JPS63111465A - Enzyme immunoassay method for leucine enkephaline - Google Patents
Enzyme immunoassay method for leucine enkephalineInfo
- Publication number
- JPS63111465A JPS63111465A JP25719086A JP25719086A JPS63111465A JP S63111465 A JPS63111465 A JP S63111465A JP 25719086 A JP25719086 A JP 25719086A JP 25719086 A JP25719086 A JP 25719086A JP S63111465 A JPS63111465 A JP S63111465A
- Authority
- JP
- Japan
- Prior art keywords
- enk
- antibody
- antigen
- dhfr
- leucine
- 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.)
- Granted
Links
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 25
- 238000003018 immunoassay Methods 0.000 title claims 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 title abstract 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 title abstract 2
- 239000000427 antigen Substances 0.000 claims abstract description 39
- 102000036639 antigens Human genes 0.000 claims abstract description 39
- 108091007433 antigens Proteins 0.000 claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- OZRNSSUDZOLUSN-LBPRGKRZSA-N dihydrofolic acid Chemical compound N=1C=2C(=O)NC(N)=NC=2NCC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-LBPRGKRZSA-N 0.000 claims abstract description 3
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- 108020001507 fusion proteins Proteins 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 239000007790 solid phase Substances 0.000 claims description 4
- 108010022337 Leucine Enkephalin Proteins 0.000 claims description 3
- URLZCHNOLZSCCA-UHFFFAOYSA-N leu-enkephalin Chemical compound C=1C=C(O)C=CC=1CC(N)C(=O)NCC(=O)NCC(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 URLZCHNOLZSCCA-UHFFFAOYSA-N 0.000 claims description 3
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- 244000063299 Bacillus subtilis Species 0.000 description 2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
ａ、産業上の利用分野
本発明はエンザイムアッセイ法（以下ＥＩＡと称する）
によるロイシンエンケファリン（以下Ｌ−Ｅｎｋと称す
る）測定法に関するものである。[Detailed description of the invention] a. Industrial application field The present invention relates to an enzyme assay method (hereinafter referred to as EIA).
The present invention relates to a method for measuring leucine enkephalin (hereinafter referred to as L-Enk).
Ｌ Ｅｎｋは生体内に存在し９モルヒネ様鎮痛作用を
示す５個のアミノ酸からなるペプチドホルモンの一種で
ある。その化学構造はＬ−チロシル−グリシル−グリシ
ル−し−フェニルアラニル−Ｌ−ロイシンであることが
確かめられており、 Ｌ−Ｅｎｋの測定は診断及び治療
などの医療分野に重要な役割を果たす。L Enk is a type of peptide hormone consisting of 5 amino acids that exists in the body and exhibits a morphine-like analgesic effect. Its chemical structure has been confirmed to be L-tyrosyl-glycyl-glycyl-phenylalanyl-L-leucine, and measurement of L-Enk plays an important role in medical fields such as diagnosis and treatment.
ｂ、従来の技術
従来９体液中及び組織中のＬ −Ｅ ｎ ｋを測定する
場合、生体反応の応答を利用したバイオアッセイ法、お
よび抗原抗体反応を応用したラジオイムノアッセイ （
以下ＲＩＡと称する）とＥＩＡとが行われていた。前者
はその操作が煩雑であり、感度も不十分であることから
、一般には感度が優れている後者のＲＩＡ及びＥＩＡが
Ｌ −Ｅｎｋの測定に利用されている。ＲＩＡ及びＥＩ
Ａの原理そのものは同じで公知であり、標識抗原として
用いる標識物質の性質が異なるだけである。以下にＥＩ
Ａについて原理を説明する。即ち、まず測定対象となる
物質に対して動物を免疫感作して抗体を調製する。つぎ
に測定対象物質と標識となる酵素とをグルタルアルデヒ
ドなどの化学試薬で共有結合させた標識抗原を調製する
。抗体溶液に標識抗原を加えると、抗原抗体反応が起こ
り、溶液中に標識抗原抗体複合物（以下Ｂと称する）と
遊離のままの標識抗原（以下Ｆと称する）が生じる。こ
のＢをＦと分離して酵素活性を測定すれば抗体に結合し
た抗原の量を知ることが出来る。従って、一定休に対し
て競争的に結合反応が起こり９反応液中の非標識抗原の
量が多いほど抗原抗体複合物中の標識抗原ｆｆｉ （Ｂ
＋は減少し酵素活性も減少する。そこで。b. Conventional technology Conventional 9 When measuring L-E n in body fluids and tissues, there are two methods: bioassay methods that utilize biological reactions, and radioimmunoassays that apply antigen-antibody reactions (
(hereinafter referred to as RIA) and EIA were conducted. Since the former is complicated to operate and has insufficient sensitivity, the latter, RIA and EIA, which have excellent sensitivity, are generally used for measuring L-Enk. RIA and EI
The principle of A itself is the same and well known, and the only difference is the properties of the labeling substance used as the labeled antigen. Below is EI
The principle of A will be explained. That is, first, an animal is immunized against a substance to be measured to prepare an antibody. Next, a labeled antigen is prepared by covalently bonding the substance to be measured and an enzyme to be labeled using a chemical reagent such as glutaraldehyde. When a labeled antigen is added to an antibody solution, an antigen-antibody reaction occurs, producing a labeled antigen-antibody complex (hereinafter referred to as B) and a free labeled antigen (hereinafter referred to as F) in the solution. By separating this B from F and measuring the enzyme activity, the amount of antigen bound to the antibody can be determined. Therefore, the binding reaction occurs competitively with respect to a fixed period of time, and the greater the amount of unlabeled antigen in the reaction solution, the more labeled antigen ffi (B
+ decreases and enzyme activity also decreases. Therefore.
各種既知濃度の非標識抗原を含む溶液を標準試料溶液と
して調製し、同様な抗原抗体反応を行うと。When a solution containing various known concentrations of unlabeled antigen is prepared as a standard sample solution and a similar antigen-antibody reaction is performed.
名種既知濃度の非標識抗原存在下で抗体と結合した標識
抗原ｍ （Ｂ）が酵素活性を指標として測定でき。Labeled antigen m (B) bound to antibody in the presence of a known concentration of unlabeled antigen can be measured using enzyme activity as an indicator.
標準曲線が求められる。この標準曲線をもとに試料中の
測定対象物質濃度を定量することができる。A standard curve is determined. Based on this standard curve, the concentration of the substance to be measured in the sample can be determined.
ＲＩＡでは、標識抗原として酵素のかわりに放射性同位
元素で標識した抗原を用いる。Ｌ−Ｅｎｋを測定する場
合には、ＲＩＡとＥＩＡが利用可能であるが、ＲＩＡで
は標識用物質として放射性同位元素を用いるため、その
使用に関して法律的制約があり、また放射性同位元素の
崩壊現象により放射能が低下して長期間保存できないと
いう欠点を有している。In RIA, an antigen labeled with a radioactive isotope is used instead of an enzyme as a labeled antigen. When measuring L-Enk, RIA and EIA can be used, but since RIA uses a radioactive isotope as a labeling substance, there are legal restrictions on its use, and due to the decay phenomenon of radioactive isotopes, It has the disadvantage that it cannot be stored for a long period of time due to decreased radioactivity.
Ｃ１発明が解決しようとする問題点
Ｌ−ＥｎｋをＥＩＡで測定する場合、ＲＩＡと異なり法
的制約はな（標識抗原も長期間保存が出来るという利点
を有しているが、その測定には標識抗原の調製という煩
雑な操作があり、また９合成された標識抗原調製物は不
均一で標識抗原の抗体に対する結合活性の低下や、標識
酵素の酵素活性の低下により、測定値の精度の低下をき
たす等の問題点があった。C1 Problems to be solved by the invention When measuring L-Enk using EIA, unlike RIA, there are no legal restrictions (labeled antigen also has the advantage of being able to be stored for a long time, but the measurement requires labeling). Preparation of the antigen is a complicated operation, and the synthesized labeled antigen preparation is heterogeneous, resulting in a decrease in the binding activity of the labeled antigen to the antibody and a decrease in the enzymatic activity of the labeled enzyme, resulting in a decrease in the accuracy of the measured value. There were some problems, such as:
ｄ０発明の構成
従って、Ｌ−ＥｎｋのＥＩＡにおいて測定感度及び再現
性の高い測定を行うためには、（１）標識抗原として用
いた酵素が標識抗原調製中に活性が低下しないこと、（
２）調製された標識抗原の抗体に対する反応性が低下し
ていないこと、（３）抗原が標識用酵素と常に同じ位置
で同じ量結合した均一な標識抗原の調製が必要である。d0 Structure of the Invention Therefore, in order to perform measurements with high sensitivity and reproducibility in L-Enk EIA, it is necessary to (1) ensure that the activity of the enzyme used as the labeled antigen does not decrease during the preparation of the labeled antigen;
2) It is necessary that the reactivity of the prepared labeled antigen with respect to the antibody is not decreased, and (3) it is necessary to prepare a homogeneous labeled antigen in which the antigen is always bound to the labeling enzyme in the same amount and at the same position.
しかし、従来化学試薬を用いた標識抗原の調製法では酵
素表面の反応部位に抗原が非特異的に結合し、抗原の結
合様式も一様でな（、その標識抗原物質の精製も容易で
はないため上記の問題点の解決は血しかった。本発明者
らは、これらの問題点を改良すべ（鋭意努力した結果、
標識抗原として遺伝子操作により標識用酵素と抗原とが
遺伝的に結合した融合タンパク質の利用を考案しＬ −
ＥｎｋのＥＩＡに成功した。However, in conventional methods for preparing labeled antigens using chemical reagents, the antigen binds nonspecifically to the reaction site on the enzyme surface, and the binding mode of the antigen is not uniform (and it is not easy to purify the labeled antigen substance). Therefore, it was very difficult to solve the above-mentioned problems.
As a labeled antigen, we devised the use of a fusion protein in which a labeling enzyme and an antigen are genetically linked through genetic manipulation.
Successful EIA of Enk.
本融合タンパク質は枯草菌由来のジヒドロ葉酸還元酵素
（以下ＤＨＦＲと称する）のカルボキシル末端側にＬ
−Ｅｎｋのアミノ末端側が結合した一本鎖のポリペプチ
ドからなるタンパク質（以下ＤＨＦＲ−Ｌ −Ｅｎｋと
称する：特許出願中）である。本融合タンパク質の特徴
的な利点として、＋ＩＩＤＨＦＲ−Ｌ −Ｅｎｋを暗号
化した遺伝子が遺伝子操作により大腸菌の遺伝子に組み
込まれており、大腸菌を増殖させることにより容易に菌
体内から精製純化でき、その構造は均一でかつ常に一定
である。（２）融合タンパク質の有するＤＨＦＲ酵素活
性は天然の枯草菌由来のＤＨＦＲと同じである。（３）
酵素反応は次式の如（表され。This fusion protein contains L on the carboxyl terminal side of dihydrofolate reductase (hereinafter referred to as DHFR) derived from Bacillus subtilis.
It is a protein (hereinafter referred to as DHFR-L-Enk: patent pending) consisting of a single-chain polypeptide to which the amino terminal side of -Enk is linked. A characteristic advantage of this fusion protein is that the gene encoding +IIDHFR-L-Enk is incorporated into the E. coli gene by genetic manipulation, and it can be easily purified from within the bacterial body by growing E. coli. is uniform and always constant. (2) The DHFR enzyme activity of the fusion protein is the same as that of natural DHFR derived from Bacillus subtilis. (3)
The enzymatic reaction is expressed as follows.
７．８−Ｈｚ−葉酸＋ＮＡＤＰＨ＋Ｈ＋−５，６，７，
８Ｈ４−葉酸＋ＮＡＤＰ”
補酵素であるＮＡＤＰＨ（還元型ニコチンアミドジヌク
レオチドリン酸）の酸化を波長３４０ ｎｍでの吸収変
化を測定することにより、容易に標識酵素であるＤＨＦ
Ｒの酵素活性を知ることが出来る。（４）本融合タンパ
ク質はそのｔ７ｇ造が明確であるため、試験管やマイク
ロプレートに本融合タンパク質を吸着させた固相ＥＩＡ
も行うことが出来る。以上のごとく多くの利点を有する
融合タンパク質を標識抗原として利用することにより、
感度の高い安定したＬ −ＥｎｋのＥＩＡ法を発明出来
た。7.8-Hz-folic acid+NADPH+H+-5,6,7,
8H4-Folic Acid + NADP” By measuring the absorption change at a wavelength of 340 nm to oxidize the coenzyme NADPH (reduced nicotinamide dinucleotide phosphate), it is easy to oxidize the labeling enzyme DHF.
You can know the enzyme activity of R. (4) Since the t7g structure of this fusion protein is clear, solid-phase EIA in which this fusion protein is adsorbed to a test tube or microplate is performed.
can also be done. By using fusion proteins as labeled antigens, which have many advantages as described above,
We were able to invent a highly sensitive and stable L-Enk EIA method.
Ｌ−ＥｎｋのＥＩＡはＧ、 Ｚａｍｂｏ ｎ ｉ （Ａ
ｎａｌｙｔｉｃａｌＢｉｏｃｈｅｍｉｓｔｒｙ １３０
．８３ ８７．１９８３）らによって報告されているが
９本発明者のように標識抗原として遺伝子操作により調
製された融合タンパク質を用いてのＥＩＡの報告はない
。本発明は前述のごとく、遺伝子操作により調製された
融合タンパク質を標識抗原として用いることを特徴とし
たＥＩＡ法であり、ＥＩＡ法の実験手技により本発明が
制限されるものではない。EIA of L-Enk is G, Zamboni (A
analytical biochemistry 130
．． 83, 87, 1983), 9 but there is no report of EIA using a fusion protein prepared by genetic engineering as a labeled antigen, as the present inventors have done. As described above, the present invention is an EIA method characterized by using a fusion protein prepared by genetic manipulation as a labeled antigen, and the present invention is not limited to the experimental techniques of the EIA method.
４、実施例 以下に実施例を挙げて本発明方法を具体的に説明する。4. Example The method of the present invention will be specifically explained below with reference to Examples.
実施例 １
（１）試薬の調製
Ｌ Ｅｎｋ、 ウサギ抗Ｌ−Ｅｎｋ抗体及びＢ、
Ｆの分離に用いたヤギ抗ウサギガンマグロブリン抗体は
すべて市販のものを使用した。Example 1 (1) Preparation of reagent L Enk, rabbit anti-L-Enk antibody and B,
All goat anti-rabbit gamma globulin antibodies used for isolation of F were commercially available.
ｆ２） ＤＨＦＲＬ Ｅｎｋの調製ＤＨＦＲＬ
Ｅｎｋを暗号化した遺伝子を有するプラスミド（特許出
願中）を含有する大腸菌を遺伝子操作により調製し、液
体培養を行った。得られた湿重ｆｆ１１３ｇの菌体を、
超音波処理、ＤＥＡＥトヨパール６５０Ｍイオン交換ク
ロマトグラフィー及びトヨパールＨＷ５５ゲルクロマト
グラフィーにて分画、精製、純化を行い、約３．７ｍ、
４．８ｑ（１，３ｍｇ／ｍＪ）のＳＤＳ電気泳動で
均一なバンドを示すＤ ＨＦ ＲＬ −Ｅ ｎ ｋを得
た。f2) Preparation of DHFRL Enk DHFRL
Escherichia coli containing a plasmid (patent pending) containing a gene encoding Enk was prepared by genetic manipulation and cultured in liquid. The obtained bacterial cells with a wet weight of 113 g were
Ultrasonic treatment, fractionation, purification, and purification were performed using DEAE Toyopearl 650M ion exchange chromatography and Toyopearl HW55 gel chromatography.
DHFRL-E n k showing a uniform band was obtained by SDS electrophoresis of 4.8q (1.3 mg/mJ).
（３）測 定
抗体、ＤＨＦＲ−Ｌ Ｅｎｋ及びＬ−Ｅｎｋに対する
希釈用緩衝液としてウシ血清アルブミン（以下ＢＳＡと
称する）を含むリン酸緩衝液（５９６ＢＳＡ、２５ｍＭ
エチレンジアミン四酢酸ナトリウム、０．１９６アジ
化ナトリウム、１０ｍＭリン酸緩衝液 ｐＨ７，５：以
下ＢＥＰＢＳと称する）を用いた。(3) Measurement A phosphate buffer (596BSA, 25mM
Sodium ethylenediaminetetraacetate, 0.196 sodium azide, 10mM phosphate buffer pH 7.5: hereinafter referred to as BEPBS) were used.
まず２０００倍希釈のウサギ抗Ｌ−Ｅｎｋ抗体溶液Ｑ、
ｌ−、５０００倍希釈のＤＨＦＲＬ−Ｅｎｋ溶液Ｑ、１
ｍｌ、およびＢＥＰＢＳ ０．１ ｒｍナイＬ １０
ｔｉｇ／ｒｎｌから１ ｐｇ／１ｍまで順次希釈したＬ
−Ｅｎｋ溶液０．１ｍｌをプラスチック製ミクロ遠沈管
にいれ４℃で一晩反応させ、つぎに３００倍希釈の正常
ウサギ血清Ｑ、 ｌ ｍｌ及び１５倍希釈のヤギ抗ウサ
ギガンマグロブリン溶液Ｑ、 ｌ ｍｌを加えてさらに
４°Ｃで一晩反応させた。反応後、エッペンドル７社の
ミクロ遠心機で５分間遠心して上清を除去し、沈査に２
０μｍの０．００５規定塩酸を加えて溶解し、さらに１
０ｍＭジチオスレイトールを含む０．５Ｍリン酸緩衝液
ΦＨ７，０）２００μｌを加えて酵素活性測定用溶液と
した。酵素活性の測定は以下のように行った。即ち。First, rabbit anti-L-Enk antibody solution Q diluted 2000 times,
l-, 5000 times diluted DHFRL-Enk solution Q, 1
ml, and BEPBS 0.1 rm Nai L 10
L serially diluted from tig/rnl to 1 pg/1m
-Pour 0.1 ml of Enk solution into a plastic microcentrifuge tube and allow to react overnight at 4°C, then add 300-fold diluted normal rabbit serum Q, 1 ml and 15-fold diluted goat anti-rabbit gamma globulin solution Q, 1 ml. was added and further reacted at 4°C overnight. After the reaction, centrifuge for 5 minutes using an Eppendorf 7 microcentrifuge to remove the supernatant, and add 2
Add 0 μm 0.005 N hydrochloric acid to dissolve, and then add 1
200 μl of 0.5 M phosphate buffer ΦH7,0) containing 0 mM dithiothreitol was added to prepare a solution for measuring enzyme activity. Enzyme activity was measured as follows. That is.
酵素活性ｔ、ＩＪ定用溶液２００μｍを１２１ＴＩＩＴ
ＩＸ７５Ｍのガラス試験管に入れ、つぎにＤ ＨＦ Ｒ
基質溶液（０，０５ｍＭジヒドロ葉酸、０．０６ｍＭ
ＮＡＤＰＨ。Enzyme activity t, IJ regular solution 200μm 121TIIT
Put it in an IX75M glass test tube, then DHF R
Substrate solution (0.05mM dihydrofolic acid, 0.06mM
NADPH.
１２ｍＭ ２−メルカプトエタノールを會む５０ｍＭ
リン酸緩衝液ｐＨ７，ｏ）ｘｒｎＩ！を加えて反応を開
始した。活性測定は反応開始直後及び３０分後の波長３
４０ｎｍにおける吸収変化を分光光度計（日本分光、Ｕ
ＶＩＤＥＣ−３２０）で測定して求めた。50mM with 12mM 2-mercaptoethanol
Phosphate buffer pH 7, o) xrnI! was added to start the reaction. Activity measurement was performed at wavelength 3 immediately after the start of the reaction and after 30 minutes.
Absorption changes at 40 nm were measured using a spectrophotometer (JASCO, U
VIDEC-320).
標準曲線は非標識抗原非存在下における抗体結合ＤＨＦ
Ｒ−Ｅｎｋ（７）酵素活性を１００％とし。Standard curve shows antibody-bound DHF in the absence of unlabeled antigen.
R-Enk (7) enzyme activity is set as 100%.
非標識抗原存在下における抗体結合ＤＨＦＲＥｎｋの酵
素活性の百分率を各既知濃度のＬ−Ｅｎｋに対して次式
のごとく求めた。The percentage of enzyme activity of antibody-bound DHFREnk in the presence of unlabeled antigen was determined for each known concentration of L-Enk using the following formula.
Ｙ＝ （Ｂ Ｎ）／ （Ｂｏ Ｎ）ｘｌｏｏＢ ：
非標識抗原存在下における抗体結合ＤＨＦＲ−Ｅｎｋの
酵素活性
Ｂｏ：非標識抗原非存在下における抗体結合ＤＨＦＲ−
Ｅｎｋの酵素活性
Ｎ ：抗Ｌ−Ｅｎｋ抗体非存在下における波長３４０ｎ
ｍでの吸収変化
得られた百分率を用いて作成した標準曲線は第１図の通
りである。Y= (BN)/(Bo N)xlooB:
Enzyme activity Bo of antibody-bound DHFR-Enk in the presence of unlabeled antigen: Antibody-bound DHFR- in the absence of unlabeled antigen
Enzyme activity N of Enk: wavelength 340n in the absence of anti-L-Enk antibody
A standard curve prepared using the obtained percentage of absorption change in m is shown in FIG.
゛（４）結 果
本ＥＩＡ法により得られたＬ−Ｅｎｋに対する標準曲線
を第１図に示した。本法ではＬ−Ｅｎｋｏ、１ｎｇ／ｍ
ｌから１μｇ／ｒｎｌの濃度範囲で容量反応曲線を得た
。(4) Results The standard curve for L-Enk obtained by this EIA method is shown in FIG. In this method, L-Enko, 1 ng/m
A dose-response curve was obtained in the concentration range from l to 1 μg/rnl.
実施例２
（１）試薬の調製
固相ＥＩＡにおいてウサギ抗Ｌ−Ｅｎｋ抗体検出に用い
たペルオキシダーゼ標識ヤギ抗ウサギがンマグロプリン
抗体は市販のものを用いた。Example 2 (1) Preparation of reagents A commercially available peroxidase-labeled goat anti-rabbit magglopurin antibody was used to detect the rabbit anti-L-Enk antibody in solid-phase EIA.
（２）ＤＨＦＲ−Ｌ Ｅｎｋ固定化マイクロプレート
の調製
９６穴マイクロプートはヌンク社のイムノプレー）ＩＩ
を用いた。ＤＨＦＲ−Ｌ−Ｅｎｋ溶液を０．１Ｍ炭酸ナ
トリウム緩衝液（ｐＨ９，５）で１０００倍に希釈し、
１穴ごと５０μｌずつ分注し、４°Ｃで２時間静置して
ＤＨＦＲＬ−Ｅｎｋを固定化した。つぎにプレートを水
で洗浄し、ＢＥＰＢＳで各穴を満たし室温で１時間放置
した後、０．０５９６ツイーン２０と０．１５Ｍ塩化ナ
トリウムを含む１０ｍＭリン酸緩衝液（ｐＨ７，５：以
下ＴＰＢＳと称する）と水でプレートを洗浄した。(2) Preparation of DHFR-L Enk-immobilized microplate The 96-well microplate is Nunc's Immunoplate) II
was used. The DHFR-L-Enk solution was diluted 1000 times with 0.1M sodium carbonate buffer (pH 9,5),
50 μl was dispensed into each well and left to stand at 4°C for 2 hours to immobilize DHFRL-Enk. Next, the plate was washed with water, and each well was filled with BEPBS and left at room temperature for 1 hour, followed by a 10 mM phosphate buffer (pH 7.5, hereinafter referred to as TPBS) containing 0.0596 Tween 20 and 0.15 M sodium chloride. ) and water.
（３）測 定
Ｌ Ｅｎｋ及びペルオキシダーゼ標識ヤギ抗ウサギガ
ンマグロブリン抗体に対する希釈用緩衝液として０．０
５９６ツイーン２０を含むＢＥＰＢＳ （アジ化ナトリ
ウムを含まない二以下ＴＢＥＰＢＳと称する）を用いた
。(3) Measurement L 0.0 as dilution buffer for Enk and peroxidase-labeled goat anti-rabbit gamma globulin antibody
BEPBS containing 596 Tween 20 (referred to as sodium azide-free TBEPBS) was used.
ＤＨＦＲ−Ｌ−Ｅｎｋ固定化マイクロプレートの穴に、
５０μｍのＴＢＥＰＢＳないし１０ μｇ／ｍｌから１
０ｐｇ／ｍ＋まで順次希釈したＬ Ｅｎｋ溶液、およ
び５０μｍの２０００倍希釈のウサギ抗Ｌ−Ｅｎｋ抗体
溶液を入れて４℃で一晩反応させた。ＴＰＢＳと水でプ
レートを洗浄して遊離状態のウサギ抗ＬＥｎｋ抗体を除
去した後、４００倍に希釈したペルオキシダーゼ標識ヤ
ギ抗ウサギガンマグロブリン抗体溶液５０μｌをくわえ
、室温で１時間反応させた。ついでＴＰＢＳと水でプレ
ートを洗浄し、固定化ＤＨＦＲＬ Ｅｎｋと結合した
ウサギ抗Ｌ−Ｅｎｋ抗体を抗原として反応したペルオキ
シダーゼ標識ヤギ抗ウサギガンマグロブリン抗体のペル
オキシダーゼ酵素活性を測定するため、基質溶液として
各穴に０．２５ｍｇ／＋＋＋Ａ！の２，２’−７ジノー
ビス（３−エチルベンゾチアゾリン−６−スルホン酸）
ト０、００２５％の過酸化水素を含む０．ＩＭのクエン
酸緩衝液（ｐＨ４，２） １００μｊを加え１反応開
始１０分後にマイクロプレート光度計（コロナ社ＭＴＰ
−２２形）を用い、波長４０５ｎｍにおける吸収増加を
測定した。Into the wells of the DHFR-L-Enk immobilized microplate,
1 from 50 μm TBEPBS or 10 μg/ml
L Enk solutions sequentially diluted to 0 pg/m+ and 50 μm rabbit anti-L-Enk antibody solution diluted 2000 times were added and reacted overnight at 4°C. After washing the plate with TPBS and water to remove the free rabbit anti-LEnk antibody, 50 μl of a 400-fold diluted peroxidase-labeled goat anti-rabbit gamma globulin antibody solution was added and allowed to react at room temperature for 1 hour. Next, the plate was washed with TPBS and water, and each well was used as a substrate solution to measure the peroxidase enzyme activity of peroxidase-labeled goat anti-rabbit gamma globulin antibody that was reacted with rabbit anti-L-Enk antibody bound to immobilized DHFRL Enk as an antigen. 0.25mg/+++A! 2,2'-7dinobis(3-ethylbenzothiazoline-6-sulfonic acid)
0.0025% hydrogen peroxide. Add 100 μj of IM citrate buffer (pH 4,2) and use a microplate photometer (Corona MTP) 10 minutes after starting one reaction.
-22 type), and the increase in absorption at a wavelength of 405 nm was measured.
標準曲線は各既知濃度の非標識抗原存在下において、固
定化ＤＨＦ Ｒ−Ｅｎｋに抗Ｌ−Ｅｎｋを介して結合し
たペルオキシダーゼ標識抗体のペルオキシダーゼ酵素活
性について、波長４０５ｎｍにおける吸収増加を測定し
て求めた。The standard curve was determined by measuring the increase in absorption at a wavelength of 405 nm for the peroxidase enzyme activity of a peroxidase-labeled antibody bound to immobilized DHF R-Enk via anti-L-Enk in the presence of each known concentration of unlabeled antigen. .
得られた標準曲線は第２図の通りである。The standard curve obtained is shown in FIG.
（４）結 果
本固相ＥＩＡ法により得られたＬ−Ｅｎｋに対する標準
曲線を第２図に示した。本法ではＬ −ＥｎｋＯ，１ｎ
ｇ／ｍｌから１０ μｇ／ｍｌの濃度範囲で容量反応曲
線を得た。(4) Results The standard curve for L-Enk obtained by this solid-phase EIA method is shown in FIG. In this method, L −EnkO,1n
A dose-response curve was obtained in the concentration range from g/ml to 10 μg/ml.
５、図の簡単な説明
第１図及び第２図は実施例により得られた既知濃度のＬ
−Ｅｎｋと酵素活性との関係を示す標準曲線である。5. Brief explanation of the figures Figures 1 and 2 show L of known concentration obtained in the example.
- It is a standard curve showing the relationship between Enk and enzyme activity.
Claims (1)
−ロイシンエンケファリン融合タンパク質（以下ＤＨＦ
Ｒ−Ｌ−Ｅｎｋと称する）を暗号化した遺伝子を大腸菌
の遺伝子に組み込み、その大腸菌により合成されたＤＨ
ＦＲ−Ｌ−Ｅｎｋを、標識抗原として用いることを特徴
としたロイシンエンケファリンを測定するためのエンザ
イムイムノアッセイ法 ２）特許請求の範囲の第１項記載のＤＨＦＲ−Ｌ−Ｅｎ
ｋを固定化抗原として用いることを特徴としたロイシン
エンケファリンを測定するための固相エンザイムイムノ
アッセイ法[Scope of Claims] 1) Using genetic recombination technology, dihydrofolate reductase-leucine enkephalin fusion protein (hereinafter referred to as DHF
A gene encoding R-L-Enk) is inserted into the E. coli gene, and DH synthesized by the E. coli
Enzyme immunoassay method for measuring leucine enkephalin characterized by using FR-L-Enk as a labeled antigen 2) DHFR-L-En according to claim 1
A solid-phase enzyme immunoassay method for measuring leucine enkephalin characterized by using K as an immobilized antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25719086A JPS63111465A (en) | 1986-10-29 | 1986-10-29 | Enzyme immunoassay method for leucine enkephaline |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25719086A JPS63111465A (en) | 1986-10-29 | 1986-10-29 | Enzyme immunoassay method for leucine enkephaline |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63111465A true JPS63111465A (en) | 1988-05-16 |
| JPH0567180B2 JPH0567180B2 (en) | 1993-09-24 |
Family
ID=17302928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP25719086A Granted JPS63111465A (en) | 1986-10-29 | 1986-10-29 | Enzyme immunoassay method for leucine enkephaline |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63111465A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005114222A1 (en) * | 2004-05-13 | 2005-12-01 | Sphingo Tec Gmbh | Use of precursors of enkephalins and/or their fragments in medical diagnostics |
-
1986
- 1986-10-29 JP JP25719086A patent/JPS63111465A/en active Granted
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005114222A1 (en) * | 2004-05-13 | 2005-12-01 | Sphingo Tec Gmbh | Use of precursors of enkephalins and/or their fragments in medical diagnostics |
| EP2293079A3 (en) * | 2004-05-13 | 2011-03-30 | B.R.A.H.M.S GmbH | Use of precursors of enkephalins and/or their fragments in medical diagnostics |
| US8013123B2 (en) | 2004-05-13 | 2011-09-06 | B.R.A.H.M.S. Gmbh | Use of precursors of enkephalins and/or their fragments in medical diagnostics |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0567180B2 (en) | 1993-09-24 |
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