JPH0833394B2 - Method for producing enzyme-labeled hapten - Google Patents
Method for producing enzyme-labeled haptenInfo
- Publication number
- JPH0833394B2 JPH0833394B2 JP2267188A JP26718890A JPH0833394B2 JP H0833394 B2 JPH0833394 B2 JP H0833394B2 JP 2267188 A JP2267188 A JP 2267188A JP 26718890 A JP26718890 A JP 26718890A JP H0833394 B2 JPH0833394 B2 JP H0833394B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- labeled
- general formula
- hapten
- reaction
- 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 - Lifetime
Links
- 102000004190 Enzymes Human genes 0.000 title claims description 39
- 108090000790 Enzymes Proteins 0.000 title claims description 39
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 3
- 229940088598 enzyme Drugs 0.000 description 36
- 238000006243 chemical reaction Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000003018 immunoassay Methods 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 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 2
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 2
- 229960005156 digoxin Drugs 0.000 description 2
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 229960000890 hydrocortisone Drugs 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 2
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 2
- 239000005495 thyroid hormone Substances 0.000 description 2
- 229940036555 thyroid hormone Drugs 0.000 description 2
- 229940034208 thyroxine Drugs 0.000 description 2
- 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 description 2
- 229940035722 triiodothyronine Drugs 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-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
- IPBNQYLKHUNLQE-UHFFFAOYSA-N 8-anilinonaphthalene-1-sulfonic acid;azane Chemical compound [NH4+].C=12C(S(=O)(=O)[O-])=CC=CC2=CC=CC=1NC1=CC=CC=C1 IPBNQYLKHUNLQE-UHFFFAOYSA-N 0.000 description 1
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- WDJUZGPOPHTGOT-OAXVISGBSA-N Digitoxin Natural products O([C@H]1[C@@H](C)O[C@@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@@](C)([C@H](C6=CC(=O)OC6)CC5)CC4)CC3)CC2)C[C@H]1O)[C@H]1O[C@@H](C)[C@H](O[C@H]2O[C@@H](C)[C@@H](O)[C@@H](O)C2)[C@@H](O)C1 WDJUZGPOPHTGOT-OAXVISGBSA-N 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000007982 barbital buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- QXJJQWWVWRCVQT-UHFFFAOYSA-K calcium;sodium;phosphate Chemical compound [Na+].[Ca+2].[O-]P([O-])([O-])=O QXJJQWWVWRCVQT-UHFFFAOYSA-K 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- WDJUZGPOPHTGOT-XUDUSOBPSA-N digitoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)CC5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O WDJUZGPOPHTGOT-XUDUSOBPSA-N 0.000 description 1
- 229960000648 digitoxin Drugs 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical group Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は酵素免疫測定法，ケミルミネッセンス免疫測
定法，蛍光免疫測定法などに利用される酵素標識ハプテ
ンの製法に関する。TECHNICAL FIELD The present invention relates to a method for producing an enzyme-labeled hapten used in enzyme immunoassays, chemiluminescence immunoassays, fluorescence immunoassays, and the like.
（従来の技術） 従来、酵素標識ハプテンの製法として、例えば、過ヨ
ウ素酸酸化法〔クリニカルケミストリー（Clin.Chem.）
24,1801（1978）参照〕やグルタルアルデヒド法〔エン
ドクリノールジャパン（Endocrinol Japan）27,275（19
80）参照〕などが知られている。(Prior Art) Conventionally, as a method for producing an enzyme-labeled hapten, for example, a periodate oxidation method [Clinical Chemistry (Clin. Chem.)
24, 1801 (1978)] and glutaraldehyde method [the end click linoleic Japan (Endocrinol Japan) 27, 275 ( 19
80)]] are known.
（発明が解決しようとする課題） しかしながら、従来の製法では酵素同志のあるいはハ
プテン同志のセルフカップリングによる感度の低下、酵
素活性の低下、安定性の低下など臨床検査薬として重大
な問題を発生させる。(Problems to be Solved by the Invention) However, the conventional production method causes serious problems as a clinical test drug, such as a decrease in sensitivity due to self-coupling of enzymes or haptens, a decrease in enzyme activity, and a decrease in stability. .
（課題を解決するための手段） そこで、本発明者等はセルフカップリングの問題を解
決した酵素標識ハプテンの製法について鋭意検討した結
果、本発明に到達した。すなわち、本発明は、 一般式（Ｉ） の複合体と、一般式（II） の複合体とを反応させることを特徴とする一般式（II
I） で記載される酵素標識ハプテンの製法（式中、Ａはハプ
テンの残基、Ｄは酵素（D-NH2）の残基、Ｒは化学結合
または６員環状炭化水素残基、ｍは２〜５の正数、ｎは
０〜10の正数である。）である。(Means for Solving the Problem) Therefore, the present inventors have arrived at the present invention as a result of extensive studies on a method for producing an enzyme-labeled hapten that has solved the problem of self-coupling. That is, the present invention provides the compound represented by the general formula (I) And the general formula (II) Of the general formula (II
I) The method for producing an enzyme-labeled hapten described in (wherein A is a residue of a hapten, D is a residue of an enzyme (D-NH 2 ), R is a chemical bond or a 6-membered cyclic hydrocarbon residue, and m is 2 to 5 is a positive number and n is a positive number from 0 to 10.).
本発明において使用されるハプテン（A-NH2）は、ト
リヨードサイロニン（T3），サイロキシン（T4）等の甲
状腺ホルモン、アミノ基を導入したコルチゾール，エス
トラジオール等のステロイドホルモン、アミノ基を導入
したジゴキシン，ジギトキシン，テオフィリン等の薬物
が挙げられる。特にトリヨードサイロニン、サイロキシ
ン等の甲状腺ホルモンは分子中にアミノ基を有している
ので反応に供するのに好ましい。また、本発明において
使用される酵素は西洋ワサビペルオキシダーゼ（PO
D）、アルカリフォスファターゼ、グルコースオキシダ
ーゼ、マレートデヒドロゲナーゼ、アセチルコリンエス
テラーゼ、グルコアミラーゼ等が挙げられる。酵素標識
ハプテンは、西洋ワサビペルオキシダーゼ標識トリヨー
ドサイロニン（T3-POD），アルカリホスファターゼ標識
コルチゾール，グルコースオキシダーゼ標識ジゴキシン
等が挙げられる。As the hapten (A-NH 2 ) used in the present invention, thyroid hormones such as triiodothyronine (T3) and thyroxine (T4), cortisol having an amino group introduced, steroid hormones such as estradiol, and an amino group introduced. Examples include drugs such as digoxin, digitoxin, and theophylline. In particular, thyroid hormones such as triiodothyronine and thyroxine have an amino group in the molecule and therefore are preferable for use in the reaction. In addition, the enzyme used in the present invention is horseradish peroxidase (PO
D), alkaline phosphatase, glucose oxidase, malate dehydrogenase, acetylcholinesterase, glucoamylase and the like. Examples of the enzyme-labeled hapten include horseradish peroxidase-labeled triiodothyronine (T3-POD), alkaline phosphatase-labeled cortisol, glucose oxidase-labeled digoxin and the like.
一般式（II）中のＲの６員環状炭化水素残基としては
ベンゼン，シクロヘキサン等が挙げられる。Examples of the R 6-membered cyclic hydrocarbon residue in the general formula (II) include benzene and cyclohexane.
一般式（Ｉ）は、水、N,N−ジメチルホルムアミド（D
MF）、テトラヒドロフラン（THF）、エタノール、アセ
トニトリル等の適当な溶媒中でハプテンと一般式（IV） の化合物とを反応させて得られる。一般式（IV）中のｍ
は２〜５の正数であり、好ましくはｍ＝３（例えばｍ＝
３の時、２−イミノチオラン塩酸塩）である。The general formula (I) includes water, N, N-dimethylformamide (D
MF), tetrahydrofuran (THF), ethanol, acetonitrile in a suitable solvent and hapten and the general formula (IV) It is obtained by reacting with the compound of. M in the general formula (IV)
Is a positive number from 2 to 5, and preferably m = 3 (for example, m =
At the time of 3, it is 2-iminothiolane hydrochloride).
ハプテンと一般式（IV）のモル比は通常1:0.1〜20、
好ましくは、1:0.5〜10である。反応温度は通常10〜50
℃、好ましくは、20〜30℃である。反応時間は通常10分
〜24時間、好ましくは、30分〜10時間である。The molar ratio of the hapten to the general formula (IV) is usually 1: 0.1-20,
Preferably, it is 1: 0.5-10. Reaction temperature is usually 10-50
C., preferably 20-30.degree. The reaction time is generally 10 minutes to 24 hours, preferably 30 minutes to 10 hours.
この様にして得られた一般式（Ｉ）はそのまま次の反
応に供してもよいし、溶媒抽出、シリカゲルクロマトグ
ラフィー、逆相クロマトグラフィー等により精製した後
に次の反応に供することも、勿論可能である。The thus-obtained general formula (I) may be directly used in the next reaction, or may be purified by solvent extraction, silica gel chromatography, reverse phase chromatography and the like and then used in the next reaction. Is.
酵素（D-NH2）と一般式（Ｖ） の化合物とを反応させて得られる一般式（II）の複合体
は〔アナリティカル レターズ（ANALYTICAL LETTERS）
15(B2),147〜160（1982）〕に記載の方法で合成するこ
とができる。一般式（Ｖ）中のＲは化学結合または６員
環状炭化水素残基、ｎは０〜10の正数である。この化合
物は、例えば〔ザ ジャーナル オブ バイオケミスト
リー（The Journal of Biochemistry）79,233（197
6），ヨーロピアン ジャーナル オブ バイオケミス
トリー（European Journal of biochemistry）101,395
（1979）〕に記載の方法で合成することができるし、ま
た、市販の物（例えばｎ＝１、Ｒ＝シクロヘキサンの
時,4−（Ｎ−マレイミドメチル）シクロヘキサン−１−
カルボン酸 Ｎ−ヒドロキシスクシンイミド エステル
［４−（N-Male-imidomethyl）cyclohexane-1-carboxyl
ic Acid N-Hydroxysuccinimide ester以下CHMと略記］
を用いることもできる。Enzyme (D-NH 2 ) and general formula (V) The compound of general formula (II) obtained by reacting with the compound of [Analytical Letters (ANALYTICAL LETTERS)
15 (B2) , 147-160 (1982)]. R in the general formula (V) is a chemical bond or a 6-membered cyclic hydrocarbon residue, and n is a positive number from 0 to 10. This compound is, for example, [The Journal of Biochemistry 79 , 233 (197
6) 、 European Journal of biochemistry 101 , 395
(1979)], or a commercially available product (for example, n = 1, R = cyclohexane, 4- (N-maleimidomethyl) cyclohexane-1-).
Carboxylic acid N-hydroxysuccinimide ester [4- (N-Male-imidomethyl) cyclohexane-1-carboxyl
ic Acid N-Hydroxysuccinimide ester, abbreviated as CHM below]
Can also be used.
酵素と一般式（Ｖ）のモル比は通常1:10〜1000、好ま
しくは、1:50〜200である。反応温度は通常10〜50℃、
好ましくは20〜30℃である。反応時間は通常10分〜24時
間、好ましくは、30分〜10時間である。The molar ratio of the enzyme to the general formula (V) is usually 1:10 to 1000, preferably 1:50 to 200. The reaction temperature is usually 10 to 50 ° C,
It is preferably 20 to 30 ° C. The reaction time is generally 10 minutes to 24 hours, preferably 30 minutes to 10 hours.
本発明において、一般式（Ｉ）と一般式（II）を反応
して一般式（III）を得る。一般式（Ｉ）と一般式（I
I）のモル比は通常0.5〜20:1、好ましくは、１〜10:1で
ある。反応温度は通常10〜50℃、好ましくは、20〜30℃
である。反応時間は通常10分〜24時間、好ましくは、30
分〜10時間である。得られた反応物をPH6.0〜9.0のリン
酸バッファー，トリス塩酸バッファー，ほう酸バッファ
ー等の適当なバッファーで平衡化したゲルろ過用カラム
で精製する。In the present invention, general formula (I) and general formula (II) are reacted to obtain general formula (III). General formula (I) and general formula (I
The molar ratio of I) is usually 0.5 to 20: 1, preferably 1 to 10: 1. The reaction temperature is usually 10 to 50 ° C, preferably 20 to 30 ° C.
Is. The reaction time is usually 10 minutes to 24 hours, preferably 30
Minutes to 10 hours. The obtained reaction product is purified by a gel filtration column equilibrated with an appropriate buffer such as a phosphate buffer (pH 6.0 to 9.0), a Tris hydrochloric acid buffer, a borate buffer or the like.
一般式（III）の合成法として一般式（Ｉ）に一般式
（Ｖ）を反応させ次いで酵素を反応させるというような
逐次反応で行う方法も可能である。As a method for synthesizing the general formula (III), a method in which the general formula (I) is reacted with the general formula (V) and then an enzyme is reacted is also possible.
以下、実施例により本発明をさらに説明するが本発明
はこれに限定されるものではない。Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.
実施例１ （ａ）T3アミノ基へのSH基の導入 試験管にT3（1mg）を秤量しDMF（1ml）を加え37℃恒
温水槽に５分間浸漬させ溶解した（a-1）。試験管に２
−イミノチオラン塩酸塩（5mg）を秤量し脱イオン水1ml
を加え溶解した（a-2）。（a-1）1mlと（a-2）200μｌ
とを混合し均一な溶液とした後30℃で５時間静置反応さ
せた（a-3）。Example 1 (a) Introduction of SH group into T3 amino group T3 (1 mg) was weighed into a test tube, DMF (1 ml) was added, and the test tube was immersed in a 37 ° C. constant temperature water bath for 5 minutes to be dissolved (a-1). 2 for test tube
-Iminothiolane hydrochloride (5 mg) is weighed and deionized water 1 ml
Was added and dissolved (a-2). (A-1) 1 ml and (a-2) 200 μl
Were mixed with each other to form a uniform solution, and the mixture was allowed to react at 30 ° C. for 5 hours (a-3).
（ｂ）酵素へのマレイミド基の導入 6mgのPOD（1-C）（東洋紡製）を1mlの0.1モルリン酸
バッファー（PH7.1）に溶解し50μｌのDMFに溶かしたCH
M（4.8mg）を加えて、37℃恒温水槽中で１時間振とう反
応させた。生成した沈澱を遠心分離で除去し上清をセフ
ァデックスG-25のカラム（2.0cm2×70cm,ファルマシア
・ファインケミカル社（スウェーデン）製）に通し、0.
1モルリン酸バッファー（PH6.0）で溶出させた（b-
1）。(B) Introduction of maleimide group into enzyme 6 mg of POD (1-C) (manufactured by Toyobo) was dissolved in 1 ml of 0.1 mol phosphate buffer (PH7.1) and dissolved in 50 μl of DMF.
M (4.8 mg) was added, and a shaking reaction was carried out for 1 hour in a 37 ° C. constant temperature water bath. The formed precipitate was removed by centrifugation, and the supernatant was passed through a Sephadex G-25 column (2.0 cm 2 × 70 cm, manufactured by Pharmacia Fine Chemicals (Sweden)),
Elution with 1 molar phosphate buffer (PH6.0) (b-
1).
（ｃ）PODへのT3導入（T3-PODの合成） （a-3）と（b-1）をモル比10:1で混合、均一な溶液と
し30℃で５時間静置反応させた。0.02モルリン酸バッフ
ァー（PH7.0）で平衡化したセファデックスG-25のカラ
ム（2.0cm2×70cm）に反応物をチャージし、1mlづつ溶
出液を分取した。分取した溶出液の酵素活性,T3活性の
測定は、以下の方法で行った。すなわち、T3抗体を結合
したガラス球（以下T3抗体ビーズと略記）と403nmにお
ける吸光度＞0.1である溶出液100μｌを0.1％ウシ血清
アルブミン含有0.02モルリン酸バッファー（PH7.2）300
μ中で37℃,15分間インキュベートした。未反応酵素標
識T3を生理食塩水で洗浄，除去し、発色試薬（ｏ−フェ
ニレンジアミン／H2O2）500μｌを添加し、37℃,15分間
インキュベートした。その後、反応停止液（1.5NH2S
O4）3mlを添加した。492nmにおける吸光度＞0.1を示す
溶出フラクションを、酵素活性,T3活性の両方を有する
画分とし、酵素標識T3を得た。(C) Introduction of T3 into POD (Synthesis of T3-POD) (a-3) and (b-1) were mixed at a molar ratio of 10: 1 to form a uniform solution, and the reaction was allowed to stand at 30 ° C for 5 hours. The reaction product was charged into a column (2.0 cm 2 × 70 cm) of Sephadex G-25 equilibrated with 0.02 mol phosphate buffer (PH7.0), and the eluate was fractionated in 1 ml fractions. The enzyme activity and T3 activity of the collected eluate were measured by the following methods. That is, T3 antibody-bound glass spheres (hereinafter abbreviated as T3 antibody beads) and 100 μl of an eluent having an absorbance at 403 nm of> 0.1 were added to 0.02 mol phosphate buffer (PH7.2) 300 containing 0.1% bovine serum albumin.
Incubated in μ at 37 ° C. for 15 minutes. Unreacted enzyme-labeled T3 was washed and removed with physiological saline, and 500 μl of a coloring reagent (o-phenylenediamine / H 2 O 2 ) was added, and the mixture was incubated at 37 ° C. for 15 minutes. Then, the reaction stop solution (1.5NH 2 S
O 4 ) 3 ml was added. The elution fraction showing an absorbance of> 0.1 at 492 nm was used as a fraction having both enzyme activity and T3 activity to obtain enzyme-labeled T3.
次に、ウシ血清アルブミン含有リン酸バッファー中に
T3を0ng/dl,800ng/dlになるように添加したもの（以
下、標準T3 0ng/dl,標準T3 800ng/dlと略記）,T3抗体ビ
ーズ,8−アニリノ−１−ナフタレンスルホン酸アンモニ
ウム・ウシ血清アルブミン含有バルビタールバッファー
（以下免疫反応用緩衝液と略記），発色液，反応停止液
を用いて、実施例１（本発明の酵素標識ハプテン）と比
較例１（過ヨウ素酸酸化法で得た酵素標識ハプテン），
比較例２（グルタルアルデヒド法で得た酵素標識ハプテ
ン）の性能の比較を行った。酵素標識T3濃度は標準T3 0
ng/dlのアッセイ吸光度＝1.0になるように調製した。ア
ッセイ方法は以下に示す通りである。Next, in a phosphate buffer containing bovine serum albumin
T3 added at 0 ng / dl and 800 ng / dl (hereinafter, abbreviated as standard T3 0 ng / dl and standard T3 800 ng / dl), T3 antibody beads, 8-anilino-1-naphthalene sulfonate ammonium bovine Example 1 (enzyme-labeled hapten of the present invention) and Comparative Example 1 (periodic acid oxidation method) were obtained using a serum albumin-containing barbital buffer (hereinafter abbreviated as an immunological reaction buffer), a coloring solution, and a reaction stop solution. Enzyme-labeled hapten),
The performance of Comparative Example 2 (enzyme-labeled hapten obtained by the glutaraldehyde method) was compared. Enzyme labeled T3 concentration is standard T3 0
The assay absorbance of ng / dl was adjusted to 1.0. The assay method is as follows.
標準T3 100μｌ 免疫反応用緩衝液 300μｌ T3抗体ビーズ １ケ 37℃,15分間インキュベート 生理食塩水で洗浄 酵素標識T3 500μｌ 37℃,15分間インキュベート 生理食塩水で洗浄 発色液 500μｌ 37℃,15分間インキュベート 反応停止液 3ml 表１は実施例１、比較例1,2で得られた酵素標識T3を
用いた時の感度を示す。明かに、感度（標準T3 0ng/dl,
標準T3 800ng/dlのアッセイ吸光度差）は比較例1,2がΔ
＝0.3〜0.4に対し実施例１はΔ＝0.7と約２倍の良好な
感度を示した。Standard T3 100 μl Immune reaction buffer 300 μl T3 antibody beads 1 incubation at 37 ° C, 15 minutes Wash with physiological saline Enzyme-labeled T3 500 μl 37 ° C, 15 minutes incubation Saline wash Color development solution 500 μl 37 ° C, 15 minutes incubation Reaction Stopping solution 3 ml Table 1 shows the sensitivity when using the enzyme-labeled T3 obtained in Example 1 and Comparative Examples 1 and 2. Clearly, the sensitivity (standard T3 0ng / dl,
Standard T3 800 ng / dl assay absorbance difference)
= 0.3 to 0.4, Example 1 showed Δ = 0.7, which is about twice as good as the sensitivity.
また酵素活性（比較例1,2の酵素標識T3の希釈倍数を
実施例１の酵素標識T3の希釈倍数50000で割った値）は
表２から明かなごとく実施例１の酵素標識T3は比較例1,
2の酵素標識T3の約10倍であった。 The enzyme activity (value obtained by dividing the dilution factor of the enzyme-labeled T3 of Comparative Examples 1 and 2 by the dilution factor of 50000 of the enzyme-labeled T3 of Example 1) is clear from Table 2, and the enzyme-labeled T3 of Example 1 is a comparative example. 1,
It was about 10 times that of the enzyme-labeled T3 of 2.
実施例１で得られた酵素標識T3の安定性についても表
３から明かなごとく、凍結保存（−40℃〜−20℃）で１
年間安定であった。 Regarding the stability of the enzyme-labeled T3 obtained in Example 1, it is clear from Table 3 that the stability of the enzyme-labeled T3 was 1 when it was stored frozen (-40 ° C to -20 ° C).
It was stable for a year.
（発明の効果） 本発明の製造法により得られた酵素標識ハプテンは、
ハプテン同志のあるいは酵素同志のセルフカップリング
が起こらないため、感度の低下、酵素活性の低下、安定
性の低下が見られない。よって、本発明の酵素標識ハプ
テンは酵素免疫測定法、蛍光免疫測定法、ケミルミネッ
センス免疫測定法等の利用に適している。 (Effect of the invention) The enzyme-labeled hapten obtained by the production method of the present invention is
Since hapten or enzyme self-coupling does not occur, there is no decrease in sensitivity, enzyme activity, or stability. Therefore, the enzyme-labeled hapten of the present invention is suitable for use in enzyme immunoassay, fluorescence immunoassay, chemiluminescence immunoassay, and the like.
Claims (1)
I） で記載される酵素標識ハプテンの製法。 （式中、Ａはハプテンの残基、Ｄは酵素（D-NH2）の残
基、Ｒは化学結合または６員環状炭化水素残基、ｍは２
〜５の正数、ｎは０〜10の正数である。）1. A general formula (I) And the general formula (II) Of the general formula (II
I) The method for producing an enzyme-labeled hapten described in 1. (In the formula, A is a residue of a hapten, D is a residue of an enzyme (D-NH 2 ), R is a chemical bond or a 6-membered cyclic hydrocarbon residue, and m is 2
Is a positive number of 5 and n is a positive number of 0-10. )
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2267188A JPH0833394B2 (en) | 1990-10-03 | 1990-10-03 | Method for producing enzyme-labeled hapten |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2267188A JPH0833394B2 (en) | 1990-10-03 | 1990-10-03 | Method for producing enzyme-labeled hapten |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04144679A JPH04144679A (en) | 1992-05-19 |
| JPH0833394B2 true JPH0833394B2 (en) | 1996-03-29 |
Family
ID=17441341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2267188A Expired - Lifetime JPH0833394B2 (en) | 1990-10-03 | 1990-10-03 | Method for producing enzyme-labeled hapten |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0833394B2 (en) |
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| CN110174363A (en) * | 2019-01-09 | 2019-08-27 | 北京九强生物技术股份有限公司 | Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent |
| CN117030997B (en) * | 2023-08-01 | 2024-06-04 | 美康生物科技股份有限公司 | Homogeneous phase immunoassay method of small molecule compound |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4263277A (en) | 1979-06-20 | 1981-04-21 | American Cyanamid Company | Cold permanent wave composition and method containing 2-iminothiolane |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL171930C (en) * | 1972-05-11 | 1983-06-01 | Akzo Nv | METHOD FOR DETERMINING AND DETERMINING BITES AND TEST PACKAGING. |
-
1990
- 1990-10-03 JP JP2267188A patent/JPH0833394B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4263277A (en) | 1979-06-20 | 1981-04-21 | American Cyanamid Company | Cold permanent wave composition and method containing 2-iminothiolane |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04144679A (en) | 1992-05-19 |
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