JP2007330185A - Method for detecting two or more luciferases in multispecimen sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable the continuous assay of a multispecimen sample by achieving the improvement of light-emitting kinetics and the control of light emission intensity over a long time of ≥30 min from the start of light-emitting reaction, and separating and determining a plurality of signals having different colors of the emitted lights by a simple operation by reducing background in a reporter assay by the light-emitting reactions of three or more kinds of luciferins/luciferases having different emitted light colors. <P>SOLUTION: The system involves allowing multicolor luciferase emitting three or more colors to emit the lights simultaneously in the multispecimen sample containing two or more specimens by a reagent containing a cytolytic component to continuously carry out reporter assay. The reagent, the device for measuring the emitted light, etc., usable in the system are also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for stabilizing a luminescence reaction of a beetle luciferase characterized by a luminescent color, a method for using the method, a luminescent reagent, and a technique for separating and quantifying monochromatic light from mixed light. The system of the present invention can be applied to any luciferase assay system using a luciferase enzyme having a different luminescent color as a reporter and a signal, as well as a luciferase assay system using a multicolor simultaneous luminescence measurement system and a multi-analyte assay system (High-Throughput Screening).

  Conventionally, photoprotein genes have been used to monitor gene expression (the process of transcription and translation from gene to protein) by quantification. Usually, the gene of luciferase, a photoprotein of North American fireflies, is used. When performing a luminescence reaction with North American firefly luciferin / luciferase in vitro, the luminescence pattern is observed in the form of a flash, so the luminescence reaction must be accurately measured unless a device with a special mechanism for reagent injection is used. I could not. In order to improve this, a method of extending the half-life of luminescence to about 5 minutes and increasing the amount of luminescence by a method using thiols such as CoA as a luciferase detection method (luciferase assay system) was invented ( Patent Document 1). This method enables accurate measurement of the amount of luminescence by luciferase using a luminometer or liquid scintillation counter that does not have an automatic reagent injection device, and a reporter that measures luciferase expressed as a reporter in cultured cells with high sensitivity. Widely used in assay methods.

  On the other hand, the above-mentioned luciferase assay method requires an internal standard that can correct the amount of luminescence in consideration of the state and number of cells (Non-patent Document 1). For this reason, another type of luciferase gene (for example, Renilla luciferase) having different substrate specificities is introduced into the cell together as an internal standard reporter, and the two types of luciferase activities are individually measured, so that the target reporter for the internal standard reporter ( The transcriptional activity efficiency of firefly luciferase) is determined. This method is called a dual luciferase assay and is widely used in gene expression studies (Non-patent Document 2). However, this method uses coelenterazine (Renilla luciferin), which is extremely expensive compared to firefly luciferin, and quenches one luminescence reaction (firefly) and then measures the other luminescence reaction (Renilla). That is, a plurality of reagent injection operations are required. In addition, a technology for stably carrying out the luminescence reaction of Renilla luciferin for a long time has not been established. In other words, since an expensive automated reagent injection device is essential as a system for monitoring gene expression in multiple samples, it is currently not being applied to industrial applications such as the HTS (High-Throughput Screening) method. It is.

In contrast, luciferases derived from luminescent beetles and having different emission colors perform a luminescent reaction with a common luminescent substrate. Therefore, instead of the Renilla luciferase, for example, a red luminescence luciferase derived from a railroad worm (Non-patent Document 3) is used to simultaneously emit the yellow-green North American firefly luciferase that has been conventionally used. Can be quantified in a single measurement step. In order to apply this operation to industrial applications, efficiency of the luciferase signals emitted from multiple samples (two or more samples to be measured at one time) emitted from different samples in different emission colors is eliminated as much as possible. There was a need to develop a method for measuring well. When such a measurement system is put to practical use, the efficiency of the HTS method for the development of genome drug discovery using monochromatic light (single signal) luciferase, which is currently being actively promoted by drug discovery manufacturers, can be significantly improved. Be expected.

Japanese Patent No. 3171595 Cell engineering "Deisotope battle protocol 2 kit simple edition", Shintaro Nomura, Toshiki Watanabe, Shujunsha (1998), p.334-346. Grentzmann, G., Ingraman, J.J.A., Kelly, P.J., Gesteland, R.F., and Atkins, J.F. (1998) RNA 4, 479-486. Viviani, V.R., Bechara, E.J.H, and Ohmiya, Y. Biochemistry (1999) 38, 8271-827.Biochem. Biophys. Res.Com. 280, 1286-1291.

  In the reporter assay based on the luminescence reaction of three or more luciferins / luciferases having different luminescent colors, the present invention realizes improvement of luminescence kinetics and control of luminescence intensity over a long period of 30 minutes or more from the start of the luminescence reaction, Furthermore, by separating and quantifying a plurality of signals with different emission colors by reducing the background with a simple operation, a one-pack type luciferase luminescence method for simultaneous measurement of multicolor luminescence, which enables continuous measurement of multiple sample samples, and The object is to provide a detection system.

In order to solve the above-mentioned problems, the present inventors have studied to aim at practical application of a luminescent reaction by a new luminescent beetle other than the firefly family. As the luminescent beetles examined, the red luciferase derived from the railworm Phrixothrix hirtus (Non-patent Document 3) as the luciferase derived from the firefly family, and the green luciferase derived from Rhagophthalmidae Ohbai (Sumiya, M. , Viviani, VR, Ohba, N., And Ohmiya, Y. (1998) In Bioluminescence and Chmiluminescence.Proceeding of 10 ^th International Symposium on Bioluminescence and Chemiluminescence (Roda, A., Pazzagli, M., Kricka, L., and Stanley, PE, Erd.), Pp. 433-436, Bolona, Italy.) And its site-specific mutant orange luciferase (Viviani, VR, Uchida, A., Suenaga, N., Ryufuku, M., and Ohmiya, Y. (2001) Biochem. Biophys. Res. Com. 280, 1286-1291.), Each luminescence reaction was improved and optimized for luminescence changes (Kinetics). In addition, when the ratio of the emission intensity of the three emission colors is large, it becomes difficult to measure the emission intensity accurately due to the limitations of the measuring instrument. Therefore, the emission intensity can be controlled by controlling the emission intensity under the condition of maintaining stable emission Kinetics. We developed a luminescence reaction system that enables multicolor simultaneous luminescence measurement because of the close ratio.

  The present inventors have optimized various other components by interfering with coenzyme A (CoA) and pyrophosphate or pyrophosphate in the signal detection method using the luminescence reaction of luciferin / luciferase derived from luminescent beetles other than the firefly family Luminescent reaction reagent with a long-lasting luminescence intensity that contains a coexisting cell lysis component and has a constant luminescence intensity per unit time even after more than 30 minutes from the start of the luminescence reaction. We succeeded in constructing a luminescence reaction system that can control the above.

  Furthermore, the present inventors have added a successful technique for separating and quantifying a luminescent component with high efficiency and accuracy in a multi-sample sample in which a plurality of luminescent components are mixed, thereby providing a reporter assay using multicolor luciferase of three or more colors. In the present invention, the present invention, which is a luciferase luminescence method and detection system for simultaneous measurement of multi-color luminescence that enables continuous measurement of multi-analyte samples, has been completed.

The gist of the present invention is as follows.
(1) A system in which three or more color luciferases in two or more multi-samples are simultaneously emitted by a reagent containing a cell lysis component to continuously perform a reporter assay.
(2) The system according to (1), wherein reproducibility can be obtained within a range where the light emission ratio is 25% or less for each time change of red, green, and orange luciferase luminescence regardless of the passage of time.
(3) The reagent used in the system according to (1) or (2), which enables light emission for a long time of 30 minutes or more of a multicolor luciferase having three or more colors.
(4) The reagent used in the system according to (1) or (2), which does not require a physical cell lysis operation when emitting multicolor luciferase of three or more colors.
(5) The reagent used in the system according to (1) or (2), which enables cell lysis and light emission operation of each color of three or more multicolor luciferases by one operation of reagent addition.
(6) A method for performing a reporter assay on two or more samples continuously using the reagent according to any one of (3) to (5).
(7) In a reporter assay using the system according to (1) or (2) and / or the reagent according to any of (3) to (5), spectroscopy using a spectral filter to calculate a monochromatic light amount Method.
(8) A method of eliminating luminescence other than the luciferase emission wavelength and electrochemical noise by the spectral filter used for calculating the monochromatic light amount by the method described in (7).
(9) A luminescence measuring device used in the system according to (1) or (2), comprising a spectral filter.
(10) The apparatus according to (9), further comprising means for calculating a monochromatic light amount from the measurement value obtained by the spectral filter.
(11) A method for measuring luminescence in a continuous reporter assay for two or more multi-samples, wherein the reagent containing a cell lysis component simultaneously emits three or more multicolor luciferases in the sample. A method for measuring luminescence, comprising a step of obtaining a measurement value using a spectral filter and a step of calculating a monochromatic light quantity from the obtained measurement value.
(12) Two or more reporters or two or more reporters using the results of measurement using the system according to (1) or (2) and / or the reagent according to any one of (3) to (5) A method for measuring a difference between combinations of inducers or using a single reporter.

  In the present specification, “monochromatic light amount” refers to the amount of light emitted from one kind of luciferase when a plurality of kinds of luciferases that emit light of different wavelengths are in a mixed state.

  According to the present invention, in a reporter assay using a luminescence reaction of green luciferase derived from Iriomote butterfly selected as a multicolor luminescence luciferase, and further a red luciferase derived from a railroad worm, it is a long time of 30 minutes or more from the start of the reaction. Emission kinetics with constant emission intensity per unit time required for quantitative and simultaneous measurement in the reaction and a control system that brings the three emission intensities close to each other within the measurement range have been realized. It is possible to construct a luciferase luminescence reaction system for continuous multi-sample simultaneous multicolor luminescence measurement. Furthermore, a reporter assay system capable of separating and quantifying a plurality of signals having different emission colors by reducing the background by a simple operation, and a method and apparatus thereof can be supplied.

  The luciferase used in the present invention is a firefly luciferin derived from a beetle, that is, a multi-heterocyclic organic acid D-(−)-2- (6′hydroxy-2′-benzothiazolyl) -D2-thiazoline-4-carboxylic acid Is expressed as “luciferin” unless otherwise specified). It is an enzyme that emits photons by catalyzing it as a luminescent substrate. It is derived from luminescent beetles, such as the family Alaskaceae, Firefly, Iriomote, etc. Including all enzymes that contribute to the luminescence reaction. This includes enzymes in which the stability and luminescence properties of the enzyme protein itself have been artificially altered by recombinant DNA technology or mutation technology.

  The concentration of luciferase in the luciferin / luciferase luminescence reaction system is suitably 100 femto g / ml to 100 μg / ml, and preferably 1 ng / ml to 200 ng / ml.

  The luciferin used in the present invention is the above-mentioned beetle luciferin, including those extracted and purified directly from beetles and those chemically synthesized. Furthermore, a luminescent substrate which is a derivative of beetle luciferin and has luminescence activity after digestion with an enzyme. Such beetle luciferin derivatives include 4-methyl-D-luciferin, D-luciferyl-L-methionine, 6-O-galactopyranosyl-luciferin, DEVD-luciferin, luciferin-6 'methyl ester, luciferin 6 Examples include '-chloroethyl ester, 6'-deoxyluciferin, and luciferin 6'benzyl ester. Luciferin and its derivatives may be in the form of a salt. Examples of the salt include potassium salt and sodium salt.

The concentration of luciferin in the luciferin / luciferase luminescence reaction system is suitably 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM.
The pyrophosphate and / or pyrophosphate used in the present invention is preferably water-soluble, and it is preferable to use sodium pyrophosphate, potassium pyrophosphate, ammonium pyrophosphate, sodium hydrogen pyrophosphate, etc. it can. The use concentration of pyrophosphate and / or pyrophosphate is suitably 0.0001 mM to 100 mM, preferably 0.001 mM to 10 mM.

CoA used in the present invention is extracted and purified from yeast, and can be used in the form of lithium salt or sodium salt. The appropriate concentration of CoA is 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM.
The α-cyclodextrin used in the present invention is suitably 0.01% to 1.0%, preferably 0.05% to 0.5%.
The sodium fluoride used in the present invention is suitably 0.1 mM to 500 mM, preferably 1 mM to 100 mM.

As the saponin used in the present invention, those extracted and purified from soap bark-derived bark, soybean-derived, and tea seeds can be used. The use concentration of saponin is suitably 0.001% to 10%, preferably 0.01% to 1%.
HEPES and Tris used as buffer components in the present invention can be used as long as they are commercially available biochemical grades. Since the use concentration of HEPES and Tris has a sufficient buffer capacity, a concentration of 5 mM to 1 M is usually appropriate, preferably 10 mM to 250 mM. The pH used in these buffer components is suitably 7.0 to 9.0, but the preferred pH range is 7.5 to 8.5.

In the luciferin / luciferase luminescence reaction of the present invention, a reducing agent, adenosine triphosphate, and magnesium ion may be further added.
The reducing agent is considered to have an effect of protecting the luciferase enzyme. Examples of the reducing agent include dithiocarbamates, xanthates, thiophosphates, thiazoles and the like.

  Dithiocarbamates include pentamethylenedithiocarbamate piperidine, methylpentamethyldithiocarbamate pipecoline, dimethyldithiocarbamate, zinc diethyldithiocarbamate, zinc dibutyldithiocarbamate, zinc ethylphenyldithiocarbamate, sodium dimethyldithiocarbamate, sodium diethyldithiocarbamate, Examples include sodium dibutyldithiocarbamate, potassium dimethyldithiocarbamate, copper dimethyldithiocarbamate, iron dimethyldichocarbamate, lead ethylphenyldithicarbamate, selenium diethyldithicarbamate, and tellurium diethyldithiocarbamate. The metal salt described in the periodic table can be used according to the purpose without being limited thereto. .

Examples of xanthates include potassium xanthate, zinc butylxanthate, and sodium isopropylxanthate, but are not limited thereto, and can form salts with metals described in the periodic table. Can be used.
Examples of the thiophosphate include piperidine bis (o, o-distearyl dithiophosphate).

  Examples of thiazoles include 2-mercaptobenzothiazole, 2-mercaptobenzothiazole zinc salt, 2-mercaptobenzothiazole sodium salt, 2-mercaptobenzothiazole cyclohexylamine salt, 2-mercaptobenzothiazole copper salt, and the like. There is no limitation to this, and any metal or salt or complex described in the periodic table can be used.

  In addition, sulfhydryl compounds such as dithiothreitol, dithioerythritol, β-mercaptoethanol, 2-mercaptopropanol, 3-mercaptopropanol, 2,3-dithiopropanol, glutathione, and coenzyme A may be used.

The reducing agents can be used alone or in combination.
The concentration of the reducing agent used is suitably 0 mM to 50 mM, preferably 0.01 mM to 10 mM, but can be used at a concentration of 2 mM or less. In some cases, it is not necessary to add a reducing agent.

  Adenosine triphosphate generates luciferyl-AMP (adenylate) from luciferin by reaction with luciferase and contributes to the luminescence reaction. Adenosine triphosphate may be in the form of a salt. Examples of the salt include disodium salt, dipotassium salt, and magnesium salt.

The concentration of adenosine triphosphate used is suitably 0.01 pM to 50 mM, preferably 0.01 mM to 10 mM.
Magnesium ions are considered as a cofactor for the luminescent reaction. Examples of the compound containing magnesium ion include magnesium chloride, magnesium carbonate hydroxide, magnesium sulfate, magnesium acetate, and magnesium hydrogen phosphate.

The concentration of magnesium ion used is suitably 0.001 mM to 200 mM, preferably 0.1 mM to 100 mM.
The above components are the main components that contribute to the control of the luminescent reaction, but in addition to surfactants necessary for lysis of cultured cells, glycerin, BSA (bovine serum albumin), chelating agents, etc. Various compositions can be added accordingly.

  The luminescent reagent of the present invention may be in an aqueous solution in which each component is dissolved, or may be in a lyophilized state. The luminescent reagent in an aqueous solution state is preferably supplied as a frozen product, and is used after sufficiently thawing to room temperature. When the luminescent reagent is freeze-dried, only the components that are relatively unstable in the solution (such as luciferin, adenosine triphosphate, and CoA) are freeze-dried (referred to as reagent A). , Pyrophosphate and / or pyrophosphate, α-cyclodextrin, sodium fluoride, reducing agent, magnesium ion, saponin, buffer component, other components (for example, surfactant necessary for lysis of cultured cells, glycerin , BSA, chelating agent, etc.) are separated into aqueous solutions (reagent B) dissolved in water to the same concentration as the solution at the time of use, and freeze-dried products (reagent A) are stored frozen. The aqueous solution (reagent B) may be stored refrigerated or frozen. In this case, the reagent B is sufficiently returned to room temperature, dissolved in the reagent A and made into a solution, and then used. In addition, when the above components are divided into two types of cultured cell lysis reagent and a luminescent reagent, and a luminescent reaction is performed on the specimen, all components can be mixed. The luminescence reaction of luciferin / luciferase is preferably performed under a temperature condition of 20 ° C to 25 ° C.

  Next, from the samples in which the green luciferase derived from Iriomote butterfly family selected as the multicolor luminescent luciferase, the orange luciferase derived from Iriomote butterfly, and the red luciferase derived from the railroad worm are simultaneously emitted, each luciferase specific In order to measure the light intensity, it is preferable to use a color separation filter (spectral filter) having three or more arbitrary transmission wavelengths and transmittances.

  When the emission spectrum is analyzed, a green light emitting luciferase derived from Iriomote botulinum has a wavelength of 550 nm, an orange luciferase derived from Iriomote botulata has a maximum of 580 nm, and a red luciferase derived from a railworm exhibits a maximum curve of 620 nm. However, a signal as noise appears due to external factors such as temperature and magnetic field at wavelengths shorter than 470 nm where the emission of green luciferase disappears (Figure 1). In the measurement of monochromatic light, such as a dual luciferase system, it is rarely a problem. However, in the monochromatic separation system in this system, this noise causes an error in the measurement result. Therefore, a spectral filter that cuts light on the short wavelength side from any wavelength from 520 nm to 370 nm, which can cover the full width at half maximum of green in this system and can cut electrochemical noise, is used. It is essential. Let F1 be a spectral filter that cuts this short wavelength signal. In addition, two types of spectral filters on the longer wavelength side than 520 nm are set. As an example, the spectral filter that cuts the wavelength signal shorter than 560 nm is F2, and the spectral filter that cuts the wavelength signal shorter than 600 nm is F3.

  In addition, the signal values obtained from F1, F2, and F3 do not directly represent eigenvalues (individual light emission amounts) of the respective colors. Therefore, we have devised a calculation method for separating and quantifying color components when these spectral filters are used.

Three color components G (light emission of green luciferase from Iriomote butterfly), O (light emission of orange luciferase from Iriomote butterfly), R (light emission of red luciferase from railworm) individually with three spectral filters Shows the measurement method. First, signal values of the luminescent components GO and R are acquired from a detector such as a photomultiplier tube when the spectral filter F1 is used. This value covers a signal value of 80% or more in G and 100% emission spectrum in O and R. Therefore, it can be regarded as the total light value of G, O, and R. Next, obtain the G · O · R signal value when using the spectral filter F2 and divide by the F1 signal value to obtain the transmittance fg2, · fo2 · fr2 of the G · O · R spectral filter F2. . Similarly, the transmittances fg3, fo3, and fr3 are obtained when the spectral filter F3 is used. When the spectral filter F1 is used in a real sample where g is the detector signal value due to component G emission, o is the detector signal value due to component O emission, and r is the detector signal value due to component R emission. If the signal value is L1, the signal value when using the spectral filter F2 is L2, and the signal value when using the spectral filter F3 is L3, the signal value is proportional to the transmittance of the spectral filter.
Simultaneous equations

Can be established. Since the green, orange, and red signal values in the mixed light are g, o, and r, respectively, it is possible to obtain them by solving the simultaneous equations for obtaining these values. In solving these simultaneous equations, the solution can also be obtained by handwriting. In addition, there are various methods for solving simultaneous equations, such as Gauss-Seidel method, Gaussian elimination method, Thomas method, modified Koresky method, and calculation method using inverse matrix. As long as is taken, the value of g · o · r can be solved.

  Although the above is limited to three colors, the number of light emitting components can be increased infinitely, and the same number of spectral filters as the number of light emitting components may be prepared.

The luminescence measuring device for realizing this method is only required to have a photomultiplier tube or CCD suitable for weak luminescence measurement and to be equipped with a spectral filter. As the spectral filter, a commercially available filter for measuring fluorescence wavelength or a long wavelength transmission filter can be used. However, it is desirable to select a spectral filter that efficiently transmits a component having a small light emission amount. The light emission amount may be measured at a desired time interval over a desired period. However, in the multicolor luminescence luciferase assay of a multi-sample, the amount of luminescence from each sample is measured per 0.1 second to 10 seconds from the start of the luminescent reaction to 8 hours. In this system, since the measurement is performed after switching the spectral filter, a time difference occurs in the measurement as the number of the spectral filters increases. However, in the present invention, since the luminescence kinetics is stabilized by the luminescent reagent, there is no problem. .
A schematic diagram of an example of a luminescence measuring apparatus is shown in FIG.

  A schematic diagram of an example of a luminescence measuring apparatus is shown in FIG. A cell culture plate into which a plurality of luminescent genes are introduced is placed on a sample stage. In order to obtain light emission by mixing the detection reagent with the sample, the detection reagent is sent to the dispensing device through the liquid feed pipeline by the power of the liquid feed pump. The dispensing device adjusts the amount of the detection reagent with respect to the sample. The amount dispensed can be controlled by a computer. The dispensing operation is performed on all samples before measurement. When the motor 1 is driven and the sample stage moves in the horizontal direction, an equal amount of detection reagent is mixed with the specimen. Further, after dispensing, the sample stage can be vibrated in the horizontal direction by the motor 1, and the sample and the detection reagent can be mixed evenly. The light emitted from the specimen is condensed on the spectral filter by the condenser lens. The filter mounting plate is disk-shaped, a plurality of filters can be fixed, and the filter desired by the measurer can be selected by rotating the motor 2. In addition, the selection of spectral filters is controlled by a computer so that different spectral filters can be switched quickly. The light transmitted through the spectral filter is introduced into the detector. In the detector, the luminescence signal is converted into an electrical signal and the value is transferred to a computer. The computer also has a program for calculating the value of each light emitting component based on the transferred data.

  When the value of L1 was measured without using the spectral filter F1, the orange luciferase was allowed to emit light alone and examined. As described above, electrical noise tends to be noticeable particularly when green / orange luciferase emits light in the wavelength range of 520 nm or less. When the monochromatic light value is calculated without using the spectral filter F1, the luminescence value of green luciferase that should not be calculated is calculated. This value is about 1% of the luminescence value of orange luciferase emitting alone (FIG. 2).

  The present invention can be used for measurement of a beetle luciferase amount (luciferase assay) in which multicolor emission is performed in a multi-sample. The luciferase assay is a method for examining the transcriptional activity of a gene by measuring the luminescence activity using a luciferase gene as a reporter gene. For example, a cultured cell is transfected with a vector in which a luciferase gene is bound downstream of the promoter to be analyzed, and cultured. In the cell growth process, if the transcriptional activity of the promoter is strong, many luciferase enzymes are produced in the cell, and if the activity is weak, the amount of luciferase enzyme produced decreases. The amount of luciferase enzyme can be quantified with high sensitivity and ease by the luminescence reaction of luciferin / luciferase according to the present invention. Luciferase assay includes gene expression analysis (promoter and enhancer transcriptional activity analysis), analysis of mRNA action mechanism in cells, elucidation of the structure and action mechanism of proteins such as receptors and gene regulatory functions, in transgenic plants It can be used to analyze organ-specific expression patterns.

  A multicolor luciferase measurement system based on simultaneous multicolor luminescence measurement has been devised to further enhance the functionality of this luciferase assay. In conventional luciferase assay methods using a single signal, it has been difficult to evaluate assay data due to variations in the luminescence reaction itself. For this reason, a different kind of luminescent reaction system that does not interact with each other is introduced, which is completely different from fireflies, and the two types of luciferase activities are individually measured, so that the transcription activity efficiency of the target reporter relative to the internal standard reporter is determined. A luciferase assay was introduced. However, this method has not been applied to industrial uses such as the HTS (High-Throughput Screening) method in terms of cost and operability due to the necessity of performing an expensive luminescent substrate and a two-step reaction.

  In contrast, luciferases derived from luminescent beetles and having different emission colors perform a luminescent reaction with a common luminescent substrate. Therefore, if luciferases having different emission colors are simultaneously emitted and the emission spectra indicating the difference in color tone and the emission amount can be separated by the spectral filter, each emission amount can be quantified in one measurement step. A multicolor luciferase luminescence simultaneous measurement system makes it possible to measure a plurality of signals from one reaction (one step). With this system, two types of transcriptional activity are measured simultaneously from the amount of luminescence of two colors, while the amount of luminescence of the remaining one color is used as an internal standard, so that the two types of transcriptional activity measured are corrected for more accuracy. Multiple transcriptional activities can be evaluated.

  The present invention also provides a multi-analyte assay kit comprising the luminescent reagent of the present invention. In addition to the luminescent reagent of the present invention, the kit of the present invention includes an instruction manual, a flow chart of an operating method showing rough usage, a document describing the principle of calculating monochromatic light from mixed light, and a program containing a calculation formula Etc.

  The instruction manual should include an outline of the luminescent reaction, measurement principle, product characteristics, storage conditions, reagent preparation and operation methods, related products, troubleshooting, and the like. The kit of the present invention may further contain a luciferase enzyme (when used in a luciferase assay) as a standard product, depending on the application.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

PEX-Red expressing the red luciferase gene (RED) from the firefly Phrixothrix hirtus, pEX-ROL expressing the green luciferase (ROL) gene from Rhagophthalmidae Ohbai, Rhagophthalmidae Ohbai Plasmids of three types of luciferase expression vectors, pEX-ROLO, which expresses an orange luciferase (ROLO) gene, which is a site-specific mutant of the green luciferase (ROL) gene derived from the luciferase, were prepared. Each expression vector was prepared by replacing the luc + gene on the vector with the RED, ROL, and ROLO genes using the commercially available Picker Gene Control Vector 2 (Production Number: PGV-C2: manufactured by Toyo Ink Co., Ltd.). did.
Use CHO cells (Chinese hamster ovary cells) cultured in Ham's F-12 medium (GIBCO) / 10% fetal bovine serum (FBS, Nippon Pharmaceutical) under conditions of 37 ° C and 5% CO ₂ It was. Cultivate CHO cells to 2 × 10 ⁴ cells / well / 100 μl in a 96-well plate (Corning No. 3610) and use lipofectamine plus reagent (Invitrogen) to prepare 3 types of 200 ng. Plasmids pEX-Red, pEX-ROL, and pEX-ROLO were introduced (transfected) into CHO cells. 100 μl of luciferase luminescence reagent for single-color multicolor luminescence [1.06 mM ATP / 1.88 mM luciferin / 540 μM CoA / 4.9 mM DTT (dithiothreitol) for CHO cell culture cultured for 24 hours after transfection /5.34 mM MgSO ₄ /7.5% α-cyclodextrin / 0.01% Triton X-100 / 100 mM Tris-hydrochloric acid (pH 7.8)] was added. Next, the 96 well plate was stirred for 5 minutes using a plate shaker (TAIYO MICRO MIXER S-5, manufactured by TAITEC), and then the luminescence obtained by the luminometer (LB96V, manufactured by Berthold) FIG. 3 shows the results of comparing changes over time in the emission intensity of the three colors (green, orange, red) in 130 minutes from the start of the reaction. The emission half-lives were 4.5 hours, 2.25 hours, and 2.5 hours for green, orange, and red, respectively.

Site-specific red luciferase gene (RED) from the firefly Phrixothrix hirtus, green luciferase (ROL) gene from Rhagophthalmidae Ohbai, green luciferase (ROL) gene from Rhagophthalmidae Ohbai from Iriomote family The mutant orange luciferase (ROLO) gene was obtained using a baculovirus / silkworm protein expression system (Superworm System, Katakura Industry Co., Ltd.), and three silkworm-produced luciferase crude enzyme preparations were obtained.
Three silkworm-produced luciferase crude enzyme preparations were mixed in the following ratios.
(1) System using green luciferase as a control Green: Red: Orange = 100: 100: 0
2. Green: Red: Orange = 100: 75: 25
3. Green: Red: Orange = 100: 50: 50
4). Green: Red: Orange = 100: 25: 75
5). Green: Red: Orange = 100: 0: 100
(2) System using red luciferase as control green: red: orange = 100: 100: 0
Green: Red: Orange = 75: 100: 25
Green: Red: Orange = 50: 100: 50
Green: Red: Orange = 25: 100: 75
Green: Red: Orange = 0: 100: 100
20 μl of the mixed lysate was dispensed into a 96 well plate (Corning No. 3610). As a luciferase luminescence reagent for multicolor luminescence, 530 μM ATP / 470 μM luciferin / 2 mM AED / 5 mM MgSO ₄ /0.1% α-cyclodextrin / 0.1 mM potassium pyrophosphate / 0.01% saponin / 20 mM NaF / 100 mM Tris-phosphate (pH 8.0) was prepared. Sharp cut filter L37 (manufactured by HOYA OPTRONICS) is a filter that transmits light of 370 nm or more as spectral filter F1, and sharp cut filter O56 (HOYA OPTRONICS) is a filter that transmits light of 560 nm or more as spectral filter F2. A sharp cut filter R60 (made by HOYA OPTRONICS) that transmits light of 600 nm or more as a spectral filter F3 is mounted on a luminometer with filter switching function (Mithras made by Berthold) and automatically controlled by a control program Settings that can be switched were made. Next, the luminescent reagent was added to a 96-well plate, and the 96-well plate was stirred for 5 minutes using a plate shaker (TAIYO MICRO MIXER S-5 manufactured by TAITEC). Signals transmitted from the respective spectral filters were measured for 30 seconds each, monochromatic light was separated from the mixed light, and the luminescence value of each color was plotted with the luminescence value at a mixing ratio of 100 as 100 (FIG. 4). In addition, as another experimental system, the spectral filter F1 was changed to a sharp cut filter Y44 (made by HOYA OPTRONICS) that is a filter that transmits light of 440 nm or more, and F1, F2, and F3 were transmitted in the same manner as described above. The signal value was measured, monochromatic light was separated from the mixed light by calculation, and the luminescence value of each color was plotted with the luminescence value at a mixing ratio of 100 as 100 (FIG. 5). Regardless of whether the spectral filter F1 is L37 or Y44, it is possible to distinguish the light emission amounts of the other two colors in the reaction system that emits light at the same time, and the same value as the enzyme mixing ratio can be calculated. It was confirmed (Figures 6 and 7). Further, even when green and red luciferases were used as controls, it was possible to obtain the same value as the mixing ratio of the enzymes.
In the experiment of this example, as the spectral filter F1, a filter that transmits light of 380 nm or more, a filter that transmits light of 390 nm or more, a filter that transmits light of 420 nm or more, and a light that transmits 480 nm or more Filters that transmit light above 500 nm, filters that transmit light above 520 nm, and similar results. Even when orange luciferase is used as a control, a monochromatic light value can be obtained in the same manner as described above.

PEX-Red-SV40 designed to express the red luciferase gene (RED) from the firefly Phrixothrix hirtus constitutively under the control of the SV40 promoter, green luciferase (ROL) from Rhagophthalmidae Ohbai PEX-ROL-CRE with transcription factor binding sequence CRE that induces expression by forskolin, site-specific variant of orange luciferase (ROLO) gene from green luciferase (ROL) gene derived from Rhagophthalmidae Ohbai PEX-ROLO-NF, which binds a transcription factor binding sequence NFκB that induces expression by tumor necrosis factor-alpha (TNF-α), was prepared as a plasmid for each of the three luciferase expression vectors.
NIH3T3 cells cultured in Dulbecco's modified Eagle medium (DMEM, manufactured by SIGMA) / 10% fetal bovine serum (FBS, manufactured by Nippon Pharmaceutical Co., Ltd.) under the conditions of 37 ° C. and 5% CO ₂ were used. NIH3T3 cells are cultured in a 96-well plate (Corning No. 3610) at 2 × 10 ⁴ cells / well / 100 μl and pEX-Red-SV40wo using Lipofectamine 2000 reagent (Invitrogen).
Plasmids of 100 ng, pEX-ROL-CRE 50 ng, and pEX-ROLO-NF 50 ng were introduced (transfected) into NIH3T3. After incubating for 24 hours, in order to induce the expression of the above gene, it was replaced with Dulbecco's modified Eagle medium containing 10 μM forskolin or 10 μg / ml TNF-α. As a control, an experimental group was set in which only the operation of exchanging with a Dulbecco's modified Eagle medium containing no drug was performed. The cells are cultured for 8 hours under conditions of 37 ° C. and 5% CO ₂ , and luciferase luminescence reagent for multicolor luminescence [530 μM ATP / 470 μM luciferin / 2 mM AED / 5 mM MgSO ₄ / 0.1% α-cyclodextrin / 0.1 mM potassium pyrophosphate / 2% cyclodextrin α / 20 mM NaF / 100 mM tris-phosphate (pH 8.0)] was added. Next, the 96-well plate was stirred for 5 minutes using a plate shaker (TAIYO MICRO MIXER S-5, manufactured by TAITEC), and then the light of 370 nm or more was transmitted as the spectral filter F1. Sharp cut filter L37 (manufactured by HOYA OPTRONICS), a filter that transmits light of 560 nm or more as a spectral filter F2, Sharp cut filter O56 (manufactured by HOYA OPTRONICS), as a spectral filter F3, and 600 nm or more as a spectral filter F3 A sharp cut filter R60 (made by HOYA OPTRONICS), a filter that transmits light, was mounted on a luminometer with filter switching function (Mithras, manufactured by Berthold), and a setting that allows automatic switching by a control program was performed. Signals transmitted from the three types of spectral filters in each well were measured for 10 seconds, and monochromatic light was separated and quantified from the obtained values. The luminescence value of green / orange luciferase was divided by the luminescence value of red luciferase, and the value in the control group was taken as 1. In the forskolin or TNF-α induction group, the luminescence value of green / orange luciferase was divided by the value of red luciferase to obtain a value. When these were compared, it was confirmed that the luminescence value of green luciferase was significantly increased in the forskolin induction group and the orange luciferase was increased in the TNF-α induction group (FIG. 8). Therefore, CRE in the forskolin-induced zone and NFκB sequence in the TNF-α-induced zone were consistent with the reported experimental results (Gonzalez GA, Yamamoto KK, Fischer WH, Karr D, Menzel P , Biggs W 3rd, Vale WW, and Montminy MR. Nature (1989) 337, 749-52. And Ghosh S, May MJ and Kopp EB. Annu Rev Immunol. (1998) 16,225-60.).

  The luciferase emission method and monochromatic light separation and quantification system for simultaneous multicolor emission measurement of the present invention can simultaneously measure the amount of luminescence by a plurality of luciferases with different emission colors by performing a luminescence reaction once, which is 2 to 3 times the conventional amount. It can be used for a highly functional luciferase assay for obtaining a signal of Therefore, by using the luciferase luminescence method for simultaneous multicolor luminescence measurement of the present invention, simultaneous measurement of the transcriptional activity of a plurality of genes can be easily performed, and the possibility of application development in the field of genomic drug discovery and the like is expanded.

The emission spectrum from 320 nm to 750 nm of a red luciferase derived from a railway insect, a green luminescent luciferase derived from Iriomote botaru, and an orange luminescent luciferase derived from Iriomote botaru is shown. Results of separation and quantification of monochromatic light from composite light in the case of using the spectral filter F1 (upper figure in Fig. 2) and in the case of not using the spectral filter (lower figure in Fig. 2) in the luminescence reaction by the orange luciferase derived from Iriomote butterfly Indicates. The luminescence intensity and time course of green, orange, and red luciferases when using a luminescent reagent for multicolor luminescence with a luminescence half-life of 2 hours or more in a one-liquid system are shown. The quantification of the luminescence reaction in dilution series of red luminescence luciferase and orange luminescence luciferase when using green luminescence luciferase as a control during simultaneous three-color luminescence using a multicolor luminescence luminometer. A filter that transmits a wavelength of 370 nm or more was used as the spectral filter F1. The quantification of the luminescence reaction in dilution series of red luminescence luciferase and orange luminescence luciferase when using green luminescence luciferase as a control during simultaneous three-color luminescence using a multicolor luminescence luminometer. A filter that transmits a wavelength of 440 nm or more was used as the spectral filter F1. The quantification of the luminescence reaction in the dilution series of green luminescence luciferase and orange luminescence luciferase using red luminescence luciferase as a control during simultaneous three-color luminescence using a multicolor luminescence luminometer. A filter that transmits a wavelength of 370 nm or more was used as the spectral filter F1. The quantification of the luminescence reaction in the dilution series of green luminescence luciferase and orange luminescence luciferase using red luminescence luciferase as a control during simultaneous three-color luminescence using a multicolor luminescence luminometer. A filter that transmits a wavelength of 440 nm or more was used as the spectral filter F1. In order to constitutively express a green luciferase-conjugated NFκB plasmid, an orange luciferase-linked CRE sequence, and a red luciferase, a SV40 promoter-bound plasmid was simultaneously transfected into HEK293 cells, and each spectrum after drug induction. The amount of luminescence from the filter was calculated, and the amount of luminescence in the experimental group where the drug induction was not performed was defined as the relative luminescence amount 1. It is the schematic of an example of the light emission measuring apparatus of this invention.

Claims (12)

A system in which a multicolor luciferase of three or more colors in two or more multisamples is simultaneously light-emitted by a reagent containing a cell lysis component, and a reporter assay is continuously performed. 2. The system according to claim 1, wherein reproducibility of each time change of red, green and orange luciferase luminescence is obtained within a range where the luminescence ratio is 25% or less regardless of the passage of time. The reagent used in the system according to claim 1 or 2, which enables light emission for a long time of 30 minutes or more of a multicolor luciferase having three or more colors. The reagent used in the system according to claim 1 or 2, which does not require a physical cell lysis operation when emitting multicolor luciferase having three or more colors. The reagent used in the system according to claim 1 or 2, which enables cell lysis and light emission operation of each color of polychromatic luciferase of 3 or more colors by a single operation of reagent addition. A method for continuously performing reporter assay on two or more samples using the reagent according to claim 3. A spectroscopic method using a spectroscopic filter for calculating a monochromatic light amount in a reporter assay using the system according to claim 1 or 2 and / or the reagent according to any one of claims 3 to 5. A method for eliminating luminescence and electrochemical noise other than the luciferase emission wavelength by the spectral filter used for calculating the monochromatic light quantity by the method according to claim 7. The luminescence measuring apparatus used in the system according to claim 1 or 2, further comprising a spectral filter. The apparatus according to claim 9, further comprising means for calculating a monochromatic light amount from a measurement value obtained by the spectral filter. A method for measuring luminescence in a continuous reporter assay for two or more multi-samples, the step of simultaneously emitting three or more colors of multi-color luciferase in a sample with a reagent containing a cell lysis component, a spectral filter A method for measuring luminescence, comprising a step of obtaining a measurement value using the method and a step of calculating a monochromatic light quantity from the obtained measurement value. Using the results measured using the system according to claim 1 or 2 and / or the reagent according to any of claims 3 to 5, between a combination of two or more reporters or two or more inducers A method of measuring the difference or the difference when using a single reporter.