Body fluid-derived samples

Abstract

To provide a technique that enhances accuracy of detecting chemical substances contained in body fluid.SOLUTION: A sample derived from a body fluid is provided, the sample having a lower concentration of a factor that increases intracytoplasmic calcium concentration than that in the body fluid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to samples derived from body fluids and the like. [Background technology]

[0002] Groups of odorants that characterize specific human diseases and mental states have been identified, and because of their high utility as test markers, the development of various odor sensors targeting these has become active. Because biological olfactory receptors have superior properties in terms of diversity, sensitivity, selectivity, etc. that are not found in conventional odor sensor elements such as semiconductors, there are high expectations for the development of new odor sensors that use olfactory receptors as sensor elements.

[0003] Patent Document 1 discloses the use of cells expressing modified olfactory receptors or lipid bilayer membranes comprising modified olfactory receptors as odor sensors. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2022 / 024902 Summary of the Invention [Problem to be solved by the invention]

[0005] When using chemical substances such as odorants as test markers, it is necessary to detect trace amounts of chemical substances contained in body fluids such as urine. However, the inventors' research has revealed that cells respond to the body fluid itself, preventing concentration-dependent responses to the target chemical substances such as odorants.

[0006] An object of the present disclosure is to provide a technology that further improves the accuracy of detecting chemical substances contained in body fluids. [Means for solving the problem]

[0007] In view of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by using a sample derived from a body fluid, characterized in that the concentration of a factor that increases intracytoplasmic calcium concentration is lower than the concentration of the factor in the body fluid.

[0008] Item 1. A sample derived from a body fluid, wherein the concentration of a factor that increases intracytoplasmic calcium concentration is lower than the concentration in the body fluid.

[0009] Item 2. The sample according to Item 1, wherein the factor is a substance that is ultrafiltered.

[0010] Item 3. The sample according to Item 2, wherein the ultrafiltration is ultrafiltration with a molecular weight cutoff of 1 to 100 kDa.

[0011] Item 4. The sample according to Item 1, obtained by subjecting the body fluid to a treatment comprising at least one method selected from the group consisting of ultrafiltration, dialysis, and gel filtration chromatography.

[0012] Item 5. The sample according to Item 4, wherein the treatment includes ultrafiltration.

[0013] Item 6. The sample according to Item 1, wherein the cells are insect cells.

[0014] Item 7. The sample according to Item 1, wherein the body fluid is at least one selected from the group consisting of urine, blood, saliva, sweat, tears, interstitial fluid, synovial fluid, follicular fluid, cerebrospinal fluid, semen, milk, and vaginal fluid.

[0015] Item 8. The sample according to Item 1, wherein the body fluid is urine.

[0016] Item 9. The sample according to Item 1, which is a sample for measuring the activity of a sensor protein.

[0017] Item 10. The sample according to Item 9, wherein the sensor protein is an olfactory receptor protein.

[0018] Item 11. A method for measuring the activity of a sensor protein, comprising adding the sample according to any one of items 1 to 10 to a compartment containing the sensor protein. [Effects of the Invention]

[0019] According to the present invention, it is possible to further improve the accuracy of detecting chemical substances contained in body fluids. Specifically, it is possible to provide a sample derived from body fluid, a method for producing the sample, a method for measuring the activity of a sensor protein using the sample, etc. [Brief explanation of the drawings]

[0020] [Figure 1] This shows the area under the curve (vertical axis) of luminescence intensity when a test solution (horizontal axis) was added to cells in Test Example 1. On the horizontal axis, HO: water, HH: HH buffer solution (Hanks' Balance Salt Solution with 20 mM HEPES), and Urine 1-10: human urine samples collected from 10 subjects. [Figure 2] The graph shows the integrated luminescence intensity (vertical axis) when test solutions (A: HH buffer solution, B: human urine sample) were added to cells expressing each olfactory receptor (horizontal axis) in Test Example 2. The legend indicates the concentration in the test solution of the substance to which each olfactory receptor responds. [Figure 3] In Test Example 3, the area under the curve (vertical axis) of luminescence intensity is shown when test solutions (HH: HH buffer, U1 / U2: human urine samples collected from two subjects) (horizontal axis) are added to cells (horizontal axis) into which expression plasmids for various factors have been introduced. The factors encoded by the introduced expression plasmids are shown below the horizontal axis, with GFP-AEQ representing GAP (GFP-Aequorin), Orco representing an insect olfactory co-receptor, and OR-A representing an insect olfactory receptor. No TF on the horizontal axis indicates no introduction of the expression plasmid. [Figure 4]The vertical axis shows the integrated luminescence intensity when the test solution was added to cells expressing each olfactory receptor (legend) in Test Example 4. On the horizontal axis, Urine 1 to 10: human urine samples collected from 10 subjects, Untreated: human urine sample without ultrafiltration treatment, 10k Treatment: ultrafiltration of human urine sample. [Figure 5] In Test Example 5, the integrated luminescence intensity (vertical axis) is shown when a test solution (horizontal axis) is added to cells expressing each olfactory receptor (horizontal axis). On the horizontal axis, HH represents HH buffer solution, and Urine represents a human urine sample. In Panel A, the test solution was either an HH buffer solution to which a substance to which each olfactory receptor responds was added so that the test solution concentration indicated in the legend, or a human urine sample. In Panel B, the test solution was either an HH buffer solution to which a substance to which each olfactory receptor responds was added so that the test solution concentration indicated in the legend, or a solution obtained by ultrafiltration of a human urine sample. DETAILED DESCRIPTION OF THE INVENTION

[0021] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0022] In this specification, any combination of upper and lower limits of a certain numerical range is also directly and unambiguously disclosed.

[0023] In one aspect, the present disclosure relates to a sample derived from a body fluid, characterized in that the concentration of a factor that increases intracytoplasmic calcium concentration is lower than the concentration in the body fluid (sometimes referred to herein as a "sample of the present disclosure"). This will be described below.

[0024] The body fluid contains a chemical substance to be detected, and is not particularly limited.

[0025] The organisms (subjects) from which the body fluids are derived are not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, horses, cows, and pigs, with humans being preferred.

[0026] The subject is not particularly limited, and examples include a subject whose presence or absence of the disease being tested is unknown, a subject determined to have the disease being tested, a subject determined not to have the disease being tested, a subject for which information regarding the disease or condition is unknown, etc. When the subject is a human, any person can be the subject, regardless of their past medical history, including those who are considered to be healthy, and for example, subjects can include those who undergo health checkups or comprehensive medical examinations, and those who have undergone tests for the disease of interest using interview methods, questionnaire tests, score methods, pathological diagnostic methods, imaging diagnostic methods, blood biochemistry tests, etc.

[0027] Examples of body fluids include urine, blood, saliva, sweat, tears, tissue fluid, synovial fluid, follicular fluid, cerebrospinal fluid, semen, milk, vaginal fluid, etc. Among these body fluids, urine is preferred from the viewpoint that it is more suitable as a body fluid from which the sample of the present disclosure is derived.

[0028] Body fluids can be collected from living organisms according to or in accordance with known methods. The collected body fluids can be used immediately for preparing samples of the present disclosure, or can be stored (e.g., refrigerated or frozen) before being used for preparing samples of the present disclosure.

[0029] A sample "derived from a body fluid" is not a body fluid itself, but rather a sample obtained by subjecting a body fluid to some treatment that affects the component composition within the body fluid, and is not particularly limited thereto. Such treatments can include various processes such as dilution with a solvent or solution and purification. However, purification is preferred from the viewpoint of suppressing a decrease in the concentration of the target chemical substance during the treatment process. Furthermore, from this viewpoint, the ratio of the volume of the sample to the volume of the body fluid used to prepare the sample of the present disclosure is preferably less than 10, 9.9 or less, 9.8 or less, 9.7 or less, 9.6 or less, 9.5 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, or 1.1 or less. The ratio is, for example, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more.

[0030] The sample of the present disclosure is characterized in that the concentration of a factor that increases intracytoplasmic calcium concentration is lower than the concentration in the body fluid. That is, the sample of the present disclosure has a lower concentration of a factor that increases intracytoplasmic calcium concentration than the body fluid used to prepare the sample, not than body fluids in general. More specifically, for example, sample a of the present disclosure derived from body fluid a (e.g., urine a) collected from a certain subject (subject a) has a lower concentration of a factor that increases intracytoplasmic calcium concentration than body fluid a (e.g., urine a), and sample b of the present disclosure derived from body fluid b (e.g., urine b) collected from a subject (subject b) different from subject a has a lower concentration of a factor that increases intracytoplasmic calcium concentration than body fluid b (e.g., urine b).

[0031] The factor that increases the calcium concentration in the cytoplasm is not particularly limited, as long as it is a factor contained in a body fluid and can increase the calcium concentration in the cytoplasm by contacting the factor with a cell. The increase in the calcium concentration in the cytoplasm can occur either by calcium influx from the outside of the cell into the cytoplasm or by calcium release from an intracellular calcium store such as the endoplasmic reticulum of the cell.

[0032] As will be described in the Examples below, it has been discovered that factors that increase intracytoplasmic calcium concentrations can be removed or reduced by ultrafiltration. Therefore, in one embodiment of the present invention, the factors can be ultrafiltered. Ultrafiltered substances are substances that can be captured by an ultrafiltration membrane through ultrafiltration. From the viewpoint of the efficiency of factor removal / reduction, the molecular weight cutoff (nominal molecular weight cutoff) of this ultrafiltration is preferably 1 to 100 kDa, more preferably 1 to 70 kDa, even more preferably 1 to 50 kDa, even more preferably 1 to 30 kDa, particularly preferably 1 to 20 kDa, and particularly preferably 1 to 15 kDa. The material of the ultrafiltration membrane is not particularly limited, as long as it is a material whose pore size can be controlled for ultrafiltration. Examples of such materials include cellulose (e.g., regenerated cellulose), cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, and polyacrylonitrile.

[0033] In addition to ultrafiltration, factors that increase intracytoplasmic calcium concentration can be removed or reduced by treatments that can remove or reduce substances with molecular weights similar to those of ultrafiltration (e.g., treatments with the above-mentioned molecular weight cutoff). In one aspect, the sample of the present invention can be a sample obtained by treating a body fluid with at least one treatment selected from the group consisting of ultrafiltration, dialysis, and gel filtration chromatography (particularly preferably, ultrafiltration). Ultrafiltration is as described above. Dialysis and gel filtration chromatography can also be performed in accordance with the above-mentioned molecular weight cutoff for ultrafiltration.

[0034] The cells are not particularly limited. From the viewpoint of suitability for detecting chemical substances, animal cells such as insect cells and mammalian cells are preferred. Among these, insect cells are preferred from the viewpoint that a factor that increases the calcium concentration in the cytoplasm increases the calcium concentration more significantly.

[0035] Examples of insect cells include Sf cells, MG1 cells, and High Five cells.TM Examples of Sf cells that can be used include Sf9 cells (ATCC CRL1711) and Sf21 cells. Among insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0036] The cells derived from an insect of the family Arctiidae are primary cultured cells or established cell lines of cells derived from an insect of the family Arctiidae that constitute the living body, and are not particularly limited as long as they are so.

[0037] Examples of the Arctiidae family include the Arctiinae subfamily (Arctiinae), the Lithosiinae subfamily (Lithosiinae), and the Syntominae subfamily (Syntominae), with the Arctiinae subfamily being preferred among these. Examples of the Arctiinae subfamily include the genera Spilosoma (the genus Spilarctia), Spilarctia, and Rhagonis, with the genus Spilasoma being particularly preferred. The genus Spilasoma is not particularly limited, but the genus Spilasoma imparilis is particularly preferred.

[0038] Cells derived from insects of the family Arctiidae can be obtained from known biobanks, or can be obtained by collecting and culturing live insects of the family Arctiidae according to or in accordance with known methods, and can also be established as lines if necessary.

[0039] Examples of cells derived from Mulberry butterfly include FFPRI-SpIm-2AM-SF cells (MAFF number: 275052) and FFPRI-SpIm-2AM-IPL411 cells (MAFF number: 275053) from the National Agriculture and Food Research Organization Genebank.

[0040] The sample of the present disclosure can be a sample (=sample composition) for measuring the activity of a sensor protein. By measuring the activity of a sensor protein, chemical substances that can be recognized by the sensor protein can be detected. The sample of the present disclosure can be used to contact the sensor protein.

[0041] The sensor protein may be selected from proteins involved in a reaction that detects the presence of a chemical substance and allows cations to flow into cells, and may be, for example, a receptor protein that uses the chemical substance as a ligand. The chemical response activity of the sensor protein can be evaluated by using the amount of cations that flow into cells due to a reaction that occurs after the detection of the chemical substance as an indicator. The sensor protein is preferably an olfactory receptor protein, and particularly preferably an insect olfactory receptor protein. The sensor protein preferably exhibits calcium ion channel activity.

[0042] Insect olfactory receptor proteins are membrane proteins with seven transmembrane domains that function as odor sensors in living organisms. From the amino terminus (hereinafter sometimes referred to as the "N-terminus") to the carboxyl terminus (hereinafter sometimes referred to as the "C-terminus") of an olfactory receptor protein, they are composed of the N-terminal region (NT), the first transmembrane domain (TM1), the first extracellular loop (EC1), the second transmembrane domain (TM2), the first intracellular loop (IC1), the third transmembrane domain (TM3), the second extracellular loop (EC2), the fourth transmembrane domain (TM4), the second intracellular loop (IC2), the fifth transmembrane domain (TM5), the third extracellular loop (EC3), the sixth transmembrane domain (TM6), the third intracellular loop (IC3), the seventh transmembrane domain (TM7), and the C-terminal region (CT). In the present disclosure, each region is determined by structure prediction (default conditions) using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane spanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html).

[0043] Insects from which insect olfactory receptor proteins are derived preferably include Diptera insects such as Culicidae and Drosophilidae, Lepidoptera insects such as Bombycidae, Hymenoptera insects such as Apidae, Orthoptera insects such as Acrididae, and Hemiptera insects such as Cimex, and more preferably Diptera insects such as Culicidae and Drosophilidae, Orthoptera insects such as Acrididae, and Hemiptera insects such as Cimex. Examples of Culicidae insects include Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Examples of insects in the Drosophilidae family include Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virillis. Examples of insects in the Bombycidae family include Bombyx mori, Bombyx mandarina, and Trilocha varians. Examples of insects in the Apidae family include Apis mellifera, Apis florea, Apis dorsata, and Bombus terrestris. Examples of insects belonging to the Acrididae family include the migratory locust (Locusta migratoria), and examples of insects belonging to the Cimex family include the bedbug (Cimex lectularius).

[0044] Specific examples of wild-type insect olfactory receptor proteins include AaOR1, AaOR2, AaOR4, AaOR5, AaOR6, AaOR8, AaOR9, AaOR10, AaOR15, AaOR22, AaOR24, AaOR25, AaOR26, AaOR27, AaOR28, AaOR30, AaOR34, AaOR36, AaOR38, AaOR41, AaOR42, AaOR43, AaOR44, AaOR47, AaOR49, AaOR50, AaOR52, AaOR54, AaOR58, AaOR59, AaOR60, AaOR61, Aa OR64, AaOR65, AaOR66, AaOR67, AaOR69, AaOR70, AaOR71, AaOR72, AaOR73, AaOR74, AaOR75, AaOR77, AaOR78, AaOR79, AaOR81, AaOR83, AaOR84, AaOR85, AaOR86, AaOR87, AaOR91, AaOR95, AaOR97, AaOR96, AaOR99, AaOR100, AaOR102, AaOR103, AaOR104, AaOR105, AaOR107, AaOR108, AaOR109, AaOR110, AaOR 112, AaOR114, AaOR116, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR8, AgOR9, AgOR10, AgOR11a, A gOR12, AgOR12, AgOR13, AgOR14, AgOR15, AgOR16, AgOR17, AgOR18, AgOR20, AgOR21, AgOR23, AgOR25, AgOR26, AgOR27, AgOR28, AgOR30, AgOR34, AgOR36, AgOR37, AgOR38, AgOR39, AgOR40, AgOR42, AgOR44, AgOR45, AgOR46, AgOR47, AgOR49, AgOR50, AgOR54, AgOR56, AgOR57, AgOR60, AgOR61, AgOR62, AgOR6 3, AgOR64, AgOR65, AgOR69, AgOR70, AgOR71, AgOR72, AgOR74, AgOR75, AgOR76, AmOR1, AmOR3, AmOR9, AmOR10, AmOR13, AmOR41, AmOR51, AmOR52, AmOR55,AmOR71, AmOR73, AmOR78, AmOR85, AmOR89, AmOR90, AmOR114, AmOR115, AmOR118, AmOR120, AmOR121, AmOR161, BmOR1, BmOR2, BmOR3, BmOR4, BmOR5, BmOR8, BmOR9, BmOR10, B mOR13, BmOR17, BmOR18, BmOR23, BmOR24, BmOR25, BmOR35, BmOR36, BmOR42, BmOR45, B mOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmOR1a, DmOR9a, DmOR19a, DmOR22a , DmOR22b, DmOR22c, DmOR24a, DmOR30a, DmOR33a, DmOR33b, DmOR33c, DmOR35a, DmOR42b, DmOR43a, DmOR45a, DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmOR65c, DmOR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83c, DmOR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, DmOR98b, etc.

[0045] In this specification, OR indicates an olfactory receptor, Dm indicates that it is derived from Drosophila melanogaster, Bm indicates that it is derived from Bombyx mori, Ag indicates that it is derived from Anopheles gambiae, Aa indicates that it is derived from Aedes aegypti, and Am indicates that it is derived from Apis mellifera. The amino acid sequences and coding sequences of various olfactory receptor proteins, including these, are publicly known or can be easily identified by sequence identity searches based on publicly known sequences.

[0046] The sensor protein may contain amino acid mutations relative to the wild-type amino acid sequence, as long as the chemical response activity is not significantly reduced. "Not significantly reduced" means, for example, that the chemical response activity of the sensor protein containing the amino acid mutation is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more of the chemical response activity of the wild-type sensor protein (100%).

[0047] The amino acid mutation is, for example, an amino acid substitution, insertion, addition, or deletion, preferably a substitution, and particularly preferably a conservative substitution.

[0048] The sensor protein can include a wild-type amino acid sequence, or an amino acid sequence that has, for example, 70% or more identity to the wild-type amino acid sequence, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity.

[0049] As used herein, chemical response activity refers to the property of a sensor protein recognizing a chemical substance and exhibiting signal transduction activity (e.g., ion channel activity) either alone or in conjugation with other proteins. When the sensor protein is an olfactory receptor, it may be a G protein-coupled receptor or an ion channel receptor. In the case of an insect olfactory receptor, however, the term refers to the property of the olfactory receptor recognizing a chemical substance and activating the olfactory receptor complex formed by the olfactory receptor and an olfactory receptor co-receptor, thereby exhibiting ion channel activity. The chemical response activity of a sensor protein can be measured using as an indicator the signal transduction activity of the sensor protein in contact with a chemical substance (for example, by quantifying and evaluating the amount of signal molecules). In the case of an insect olfactory receptor, the chemical response activity of the olfactory receptor can be measured using as an indicator the ion channel activity of the olfactory receptor complex formed by the olfactory receptor in contact with a chemical substance and an olfactory receptor co-receptor. For example, cells expressing proteins that emit fluorescence or luminescence in response to ions (such as calcium ions) that flow into the cells when (a) olfactory receptors, (b) olfactory receptor co-receptors, and (c) olfactory receptor complexes respond are contacted with a chemical substance, and the amount of luminescence from the cells is measured. The greater the measured amount of luminescence, the higher the response activity of the olfactory receptor to the chemical substance is determined to be. Specifically, measurements can be performed according to the method described in Patent Document 1.

[0050] The sensor protein is preferably in a state that is retained by cells. The sample of the present disclosure can be used to contact cells that retain the sensor protein.

[0051] The cells preferably contain an exogenous polynucleotide containing a coding sequence for a sensor protein, which allows the expression of any sensor protein and increases the expression level of the target sensor protein, thereby enhancing the detection sensitivity of the target chemical substance.

[0052] An exogenous polynucleotide is a polynucleotide containing a base sequence that is not derived from the genomic DNA (particularly chromosomal genomic DNA) of a cell, and is not particularly limited insofar as it is so.

[0053] When the sensor protein is an insect olfactory receptor, the exogenous polynucleotide preferably contains a coding sequence for an insect olfactory receptor co-receptor. Insect olfactory receptor co-receptors are membrane proteins with a seven-transmembrane structure, similar to olfactory receptors, and function by forming a heterocomplex with the olfactory receptor. The olfactory receptor complex, which is a heterocomplex composed of an olfactory receptor and an olfactory receptor co-receptor, has ion channel activity that is activated by odorants, and when activated, it transports sodium ions (Na + ), calcium ions (Ca 2+ ) into the cell.

[0054] The exogenous polynucleotide preferably contains a coding sequence for a protein that emits fluorescence or light in response to ions (such as calcium ions) that flow into the cell when the sensor protein (particularly, the olfactory receptor protein) responds. Examples of such proteins include aequorin, yellow camelon, and GCaMP. Alternatively, the cells of the present disclosure preferably contain an ion-dependent fluorescent dye such as a calcium ion-dependent fluorescent dye (e.g., Fura-2, Fluo-3, Fluo-4, etc.).

[0055] Coding sequences such as those for insect olfactory receptor co-receptors, fluorescent or luminescent proteins, and drug resistance genes are preferably contained in the exogenous polynucleotide in the form of an expression cassette. The structure of the expression cassette is similar to that of the expression cassette for the sensor protein. The promoter of the expression cassette can be shared among multiple coding sequences.

[0056] In one aspect, the present disclosure relates to a method for measuring the activity of a sensor protein, comprising adding a sample of the present disclosure to a compartment containing the protein.

[0057] The compartment can be, for example, a compartment within a cell chip that comprises cells and a device for holding the cells (such as a dish or well plate).

[0058] The shape of the compartment is not particularly limited as long as it is capable of retaining cells. From the viewpoint of cell retention, production efficiency, and chemical substance detection, the compartment is preferably in the form of a well.

[0059] The material of the compartment is not particularly limited as long as it can hold cells, and can be, for example, resin, metal, etc.

[0060] The area of ​​the compartment is preferably 0.5 to 100 mm from the viewpoint of detection sensitivity or production efficiency. 2 , more preferably 1 to 50 mm 2 , and more preferably 1.5 to 40 mm 2 , and even more preferably 2 to 40 mm 2 In a preferred embodiment of the present disclosure, the area is 50 mm 2 Below, 35mm 2 or less, or 15 mm 2 The following is the result.

[0061] From the viewpoint of detection sensitivity or production efficiency, the number of compartments is preferably 1 to 2000, more preferably 4 to 1600, and even more preferably 8 to 400. In a preferred embodiment of the present disclosure, the number is 10 or more, 20 or more, or 50 or more.

[0062] From the viewpoint of detection sensitivity, a compartment usually contains multiple cells. 2 The number of cells per unit area is, for example, 50 to 20,000 cells / mm 2 From the viewpoint of detection sensitivity, cell viability, etc., it is preferably 100 to 15,000 cells / mm 2 , more preferably 100 to 10,000 cells / mm 2 , and more preferably 200 to 10,000 cells / mm 2 , and even more preferably 500 to 7000 cells / mm2 , and particularly preferably 1000 to 5000 cells / mm 2 is.

[0063] The cell chip may contain only cells of one type of sensor protein, but preferably contains cells of two or more types (more preferably three or more, even more preferably four or more, even more preferably five or more, ten or more, fifteen or more, or twenty or more) of different types of sensor protein. [Example]

[0064] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0065] Test Example 1: Analysis of cellular responsiveness to body fluids SpIm cells derived from the mulberry butterfly (Spilosoma imparilis) were maintained in Sf900-III (Thermo Fisher) medium at 27°C under atmospheric conditions. SpIm cells were transfected with expression plasmids containing the GAP (GFP-Aequorin) coding sequence, the insect olfactory co-receptor Orco coding sequence, and the insect olfactory receptor OR-A coding sequence. Transfection of SpIm cells was performed using FuGENE HD Reagent (Promega). Specifically, 1.0 x 10 cells were transfected into a 6-well plate. 6 After incubation at 27°C for 24 hours under atmospheric conditions, transfection was performed at a ratio of 2 μg of Plasmid DNA to 4 μL of Reagent per well. After incubation at 27°C for 48 hours under atmospheric conditions, the cells were transfected at a ratio of 1.0 x 10 cells / well in a 96-well plate using Sf900-III medium containing 2.0 μM EnduRen (Promega). 5Cells were replated at 1000 cells / well (medium volume: 80 μL / well). After 24 hours of incubation at 27°C under atmospheric conditions, luminescence measurements were performed using a Flexstation 3 (Molecular Devices). 20 seconds after the start of measurement in Flex mode, 20 μL of human urine samples collected from 10 subjects or HH buffer (Hanks' Balance Salt Solution with 20 mM HEPES) was added, and luminescence intensity was measured every 2 seconds until 90 seconds after the start of measurement. The area under the curve passing through the luminescence intensity at each measurement point was calculated and used as the measurement value for each experiment.

[0066] The results are shown in Figure 1. When water or HH buffer was added to cells expressing GAP (GFP-Aequorin), the insect olfactory co-receptor Orco, and the insect olfactory receptor OR-A, background luminescence remained low, but when body fluid was added, a high luminescence response was observed in all samples.

[0067] Test Example 2: Analysis of the influence of the type of olfactory receptor on the cellular response to body fluids SpIm cells were transfected in the same manner as in Test Example 1, except that the coding sequence for the insect olfactory receptor OR-A was replaced with the coding sequence for another insect olfactory receptor (OR-B, OR-C, OR-D, OR-E, OR-F, OR-G, OR-H, or OR-I), and a stable expression strain was prepared according to standard methods. The resulting cells were plated at 3.5 x 10 in a 384-well plate using Sf900-III medium containing 2.0 μM EnduRen. 4 Cells were seeded at 40 μL / well (medium volume 40 μL / well). After 24 hours of incubation at 27°C under atmospheric conditions, luminescence measurements were performed using an FDSS / μCELL (Hamamatsu Photonics). 20 seconds after the start of measurement, 10 μL of human urine sample or HH buffer solution was added, and the luminescence intensity was measured every 2 seconds until 90 seconds after the start of measurement. The integrated value of the luminescence intensity at each measurement point was used as the measured value. In this test, human urine sample or HH buffer solution was used after adding a substance to which the insect olfactory receptor expressed in the cells responds at concentrations of 0.1 μM, 1 μM, or 10 μM, or after adding only the solvent DMSO.

[0068] The results are shown in Figure 2. Cells expressing GAP (GFP-Aequorin), the insect olfactory co-receptor Orco, and insect olfactory receptors exhibited high luminescence responses to body fluids, regardless of the type of olfactory receptor expressed. Furthermore, when HH buffer was used, luminescence responses were dependent on the concentration of the substance to which each olfactory receptor responded, but when body fluids were used, no such concentration-dependent luminescence responses were observed.

[0069] Test Example 3. Searching for the cause of cellular humor responsiveness 1 To SpIm cells, An expression plasmid containing the GAP (GFP-Aequorin) coding sequence, an expression plasmid containing the GAP (GFP-Aequorin) coding sequence and the insect olfactory co-receptor Orco coding sequence, or Expression plasmid containing the GAP (GFP-Aequorin) coding sequence, the insect olfactory co-receptor Orco coding sequence, and the insect olfactory receptor OR-A coding sequence were transfected in the same manner as in Test Example 1. The resulting cells or SpIm cells into which no expression plasmid had been introduced were treated in the same manner as in Test Example 1, and the area under the curve of luminescence intensity when a human urine sample or HH buffer solution was added was measured.

[0070] The results are shown in Figure 3. Cells without the expression plasmid did not emit light when body fluid was added, but a luminescence response to body fluid was observed in all cells expressing GFP-AEQ. Since a response was observed even in cells expressing only GFP-AEQ, it was found that the body fluid responsiveness of the cells was not due to the expressed olfactory receptor or a substance in the body fluid to which the olfactory receptor responded, but rather that an intrinsic factor in the cell caused an increase in the calcium concentration in the cytoplasm.

[0071] Test Example 4. Searching for the cause of cellular humor responsiveness 2 The stable expression clone obtained in Test Example 2 was cultured in a 384-well plate at 3.5 x 10 cells / well using Sf900-III medium containing 2.0 μM EnduRen. 4Cells were seeded at 40 μL / well (medium volume 40 μL / well). After 24 hours of incubation at 27°C under atmospheric conditions, luminescence measurements were performed using the FDSS / μCELL. 20 seconds after the start of measurement, 10 μL of each human urine sample collected from 10 subjects or its ultrafiltrate was added, and luminescence intensity was measured every 2 seconds until 90 seconds after the start of measurement. The area under the curve of luminescence intensity at each measurement point was used as the measured value. The ultrafiltrate was obtained by adding each human urine sample to an Amicon Ultra 10 kD (Merck) device and centrifuging it.

[0072] The results are shown in Figure 4. Background signals were observed in almost all body fluid samples before ultrafiltration, but ultrafiltration reduced the background signals. This indicates that the cellular response to body fluids is due to factors present in body fluids that increase calcium concentrations in the cytoplasm, which act on factors within the cells to cause an increase in calcium concentrations in the cytoplasm.

[0073] Test Example 5. Evaluation of olfactory receptor responses under conditions where non-specific responses are suppressed The stable expression clone obtained in Test Example 2 was cultured in a 384-well plate at 3.5 x 10 cells / well using Sf900-III medium containing 2.0 μM EnduRen. 4 Cells were seeded at 1000 cells / well (medium volume: 40 μL / well). After 24 hours of incubation at 27°C under atmospheric conditions, luminescence measurements were performed using the FDSS / μCELL. 20 seconds after the start of measurement, 10 μL of the test solution was added, and luminescence intensity was measured every 2 seconds until 90 seconds after the start of measurement. The area under the curve for luminescence intensity at each measurement point was recorded as the measured value.

[0074] In Test A, a human urine sample or HH buffer solution was used as the test solution, to which a substance to which the insect olfactory receptor expressed in cells responds was added at a concentration of 0.1 μM, 1 μM, or 10 μM, or to which only solvent was added.

[0075] In Test B, a human urine sample or HH buffer solution was added with a substance to which the insect olfactory receptor expressed in the cells responds at a concentration of 0.1 μM, 1 μM, or 10 μM, or with the solvent alone, and then each solution was ultrafiltered (prepared in the same manner as in Test Example 4) and used as the test solution.

[0076] The results are shown in Figure 5. By reducing the concentration of the factor that increases the cytoplasmic calcium concentration, which causes a non-specific response, a concentration-dependent luminescence response was obtained for the substance to which the insect olfactory receptor expressed in the cells responded.

Claims

1. A sample derived from a body fluid, characterized in that the concentration of a factor that increases intracytoplasmic calcium concentration is lower than said concentration in said body fluid.

2. The sample of claim 1 , wherein the agent is an ultrafiltered substance.

3. The sample according to claim 2, wherein the ultrafiltration is ultrafiltration with a molecular weight cutoff of 1 to 100 kDa.

4. The sample according to claim 1, obtained by subjecting the body fluid to a treatment comprising at least one selected from the group consisting of ultrafiltration, dialysis, and gel filtration chromatography.

5. The sample of claim 4 , wherein the treatment comprises ultrafiltration.

6. The sample of claim 1 , wherein the cells are insect cells.

7. The sample according to claim 1, wherein the body fluid is at least one selected from the group consisting of urine, blood, saliva, sweat, tears, interstitial fluid, synovial fluid, follicular fluid, cerebrospinal fluid, semen, milk, and vaginal fluid.

8. The sample of claim 1 , wherein the body fluid is urine.

9. The sample according to claim 1, which is a sample for measuring the activity of a sensor protein.

10. The sample according to claim 9 , wherein the sensor protein is an olfactory receptor protein.

11. A method for measuring the activity of a sensor protein, comprising adding the sample according to any one of claims 1 to 10 to a compartment containing the sensor protein.

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