JP2009153444A - Aqueous solution of luminescent substrate and method for producing the same - Google Patents
Aqueous solution of luminescent substrate and method for producing the same Download PDFInfo
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- 239000007864 aqueous solution Substances 0.000 title claims abstract description 72
- 238000004519 manufacturing process Methods 0.000 title claims description 16
- 239000003960 organic solvent Substances 0.000 claims abstract description 56
- 239000000243 solution Substances 0.000 claims abstract description 52
- 239000005089 Luciferase Substances 0.000 claims abstract description 36
- 108060001084 Luciferase Proteins 0.000 claims abstract description 35
- 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 claims description 76
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 60
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 claims description 14
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 239000002504 physiological saline solution Substances 0.000 claims description 11
- 238000003556 assay Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 claims description 8
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 claims description 8
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 claims description 8
- ZWPWSXGBDMGKKS-ZDUSSCGKSA-N Cypridina luciferin Chemical compound C1=CC=C2C(C=3NC(CCCNC(N)=N)=C4N=C(C(N4C=3)=O)[C@@H](C)CC)=CNC2=C1 ZWPWSXGBDMGKKS-ZDUSSCGKSA-N 0.000 claims description 6
- JREHDCFHGRHVKG-ZDUSSCGKSA-N Cypridina luciferin Natural products CC[C@H](C)C1=NC2=C(CCCNC(=N)N)NC(=CN2C1=O)c3cc4ccccc4[nH]3 JREHDCFHGRHVKG-ZDUSSCGKSA-N 0.000 claims description 6
- QUHVVVWAQMRCSJ-IXXPHHLHSA-N dinoflagellate luciferin Chemical compound N1C(CC2=C(C=3C(=O)CC(/C=3N2)=C/2[C@H]([C@H](C)[C@H](N\2)C(O)=O)CCC(O)=O)C)=C(CC)C(C)=C1CC1NC(=O)C(C)=C1C=C QUHVVVWAQMRCSJ-IXXPHHLHSA-N 0.000 claims description 6
- 238000004020 luminiscence type Methods 0.000 description 20
- 239000012472 biological sample Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
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- 239000012491 analyte Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
本発明は、ルシフェラーゼの発光に用いる発光基質水溶液、その製造方法、それを用いたレポーターアッセイ法、および発光基質水溶液キットに関する。 The present invention relates to a luminescent substrate aqueous solution used for luminescence of luciferase, a method for producing the same, a reporter assay method using the same, and a luminescent substrate aqueous solution kit.
ルシフェラーゼは原核細胞生物である発光細菌から高等真核細胞生物であるホタルまでの発光生物において、酸素存在下で発光基質（ルシフェリン）が酸化して、光を放つ化学反応を触媒する蛋白質の総称である。発光基質としてセレンテラジン、ホタルルシフェリン、ウミホタルルシフェリン、渦鞭毛ルシフェリン、オキアミルシフェリン及びその誘導体などが知られている。特にセレンテラジンを発光基質とするルシフェラーゼとして、レニラルシフェラーゼ、ガウシアルシフェラーゼ、オプロフォーラスルシフェラーゼ、メトリディアルシフェラーゼ、ペリフェラルシフェラーゼ、コンケシアルシフェラーゼ等が知られている。 Luciferase is a generic term for proteins that catalyze chemical reactions that emit light when a luminescent substrate (luciferin) is oxidized in the presence of oxygen in luminescent organisms from prokaryotic luminescent bacteria to higher eukaryotic organisms fireflies. is there. Coelenterazine, firefly luciferin, Cypridina luciferin, dinoflagellate luciferin, Okiamyl luciferin and derivatives thereof are known as luminescent substrates. In particular, Renilla luciferase, Gaussia luciferase, Oproforus luciferase, Metrical luciferase, Peripheral luciferase, Conchec luciferase and the like are known as luciferases using coelenterazine as a luminescent substrate.
このルシフェラーゼとその発光基質との発光伴う化学反応は、検査・診断薬やレポーターアッセイ法に代表される医薬原体のスクリーニング方法などに応用されている。
検査・診断薬や、医薬原体のスクリーニングにおいて、ルシフェラーゼの発光は、発光強度が高く、かつ発光時間が長いことが求められている。殊に、マウスやラットなどの小動物を生体試料として使用し、被検体物質の生物学的活性を、ルシフェラーゼをレポーターとし、ルシフェリン−ルシフェラーゼ反応を利用した発光によって検出するレポーターアッセイ法においては、生体試料と被検体物質とを画像化し、生体内外における動的変化を経時的に観察する必要性が高いことから、より高い発光強度、より長い発光時間が求められている。更に、生体への影響を考慮すると、少量の溶液でより多くの発光基質を注入することが望ましく、高濃度のルシフェリンを調製する必要があった。 In screening of diagnostic / diagnostic drugs and drug substances, luciferase is required to have high luminescence intensity and long luminescence time. In particular, in a reporter assay method in which a small animal such as a mouse or a rat is used as a biological sample, and the biological activity of the analyte is detected by luminescence using a luciferase as a reporter and a luciferin-luciferase reaction, Therefore, higher luminescence intensity and longer luminescence time are required. Furthermore, considering the effect on the living body, it is desirable to inject a larger amount of the luminescent substrate with a small amount of solution, and it was necessary to prepare a high concentration of luciferin.
レニラルシフェラーゼをレポーターとして用いマウスの全身を可視化した、非特許文献１について例示すると、レニラルシフェラーゼを生産する細胞を移植したマウスに、メタノールにて溶解した発光基質（セレンテラジン）をリン酸緩衝液にて希釈して、尾静脈注射にて注入し、麻酔した上で、得られる発光をＣＣＤカメラにて撮影するといった手法が用いられている。しかしながら、上記条件では、発光基質（セレンテラジン）溶液における発光基質の濃度は最大でも０．２ｍｇ／ｍｌ（２５℃）にしかならず、観察に必要な発光強度と発光時間が充分に達成されているとは言い難かった。 As an example of Non-Patent Document 1, which visualizes the whole body of a mouse using renilla luciferase as a reporter, a luminescent substrate (coelenterazine) dissolved in methanol is phosphate buffered in a mouse transplanted with cells that produce renilla luciferase. A method is used in which the obtained light is diluted with a caudal vein, injected, and anesthetized, and the resulting luminescence is photographed with a CCD camera. However, under the above conditions, the concentration of the luminescent substrate in the luminescent substrate (coelenterazine) solution is only 0.2 mg / ml (25 ° C.) at the maximum, and the luminescence intensity and luminescence time necessary for observation are sufficiently achieved. It was hard to say.
なお、単に発光基質水溶液における発光基質の濃度を上げるだけであれば、発光基質水溶液に含まれる有機溶剤の濃度を上げればよいが、有機溶剤濃度が高まるほど、ルシフェラーゼの発光活性や、生体試料に対する影響が高まることから、有機溶剤濃度を上げる場合であっても限度があった。 If the concentration of the luminescent substrate in the luminescent substrate aqueous solution is simply increased, the concentration of the organic solvent contained in the luminescent substrate aqueous solution may be increased. However, as the organic solvent concentration increases, the luminescence activity of luciferase and Due to the increased effect, there was a limit even when the organic solvent concentration was increased.
本発明者らは上記課題を解決すべく鋭意研究を重ねた結果、ルシフェラーゼの発光基質を有機溶媒に溶解した発光基質水溶液と、有機溶媒を含有する水溶液とを混合することにより、最終的に得られる発光基質水溶液の有機溶剤濃度が同じであっても、より多くの発光基質が溶解可能であることを知見し、本発明を完成させた。 As a result of intensive studies to solve the above problems, the present inventors finally obtained by mixing a luminescent substrate aqueous solution in which a luciferase luminescent substrate is dissolved in an organic solvent and an aqueous solution containing the organic solvent. Even when the organic solvent concentration of the resulting luminescent substrate aqueous solution is the same, more luminescent substrates can be dissolved, and the present invention has been completed.
本発明は以下の構成を有する。
（１）ルシフェラーゼの発光基質を有機溶媒に溶解した発光基質溶液と、有機溶媒を含有する水溶液とを混合することにより得られる発光基質水溶液。
（２）ルシフェラーゼの発光基質が、セレンテラジン、ホタルルシフェリン、ウミホタルルシフェリン、渦鞭毛ルシフェリン、オキアミルシフェリン、およびそれらの誘導体から選ばれた１種以上である、前記第１項に記載の発光基質水溶液。
（３）有機溶媒が、ジメチルスルホキシド、ジメチルホルムアミド、メタノール、およびエタノールから選ばれた１種以上である、前記第１項に記載の発光基質水溶液。
（４）水溶液が、生理食塩水または塩化ナトリウムを含有するリン酸緩衝液である、前記第１項に記載の発光基質水溶液。
（５）有機溶媒を含有する水溶液における、有機溶媒の濃度が、有機溶媒含有水溶液に対して３〜１５重量％の範囲である、前記第１項に記載の発光基質水溶液。
（６）ルシフェラーゼの発光基質を有機溶媒に溶解した発光基質溶液と、有機溶媒を含有する水溶液とを混合することを特徴とする発光基質水溶液の製造方法。
（７）ルシフェラーゼの発光基質が、セレンテラジン、ホタルルシフェリン、ウミホタルルシフェリン、渦鞭毛ルシフェリン、オキアミルシフェリン、およびそれらの誘導体から選ばれた１種以上である、前記第６項に記載の発光基質水溶液の製造方法。
（８）有機溶媒が、ジメチルスルホキシド、ジメチルホルムアミド、メタノール、およびエタノールから選ばれた１種以上である、前記第６項に記載の発光基質水溶液の製造方法。
（９）水溶液が、生理食塩水または塩化ナトリウムを含有するリン酸緩衝液である、前記第６項に記載の発光基質水溶液の製造方法。
（１０）有機溶媒を含有する水溶液における、有機溶媒の濃度が、有機溶媒含有水溶液に対して３〜１５重量％の範囲である、前記第６項に記載の発光基質水溶液の製造方法。
（１１）レポーターアッセイ法において、ルシフェラーゼを生産する生物を発光させるにあたり、前記第１項〜第５項の何れか１項に記載の発光基質水溶液を用いることを特徴とする、レポーターアッセイ法。
（１２）ルシフェラーゼの発光基質、有機溶媒、および有機溶媒を含有する水溶液から構成される発光基質水溶液キット。
The present invention has the following configuration.
(1) A luminescent substrate aqueous solution obtained by mixing a luminescent substrate solution obtained by dissolving a luciferase luminescent substrate in an organic solvent and an aqueous solution containing the organic solvent.
(2) The aqueous luminescent substrate solution according to the above item 1, wherein the luminescent substrate of luciferase is at least one selected from coelenterazine, firefly luciferin, Cypridina luciferin, dinoflagellate luciferin, okiamyl luciferin, and derivatives thereof.
(3) The luminescent substrate aqueous solution according to the above item 1, wherein the organic solvent is at least one selected from dimethyl sulfoxide, dimethylformamide, methanol, and ethanol.
(4) The luminescent substrate aqueous solution according to item 1, wherein the aqueous solution is a phosphate buffer containing physiological saline or sodium chloride.
(5) The luminescent substrate aqueous solution according to the above item 1, wherein the concentration of the organic solvent in the aqueous solution containing the organic solvent is in the range of 3 to 15% by weight with respect to the organic solvent-containing aqueous solution.
(6) A method for producing an aqueous luminescent substrate solution, comprising mixing a luminescent substrate solution obtained by dissolving a luminescent substrate of luciferase in an organic solvent and an aqueous solution containing the organic solvent.
(7) The production of the aqueous luminescent substrate solution according to the above item 6, wherein the luminescent substrate of luciferase is one or more selected from coelenterazine, firefly luciferin, Cypridina luciferin, dinoflagellate luciferin, okiamyl luciferin, and derivatives thereof. Method.
(8) The method for producing an aqueous luminescent substrate solution according to the above item 6, wherein the organic solvent is at least one selected from dimethyl sulfoxide, dimethylformamide, methanol, and ethanol.
(9) The method for producing an aqueous luminescent substrate solution according to the above item 6, wherein the aqueous solution is a phosphate buffer containing physiological saline or sodium chloride.
(10) The method for producing an aqueous luminescent substrate solution according to Item 6, wherein the concentration of the organic solvent in the aqueous solution containing the organic solvent is in the range of 3 to 15% by weight with respect to the aqueous solution containing the organic solvent.
(11) In the reporter assay method, the luminescent substrate aqueous solution described in any one of the above items 1 to 5 is used for causing the organism producing luciferase to emit light.
(12) A luminescent substrate aqueous solution kit comprising a luciferase luminescent substrate, an organic solvent, and an aqueous solution containing the organic solvent.
本発明の発光基質水溶液は発光基質濃度が高い。本発明の発光基質水溶液の製造方法は、同一の有機溶剤濃度であれば、より多くの発光基質の溶解を可能とする。本発明の発光基質水溶液は、発光基質濃度が高く、ルシフェラーゼの発光に用いた場合には、高い発光強度と長い発光時間を実現させることが可能であることから、レポーターアッセイ法に好適に使用することができる。 The luminescent substrate aqueous solution of the present invention has a high luminescent substrate concentration. The method for producing an aqueous luminescent substrate solution of the present invention enables more luminescent substrates to be dissolved if the organic solvent concentration is the same. Since the luminescent substrate aqueous solution of the present invention has a high luminescent substrate concentration and can be used for luciferase luminescence, it can realize a high luminescence intensity and a long luminescence time. be able to.
以下に、本発明の実施の形態において実施例を挙げながら具体的かつ詳細に説明するが、本発明はこれらに限定されるものではない。なお、本発明の目的、特徴、利点、及びそのアイデアは、本明細書の記載により、当業者には明らかであり、本明細書の記載から、当業者であれば、容易に本発明を再現できる。以下に記載された発明の実施の形態及び具体的な実施例などは、本発明の好ましい実施態様を示すものであり、例示又は説明のために示されているのであって、本発明をそれらに限定するものではない。本明細書で開示されている本発明の意図ならびに範囲内で、本明細書の記載に基づき、様々に修飾ができることは、当業者にとって明らかである。 Hereinafter, the present invention will be described specifically and in detail with reference to examples, but the present invention is not limited thereto. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.
＜１．本発明に用いられる材料＞
本発明に用いられる発光基質は、ルシフェラーゼの発光基質であれば特に限定されるものではない。具体的には、セレンテラジン、ホタルルシフェリン、ウミホタルルシフェリン、渦鞭毛ルシフェリン、オキアミルシフェリン、およびそれらの誘導体などが挙げられる。本発明においては、それらから選ばれた１種を用いても良く、２種以上を使用しても良い。本発明においては、セレンテラジンが好ましく用いられる。
<1. Materials used in the present invention>
The luminescent substrate used in the present invention is not particularly limited as long as it is a luciferase luminescent substrate. Specific examples include coelenterazine, firefly luciferin, Cypridina luciferin, dinoflagellate luciferin, Okiamyl luciferin, and derivatives thereof. In this invention, 1 type chosen from them may be used and 2 or more types may be used. In the present invention, coelenterazine is preferably used.
本発明に用いられる有機溶媒は、ルシフェラーゼの発光基質を溶解することができれば特に限定されない。具体的には、ジメチルスルホキシド、ジメチルホルムアミド、メタノール、エタノール等が挙げられる。発光基質がセレンテラジンである場合には、セレンテラジンの溶解度の高さの点から、ジメチルスルホキシドおよびメタノールが好ましく用いられる。 The organic solvent used in the present invention is not particularly limited as long as it can dissolve the luciferase luminescent substrate. Specific examples include dimethyl sulfoxide, dimethylformamide, methanol, ethanol and the like. When the luminescent substrate is coelenterazine, dimethyl sulfoxide and methanol are preferably used from the viewpoint of the high solubility of coelenterazine.
本発明に用いられる水溶液は、通常生体試料への注入に用いられる水溶液であれば特に限定されない。具体的には、生理食塩水、グルコース溶液、リン酸緩衝液、塩化ナトリウムを含有するリン酸緩衝液などが好ましく用いられる。本発明においては、生理食塩水および塩化ナトリウムを含有するリン酸緩衝液が特に好ましく使用することができる。本発明に緩衝液を用いる場合、緩衝液は弱酸から弱アルカリに緩衝能を有するものであることが好ましい。
なお、本発明においては、水溶液が単なる水である場合であっても、本発明の効果が得られる。この単なる水は、Ｃａイオンを含まないか、含んでいたとしてもＣａの含有量は本発明の効果を損なわない程度の微量であることが好ましい。
The aqueous solution used for this invention will not be specifically limited if it is the aqueous solution normally used for injection | pouring to a biological sample. Specifically, physiological saline, glucose solution, phosphate buffer, phosphate buffer containing sodium chloride, and the like are preferably used. In the present invention, a phosphate buffer containing physiological saline and sodium chloride can be particularly preferably used. When a buffer solution is used in the present invention, the buffer solution preferably has a buffer capacity from a weak acid to a weak alkali.
In the present invention, the effect of the present invention can be obtained even when the aqueous solution is simply water. It is preferable that this simple water does not contain Ca ions, or even if it contains Ca ions, the Ca content is so small that the effects of the present invention are not impaired.
＜２．発光基質水溶液の製造方法＞
本発明の発光基質水溶液（以下「第３溶液」と言うことがある）の製造方法は、ルシフェラーゼの発光基質を有機溶媒に溶解した発光基質溶液（以下「第１溶液」と言うことがある）と、有機溶媒を含有する水溶液（以下「第２溶液」と言うことがある）とを混合することを特徴とする。
第１溶液における発光基質の濃度は、発光基質の溶解度を満たすものであれば特に限定されない。第１溶液の調製の際、多量の有機溶媒で発光基質を溶解すると、第３溶液に含まれる有機溶媒の最終濃度が高まる。第３溶液が生体試料内に注入されるような場合、有機溶媒濃度が低い方が、生体試料が受ける有機溶媒の影響が少ない傾向にある。このような点を考慮すると、第１溶液における当該発光基質の濃度は、１〜８ｍｇ／ｍｌの範囲であることが好ましく、より好ましくは２〜６ｍｇ／ｍｌの範囲である。
<2. Method for producing luminescent substrate aqueous solution>
The method for producing an aqueous luminescent substrate solution (hereinafter sometimes referred to as “third solution”) of the present invention comprises a luminescent substrate solution obtained by dissolving a luciferase luminescent substrate in an organic solvent (hereinafter sometimes referred to as “first solution”). And an aqueous solution containing an organic solvent (hereinafter sometimes referred to as “second solution”).
The concentration of the luminescent substrate in the first solution is not particularly limited as long as it satisfies the solubility of the luminescent substrate. When preparing the first solution, the final concentration of the organic solvent contained in the third solution is increased by dissolving the luminescent substrate with a large amount of the organic solvent. When the third solution is injected into the biological sample, the lower the organic solvent concentration, the less the influence of the organic solvent that the biological sample receives. Considering such points, the concentration of the luminescent substrate in the first solution is preferably in the range of 1 to 8 mg / ml, more preferably in the range of 2 to 6 mg / ml.
第２溶液における有機溶媒の濃度は、第１溶液を加えた際に発光基質が析出せず、且つ第３水溶液の有機溶媒濃度が２０重量％を超えなければ、特に限定されない。具体的には３〜１５重量％の範囲である。 The concentration of the organic solvent in the second solution is not particularly limited as long as the luminescent substrate does not precipitate when the first solution is added and the organic solvent concentration of the third aqueous solution does not exceed 20% by weight. Specifically, it is in the range of 3 to 15% by weight.
なお、第１溶液に使用する有機溶媒と、第２溶液に使用する有機溶媒の種類は、同じであっても違っていても良いが、本発明においては、同じであることが好ましい。 In addition, although the kind of the organic solvent used for a 1st solution and the organic solvent used for a 2nd solution may be the same or different, it is preferable that it is the same in this invention.
第３溶液における有機溶媒の濃度は特に限定されるものではない。溶媒濃度が高いほど、より多くの発光基質を第３溶液に溶解させることが可能である。一方、溶媒濃度が低いほど、生体試料に注入した際の、有機溶媒の影響が少なくなり、検査、診断、スクリーニングの精度が高くなる傾向がある。こういった点に鑑みると、第３溶液における有機溶媒の濃度は、１０〜２０重量％以下であることが好ましい。 The concentration of the organic solvent in the third solution is not particularly limited. The higher the solvent concentration, the more luminescent substrate can be dissolved in the third solution. On the other hand, the lower the solvent concentration, the less the influence of the organic solvent when injected into the biological sample, and the higher the accuracy of examination, diagnosis and screening. In view of these points, the concentration of the organic solvent in the third solution is preferably 10 to 20% by weight or less.
本発明においては、発光基質と有機溶媒と水溶液の組み合わせは特に限定されるものではない。本発明において好ましい組み合わせとしては、セレンテラジン、ジメチルスルホキシド、および生理食塩水の組み合わせ、セレンテラジン、ジメチルスルホキシド、および塩化ナトリウムを含有するリン酸緩衝液の組み合わせ、セレンテラジン、メタノール、および生理食塩水の組み合わせを挙げることが出来る。 In the present invention, the combination of the luminescent substrate, the organic solvent and the aqueous solution is not particularly limited. Preferred combinations in the present invention include a combination of coelenterazine, dimethyl sulfoxide, and physiological saline, a combination of coelenterazine, dimethyl sulfoxide, and a phosphate buffer containing sodium chloride, a combination of coelenterazine, methanol, and physiological saline. I can do it.
＜３．本発明発光基質溶液の用途＞
本発明の発光基質水溶液の用途は特に限定されるものではないが、本発明の発光基質水溶液は、発光基質濃度が高く、ルシフェラーゼの発光に用いた場合には、高い発光強度と長い発光時間を実現させることが可能であることから、レポーターアッセイ法に好適に使用することができる。
<3. Use of the luminescent substrate solution of the present invention>
The use of the luminescent substrate aqueous solution of the present invention is not particularly limited, but the luminescent substrate aqueous solution of the present invention has a high luminescent substrate concentration, and when used for luciferase luminescence, has high luminescence intensity and long luminescence time. Since it can be realized, it can be suitably used for a reporter assay.
現在、生物学分野や医学分野の研究において、細胞等の生体試料の生物学的活性を、ルシフェラーゼをレポーターとして、ルシフェリン−ルシフェラーゼ反応を利用した発光によって検出する技術（レポーターアッセイ法）が広く利用されている。さらに、レポーターアッセイ法と高感度発光検出装置とを併用する方法であれば、生細胞あるいはマウスやラットなどの小動物を生体試料とした個体レベルにおいて、種々の生物学的活性を可視化することができるので、より効率的な医療用薬剤の研究、開発が可能となる。本発明の発光基質水溶液は、このレポーターアッセイ法と高感度発光検出装置とを併用する方法において特に好ましく使用することができる。 Currently, in biological and medical research, a technique (reporter assay) that detects the biological activity of a biological sample such as a cell by using luciferase as a reporter and luminescence using a luciferin-luciferase reaction is widely used. ing. Furthermore, if the reporter assay method is used in combination with a high-sensitivity luminescence detection device, various biological activities can be visualized at the individual level using living cells or small animals such as mice and rats as biological samples. Therefore, more efficient research and development of medical drugs becomes possible. The luminescent substrate aqueous solution of the present invention can be particularly preferably used in a method in which this reporter assay method and a highly sensitive luminescence detection apparatus are used in combination.
本出願におけるもう１つの発明は、発光基質水溶液キットである。本発明の発光基質水溶液キットは、ルシフェラーゼの発光基質、有機溶媒、および水溶液、もしくは有機溶媒を含有する水溶液から構成されるが、本発明の効果を損なわない限り、それら以外の成分を含むものであってもよい。 Another invention in the present application is a luminescent substrate aqueous solution kit. The luminescent substrate aqueous solution kit of the present invention is composed of a luciferase luminescent substrate, an organic solvent, and an aqueous solution, or an aqueous solution containing an organic solvent, and contains other components unless the effects of the present invention are impaired. There may be.
本発明の発光基質水溶液キットの組成は特に限定されるものではないが、キットを用いて得られる発光基質水溶液の組成が、前述の発光基質水溶液の製造方法において述べた有機溶媒濃度、発光基質濃度の範囲になるようなものであることが好ましい。 The composition of the luminescent substrate aqueous solution kit of the present invention is not particularly limited, but the composition of the luminescent substrate aqueous solution obtained using the kit is the same as the organic solvent concentration and luminescent substrate concentration described in the method for producing the luminescent substrate aqueous solution described above. It is preferable that it is in the range.
以下実施例をもって本発明を具体的に説明するが、本発明は以下の実施例に限定されるものではない。なお、本実施例において「％」は、特に断りがない限り「重量％」を意味する。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. In this example, “%” means “% by weight” unless otherwise specified.
実施例１
２０μｌのジメチルスルホキシド（ＤＭＳＯ、和光純薬社製）に、１００μｇのセレンテラジン（チッソ社製）を加えて溶解した。このセレンテラジンのＤＭＳＯ溶液を、１０μｌのＤＭＳＯを加えた１３７ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ含有１０ｍＭリン酸緩衝液（シグマ社製）２８０μｌに加え、発光基質水溶液を調整した。この発光基質水溶液のセレンテラジンの溶解性等を表１に示した。
Example 1
To 20 μl of dimethylsulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.), 100 μg of coelenterazine (manufactured by Chisso) was added and dissolved. This DMSO solution of coelenterazine was added to 280 μl of 137 mM NaCl and 2.7 mM KCl-containing 10 mM phosphate buffer (manufactured by Sigma) supplemented with 10 μl of DMSO to prepare an aqueous luminescent substrate solution. The solubility of coelenterazine in this luminescent substrate aqueous solution is shown in Table 1.
実施例２
２０μｌのジメチルスルホキシド（ＤＭＳＯ、和光純薬社製）に、１００μｇのセレンテラジン（チッソ社製）を加えて溶解した。このセレンテラジンのＤＭＳＯ溶液を、１０μｌのＤＭＳＯを含有する生理食塩水（大塚製薬製）２８０μｌに加え、この発光基質水溶液のセレンテラジンの溶解性等を表１に示した。
Example 2
To 20 μl of dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.), 100 μg of coelenterazine (manufactured by Chisso) was added and dissolved. The DMSO solution of coelenterazine was added to 280 μl of physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) containing 10 μl of DMSO, and the solubility of coelenterazine in this luminescent substrate aqueous solution is shown in Table 1.
実施例３
７０μｌのメタノール（和光純薬社製）に、２００μｇのセレンテラジン（チッソ社製）を加えて溶解した。このセレンテラジンのメタノール溶液を、７０μｌのメタノールを含有する生理食塩水（大塚製薬製）６３０μｌに加え、発光基質水溶液を調整した。この発光基質水溶液のセレンテラジンの溶解性等を表１に示した。
Example 3
To 70 μl of methanol (manufactured by Wako Pure Chemical Industries, Ltd.), 200 μg of coelenterazine (manufactured by Chisso Corporation) was added and dissolved. This methanol solution of coelenterazine was added to 630 μl of physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) containing 70 μl of methanol to prepare a luminescent substrate aqueous solution. The solubility of coelenterazine in this luminescent substrate aqueous solution is shown in Table 1.
比較例１
３０μｌのジメチルスルホキシド（ＤＭＳＯ、和光純薬社製）に、１００μｇのセレンテラジン（チッソ社製）を加えて溶解した。このセレンテラジンのＤＭＳＯ溶液を、１３７ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ含有１０ｍＭリン酸緩衝液（シグマ社製）２７０μｌに加え、発光基質水溶液を調整した。この発光基質水溶液のセレンテラジンの溶解性等を表１に示した。
Comparative Example 1
To 30 μl of dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.), 100 μg of coelenterazine (manufactured by Chisso) was added and dissolved. This DMSO solution of coelenterazine was added to 270 μl of 137 mM NaCl, 2.7 mM KCl-containing 10 mM phosphate buffer (Sigma) to prepare a luminescent substrate aqueous solution. Table 1 shows the solubility of coelenterazine in the aqueous luminescent substrate solution.
比較例２
１４０μｌのメタノール（和光純薬社製）に、２００μｇのセレンテラジン（チッソ社製）を加えて溶解した。このセレンテラジンのメタノール溶液を、生理食塩水（大塚製薬製）５６０μｌに加え、発光基質水溶液を調整した。この発光基質水溶液のセレンテラジンの溶解性等を表１に示した。
Comparative Example 2
To 140 μl of methanol (manufactured by Wako Pure Chemical Industries, Ltd.), 200 μg of coelenterazine (manufactured by Chisso Corporation) was added and dissolved. This methanol solution of coelenterazine was added to 560 μl of physiological saline (manufactured by Otsuka Pharmaceutical) to prepare an aqueous luminescent substrate solution. Table 1 shows the solubility of coelenterazine in the aqueous luminescent substrate solution.
ＰＢＳ：１３７ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ含有１０ｍＭリン酸緩衝液
比較例１のように、あらかじめＤＭＳＯを加えない水溶液では、セレンテラジンは溶解せず懸濁した。実施例１、２では、ＤＭＳＯで５ｍｇ／ｍｌのセレンテラジン溶液を作製し、混合する水溶液中にあらかじめ３．６重量％のＤＭＳＯを加えたところ、セレンテラジンは溶解した。これにより、従来では溶解しなかったセレンテラジンの濃度で水溶液が作製できることが示された。 As in Comparative Example 1, coelenterazine was not dissolved but suspended in an aqueous solution to which DMSO was not added in advance. In Examples 1 and 2, a 5 mg / ml coelenterazine solution was prepared with DMSO, and 3.6 wt% DMSO was added in advance to the aqueous solution to be mixed, whereby the coelenterazine was dissolved. Thus, it was shown that an aqueous solution can be produced at a concentration of coelenterazine that has not been dissolved in the past.
比較例２のように、あらかじめメタノールを加えない水溶液では、セレンテラジンは溶解せず懸濁した。実施例３では、メタノールで２．９ｍｇ／ｍｌのセレンテラジン溶液を作製し、混合する水溶液中にあらかじめ１２．５重量％のメタノールを加えたところ、セレンテラジンは溶解した。これにより、従来では溶解しなかったセレンテラジンの濃度で水溶液が作製できることが示された。 As in Comparative Example 2, in the aqueous solution to which methanol was not added in advance, coelenterazine was not dissolved but suspended. In Example 3, a coelenterazine solution of 2.9 mg / ml was prepared with methanol, and 12.5% by weight of methanol was added in advance to the aqueous solution to be mixed, whereby the coelenterazine was dissolved. Thus, it was shown that an aqueous solution can be produced at a concentration of coelenterazine that has not been dissolved in the past.
本発明の発光基質水溶液は、ルシフェラーゼの発光に用いることができ、特に、ルシフェラーゼの発光を利用したレポーターアッセイ法に好適に使用することができる。 The aqueous luminescent substrate solution of the present invention can be used for luminescence of luciferase, and can be particularly suitably used for a reporter assay method using luminescence of luciferase.
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