQVDUKEQYOJNR-VZXSFKIWSA-N 0.000 abstract 2
- 241000512297 Siphonariidae Species 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 108010094139 tumor-globulin Proteins 0.000 description 3
- HVWQTEPEBQYIFB-PXXJPSRFSA-N (1s,3s,4r,5r)-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]octane-4-carboxylic acid;hydrochloride Chemical compound [Cl-].C1[C@H](O)[C@H](C(O)=O)[C@H]2CC[C@@H]1[NH+]2C HVWQTEPEBQYIFB-PXXJPSRFSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical group C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 241000238413 Octopus Species 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- WHMDPDGBKYUEMW-UHFFFAOYSA-N pyridine-2-thiol Chemical compound SC1=CC=CC=N1 WHMDPDGBKYUEMW-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明（よ 抗コカイン抗体作製に使用可能な免疫原
すなわちエクゴニン・タンパク質コンジュゲート、およ
びその定量に必要不可欠な誘導体すなわちエクゴニン・
タンパク質コンジュゲート誘導体に関するものであム
従来の技術
］カインの免疫的測定に有用な抗コカイン抗体作製に必
要となるエクゴニン誘導体の合成例としては 例えば
シ゛ヤーナル オフゝ ７アーマロシ゛イ アンド イ
クスベリメンタル ヤラビューティクス第１９９巻第１
７１頁 （Ｊ、 Ｐｈａｒｍａｃｏｌ。[Detailed description of the invention] Industrial application field of the present invention (Immunogen that can be used for producing anti-cocaine antibodies)
Namely, ecgonine-protein conjugate and derivatives essential for its quantification, namely ecgonine-protein conjugate.
Conventional techniques related to protein conjugate derivatives] Examples of the synthesis of ecgonine derivatives necessary for the production of anti-cocaine antibodies useful for immunoassay of Caine include: Volume 199 No. 1
Page 71 (J, Pharmacol.
Ｅｘｐ、 Ｔｈｅｒ、、１９９． ｐ１７１ （１９７
６））に記載されているようく ヒツジｒ−グロブリン
にエクゴニンを導入したものがあム
発明が解決しようとする課題
特異性の高い抗コカイン抗体を得るに（友 より強い免
疫惹起能力を有するコンジュゲートを作製することが必
要である。Exp, Ther, 199. p171 (197
6)) Introducing ecgonine into sheep r-globulin is the best way to obtain a highly specific anti-cocaine antibody, which is the problem that the invention aims to solve. It is necessary to create a gate.
また 上述のヒツジγ−グロブリンにエクゴニンを導入
する従来の技術によって得られるコンジュゲートで（ｉ
タンパク質に導入されたエクゴニン分子数の定量が困
難であり、これを正確に知ることはできないという課題
があっな
本発明は係る従来の課題に対してなされたもので、特異
性が高く強い免疫惹起能力を有し 定量が可能な抗コカ
イン抗体を提供することにあ４課題を解決するための手
段
本発明は上記課題を解決するために チキンＴ−グロブ
リス ウシ血清アルブミンまたはスカシ貝ヘモシアニン
の内の何れかのタンパク質の少なくとも１つのアミン基
と、エクゴニンのカルボキシル基とが結合しているエク
ゴニン・タンパク質コンジュゲートおよびその誘導体を
提供するものであ４
作用
本発明によるエクゴニン・タンパク質コンジュゲート（
よ コカインの分解生成物であるエクゴニンを使用して
いるた敦 動物に免疫することによりコカインに対して
高いアフィニティを有すムまな エクゴニン・タンパク
質コンジュゲートのタンパク質の未反応のアミン基を、
３−（２−ピリジルジチオ）−１−プロピノイル基の
カルボニル基と結合させた誘導体を作製することによっ
て、 ３−（２−ピリジルジチオ）−１−プロピノイル
基に帰属する吸光度が容易に測定できるた感タンパク質
ｊ分子に導入されたエクゴニン分子数の定量が容易とな
る。In addition, a conjugate obtained by the conventional technique of introducing ecgonine into the above-mentioned sheep γ-globulin (i
There is a problem in that it is difficult to quantify the number of ecgonine molecules introduced into a protein, and it is impossible to know this accurately. The present invention aims to solve the above-mentioned problems by providing an anti-cocaine antibody capable of being quantified. The present invention provides ecgonine-protein conjugates and derivatives thereof, in which at least one amine group of the above protein and a carboxyl group of ecgonine are bonded.
Atsushi uses ecgonine, a degradation product of cocaine, which has a high affinity for cocaine by immunizing animals with unreacted amine groups in the protein of the ecgonine-protein conjugate.
By preparing a derivative in which the carbonyl group of 3-(2-pyridyldithio)-1-propinoyl group is bonded, the absorbance attributed to 3-(2-pyridyldithio)-1-propinoyl group can be easily measured. It becomes easy to quantify the number of ecgonine molecules introduced into sensitive protein j molecules.
実施例
本発明は 抗コカイン抗体を作成するた碌 コカインの
分解生成物であるエクゴニン分子を用し＼エクゴニンの
カルボキシル基と、タンパク質のアミン基とを結合させ
たコンジュゲートである。EXAMPLE The present invention is a conjugate in which the carboxyl group of ecgonine and the amine group of a protein are bonded using ecgonine molecule, which is a decomposition product of cocaine.
本発明に供されるタンパク質源としては チキンγ−グ
ロブリン、ウシ血清アルブミンまたはスカシ貝ヘモシア
ニンの何れかであム これらのタンパク質源は何れも免
疫惹起能力が高いため好ましく、特にウシ血清アルブミ
ンは水に溶は易いため取扱いが容易である。The protein source used in the present invention is chicken γ-globulin, bovine serum albumin, or keyhole keyhole hemocyanin. All of these protein sources are preferable because they have a high ability to induce immunity. In particular, bovine serum albumin is preferable in water. It is easy to handle because it melts easily.
また これらタンパク質源のアミノ基の内生なくとも１
つと、エクゴニンのカルボキシル基とが結合すれば良し
Ｘ、。In addition, the endogenous amino groups of these protein sources are at least 1
It is good if X and the carboxyl group of ecgonine bond together.
節板 本発明のコンジュゲートζ瓜 エクゴニンの作用
で特異性が高い抗コカイン性が発揮されまた タンパク
質の作用で強い免疫惹起能力を有すム
さら凶 本発明のエクゴニン・タンパク質コンジュゲー
ト（飄 エクゴニンのカルボキシル基と未結合の上記何
れがのタンパク質のアミノ基力（３−（２−ピリジルジ
チオ）−１−プロピノイル基のカルボニル基と結合した
エクゴニン・タンパク質コンジュゲート誘導体を用いる
と、タンパク質１分子に結合したエクゴニン分子数が定
量できるため好ましｔも
以下番ミ 本発明の具体的実施例について説明すも
ん エクゴニン塩酸塩の作製方法
コカイン塩酸塩１． Ｏｇ（２，９ｍｍｏｌ）を、２０
ｍ１のＩＮ塩酸に溶解し 油浴温度１１０〜１２０℃で
１５時間還流し九この溶液を室温にもどａ エーテルで
３回洗浄した、
水層を減圧下で濃縮して、６４９ｍｇのエクゴニン塩酸
塩を得九 収率は９９％であった
Ｂ、エクゴニン・タンパク質コンジュゲートの作製方法
タンパク質がチキンＴ−グロブリン（以下ＣＧＧと略す
）の場合を例として以下に示す。The conjugate of the present invention exhibits highly specific anti-cocaine properties due to the action of ecgonine, and has a strong immune-inducing ability due to the action of the protein. When using an ecgonine protein conjugate derivative that is bonded to the carbonyl group of the amino group (3-(2-pyridyldithio)-1-propinoyl group) of any of the above proteins that is not bonded to the carboxyl group, it can be bonded to one protein molecule. Since the number of ecgonine molecules can be quantified, it is preferable that
The solution was dissolved in 1 m of IN hydrochloric acid and refluxed for 15 hours at an oil bath temperature of 110-120°C.The solution was returned to room temperature and washed three times with ether.The aqueous layer was concentrated under reduced pressure to obtain 649 mg of ecgonine hydrochloride. The yield was 99%. B. Method for producing ecgonine-protein conjugate The case where the protein is chicken T-globulin (hereinafter abbreviated as CGG) will be described below as an example.
エクゴニン塩酸塩４０ｍｇ（０，１８ｍｍｏｌ）を２ｍ
ｌの純水に溶解し 室温で撹拌しながら２＄１の純水に
溶解した１−エチル−３−（３−ジメチルアミノプロピ
ル）−カルボジイミド塩酸塩（以下ＥＤＣＩと略す）
４０ｍｇ（０，２１ｍｍｏｌ）をゆっくり滴下しれ室温
で１時間撹拌した後、この溶液を２００ｍｇのＣＧＧを
溶かしたＯ、 ＩＭのＮａＣ１を含む０．１Ｍのリン酸
バッファーサリン（以下ＰＢＳと略す）溶液４ｍｌ中に
滴下し九
５℃で一晩撹拌した後、生じた沈澱物を遠心分離により
除い九
得られた上溝を、直径２ｃｍ長さ８０ｃｒｎのセファデ
ックス０２５カラム（ファルマシア社製以下同じ）でゲ
ル濾過上 エクゴニン・ＣＧＧのＰＢＳ溶液３２ｍ１を
得なＣｏＣＧＧｌ分子あたりのエクゴニン分子数の定量
ＣＧＧおよびエクゴニン・ＣＧＧのアミノ基に それぞ
れＮ−サクシンイミジル−３−（２−ピリジルジチオ）
プロピオネート（以下５ＰＤＰと略す）を導入し その
導入数の差をＣ００１分子あたりのエクゴニン分子数と
しな
以下にその測定方法を示も
（イ） ＣＧＧ−５ＰＤＰの定量
２ｍｇ／ｍｌに調整したＣＧＧのＰＢＳ溶液２５ｍ１中
に １ｍｌのエタノールに溶解したＳＰＤＰｌｏｍｇを
、室温でゆっくりと滴下しな
５℃で一晩撹拌した後、生じた沈澱物を遠心分離により
除いな
得られた上清をセファデックス０２５カラムでゲル濾過
Ｌ ＣＧＧ−３ＰＤＰ溶液３２ｍ１を得たこの溶液を
ＰＢＳで４．６５倍に希釈し そのうち１ｍｌを用いて
２８０ｎｍにおける吸光度を測定し その吸光度は０．
１７６であっち
これに１００ｍＭのジチオスレイトール（以下ＤＴＴと
略す）水溶液５０μｍを加え ５分間放置した後３４３
ｎｍにおける吸光度を測定し その吸光度は０．１０３
であっち
ＤＴＴ還元によって放出されたピリジン−２−チオンの
３４３ｎｍにおける分子吸光係数を８．０８Ｘ１０”と
すると、その濃度は次のように計算することができも
＝ １．２７７ｘ １０−’ Ｍ
この濃度はＣＧＧに導入された５ＰＤＰの濃度に等し１
．％まＡ ５ｐＩ）ｐの２−ピリジルジスルフィド基
が２８０ｎｍにおける吸光度に寄与するので、ＣＧＧ濃
度の計算には次のような補正が必要であム
ＣＧＧに起因する２８０ｎｍにおける吸光度（ＡｂＳ：
：：）は次のようになる。40 mg (0.18 mmol) of ecgonine hydrochloride in 2 m
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (hereinafter abbreviated as EDCI) dissolved in 2 $1 of pure water while stirring at room temperature.
After 40 mg (0.21 mmol) was slowly added dropwise and stirred at room temperature for 1 hour, this solution was mixed with 4 ml of a 0.1 M phosphate buffer saline (hereinafter abbreviated as PBS) solution containing 200 mg of CGG dissolved in O, IM NaCl. After stirring overnight at 95°C, the resulting precipitate was removed by centrifugation, and the resulting upper groove was gel-filtered using a Sephadex 025 column (manufactured by Pharmacia Co., Ltd., hereinafter the same) with a diameter of 2 cm and a length of 80 crn. Quantification of the number of ecgonine molecules per CoCGGl molecule using 32 ml of a PBS solution of ecgonine/CGG.
Propionate (hereinafter abbreviated as 5PDP) is introduced, and the difference in the number of introduced molecules is taken as the number of ecgonine molecules per C00 molecule.The measurement method is shown below.(a) Quantification of CGG-5PDP CGG PBS adjusted to 2 mg/ml Slowly add SPDPlomg dissolved in 1 ml of ethanol to 25 ml of solution at room temperature. After stirring overnight at 5°C, remove the resulting precipitate by centrifugation. Transfer the resulting supernatant to a Sephadex 025 column. Gel filtration L 32 ml of CGG-3PDP solution was obtained. This solution was diluted 4.65 times with PBS, and 1 ml of the solution was used to measure the absorbance at 280 nm. The absorbance was 0.
176, add 50 μm of 100 mM dithiothreitol (hereinafter abbreviated as DTT) aqueous solution here and there, leave it for 5 minutes, and then 343
Measure the absorbance at nm and the absorbance is 0.103
If the molecular extinction coefficient at 343 nm of pyridine-2-thione released by DTT reduction is 8.08 x 10'', its concentration can be calculated as follows = 1.277 x 10' M This concentration is equal to the concentration of 5PDP introduced into CGG, which is 1
．． Since the 2-pyridyl disulfide group of AbS contributes to the absorbance at 280 nm, the following correction is necessary to calculate the CGG concentration.
::) becomes as follows.
Ａｂｓ：：：＝ ０．１７６−（１，２７７ｘ １Ｏ−
５ｘ ５． ｌｘ １０３）＝０．１１０７
但し２−ピリジルジスルフィド基（Ｄ、 ２８０ｎｍ
における分子吸光係数を５．ｌＸ１０”とする。Abs:::= 0.176-(1,277x 1O-
5x 5. lx 103) = 0.1107 However, 2-pyridyl disulfide group (D, 280 nm
The molecular extinction coefficient at 5. 1×10”.
従ってＣＧＧの濃度 およびＣＧＧ １分子あたりに導
入された５ＰＤＰの分子数は次のようになム＝ ５．５
６１ｘ １０−’ Ｍ
（ロ）エクゴニン・ＣＧＧ −５ＰＤＰの定量２ｍｇ／
ｍｌに調整したエクゴニン−ＣＧＧのＰＢＳ溶液５ｍｌ
中へ１の１のエタノールに溶解した５ＰＤＰ９．６４ｍ
ｇを室温でゆっくりと滴下し九
５℃で一晩撹拌した夜 生じた沈澱物を遠心分離により
除い九
得られた上清をセファデックス０２５カラムでゲル濾過
し エクゴニン・ＣＧＧ −５ＰＤＰ溶液２４ｍ１を得
たこの溶液１ｍｌを用いて、２８０ｎｍにおける吸光度
を測定し 吸光度は０．２０６７であっ通この溶液に１
００ｍＭのＤＴＴ水溶液５０μｌを加７−．５分間放置
した後、３４３ｎｍにおける吸光度を測定し 吸光度は
０．０８３２であった
ＣＧＧ−５ＰＤＰの場合と同様の計算方法によって、エ
クゴニン・ＣＧＧ １分子あたりに導入された５ＰＤＰ
の分子数を求めることができる。Therefore, the concentration of CGG and the number of molecules of 5PDP introduced per molecule of CGG are as follows: = 5.5
61x 10-' M (b) Quantification of ecgonine/CGG-5PDP 2mg/
5 ml of PBS solution of ecgonine-CGG adjusted to ml
5PDP9.64m dissolved in 1 part ethanol
The resulting precipitate was removed by centrifugation, and the resulting supernatant was gel-filtered through a Sephadex 025 column to obtain 24 ml of ecgonine/CGG-5PDP solution. Using 1 ml of the octopus solution, the absorbance at 280 nm was measured, and the absorbance was 0.2067.
Add 50μl of 00mM DTT aqueous solution 7-. After standing for 5 minutes, the absorbance at 343 nm was measured and the absorbance was 0.0832. Using the same calculation method as in the case of CGG-5PDP, 5PDP introduced per ecgonine/CGG molecule was measured.
The number of molecules can be found.
その計算式を次に示す。The calculation formula is shown below.
＝１，０３０ｘｌＯ”” Ｍ
コノ濃ｆＬｔ、 エクゴニン・ＣＧＧに導入された５
ＰＤＰの濃度に等しく■
エクゴニン・ＣＧＧに起因する２８０ｒ＋ｍにおける吸
光度を、ＡｂＳ台言−エ）、ＣＧＧとすると、次のよう
になもＡｂｓ台言−：、、ｃ、、 ＝ ０．２０７−（
１，０３０ｘ １Ｏ−ＳＸ ５．１ ｘ １０’）＝
０．１５４
＝ ７．７４８ｘ １０−’ Ｍ
［エクゴニン・ＣＧＧ］ ７．７４８ｘ １０−
’＝１３．３個
（イ）および（ロ）の結果より、ＣＧｏ １分子あたり
に導入されたエクゴニンの分子数を計算すると次のよう
になム
２２、９−１３．３＝ ９．６個
なお 上記実施例ではチキンＴ−グロブリンの例を示し
た力交 本発明のエグゴニン・タンパク質コンシュケー
トには ウシ血清アルブミンやスカシ貝ヘモシアニンで
も同様にコンジュゲートを形成させることができる。= 1,030xlO"" M Konoko fLt, 5 introduced into ecgonine/CGG
Equal to the concentration of PDP ■ If the absorbance at 280r+m due to ecgonine/CGG is AbS table-e) and CGG, then Namo Abs table-: , c, , = 0.207-(
1,030x 1O-SX 5.1 x 10')=
0.154 = 7.748x 10-' M [ecgonine/CGG] 7.748x 10-
' = 13.3 From the results of (a) and (b), the number of ecgonine molecules introduced per molecule of CGo is calculated as follows: 22,9-13.3 = 9.6 In the above examples, chicken T-globulin was used as an example, but the eggonin-protein consinate of the present invention can be similarly formed into a conjugate using bovine serum albumin or keyhole keyhole hemocyanin.
また 上記実施例で記載した作製方法（よ 一実施例で
あり、他の手法も当然考えられる力（本発明のコンジュ
ゲートを作製するに法 上記の方法が現在のところ一番
簡単なプロセスであ４さら置 上記実施例ではコンジュ
ゲート誘導体として５ＰＤＰを用いた例を示しためｔ
本発明の誘導体の製造にはこれに限定されるものではな
く、 ３−（２−ピリジルジチオ）−１−プロピノイル
基を含んでいれば良しも
発明の効果
以上のように本発明？、１． チキンＴ−グロブリン
、ウシ血清アルブミンまたはスカシ貝ヘモシアニンの内
の何れかの化合物の少なくとも１つのアミノ基と、エク
ゴニンのカルボキシル基とが結合しているエクゴニン・
タンパク質コンジュゲートであるた嵌 容易に抗コカイ
ン抗体の作製が可能となるコンジュゲートが得られも
さらに このコン・シュゲートを３−（２−ピリジルジ
チオ）−１−プロピノイル基のカルボニル基と反応させ
ることにより、タンパク質１分子に結合したエクゴニン
を容易に定量することのできるエクゴニン・タンパク質
コンジュゲート誘導体が提供できる効果があもIn addition, the production method described in the above example is just one example, and other methods are naturally possible. 4. In the above example, 5PDP was used as the conjugate derivative.
The production of the derivative of the present invention is not limited to this, but it is sufficient if it contains a 3-(2-pyridyldithio)-1-propinoyl group. , 1. Ecgonine, in which at least one amino group of a compound of chicken T-globulin, bovine serum albumin or keyhole keyhole hemocyanin is bonded to the carboxyl group of ecgonine.
Although a protein conjugate can be obtained that makes it possible to easily produce an anti-cocaine antibody, it is further possible to react this protein conjugate with the carbonyl group of 3-(2-pyridyldithio)-1-propinoyl group. As a result, ecgonine-protein conjugate derivatives that can easily quantify ecgonine bound to one protein molecule are more effective.
Claims (2)
はスカシ貝ヘモシアニンの内の何れかのタンパク質の少
なくとも１つのアミノ基と、エクゴニンのカルボキシル
基とが結合していることを特徴とするエクゴニン・タン
パク質コンジュゲート。(1) An ecgonine-protein conjugate characterized in that at least one amino group of a protein selected from chicken γ-globulin, bovine serum albumin, or keyhole keyhole hemocyanin is bonded to a carboxyl group of ecgonine. .
はスカシ貝ヘモシアニンの内の何れかのタンパク質の少
なくとも１つのアミノ基と、エクゴニンのカルボキシル
基とが結合し、前記カルボキシル基と未反応の前記化合
物のアミノ基が、３−（２−ピリジルジチオ）プロピノ
イル基のカルボニル基と結合していることを特徴とする
エクゴニン・タンパク質コンジュゲート誘導体。(2) At least one amino group of any protein of chicken γ-globulin, bovine serum albumin, or keyhole limpet hemocyanin is bonded to the carboxyl group of ecgonine, and the amino group of the compound that has not reacted with the carboxyl group is bonded to the carboxyl group of ecgonine. An ecgonine protein conjugate derivative, wherein the group is bonded to a carbonyl group of a 3-(2-pyridyldithio)propinoyl group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30977490A JPH04182499A (en) | 1990-11-14 | 1990-11-14 | Ecgonine-protein conjugate and derivative thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30977490A JPH04182499A (en) | 1990-11-14 | 1990-11-14 | Ecgonine-protein conjugate and derivative thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04182499A true JPH04182499A (en) | 1992-06-30 |
Family
ID=17997098
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP30977490A Pending JPH04182499A (en) | 1990-11-14 | 1990-11-14 | Ecgonine-protein conjugate and derivative thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04182499A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0613899A2 (en) * | 1993-03-04 | 1994-09-07 | Matsushita Electric Industrial Co., Ltd. | Cocaine derivative, protein conjugate thereof, monoclonal antibody producing cell line, method for preparing the cell line and monoclonal antibody |
| WO1996030049A3 (en) * | 1995-03-31 | 1997-03-06 | Immulogic Pharma Corp | Hapten-carrier conjugates for use in drug-abuse therapy |
| EP1782836A3 (en) * | 1995-03-31 | 2008-05-14 | Xenova Research Limited | Hapten-carrier conjugates for use in drug-abuse therapy (cocaine) |
-
1990
- 1990-11-14 JP JP30977490A patent/JPH04182499A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0613899A2 (en) * | 1993-03-04 | 1994-09-07 | Matsushita Electric Industrial Co., Ltd. | Cocaine derivative, protein conjugate thereof, monoclonal antibody producing cell line, method for preparing the cell line and monoclonal antibody |
| EP0613899A3 (en) * | 1993-03-04 | 1995-01-18 | Matsushita Electric Ind Co Ltd | Cocaine derivative, protein conjugate thereof, monoclonal antibody producing cell line, method for preparing the cell line and monoclonal antibody. |
| WO1996030049A3 (en) * | 1995-03-31 | 1997-03-06 | Immulogic Pharma Corp | Hapten-carrier conjugates for use in drug-abuse therapy |
| EP1782836A3 (en) * | 1995-03-31 | 2008-05-14 | Xenova Research Limited | Hapten-carrier conjugates for use in drug-abuse therapy (cocaine) |
| EP1782835A3 (en) * | 1995-03-31 | 2008-06-11 | Xenova Research Limited | Hapten-carrier conjugates for use in drug-abuse therapy |
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