JPH02253162A - Detection using aequorin conjugated with material having specific conjugatability - Google Patents
Detection using aequorin conjugated with material having specific conjugatabilityInfo
- Publication number
- JPH02253162A JPH02253162A JP7474289A JP7474289A JPH02253162A JP H02253162 A JPH02253162 A JP H02253162A JP 7474289 A JP7474289 A JP 7474289A JP 7474289 A JP7474289 A JP 7474289A JP H02253162 A JPH02253162 A JP H02253162A
- Authority
- JP
- Japan
- Prior art keywords
- aequorin
- substance
- igg
- protein
- specific binding
- 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
- 108010000239 Aequorin Proteins 0.000 title claims abstract description 58
- 238000001514 detection method Methods 0.000 title claims description 19
- 239000000463 material Substances 0.000 title abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 26
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- -1 for example Substances 0.000 claims abstract description 3
- 239000000126 substance Substances 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 28
- 230000009870 specific binding Effects 0.000 claims description 27
- 239000013076 target substance Substances 0.000 claims description 18
- 230000027455 binding Effects 0.000 claims description 14
- 238000009739 binding Methods 0.000 claims description 14
- 230000000694 effects Effects 0.000 claims description 12
- 108020004414 DNA Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 4
- 108091008324 binding proteins Proteins 0.000 claims description 3
- 239000005515 coenzyme Substances 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims description 2
- 101710096438 DNA-binding protein Proteins 0.000 claims description 2
- 101710120037 Toxin CcdB Proteins 0.000 claims description 2
- 102000023732 binding proteins Human genes 0.000 claims 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 abstract description 8
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 4
- 239000004793 Polystyrene Substances 0.000 abstract description 4
- 239000002981 blocking agent Substances 0.000 abstract description 4
- 229910052791 calcium Inorganic materials 0.000 abstract description 4
- 239000011575 calcium Substances 0.000 abstract description 4
- 229920002223 polystyrene Polymers 0.000 abstract description 4
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 abstract description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 abstract description 2
- 239000007790 solid phase Substances 0.000 abstract description 2
- 230000001268 conjugating effect Effects 0.000 abstract 2
- 239000013077 target material Substances 0.000 abstract 2
- 230000021615 conjugation Effects 0.000 abstract 1
- YHIPILPTUVMWQT-UHFFFAOYSA-N Oplophorus luciferin Chemical compound C1=CC(O)=CC=C1CC(C(N1C=C(N2)C=3C=CC(O)=CC=3)=O)=NC1=C2CC1=CC=CC=C1 YHIPILPTUVMWQT-UHFFFAOYSA-N 0.000 description 8
- 238000004020 luminiscence type Methods 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000006180 TBST buffer Substances 0.000 description 6
- 108020001507 fusion proteins Proteins 0.000 description 6
- 102000037865 fusion proteins Human genes 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 108010041089 apoaequorin Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 241000242583 Scyphozoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000012521 purified sample Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 1
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔技術の分野〕
本発明は、特異的結合能を有する物質と結合したエクオ
リンを用いた標的物の検出法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Technology] The present invention relates to a method for detecting a target using aequorin bound to a substance having specific binding ability.
発光蛋白エクオリンは、米国ワシントン州フライデーハ
ーバ−島近郊の海洋に生息する発光オワンクラゲより単
離されたカルシウム結合タンパク質である。エクオリン
は、自然界においてはタンパク貿部分のアポエクオリン
と、基質部分であるセレンテラジンが、分子状酸素を介
して複合体を形成しており、この複合体にカルシウムが
結合することにより発光することを特徴とする。この発
光を利用してカルシウム濃度を測定することができる。The photoprotein aequorin is a calcium-binding protein isolated from the luminescent aequorin jellyfish that lives in the ocean near Friday Harbor Island, Washington, USA. Aequorin is characterized by the fact that in nature, apoaequorin, which is a protein trading part, and coelenterazine, which is a substrate part, form a complex via molecular oxygen, and when calcium binds to this complex, it emits light. shall be. Calcium concentration can be measured using this luminescence.
本発明者は組換えＤＮＡの手法を用いて、発光オワンク
ラゲよりアポエクオリンのｃＤＮＡをクローニングし、
その１次構造を決定したく特開昭６１−１３５．５８６
）。次いで、このｃＤＮＡを用いて大腸菌を宿主とし
、その菌体内及び菌体外でのアポエクオリンの生産に成
功した（特願昭６０−２８０．２５９．６１−２４９．
０９８ ）。さらに、機能遺伝子と結合したエクオリン
遺伝子を作成し、その融合タンパク質の生産に成功した
く特願＠ ６２−１９６．θ３１ ）。また、エクオリ
ンの発光を利用した金属の検出方法を開発した（特願昭
６１−１０３，８４９ ）。そして、特異的結合タンパ
ク質の遺伝子と結合したエクオリン遺伝子を作成し、そ
の融合タンパク貿の生産に成功した（特願昭［１３−３
０８，４２４）。The present inventor used recombinant DNA techniques to clone the cDNA of apoaequorin from the luminescent Aequorina jellyfish,
To determine its primary structure, JP-A-61-135.586
). Next, using this cDNA, we successfully produced apoaequorin inside and outside the bacterial cell using Escherichia coli as a host (Japanese Patent Application No. 60-280-259-61-249).
098). In addition, we would like to create an aequorin gene combined with a functional gene and successfully produce a fusion protein using the aequorin gene @62-196. θ31). He also developed a method for detecting metals using the luminescence of aequorin (Japanese Patent Application No. 103,849/1984). Then, they created an aequorin gene combined with a specific binding protein gene and succeeded in producing the fusion protein (Tokugan Sho [13-3
08,424).
さらに、酵素免疫測定法に利用すべく、その融合タンパ
ク質の高純度精製標品の調製法を確立した（
）。Furthermore, we established a method for preparing a highly purified sample of the fusion protein for use in enzyme immunoassay (
).
しかし、未だエクオリンの発光を利用した金属以外の検
出技術の報告はなく、本発明は特異的結合能を有する物
質と結合したエクオリンを用いた検出技術への応用を実
証した最初の報告である。However, there have been no reports yet of a detection technique for detecting substances other than metals using the luminescence of aequorin, and the present invention is the first report demonstrating the application of aequorin to a detection technique using a substance bound to a substance with specific binding ability.
ところで、エクオリンの有用性は当業者に周知であり、
エクオリンを特異的結合能を有する物質を介して標的１
に結合することが可能になれば、標的物を特異的に発光
で検出することができる。By the way, the usefulness of Aequorin is well known to those skilled in the art.
Target 1 via a substance with specific binding ability to aequorin
If it becomes possible to bind to the target substance, the target substance can be specifically detected by luminescence.
ここで、特異的結合とは抗原抗体反応、酵素反応、レセ
プターへの特異的結合、核酸とタンパク貿の特異的結合
、核酸のハイブリッド形成等を利用したものである。す
なわち、特異的結合能を有する物質と結合したエクオリ
ンは、酵素免疫測定法やＤＮＡプローブなどのあらゆる
測定系に応用できるものであり、上述した機能から診断
薬等の検査薬として有用であることが予測される。Here, specific binding refers to the use of antigen-antibody reactions, enzyme reactions, specific binding to receptors, specific binding between nucleic acids and proteins, hybridization of nucleic acids, and the like. In other words, aequorin bound to a substance with specific binding ability can be applied to all measurement systems such as enzyme-linked immunosorbent assays and DNA probes, and the above-mentioned functions indicate that it is useful as a test agent such as a diagnostic agent. is expected.
本発明者は上述の技術的事情にかんがみ、研究の結果、
特異的結合能を有する物質と結合したエクオリンを用い
た発光による検出法を開発することができた。以上の説
明から明らかなように、本発明の目的はエクオリンを用
いた新しい検出技術を提供することである。In view of the above-mentioned technical circumstances, as a result of research, the present inventor has
We were able to develop a luminescent detection method using aequorin bound to a substance with specific binding ability. As is clear from the above description, an object of the present invention is to provide a new detection technique using aequorin.
本発明（１発明）は、下記 （１）〜（５）の構成を有
する。The present invention (1 invention) has the following configurations (1) to (5).
（１） Ｖ異的結合能を有する物質とエクオリンを結合
させ、かくして得られた結合物を標的物質に結合させる
ことを特徴とする標的物質の検出法。(1) A method for detecting a target substance, which comprises binding aequorin to a substance having V-differential binding ability, and binding the thus obtained bound product to the target substance.
（２）結合物がエクオリン活性および抗体結合能を有す
る物質である前記第（＋、）項に記載の検出法。(2) The detection method according to item (+) above, wherein the bound substance is a substance having aequorin activity and antibody binding ability.
（３）特異的結合能を有する物質が、酵素、抗体、プロ
ティンＡ、プロティンＧ、 ＤＮＡ 、 ＲＮＡ 、
ＤＮＡ結合タンパク貿若しくはレセプターである前記
第（１）項に記載の検出法。(3) Substances with specific binding ability include enzymes, antibodies, protein A, protein G, DNA, RNA,
The detection method according to item (1) above, which is a DNA binding protein or receptor.
（４）標的物質が、基質、補酵素、補欠分子族、抗原、
抗体、ＤＮＡ、 ＲＮＡ 、ホルモン若しくはトランス
ミツターである前記第（１）項に記載の検出法。(4) The target substance is a substrate, a coenzyme, a prosthetic group, an antigen,
The detection method according to item (1) above, which is an antibody, DNA, RNA, hormone, or transmitter.
（５）プロティンＡをエクオリンと結合させ、かくして
得られた結合物を抗体と結合させることを特徴とする抗
体の検出法。(5) A method for detecting antibodies, which comprises binding protein A to aequorin and binding the thus obtained bound product to an antibody.
本発明の構成と効果につき以下に詳述する。The configuration and effects of the present invention will be explained in detail below.
本発明はエクオリン活性を保持した特異的結合能を有す
る物質と結合したエクオリンを用いた標的物の検出法で
あり、例えば後述の実施例に示す方法で行うことができ
る。The present invention is a method for detecting a target using aequorin bound to a substance having specific binding ability that retains aequorin activity, and can be carried out, for example, by the method shown in the Examples below.
本発明の方法において°°特異的結合能を有する°′物
質とは、標的物（後述）と特異的すなわち選択的に結合
する物質をいうのであって、例えば、抗原−抗体反応に
おける抗体を意味する。In the method of the present invention, the term "substance with specific binding ability" refers to a substance that specifically or selectively binds to a target (described later), and for example, refers to an antibody in an antigen-antibody reaction. do.
また、標的物とは、本発明方法の対象となる被検出物を
いい、例えば、抗原−抗体反応における抗原を意味する
。Further, the target substance refers to a substance to be detected that is a target of the method of the present invention, and refers to, for example, an antigen in an antigen-antibody reaction.
したがって、標的物と特異的結合能を有する物質（以下
特異結合能物質ということがある）とは、場合により相
対的に互換性がある。か＼る標的物と特異結合能物質と
の特異結合関係を例示すると下表のとおりである。Therefore, a target substance and a substance having a specific binding ability (hereinafter sometimes referred to as a specific binding ability substance) are relatively compatible depending on the case. An example of the specific binding relationship between the target substance and the substance capable of specific binding is shown in the table below.
特異結合能物質とエクオリンとの結合については、本発
明者等の発明に係る特願昭６３−３０８．４２４号にも
述べられているが、次のとおりである。The binding between a specific binding substance and aequorin is also described in Japanese Patent Application No. 308.424/1989, which was filed by the present inventors, and is as follows.
１つの方法は、化学合成の手法を用いる場合で、たとえ
ば、マレイミド法やグルタルアルデヒド法などにより特
異結合能物質やエクオリンを修飾し、両者を結″合せし
める。この方法は特異結合能物質がタンパク質以外の物
質、たとえばＤＮＡやＲＮＡなとでも結合することがで
き、また、種々の特異結合能物質とエクオリンを容易に
短時間で結合することができ、応用範囲が広い結合方法
である。もう１つの方法は、組換えＤＮＡの手法を用い
る場合で、特異結合能物質はタンパク質にかぎられ、エ
クオリンとの結合は融合という形をとり、１分子の融合
タンパク質として発現される。特異結合能物質としてプ
ロティンＡを用いる場合、エクオリン発現ベクターｐｉ
ＰＨＥ（特開昭６３−１０２，６９５）からＥｃｏＲＩ
消化によりエクオリンｃＤＮＡの断片を分離する。次に
その断片をプロティンＡ融合発現ベクターｐＲＩＴ５
（ファルマシア社（株）製）のＥｃｏＲＩサイトに挿
入し、 ｐＡＡＱｌを作成する（特願昭６３−３０８，
４２４号の第１図参照）。プロティンＡ−エクオリン融
合タンパク質発現ベクターｐＡＡＱ１を大腸菌ＪＭ８３
株に形質転換して得られた形質転換株＋１ＡＡＱＩ／Ｊ
Ｍ８３を培養することにより発現されるプロティンＡ−
エクオリン融合タンパク質を以下に述べるエクオリン標
識プロティンとして使用する。One method is to use a chemical synthesis method, for example, by modifying the specific binding ability substance or aequorin using the maleimide method or the glutaraldehyde method, and binding the two.In this method, the specific binding ability substance is a protein. It is a binding method that has a wide range of applications, as it can bind to other substances such as DNA and RNA, and can easily bind aequorin to various substances with specific binding ability in a short time. One method is to use recombinant DNA techniques, in which the substance with specific binding ability is limited to proteins, and the binding with aequorin takes the form of fusion, and it is expressed as a single molecule of fusion protein. When using protein A, aequorin expression vector pi
EcoRI from PHE (JP 63-102,695)
Aequorin cDNA fragments are separated by digestion. Next, the fragment was inserted into the protein A fusion expression vector pRIT5.
(manufactured by Pharmacia Co., Ltd.) to create pAAQl (Patent application 1986-308,
(See Figure 1 of No. 424). Protein A-equorin fusion protein expression vector pAAQ1 was transferred to E. coli JM83.
Transformed strain +1AAQI/J obtained by transforming the strain
Protein A- expressed by culturing M83
Aequorin fusion protein is used as the aequorin-labeled protein described below.
第１図は、エクオリン標識プロティンＡのエクオリン活
性に対するＩｇＧ濃度の影響を示したものである。FIG. 1 shows the influence of IgG concentration on the aequorin activity of aequorin-labeled protein A.
第２図は、エクオリン標識プロティンＡを用いた抗体の
検出法（サンドイツチ法）を模式的に示したものである
。FIG. 2 schematically shows an antibody detection method (Sand-Deutsch method) using aequorin-labeled protein A.
すなわち、固相（例えば、ポリスチレン）に、Ｆｃ断片
を含まないＦ（ａｂ’）２（抗原）を吸着させる。未吸
着Ｆ（ａｂ’）２を除いた後、ブロッキング剤（ＢＳＡ
）でブロッキングする。ブロッキング剤を除いた後、種
々の濃度のＩｇＧとＦ（ａｂ’）”を接触させる。未吸
着のＩｇＧを除いた後、エクオリン標識プロティンＡと
ＩｇＧを接触させる。未吸着のエクオリン標識プロティ
ンＡを除いた後、セレンテラジン及び２−メルカプトエ
タノール存在下で、エクオリンを再生させる。大過剰の
カルシウムを加えて発光させ、その発光量からＩｇＧを
定量できる。That is, F(ab')2 (antigen) that does not contain an Fc fragment is adsorbed onto a solid phase (eg, polystyrene). After removing unadsorbed F(ab')2, blocking agent (BSA
) to block. After removing the blocking agent, IgG at various concentrations is brought into contact with F(ab')''. After removing unadsorbed IgG, IgG is brought into contact with aequorin-labeled protein A. After removal, aequorin is regenerated in the presence of coelenterazine and 2-mercaptoethanol.A large excess of calcium is added to cause luminescence, and IgG can be quantified from the amount of luminescence.
第３図は、エクオリン標識プロティンＡを用いた酵素免
疫測定法（サンドイツチ法）の検出感度を示す。FIG. 3 shows the detection sensitivity of enzyme immunoassay (Sand-Deutsch method) using aequorin-labeled protein A.
（発明の効果）
本発明の検出法によれば、対象物中の補酵素、抗原、抗
体、ＤＮＡ 、 ＲＮＡ 、ホルモン若しくはトランス
ミツター等の標的物質と特異的結合能を有する物質を選
択してエクオリンで標識し、該標識物を上述の標的物質
と結合せしめた後カルジム添加によりエクオリンを発光
せしめるので、被検体中の標的物質の濃度が極めて微量
であっても検出できる。(Effects of the Invention) According to the detection method of the present invention, a substance that has the ability to specifically bind to a target substance such as a coenzyme, antigen, antibody, DNA, RNA, hormone or transmitter in a target substance is selected. Since the aequorin is labeled with aequorin, the labeled substance is bound to the above-mentioned target substance, and the aequorin is made to emit light by adding cardiom, it can be detected even if the concentration of the target substance in the specimen is extremely small.
また、エクオリンで標識される物質は、標的物に対し、
特異的結合能を有する物質であるので、被検体中の標的
物質以外の物質と結合することがなく、検出感度が極め
て高い。In addition, substances labeled with aequorin can
Since it is a substance that has specific binding ability, it does not bind to substances other than the target substance in the analyte, and has extremely high detection sensitivity.
更にまた、標的物質の種類により、対応する特異的結合
能を有する物質を選択できるので、極めて広範囲の対象
（標的物質）に対して適用可能で、その利用範囲は極め
て広い。Furthermore, since a substance having a corresponding specific binding ability can be selected depending on the type of target substance, it can be applied to an extremely wide range of objects (target substances), and its range of use is extremely wide.
以下に実施例によって本発明を説明する。The present invention will be explained below by way of examples.
実施例１［エクオリン標識プロティンＡのエクオリン活
性に対するＩｇＧ濃度の影響コ
ゲルろ過精製エクオリン標識プロティンＡ（９，６μｇ
／ｌ１ｌｆｌ ） １０μρ、抗ウサギＩｇＧである
ヤギｒｇＧ （種々濃度）１０μβ、２−メルカプト
エタノール１μ℃、セレンテラジン（２ｍｇ／ｍＪ２）
１μ℃、３０ｍＭ ＴｒｉｓＪＩＣＪ２．１０ｍＭ Ｅ
ＤＴＡ （ｐＨ７，８）バッファー７８μにすなわち、
全容量１００μｋにて４℃、１５時間放置して、エクオ
リンを再生させた。Example 1 [Effect of IgG concentration on aequorin activity of aequorin-labeled protein A] Cogel filtration purified aequorin-labeled protein A (9.6 μg
/l1lfl) 10μρ, anti-rabbit IgG goat rgG (various concentrations) 10μβ, 2-mercaptoethanol 1μ℃, coelenterazine (2mg/mJ2)
1μ℃, 30mM TrisJICJ2.10mM E
DTA (pH 7,8) buffer 78μ, i.e.
Aequorin was regenerated by standing at 4° C. for 15 hours at a total volume of 100 μk.
反応液の一部をルミフォトメーター（ＴＤ−４０００、
ラボサイエンス社）のキュベツト中に移し、３０ｍＭＣ
ａＣｊ２２を１００μρ注入して、その発光量を測定し
た。A portion of the reaction solution was measured using a Lumiphotometer (TD-4000,
Transfer to a 30mMC cuvette (Labo Science).
100 μρ of aCj22 was injected, and the amount of luminescence was measured.
その結果をまとめたものが、第１図である。エクオリン
活性はＩｇＧ濃度１０−’ｍｇ／ｍｌ付近から徐々に低
下するもののＩｇＧ濃度０．２８ｍｇ／ｍｕでも８５％
の活性を保持していた。これはエクオリン標識プロティ
ンＡがＩｇＧに結合した状態あるいはＩｇＧと共存した
状態でもゴエクオリン活性が十分であることを示すもの
であり、酵素免疫測定法への応用が可能であることを強
く示唆するものである。Figure 1 summarizes the results. Aequorin activity gradually decreases from around 10-'mg/ml IgG concentration, but remains 85% even at 0.28 mg/mu IgG concentration.
retained its activity. This shows that goaequorin activity is sufficient even when aequorin-labeled protein A is bound to IgG or coexists with IgG, and strongly suggests that it can be applied to enzyme immunoassay. be.
実施例２［サンドイツチ法に基づいたエクオリン標識プ
ロティンＡによるＩｇＧの検出］ポリスチレン・チュー
ブ（１０φｘ ６５ｍｍ）を２ＮＮａＯＨ１６０℃に３
０分間浸して、洗浄した。水でＮａＯＨを洗い流した後
、５０℃にて乾燥した。第２図に示すようにＮａ０）Ｉ
処理ポリスチレン・チューブ中に２６μｇ／ｍＪ２ウサ
ギＦ （ａｂ’）２抗ヤギＩｇＧ 、 １００ μｕを
側壁に付かないように添加し、３７℃で３時間インキュ
ベーションした。Example 2 [Detection of IgG using aequorin-labeled protein A based on the Sanderuch method] A polystyrene tube (10φ x 65 mm) was heated with 2N NaOH at 160°C for 3 hours.
Soaked for 0 minutes and washed. After washing off NaOH with water, it was dried at 50°C. As shown in Figure 2, Na0)I
100 μu of 26 μg/mJ2 rabbit F (ab′)2 anti-goat IgG was added to the treated polystyrene tube without sticking to the side wall and incubated at 37° C. for 3 hours.
ウサギＦ （ａｂ’）２抗ヤギＩｇＧを除いた後、ブロ
ッキング・バッファー（１，０％牛血清アルブミンｉｎ
２０ｍＭ Ｔｒｉｓ−ＨＣｊＬ ｐＨ７，５，５００ｍ
Ｍ ＮａＣｌ［ＴＢＳ］ ） ２．５＋ｎｆＬを添加し
、３７℃で２．５時間インキュベーションした。After removing rabbit F (ab')2 anti-goat IgG, blocking buffer (1.0% bovine serum albumin in
20mM Tris-HCjL pH7,5,500m
M NaCl[TBS] ) 2.5+nfL was added and incubated at 37°C for 2.5 hours.
ブロッキング・バッファーを除いた後、２０ｍＭＴｒｊ
ｓ−ＨＣｌ２ ｐ）１７．５．５００ｍ１Ｊ ＮａＣ
Ｊ２．０．０５％Ｔｗｅｅｎ−２０［ＴＴＢＳ］、２．
５ｍＪ２にて１０回洗浄した。After removing the blocking buffer, 20mM Trj
s-HCl2 p) 17.5.500m1J NaC
J2.0.05% Tween-20 [TTBS], 2.
Washed 10 times with 5 mJ2.
遠心して完全にＴＴＢＳを除いた後、種々濃度（２，５
μｇ ／ｍｆ１〜２５０μｇ／ＩＩＩＪｌ）のヤギＩｇ
Ｇ抗ウサギＩｇＧ、 １００μｋを添加し、３７℃で１
５時間インキュベーションした。ヤギＩｇＧ抗ウサギＩ
ｇＧを除いた後、ＴＴＢＳ ２．５ ｌｌ１ｉｔにて１
０回洗浄した。After completely removing TTBS by centrifugation, various concentrations (2, 5
μg/mf1-250μg/IIIJl) of goat Ig
Add 100 μk of G anti-rabbit IgG and incubate at 37°C for 1 hour.
Incubation was for 5 hours. Goat IgG anti-rabbit I
After removing gG, 1 at TTBS 2.5 ll1it
Washed 0 times.
遠心して完全にＴＴＢＳを除いた後、９ｆｉｎｇ／ｍｆ
！、エクオリン標識プロティンＡ（ゲルろ過精製サンプ
ル）１００μｍを添加し、３７℃にて１．５時間インキ
ュベーションした。エクオリン標識プロティンＡを除い
た後、ＴＴＢＳ ２．５ ｌｌｌ１．にて１０回洗浄し
た。After centrifugation to completely remove TTBS, 9fing/mf
! , 100 μm of aequorin-labeled protein A (gel filtration purified sample) was added, and the mixture was incubated at 37° C. for 1.5 hours. After removing aequorin-labeled protein A, TTBS 2.5 lll1. Washed 10 times.
遠心して完全にＴＴＢＳを除いた後１０μｇ ７ｍ１ｌ
セレンテラジン、０．５％２−メルカプトエタノール、
＋ｎ３０ｍＭ Ｔｒｉｓ−ＨＣｌ２ （ｐＨ７，６）
、１０ｍＭ ＥＤＴＡ、 １００μ４１添加し、４℃に
１５時間放置した。After centrifugation to completely remove TTBS, 10μg 7ml
coelenterazine, 0.5% 2-mercaptoethanol,
+n30mM Tris-HCl2 (pH7,6)
, 10mM EDTA, 100μ41 were added, and the mixture was left at 4°C for 15 hours.
ルミフォトメーター（ＴＤ−４０００、ラボサイエンス
社）にチューブを移し、３０＋ｎＭ ＣａＣ１２を１０
０μＪ２注入し、その発光量を測定した。その結果を第
３図に示した。抗体量（、ＩｇＧ ）が１０−’ 〜１
ｕ ｇ／１ｕｂｅに関して、発光量との比例関係が認
められ、その範囲内において抗体の定量が可能であるこ
とがわかる。Transfer the tube to a Lumiphotometer (TD-4000, Labo Science) and add 30+nM CaC12 for 10 minutes.
0 μJ2 was injected and the amount of light emitted was measured. The results are shown in Figure 3. Antibody amount (IgG) is 10-' to 1
Regarding ug/1ube, a proportional relationship with the amount of luminescence was observed, and it was found that the antibody could be quantified within this range.
本検出法は、種々の特異的結合を利用することにより、
広範囲に適用できる。また、エクオリン標識特異的結合
物質は、種々の結合法（マレイミド法やグルタルアルデ
ヒド法など）を用いることにより、容易に調製すること
が可能である。さらに基質（発光体）や、発光検出機の
改善に伴ない、より感度の高い検出システムになると考
えられる。This detection method uses various specific bindings to
Applicable to a wide range of areas. Furthermore, aequorin-labeled specific binding substances can be easily prepared by using various binding methods (maleimide method, glutaraldehyde method, etc.). Furthermore, improvements in substrates (luminescent bodies) and luminescent detectors are expected to lead to more sensitive detection systems.
第１．２．３図は本発明の説明図である。
第１図は、エクオリン標識プロティンＡのエクオリン活
性に対するＩｇＧ濃度の影響を示したものである。
第２図は、エクオリン標識プロティンＡを用いたＩｇＧ
の検出法（サンドイツチ法）を模式的に示したものであ
る。
第３図は、エクオリン標識プロティンＡを用いた酵素免
疫測定法（サンドイツチ法）の検出感度を示したもので
ある。
以上FIG. 1.2.3 is an explanatory diagram of the present invention. FIG. 1 shows the influence of IgG concentration on the aequorin activity of aequorin-labeled protein A. Figure 2 shows IgG using aequorin-labeled protein A.
This is a schematic diagram of the detection method (Sandermanch method). FIG. 3 shows the detection sensitivity of enzyme immunoassay (Sand-Deutsch method) using aequorin-labeled protein A. that's all
Claims (5)
せ、かくして得られた結合物を標的物質に結合させるこ
とを特徴とする標的物質の検出法。(1) A method for detecting a target substance, which comprises binding aequorin to a substance having specific binding ability, and binding the thus obtained bound substance to the target substance.
る物質である特許請求の範囲第（１）項に記載の検出法
。(2) The detection method according to claim (1), wherein the bound substance is a substance having aequorin activity and antibody binding ability.
テインＡ、プロテインＧ、ＤＮＡ、ＲＮＡ、ＤＮＡ結合
タンパク質若しくはレセプターである特許請求の範囲第
（１）項に記載の検出法。(3) The detection method according to claim (1), wherein the substance having specific binding ability is an enzyme, antibody, protein A, protein G, DNA, RNA, a DNA binding protein, or a receptor.
抗体、ＤＮＡ、ＲＮＡ、ホルモン若しくはトランスミッ
ターである特許請求の範囲第（１）項に記載の検出法。(4) The target substance is a substrate, a coenzyme, a prosthetic group, an antigen,
The detection method according to claim (1), which is an antibody, DNA, RNA, hormone, or transmitter.
得られた結合物を抗体と結合させることを特徴とする抗
体の検出法。(5) A method for detecting antibodies, which comprises binding protein A to aequorin and binding the thus obtained bound product to an antibody.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7474289A JPH02253162A (en) | 1989-03-27 | 1989-03-27 | Detection using aequorin conjugated with material having specific conjugatability |
| EP19890121881 EP0372352B1 (en) | 1988-12-06 | 1989-11-27 | Aequorin fused with a protein having a specific-binding activity, its preparation, its purification and detection method by its use |
| DE1989622971 DE68922971T2 (en) | 1988-12-06 | 1989-11-27 | Aequorin fused to a protein with specific binding activity, its production, purification and detection method. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7474289A JPH02253162A (en) | 1989-03-27 | 1989-03-27 | Detection using aequorin conjugated with material having specific conjugatability |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02253162A true JPH02253162A (en) | 1990-10-11 |
Family
ID=13556003
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7474289A Pending JPH02253162A (en) | 1988-12-06 | 1989-03-27 | Detection using aequorin conjugated with material having specific conjugatability |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02253162A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH044872A (en) * | 1989-06-26 | 1992-01-09 | Commiss Energ Atom | Hybrid protein containing extracellular enzyme and at least one other protein and preparation thereof |
| US5486455A (en) * | 1993-02-12 | 1996-01-23 | Sealite Sciences, Inc. | Photoprotein conjugates and methods of use thereof |
| US5648218A (en) * | 1993-02-12 | 1997-07-15 | Sealite Sciences, Inc. | Preparation of photoprotein conjugates and methods of use thereof |
| JP2008048607A (en) * | 2006-08-22 | 2008-03-06 | Chisso Corp | FUSION PROTEIN OF Fc-BINDING DOMAIN AND CALCIUM-BINDING PHOTOPROTEIN, GENE ENCODING THE SAME AND USE THEREOF |
| EP1145002B2 (en) † | 1998-07-06 | 2008-12-10 | PerkinElmer Cellular Sciences Belgium BVBA | Bioluminescent assay for agonists or antagonists of a calcium-coupled receptor |
| JP2009236905A (en) * | 2008-03-04 | 2009-10-15 | Kino Pharma:Kk | Screening method of phosphate enzyme inhibitor using light emitting protein |
| US7947509B2 (en) | 2001-02-07 | 2011-05-24 | Massachusetts Institute Of Technology | Optoelectronic detection system |
| US8067184B2 (en) | 2001-02-07 | 2011-11-29 | Massachusetts Institute Of Technology | Optoelectronic detection system |
| US8216797B2 (en) | 2001-02-07 | 2012-07-10 | Massachusetts Institute Of Technology | Pathogen detection biosensor |
| US8722347B2 (en) | 1997-12-09 | 2014-05-13 | Massachusetts Institute Of Technology | Optoelectronic sensor |
| JP2015091873A (en) * | 2015-01-30 | 2015-05-14 | Jnc株式会社 | Fusion protein of fc-binding domain and calcium binding luminescent protein, gene encoding the same and use thereof |
-
1989
- 1989-03-27 JP JP7474289A patent/JPH02253162A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH044872A (en) * | 1989-06-26 | 1992-01-09 | Commiss Energ Atom | Hybrid protein containing extracellular enzyme and at least one other protein and preparation thereof |
| US5486455A (en) * | 1993-02-12 | 1996-01-23 | Sealite Sciences, Inc. | Photoprotein conjugates and methods of use thereof |
| US5648218A (en) * | 1993-02-12 | 1997-07-15 | Sealite Sciences, Inc. | Preparation of photoprotein conjugates and methods of use thereof |
| US8722347B2 (en) | 1997-12-09 | 2014-05-13 | Massachusetts Institute Of Technology | Optoelectronic sensor |
| EP1145002B2 (en) † | 1998-07-06 | 2008-12-10 | PerkinElmer Cellular Sciences Belgium BVBA | Bioluminescent assay for agonists or antagonists of a calcium-coupled receptor |
| US7947509B2 (en) | 2001-02-07 | 2011-05-24 | Massachusetts Institute Of Technology | Optoelectronic detection system |
| US8067184B2 (en) | 2001-02-07 | 2011-11-29 | Massachusetts Institute Of Technology | Optoelectronic detection system |
| US8216797B2 (en) | 2001-02-07 | 2012-07-10 | Massachusetts Institute Of Technology | Pathogen detection biosensor |
| US8835127B2 (en) | 2001-02-07 | 2014-09-16 | Massachusetts Institute Of Technology | Optoelectronic detection system |
| US9005989B2 (en) | 2001-02-07 | 2015-04-14 | Massachusetts Institute Of Technology | Optoelectronic detection system |
| US9291549B2 (en) | 2001-02-07 | 2016-03-22 | Massachusetts Institute Of Technology | Pathogen detection biosensor |
| US9494579B2 (en) | 2001-02-07 | 2016-11-15 | Massachusetts Institute Of Technology | Optoelectronic detection system |
| JP2008048607A (en) * | 2006-08-22 | 2008-03-06 | Chisso Corp | FUSION PROTEIN OF Fc-BINDING DOMAIN AND CALCIUM-BINDING PHOTOPROTEIN, GENE ENCODING THE SAME AND USE THEREOF |
| JP2009236905A (en) * | 2008-03-04 | 2009-10-15 | Kino Pharma:Kk | Screening method of phosphate enzyme inhibitor using light emitting protein |
| JP2015091873A (en) * | 2015-01-30 | 2015-05-14 | Jnc株式会社 | Fusion protein of fc-binding domain and calcium binding luminescent protein, gene encoding the same and use thereof |
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