Method and system for analyzing receptor response

Abstract

To provide a high-throughput analysis method and analysis system capable of efficiently and comprehensively analyzing the response of a receptor to a target substance.SOLUTION: The method and system for analyzing the receptor response comprise a process for labeling a cell containing one or more kinds of receptors with one or more kinds of indicators, a process for administering a ligand to a culture solution containing the cell, a process for measuring the spectrum of the culture solution, and a process for specifying the indicator and / or the receptor responding to the ligand by analyzing the spectrum.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method and system for analyzing receptor responses using an indicator. [Background technology]

[0002] In recent years, research has been progressing into the mechanisms of smell and taste, which are said to be the most difficult of the five human senses to quantify.

[0003] The perception of smell and taste is triggered by the binding of odorant and tastant (ligand) to olfactory receptors expressed in olfactory neurons in the olfactory organs and taste receptors expressed in taste cells in the taste organs, respectively.

[0004] Olfactory and taste receptors include G protein-coupled receptors (GPCRs) and ionotropic receptors. When these receptors bind to odorants or taste substances, the signals generated are transmitted to the brain, inducing the sensations of smell and taste.

[0005] Currently, calcium imaging is the main method used to analyze the binding (response) between these receptors and ligands. Calcium imaging is a method in which a protein or calcium fluorescent indicator whose fluorescence intensity changes when it binds to calcium ions is introduced into cells, and changes in calcium ion concentration are detected by the change in fluorescence intensity.

[0006] The above-mentioned olfactory and gustatory receptors are characterized by their ability to change intracellular calcium concentrations upon activation. Therefore, intracellular calcium imaging of receptor-expressing cells can be used to analyze the presence or absence of receptor responses.

[0007] In addition, methods for efficiently analyzing receptor responses include the creation of cell lines that stably express the target receptor, and methods for analyzing receptor responses by measuring phosphorescence or luminescence, in addition to calcium imaging.

[0008] As a prior art document relating to the analysis of such receptor responses, for example, Patent Document 1 describes a sweet taste receptor expression construct, a cell body expressing the construct, and uses thereof. Specifically, a sweet receptor expression construct has been disclosed in which the genes encoding the sweet receptor subunits T1R2 and T1R3 and the G protein α subunit are inserted into the same plasmid, and in which a gene encoding the G protein α subunit is linked via an IRES sequence immediately after the gene encoding the sweet receptor subunit T1R2, which is located downstream of the EF-1α promoter, and a gene encoding the G protein α subunit is linked via an IRES sequence immediately after the gene encoding the sweet receptor subunit T1R3, which is located downstream of the CMV promoter located downstream of the EF-1α promoter.It has also been disclosed that a cell line in which the above sweet receptor expression construct has been genetically introduced into HEK-293 cells containing a single FRT (Flippase Recognition Target) sequence in the genome to simultaneously express the sweet receptor subunits T1R2 and T1R3 and the G protein α subunit, and that the use of the above cell line makes it possible to measure physiological responses to sweet substances.

[0009] Furthermore, Non-Patent Document 1 discloses a multicolor imaging technique in which living cells are marked with multiple fluorescent indicators and the fluorescent signals are separated and analyzed.

[0010] Furthermore, Non-Patent Document 2 reports the results of a study in which a genetic barcoding method utilizing a next-generation sequencer was used to analyze the interactions between multiple olfactory receptors and ligands through multiple assays, and the response of each olfactory receptor was chemically mapped. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 5905187 [Non-patent literature]

[0012] [Non-Patent Document 1] Yusuke Niino, Kotaro Oka, "Visualization of Cellular Functions by Multicolor Imaging Method," Journal of the Japan Society for Laser Surgery and Medicine, NPO Japan Society for Laser Surgery and Medicine, 2006, Vol. 26, No. 4, pp. 338-345 [Non-patent document 2] Cell Syst.,2019,Vol.8,Issue.3,p.254-260 Summary of the Invention [Problem to be solved by the invention]

[0013] It has been reported that there are more than 30 types of taste receptors and approximately 400 types of olfactory receptors (the number increases when single nucleotide polymorphisms (SNPs) are taken into account) involved in human sensory reception, each of which has its own unique response characteristics to ligands (smell substances and taste substances). Furthermore, the number of ligands far exceeds the number of receptor types. In addition to the existence of agonists and antagonists, allosteric effects have been reported for these receptors, and complex synergistic and counteracting effects can be exerted by combining multiple compounds. This gives the tastes and smells we perceive a rich variety and complexity, making it difficult to design and control them as desired.

[0014] As an example of how to decipher the complexity of tastes and smells, one possible method would be to have a machine learn smells. One method would be to quantify the smell as a response pattern of olfactory receptors and build a model to predict this. However, for the reasons mentioned above, in order to predict how receptors will recognize smell patterns, it is necessary to build a predictive model that takes into account the complex interactions between factors. To achieve this, a large amount of quantified data is required as training data, which is obtained by comprehensively analyzing (actually measuring) the response patterns formed by olfactory receptors in response to various groups of compounds.

[0015] However, even when using a method such as calcium imaging as in Patent Document 1, measuring the response of receptors to ligands in a one-to-one correspondence would be both costly and time-consuming, making it unrealistic.

[0016] On the other hand, while multicolor imaging and multiplexed assays such as those described in Non-Patent Documents 1 and 2 can be used to perform analyses efficiently to a certain extent, the problem with intracellular calcium imaging using cell-permeable fluorescent indicators is that the number of indicators available for experiments is extremely limited compared to the number of receptors. In other words, comprehensive analysis of receptor responses using conventional techniques is expected to require a significant amount of time due to the limited number of indicators that can be selected. Furthermore, when considering electroporation or microinjection as a method for introducing an indicator, it is possible to select indicators that are not cell-permeable, which broadens the range of indicator candidates that can be used. However, the procedures are complicated and the required equipment is expensive, so even with these methods, the problems of cost and time cannot be solved.

[0017] The present invention has been made in view of the above circumstances, and aims to provide a high-throughput analytical method and analytical system capable of efficiently and comprehensively analyzing the responses of receptors to ligands. Specifically, the present invention aims to provide a method for comprehensively analyzing the response characteristics of receptors that form large gene families, such as olfactory receptors, and an analytical method and analytical system that can reduce the time and economic costs involved in comprehensively investigating the response patterns of receptors to numerous compounds and their mixtures. [Means for solving the problem]

[0018] The inventors have conducted extensive research and have found that a high-throughput analytical method and system can be provided that can efficiently and comprehensively analyze the response of receptors to ligands by including the steps of labeling cells containing one or more types of receptors with one or more types of indicators, administering a ligand to a culture medium containing the cells, measuring the spectrum of the culture medium, and analyzing the spectrum to identify the indicator and / or receptor that responded to the ligand, thereby completing the present invention. That is, the present invention provides the following.

[0019] (1) A first aspect of the present invention is a method for analyzing receptor responses, characterized by comprising the steps of labeling cells containing one or more types of receptors with one or more types of indicators, administering a ligand to a culture medium containing the cells, measuring the spectrum of the culture medium, and identifying the indicator and / or receptor that responded to the ligand by analyzing the spectrum.

[0020] (2) A second aspect of the present invention is the analytical method described in (1), characterized in that the cells contain one type of receptor and the indicator contains at least two or more types of indicators.

[0021] (3) A third aspect of the present invention is the analytical method according to (1), characterized in that the cells contain at least two types of receptors and the indicator is one type of indicator.

[0022] (4) A fourth aspect of the present invention is the analytical method according to (1), characterized in that the indicator contains at least a fluorescent indicator.

[0023] (5) A fifth aspect of the present invention is the analytical method according to (1), characterized in that the receptor is an olfactory receptor.

[0024] (6) A sixth aspect of the present invention is the analysis method according to (1), characterized in that the analysis of the spectrum is performed by applying mathematical optimization.

[0025] (7) A seventh aspect of the present invention is a receptor response analysis system comprising: a labeling means for labeling cells containing one or more types of receptors with one or more types of indicator; an administration means for administering a ligand to a culture solution containing the cells; a measurement means for measuring the spectrum of the culture solution; and an analysis means for identifying the indicator and / or receptor that responded to the ligand by analyzing the spectrum. [Effects of the Invention]

[0026] According to the present invention, it is possible to provide a high-throughput analytical method and analytical system that can efficiently and comprehensively analyze the response of receptors to target substances. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 10 is a schematic diagram of an analysis system according to another embodiment of the present invention. [Figure 2] FIG. 10 is a flowchart illustrating an example of the operation of an information processing device included in an analysis system according to another embodiment of the present invention. [Figure 3]1 is a schematic diagram illustrating some steps of an analysis method and analysis system according to an embodiment of the present invention. [Figure 4] 10A to 10C are schematic diagrams illustrating some steps of an analysis method and an analysis system according to another embodiment of the present invention. [Figure 5] FIG. 2 shows the fluorescence spectra of the fluorescent indicators used in the examples in the excited state and steady state. [Figure 6] FIG. 10 is a diagram showing the measured spectrum in Test 1-1, the predicted spectrum of the combined waveform, and the integration results thereof. [Figure 7] FIG. 10 is a diagram showing the measured spectrum, the predicted spectrum of the combined waveform, and the integration results thereof in Test 1-2. [Figure 8] FIG. 10 shows the measured spectra, predicted summed waveforms, and their integration results in Tests 1-3. [Figure 9] FIG. 10 is a diagram showing the measured spectrum and the predicted summed waveform spectrum in Test 2-1. [Figure 10] FIG. 10 is a diagram showing the measured spectrum and predicted summed waveform spectrum in Test 2-2. [Figure 11] FIG. 10 is a diagram showing the measured spectrum and predicted summed waveform spectrum in Test 2-3. [Figure 12] FIG. 10 is a diagram showing the measured spectrum in Test 2-4, and the integration result of the measured spectrum and the predicted summed waveform spectrum. [Figure 13] FIG. 10 shows the measured spectrum in Test 2-5, and the results of integrating the measured spectrum with two types of predicted summed waveform spectra. DETAILED DESCRIPTION OF THE INVENTION

[0028] Hereinafter, a mode for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. The following present embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be carried out by appropriately modifying it within the scope of its gist.

[0029] <Method for analyzing receptor responses> The receptor analysis method according to this embodiment comprises the steps of labeling cells containing one or more types of receptors with one or more types of indicators, administering a ligand to a culture medium containing the cells, measuring the spectrum of the culture medium containing the cells to be observed, and identifying the indicator and / or receptor that responded to the ligand by analyzing the spectrum. Each step and the substances, cells, and indicators used will be described in detail below.

[0030] (labeling process) The first step involves labeling cells containing one or more receptors with one or more indicator reagents.

[0031] (receptor) The receptors used in the analytical method according to this embodiment are not particularly limited as long as they are receptors that change the intracellular calcium concentration upon binding to a ligand, but it is preferable that they are receptor groups that form large gene families, such as olfactory receptors. The method according to the present invention is a method for comprehensively analyzing the response characteristics of such receptor groups, and is effective for those with many combinations of ligand candidates and receptor groups and complex response patterns.

[0032] The olfactory receptor may be an olfactory receptor of any organism, but is particularly intended to be an olfactory receptor of a mammal, and is more preferably a human olfactory receptor. Examples of human olfactory receptors include OR1A1, OR1A2, OR1C1, OR1D2, OR1G1, OR2A25, OR2M4, OR2T34, OR2T10, and OR5AC2. Alternatively, the olfactory receptor may be a family, subfamily, or member of these olfactory receptors. As olfactory receptors other than those of mammals, for example, olfactory receptors of insects may be used.

[0033] The taste receptor may be a taste receptor of any organism, but is preferably a mammalian taste receptor, and more preferably a human taste receptor. Examples of human taste receptors include sweet taste receptors (T1R2+T1R3), umami taste receptors (T1R1+T1R3), and bitter taste receptors (T2Rs). Furthermore, taste receptors of families, subfamilies, or members of these taste receptors may also be used.

[0034] In addition, receptors other than olfactory receptors and gustatory receptors may also be G protein-coupled receptors such as muscarinic acetylcholine receptors, adenosine receptors, adrenergic receptors, GABA receptors (type B), angiotensin receptors, cannabinoid receptors, cholecystokinin receptors, dopamine receptors, glucagon receptors, histamine receptors, opioid receptors, secretin receptors, serotonin receptors, gastrin receptors, P2Y receptors, and rhodopsin.

[0035] In addition, receptors other than G protein-coupled receptors can also be used, such as tyrosine kinase receptors such as insulin receptors, growth factor receptors, and cytokine receptors; guanylate cyclase receptors such as GC-A, GC-B, and GC-C; ion channel receptors such as nicotinic acetylcholine receptors, glycine receptors, GABA receptors (type A and type C), glutamate receptors, serotonin receptor type 3, inositol trisphosphate (IP3) receptors, ryanodine receptors, and P2X receptors.

[0036] (cell) The above receptors are preferably used in a state where they are functionally expressed on cells. In this case, there are two methods for expressing receptors on cells: stable expression (stable transfection), in which the cell's genes are rewritten, and transient expression (transient transfection), in which a plasmid (vector) is introduced into the cell. A preferred method can be used depending on the receptor and cell type used.

[0037] The cells used are not particularly limited as long as they are cells used for cell culture or gene transfer, and examples include human embryonic kidney cells (HEK293 cells), Chinese hamster cells (CHO cells), monkey cells (COS cells), HeLa cells, and Xenopus oocytes.

[0038] The receptor to be introduced into the cells may be one type, or may include two or more types of receptors. When performing qualitative receptor screening (for example, when it is desired to know whether any one of the 400 types of olfactory receptors responds), it is preferable to include three or more types of receptors, more preferably four or more types, and even more preferably five or more types of receptors.

[0039] As mentioned above, measuring the response of receptors to ligands one-to-one would be costly and time-consuming, making it unrealistic. However, by considering the combination of indicators described below, introducing two or more receptors into a single cell, the work efficiency when measuring the correspondence between a group of ligands and the corresponding receptors can be exponentially improved.

[0040] When a cell contains two or more types of receptors, the location where each receptor is expressed during transfection is not particularly limited, but is preferably a location where each receptor can be functionally expressed.

[0041] (indicator) The indicator used for cell labeling (marking) is not particularly limited as long as it is an indicator typically used in cell experiments, but a cell-permeable indicator is preferred because it is easy to label, and a fluorescent indicator is particularly preferred. The fluorescent indicator is not particularly limited as long as it emits fluorescence as a result of binding between a target substance and a receptor. Examples of the fluorescent indicator include calcium-sensitive fluorescent indicators such as Quin-2 AM, Fluo-3 AM, Rhod-2 AM, Calbryte® 520, Fluo-4 AM, Fura-2 AM, Fura Red AM, Fura-8 FF™, Indo-1 AM, Calbryte 590, and Calbryte 630.

[0042] In addition to fluorescent indicators, for example, luminescent or phosphorescent substances can also be used as indicators. Examples of luminescent reagents include luciferin and aequorin, and examples of phosphorescent substances include iridium complexes, platinum complexes, ruthenium complexes, palladium complexes, zinc sulfate, and strontium aluminate.

[0043] The indicator used may be one type, but preferably contains at least two types of indicators, more preferably contains five types of indicators, and even more preferably contains ten types of indicators.

[0044] As mentioned above, when measuring the receptor response to a ligand in a one-to-one correspondence, if there are 10 types of receptors to be measured, 10 types of indicators are required. However, since there are approximately 400 types of olfactory receptors in humans, even if 10 indicators are used simultaneously, simply calculating 400 ÷ 10 = 40 times is required to measure whether or not all olfactory receptors respond to a single target substance, which is inefficient.

[0045] It would also be possible to prepare 400 types of indicators to improve efficiency, but in the case of fluorescent indicators, for example, the indicator must have the ability to penetrate into cells, and after penetrating the cells, it must remain inside the cells, chelate with calcium ions, and be a compound that sensitively changes its specific absorption characteristics; therefore, it is not realistic to prepare 400 types (400 colors) of such indicators. However, if we use 10 types of indicators and combine two of them to label one receptor, the number of receptors that can be labeled is 10 C2 = 45 types, so 45 types of receptor responses can be measured in one measurement. In addition, the number of measurements is 400 ÷ 45 = approximately 9, which not only increases efficiency exponentially but also allows for measurements without problems even when the types of indicators that can be used for measurement are somewhat limited.

[0046] Furthermore, when two or more types of indicators are used, and the indicators include two or more types of fluorescent indicators, it is preferable that the maximum wavelengths of the respective fluorescent indicators are separated by 100 nm or more. The separation of the maximum wavelengths reduces the overlap between the respective fluorescence spectra, resulting in more accurate calculation results output in the analysis step described below. Furthermore, even when cells contain two or more types of receptors and the receptor proteins are highly homologous to each other, labeling with a combination of indicators whose maximum wavelengths are separated can prevent a decrease in data accuracy.

[0047] The procedure for labeling with an indicator may be carried out in accordance with the protocol for use of each indicator. For example, when a fluorescent indicator is used, the culture medium containing cultured cells is replaced with a buffer, the indicator is added, and the cells are incubated at a predetermined temperature for a predetermined time, so that the receptor-containing cells are labeled with the fluorescent indicator.

[0048] (Ligand administration step) The second step is to administer a ligand to the culture medium containing the cells.

[0049] (ligand) The ligand to be administered can be selected depending on the receptor contained in the cell. When the receptor is an olfactory receptor, examples of the ligand to be administered include odorants such as bombykol, 1-octen-3-ol, geosmin, phenethyl alcohol, methyl benzoate, ethyl benzoate, benzyl alcohol, methyl salicylate, benzaldehyde, pentanal, hexanal, E2-hexanal, 2-heptanone, 6-methyl-5-hepten-2-one, and 2-methylphenol.

[0050] Furthermore, when the receptor is a sweet receptor, examples of the ligand to be administered include carbohydrate-based sweeteners such as glucose, fructose, galactose, raffinose, xylose, sucrose, maltose, lactose, starch syrup, isomerized sugar, isomaltooligosaccharides, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, lactoferrin oligosaccharides, soybean oligosaccharides, trehalose, sorbitol, mannitol, maltitol, xylitol, erythritol, lactitol, isomaltol, reduced starch syrup, reduced palatinose, wasanbon, brown sugar, soft brown sugar, honey, molasses, licorice extract, and maple syrup, and non-carbohydrate sweeteners such as aspartame, saccharin, dulcin, stevioside, stevia extract, glycyrrhizin, acesulfame-K, sucralose (registered trademark), cyclamate, alitame, neotame, perillartine, monellin, and curculin (registered trademark).

[0051] It is preferable that a predetermined amount of one type of ligand is administered at once, but when measuring differences in response due to different concentrations, the same ligand may be administered multiple times and measurements may be taken after each administration. When measuring differences in response for each ligand, a mixture of multiple ligands may be administered, or a certain ligand may be administered and measured, and then a different ligand may be administered.

[0052] The method for administering the ligand to the culture medium is not particularly limited as long as it is a known method. For example, it is preferable to dispense the culture medium containing cells collected from the medium and labeled with an indicator into test tubes or microwells, and then administer a solution containing the ligand to the test tubes or microwells.

[0053] (Spectrum measurement process) The third step is to measure the spectrum of the culture medium containing the receptor-expressing cells. The type of spectrum to be measured can be appropriately selected depending on the type of indicator used. For example, when a fluorescent indicator is used, it is preferable to measure the fluorescence spectrum. Depending on the type of indicator, the absorption spectrum or excitation spectrum may also be measured. For measuring the fluorescence spectrum, known measurement methods and commercially available measurement devices can be used without any particular limitations.

[0054] In addition, the above steps preferably use a calcium imaging method using a fluorescent indicator. Specifically, for example, cellular responses can be quantified by simultaneous assay using a 96-well or 1536-well multiplate reader, and then changes in intracellular calcium concentration can be visualized using microscopy to observe whether each cell is responding. Microscopic observation using a fluorescent indicator different from that used in the simultaneous assay using the multiplate reader can confirm that the cellular response is not due to artifacts. An example of such a method is described below.

[0055] Simultaneous assays using a multiplate reader can be performed according to known methods, but automated fluorescent calcium imaging using the FlexStation® 3 (Molecular Devices) is simple and rapid, enabling high-throughput assays. The FlexStation® 3 is a multiplate reader that combines the performance of the SpectraMax M5e (Molecular Devices) with an 8-channel pipettor.

[0056] Automated fluorescence imaging using FlexStation (registered trademark) 3 (Molecular Devices) can be performed, for example, according to the following procedure. Note that the following example assumes the use of adherent cells such as HEK293T cells. First, cells expressing the target receptor are suspended in an appropriate cell culture medium and an appropriate amount (70,000 to 80,000 cells per well) is plated onto each well of a 96-well plate (Corning, CellBIND Surface).

[0057] Next, once the cells have adhered and proliferated, the medium is removed and replaced with a measurement buffer (e.g., HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)), to which a buffer containing a fluorescent calcium indicator (e.g., Fluo-4 AM) is further added. In this case, cell-permeable fluorescent indicators such as Fluo-4 AM have a lipid-soluble acetoxymethyl group introduced to facilitate their entry into cells. When added to the culture medium, they are easily taken up into cells and hydrolyzed by intracellular esterases. The hydrolyzed Fluo-4 becomes less permeable to the cell membrane and diffuses into the cells, forming a calcium complex and emitting strong fluorescence.

[0058] After waiting for the indicator to migrate into the cells and stabilize (usually, incubation at 27 to 37°C for 30 to 60 minutes will suffice), a ligand is administered thereto. Finally, the response of the target receptor to the administered ligand can be quantified by measuring the change in intracellular calcium concentration that occurs immediately after ligand administration as a fluorescence response (in the case of Fluo-4, excitation at 485 nm and fluorescence observation at 525 nm).

[0059] Fluorescence calcium imaging using a fluorescence microscope can be performed according to the following procedure. First, cells expressing the target receptor are suspended in an appropriate cell culture medium and an appropriate amount (40,000 to 80,000 cells) is plated onto each well of a 96-well plate (Greiner, Lumox).

[0060] Next, when the cells have adhered and proliferated, the medium is removed and replaced with a measurement buffer, to which a buffer containing a fluorescent calcium indicator (for example, Fura-2 AM) is further added.

[0061] After the indicator has been transferred into the cells and allowed to stabilize, any remaining extracellular fluorescent indicator is removed by buffer exchange and the cells are allowed to stand for approximately 10-20 minutes. A ligand prepared at a specific concentration is then administered, and changes in intracellular calcium concentration are observed as a fluorescent response, allowing the strength of the receptor's response to the administered ligand to be measured. When Fura-2 is used as the fluorescent indicator, excitation is performed at two wavelengths, 340 nm and 380 nm, and fluorescence is observed at a wavelength of 510 nm. As calcium ion concentration increases, the fluorescence intensity at 340 nm excitation increases and the fluorescence intensity at 380 nm excitation decreases.

[0062] Finally, the response of receptor-expressing cells to the ligand solution can be observed by capturing fluorescent images in the microscope field immediately after the addition of the ligand solution and then displaying the ratio of the two-wavelength excitation fluorescence in pseudocolor.

[0063] (Spectral analysis process) The fourth step is to identify the indicator and / or receptor that responded to the administered ligand by analyzing the spectrum.

[0064] The spectrum analysis method used here can be any known analysis method, and can also be performed using commercially available waveform separation software, but the following method applies the optimization model (hereinafter also referred to as optimization modeling) calculation function of Excel (registered trademark). Optimization modeling, also known as mathematical optimization, is a calculation technique that uses mathematical methods and algorithms to solve the problem of maximizing or minimizing a specific objective function under given conditions.

[0065] In the receptor analysis method according to this embodiment, the indicator used has a specific spectrum (in the case of a fluorescent indicator, a fluorescent spectrum) that is measured in advance according to the intracellular calcium concentration (cell response strength), and such measurement data is used that has been recorded in advance.

[0066] The spectral data for each indicator used is saved as numerical values ​​or image data. The indicator spectrum from which the spectral waveform data measured in the third step (if two or more indicators are used, the data will be the sum of the spectra from each indicator) originates can be automatically calculated using the optimization model calculation function of Excel®. For example, if one receptor is marked with two indicators, five indicators can be used to simultaneously observe 5C2 = 10 receptors. In this case, a composite waveform is created by superimposing the spectral data of the five indicators used in the ratio a:b:c:d:e (hereinafter, the composite waveform data derived by calculation in this way will be referred to as predicted data, and the spectral data obtained by experiment will be referred to as observed data). The ratio a:b:c:d:e is then automatically calculated using Excel® Solver to minimize the difference from the spectral waveform data measured in the third step (if the results vary with each run, run the calculation multiple times and use the result with the smallest difference). By performing such calculations, it is possible to identify which of the indicators used responded to changes in calcium concentration, and based on this data, it is possible to identify which receptors responded.

[0067] Another possible method for decomposing the observed fluorescence spectrum of the measurement target (in this invention, the culture medium containing receptor-expressing cells) into the fluorescence spectra of the respective fluorescent indicators and determining the abundance ratio of each fluorescent indicator is, for example, Fourier transform. Specifically, the method using the spectral deconvolution method described in US Patent Publication No. US6403332B1 can be mentioned. The calculation method is not limited to the above-described calculation method.

[0068] <Analysis system> The present invention encompasses a receptor response analysis system characterized by comprising: a labeling means for labeling cells containing one or more types of receptors with one or more types of indicator; an administration means for administering a ligand to a culture solution containing the cells; a measurement means for measuring the spectrum of the culture solution; and an analysis means for identifying the indicator and / or receptor that has responded to the ligand by analyzing the spectrum. FIG. 1 shows a schematic diagram of an analysis system 1 according to another embodiment of the present invention.

[0069] First, the analysis system 1 includes a measuring device 10 including a labeling unit 11 serving as a labeling means, an administration unit 12 serving as an administration means, and a measuring unit 13 serving as a measuring means. The measuring device 10 performs a labeling step of labeling cells containing one or more types of receptors with one or more types of indicators, an administration step of administering a ligand to a culture solution containing the cells, and a measuring step of measuring the spectrum of the culture solution. Note that in the measuring device 10, some or all of the above steps may be automated.

[0070] Furthermore, the analysis system 1 includes an information processing device 30 as an analysis means for identifying an indicator and / or a receptor that has responded to a ligand by analyzing the spectrum. The information processing device 30 includes a data acquisition unit 31, an input unit 32, an output unit 33, and a processing unit 300. The data acquisition unit 31 of the information processing device 30 is connected to the measurement unit 13 of the measurement device 10 via the network 20. When the information processing device 30 is not connected via the network 20, spectrum data output from the measurement device 10 via an external network or external memory may be input from the input unit 32.

[0071] The information processing device 30 performs an analysis step of analyzing the spectrum to identify the indicator and / or receptor that responded to the ligand. The processing unit 300 in the information processing device 30 includes an interface unit 34, a CPU 35, a memory 36, an auxiliary storage unit 37, and a bus 38.

[0072] The memory 36 stores the excited state and steady state spectra of the indicator used in the labeling unit 11. Furthermore, the auxiliary storage unit 37 stores a program for performing analysis. The processing unit 300 stores in advance in the auxiliary storage unit 37 a program for performing the processing of each step described below in FIG. 2, for example, in an executable format (for example, generated by conversion from a programming language by a compiler), and the processing unit 300 performs processing using the program stored in the auxiliary storage unit 37.

[0073] FIG. 2 is a flowchart illustrating an example of the operation of the information processing device 30 (and the processing unit 300 therein that performs information processing) according to this embodiment. Hereinafter, with reference to FIG. 2, an example of the operation of the information processing device 30 (processing unit 300) will be specifically described. Note that the example of the operation shown in FIG. 2 is merely an example, and the analysis steps and analysis means in the analysis method and analysis system of the present invention are not limited in any way to the scope of the embodiment shown in FIG. 2. In the following description, unless otherwise specified, the processing performed by the processing unit 300 means processing actually performed by the CPU 35 based on a program stored in the auxiliary storage unit 37 or the memory 36. The CPU 35 uses the memory 36 as a working area to temporarily store necessary data (intermediate data during processing, etc.), and stores data to be saved for a long period of time, such as calculation results, in the auxiliary storage unit 37 as appropriate.

[0074] First, in step 1 (S1), the processing unit 300 acquires spectral data measured by the measurement unit 13 using the data acquisition unit 31 of the information processing device 30. If the information processing device 30 and the measurement device 10 are connected via the network 20, the spectral data measured may be acquired via the network 20. However, if the information processing device 30 and the measurement device 10 are not connected via the network 20, the spectral data output from the measurement device 10 via an external network or external memory may be input from the input unit 32.

[0075] Next, in step 2 (S2), the processing unit 300 acquires spectral data of the used indicator in the excited state and the steady state. If the spectral data of the used indicator in the excited state and the steady state is stored in advance in the memory 36 of the processing unit 300, it can be acquired by reading it from the memory 36. However, if it is not stored in the memory 36, the spectral data may be input from the input unit 32. The order of steps 1 (S1) and 2 (S2) may be reversed. After acquiring the excited and steady-state spectral data of the indicator in step 2 (S2), the data may be temporarily stored in memory 36, and step 2 (S2) may be omitted in the next analysis.

[0076] Next, in step 3 (S3), the indicator and / or receptor that responded to the ligand is identified. Specifically, the spectral data measured by the processing unit 300 and the spectral data of the indicator used in the excited state and steady state are read out and analyzed. The analytical method used for identification is preferably the waveform separation system that applies the mathematical optimization described above, but other analytical methods may also be used as long as they can identify the indicator and / or receptor that responds to the ligand.

[0077] Finally, in step 4 (S4), the processing unit 300 outputs the results to the output unit 33. The output data may be in a format that describes the indicator and / or receptor that responded to the ligand, or may be in a format that outputs a sum of the spectra of the responding indicators and / or a spectrum that is an integration of such a summed spectrum and the measured spectrum as waveform data.

[0078] According to the above embodiments, it is possible to provide a high-throughput analytical method and analytical system that can efficiently and comprehensively analyze the response of receptors to target substances. By using these analytical methods and analytical systems, it is possible to achieve processing speeds at least 50 to 400 times faster than conventional methods.

[0079] A preferred embodiment of some steps in the analytical method and analytical system of the present invention is shown as a schematic diagram in Figure 3. As shown in Figure 3, according to the analytical method and analytical system of the present invention, cells labeled with multiple markers (two types of indicators in Figure 3) are placed in each well of the same observation container and subjected to simultaneous fluorescence observation, making it possible to obtain target data at a speed 10 times or more faster than conventional methods with the same effort (one fluorescence observation). The target data referred to here refers to the response relationship between a ligand and a group of receptors.

[0080] According to another embodiment of the present invention, as shown in FIG. 4, all cells to be analyzed may be measured simultaneously in a single well, and the measurement results may be analyzed to identify all indicators and / or receptors that responded to the ligand. In this case, since only one spectral data set is obtained, it is possible to obtain target data at a speed 10 times or more faster than conventional methods with the same effort (one fluorescence observation). The obtained spectral data exhibits a more complex waveform, but it is possible to identify which indicator a spectrum originates from by using the analysis method and analysis system of the present invention.

[0081] Although the present invention has been described above using embodiments, it goes without saying that the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be apparent to those skilled in the art that various modifications and improvements can be made to the above embodiments. Furthermore, it is clear from the claims that such modifications and improvements can also be included within the technical scope of the present invention. [Example]

[0082] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples shown below.

[0083] <Test 1: Observing each cell individually (single cell)> First, we expressed multiple receptors on separate cells, labeled each cell with a specific combination of indicators, and then measured and analyzed the fluorescence spectra of each cell. The test procedure was performed according to the schematic diagram (conceptual diagram) shown in Figure 3, and for more detailed procedures and conditions, we referred to the protocol described in WO2011 / 067970A1. As shown in Table 1, ten types of olfactory receptors, OR1 to OR10 (all tentative names), were used as receptors. Furthermore, as indicators, two types of each of the five fluorescent indicators (Fura-8 FF™, Quin-2, Fluo-3, Rhod-2, and Fura Red) shown in Table 1 were used to label cells (all cell membrane-permeable). The fluorescence spectra of the five fluorescent indicators in their excited state (when chelated with calcium ions) and steady state are shown in Figures 5(a) and 5(b). (In all graphs in Figures 5 to 13, the vertical axis represents fluorescence intensity, and the horizontal axis represents wavelength (nm).) The cells expressing the olfactory receptor are used in a state in which the target olfactory receptor is functionally expressed by a method such as gene transfer. [Table 1]

[0084] The test was performed according to the calcium imaging method using the fluorescent indicators described above. Specifically, cells expressing each of the olfactory receptors OR1 to OR10 were seeded into each well of a 96-well plate, and then labeled with the fluorescent indicators shown in Table 1. Finally, a ligand (aromatic substance) was added to each well, and the fluorescence spectrum was measured.

[0085] (Test 1-1: Experiment using aromatic substance A) In Test 1-1, the presence or absence of a responsive receptor was confirmed using the aromatic substance A as a ligand. In the calcium imaging procedure described above, the fluorescence spectrum of each well was measured after adding aromatic substance A, and the fluorescence spectrum shown in Figure 6(a) was detected in one well. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0086] As a result, the sum of the fluorescence spectra of Fluo-3 and Rhod-2 in their respective excited states, as shown in Figure 6(b), was derived as the predicted data with the smallest difference from the observed data. When the observed and predicted data were combined in Figure 6(c), they were nearly identical, although some differences in noise were observed. Therefore, the wells from which the observed data were detected were wells expressing cells labeled with Fluo-3 and Rhod-2 olfactory receptors. When compared with Table 1, this confirmed that OR3 responded to odorant A.

[0087] (Test 1-2: Experiment using aromatic substance B) In Test 1-2, the presence or absence of a responsive receptor was confirmed using aromatic substance B as the ligand. As in Test 1-1, the fluorescence spectrum of each well was measured after adding aromatic substance B, and the fluorescence spectrum shown in Figure 7(a) was detected in one well. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0088] As a result, the sum of the fluorescence spectra of the response patterns of Fura-8 FFTM and Quin-2 in their respective excited states, as shown in Figure 7(b), was derived as the predicted data with the smallest difference from the observed data. When the observed and predicted data were combined in Figure 7(c), they were nearly identical, although some differences due to noise were observed. Therefore, the wells in which the observed data were detected were wells in which cells expressed olfactory receptors labeled with Fura-8 FFTM and Quin-2. Comparing these results with Table 1, it was confirmed that OR1 responded to odorant B.

[0089] (Test 1-3: Experiment using aromatic substance C) In Test 1-3, the presence or absence of a responsive receptor was confirmed using the aromatic substance C as the ligand. As in Test 1-2, the fluorescence spectrum of each well was measured after adding aromatic substance C, and the fluorescence spectrum shown in Figure 8(a) was detected in one well. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0090] As a result, the sum of the fluorescence spectra of Fluo-3 and Fura Red in their respective excited states, as shown in Figure 8(b), was derived as the predicted data with the smallest difference from the observed data. When the observed and predicted data were combined in Figure 8(c), there was a near-match, although differences in the noise were observed at lower wavelengths. Therefore, the wells from which the observed data were detected were wells expressing olfactory receptors labeled with Fluo-3 and Fura Red. Comparing these results with Table 1, it was confirmed that OR10 responded to odorant C.

[0091] <Test 2: Observing all cells at once (multi-cell)> Next, multiple types of receptors were expressed on separate cells, and the same procedure as in Test 1 was followed until each cell was labeled with a predetermined combination of indicators.All the cells were then added to one well, and the fluorescence spectra were measured and analyzed to determine which receptors responded. The receptors, fluorescent indicators, and their combinations were the same as those shown in Table 1 of Test 1.

[0092] (Test 2-1: Experiment using aromatic substance D) In Test 2-1, the presence or absence of a responsive receptor was confirmed using aromatic substance D as a ligand. In the calcium imaging procedure described above, the fluorescence spectrum of the well after adding aromatic substance D was measured, and the fluorescence spectrum shown in Figure 9(a) was detected. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0093] As a result, the fluorescence spectrum detected when only OR1 responded, as shown in FIG. 9(b), was derived as predicted data with the smallest difference from the observed data.

[0094] Furthermore, the response pattern was quantified as shown in Table 2, confirming that only OR1 responded. Therefore, when fragrant substance D was used as the ligand, it was confirmed that only OR1 responded to fragrant substance D. [Table 2]

[0095] When different ligands were used, even when OR2 to OR10 responded independently, as in the above example, it was possible to determine the response from the predicted data and the numerically calculated results.

[0096] (Test 2-2: Experiment using aromatic substance E) In Test 2-2, the presence or absence of a responsive receptor was confirmed using the aromatic substance E as the ligand. In the calcium imaging procedure described above, the fluorescence spectrum of the well was measured after adding aromatic substance E, and the fluorescence spectrum shown in Figure 10(a) was detected. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0097] As a result, the fluorescence spectrum detected when OR1 and OR2 responded, as shown in Figure 10(b), was derived as predicted data with the smallest difference from the observed data. Furthermore, the numerical calculation results confirmed that OR1 and OR2 responded, as shown in Table 3. Therefore, when fragrance substance E was used as the ligand, OR1 and OR2 responded to fragrance substance E. [Table 3]

[0098] (Test 2-3: Experiment using aromatic substance F) In test 2-3, the presence or absence of a responsive receptor was confirmed using fragrant substance F as a ligand. In the calcium imaging procedure described above, the fluorescence spectrum of the well was measured after adding aromatic substance F, and the fluorescence spectrum shown in Figure 11(a) was detected. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0099] As a result, the fluorescence spectrum detected when OR2 and OR3 responded, as shown in Figure 11(b), was derived as predicted data with the smallest difference from the observed data. Furthermore, the numerical calculation results also confirmed that OR2 and OR3 responded, as shown in Table 4. Therefore, when fragrance substance F was used as the ligand, it was confirmed that OR2 and OR3 responded to fragrance substance F. [Table 4]

[0100] (Test 2-4: Experiment using aromatic substance G) In Test 2-4, the presence or absence of a responsive receptor was confirmed using the aromatic substance G as the ligand. In the calcium imaging procedure described above, the fluorescence spectrum of the well was measured after adding aromatic substance G, and the fluorescence spectrum shown in Figure 12(a) was detected. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0101] As a result, the fluorescence spectra detected when OR1, OR2, and OR3 responded were derived as predicted data with the smallest difference from the observed data, as shown in Figure 12(b) (Figure 12(b) shows the spectrum integrated with the observed data). Although the response intensity in the predicted data differs from the observed data in Figure 12(b), the characteristics of the predicted data spectrum match those of the observed data, so it was determined that the responding receptors were successfully identified. Furthermore, the numerical calculation results confirmed that OR1, OR2, and OR3 responded, as shown in Table 5. Therefore, when fragrance G was used as the ligand, OR1, OR2, and OR3 responded to fragrance G. [Table 5]

[0102] (Test 2-5: Experiment using aromatic substance H) In Test 2-5, the presence or absence of a responsive receptor was confirmed using the aromatic substance H as the ligand. In the calcium imaging procedure described above, the fluorescence spectrum of the well after adding aromatic substance H was measured, and the fluorescence spectrum shown in Figure 13(a) was detected. The fluorescence intensity at each wavelength in this fluorescence spectrum was quantified as observed data, and the response pattern of each indicator was calculated so as to minimize the difference between the observed data and the predicted data.

[0103] As a result, the fluorescence spectra detected when OR1, OR3, and OR5 or OR2, OR5, and OR6 responded, as shown in Figures 13(b) and 13(c), were derived as predicted data with the smallest difference from the observed data (both Figures 13(b) and 13(c) show spectra integrated with the observed data). Furthermore, the numerical calculation results confirmed that OR1, OR3, and OR5, or OR2, OR5, and OR6, responded, as shown in Tables 6 and 7. In other words, when fragrance substance H was used as the ligand, multiple optimal solutions were derived. Since one type of receptor is marked with multiple fluorescent reagents, multiple optimal solutions may arise if multiple receptors show similar responses (converted into the amount of spectral change in the indicator). However, since the possibility of such a case occurring is extremely low, it is presumed that in most cases the optimal solution will converge to one, making it possible to distinguish between them.

[0104] [Table 6] [Table 7]

[0105] In actual data collection in a large-scale experimental system, 0 to 10 of the 10 receptors will respond at different times, resulting in a more complex waveform pattern of the fluorescence spectrum. In such cases, assuming that 3 to 4 or more receptors respond at once, the calculations may yield multiple optimal solutions, as in Tests 2-5. In such cases, accurate prediction of results becomes difficult. However, even if 100% accuracy cannot be achieved, the analytical method and analytical system of the present invention can be an effective tool because it is possible to sufficiently narrow down the candidate receptors that respond to aroma substances.

[0106] From the above examples, it was confirmed that the present invention can provide a high-throughput analysis method and analysis system that can efficiently and comprehensively analyze the response of receptors to target substances. [Industrial Applicability]

[0107] By using the analysis method and analysis system of the present invention, it is possible to comprehensively analyze (measure) the response patterns formed by olfactory receptors in response to various groups of compounds, and obtain quantified data as learning data for machine learning of odors at a speed more than 10 times faster than conventional methods. If machine learning of smells can be achieved, it will enable the generation of many scents using fewer ingredients, and the design of desired scents can be realized with cheaper and more environmentally friendly ingredient combinations. Furthermore, a system that can generate a large number of scents using a limited number of ingredients can also be applied to technology that generates desired scents from electronic devices, which will greatly advance the digitization and digital transmission of scents.

[0108] Furthermore, since receptors are also targets for drug discovery, they can also be applied to pharmaceutical development. Furthermore, with regard to smell, it can also be applied to the development of scent prescriptions that counteract specific smells (so-called masking), and scent sensors (for example, disease prediction and health management from human breath and body odor, determining the ripeness of fresh food, or detecting dangerous and hazardous substances). [Explanation of symbols]

[0109] 1. Analysis system 10. Measuring equipment 11 Sign section 12 Administration site 13 Measuring part 20 Network 30 Information processing equipment 31 Data Acquisition Section 32 Input section 33 Output section 34 Interface section 35 CPU 36 memory 37 Auxiliary storage 38 Bus 300 Processing section

Claims

1. labeling cells containing one or more receptors with one or more indicator reagents; administering a ligand to a culture medium containing the cells; measuring the spectrum of the culture solution; analyzing the spectrum to identify an indicator and / or receptor that responded to the ligand; A method for analyzing receptor responses, comprising:

2. the cell contains one type of receptor; The analysis method according to claim 1 , wherein the indicator includes at least two types of indicators.

3. The cell contains at least two or more types of receptors, 2. The analysis method according to claim 1, wherein the indicator is one type of indicator.

4. 2. The analytical method according to claim 1, wherein the indicator includes at least a fluorescent indicator.

5. The analytical method according to claim 1 , wherein the receptor is an olfactory receptor.

6. 2. The analysis method according to claim 1, wherein the spectrum is analyzed by applying mathematical optimization.

7. a labeling means for labeling cells containing one or more receptors with one or more indicator reagents; an administration means for administering a ligand to a culture solution containing the cells; a measuring means for measuring the spectrum of the culture solution; an analysis means for identifying an indicator and / or a receptor that has responded to the ligand by analyzing the spectrum; A receptor response analysis system comprising:

Images