Sample derived from body fluid

Abstract

The present invention provides a technique for detecting chemical substances in a body fluid with higher sensitivity. This sample derived from a body fluid is characterized in that the concentration of free divalent cations is higher than the concentration in the body fluid.

Description

Samples derived from body fluids

[0001] The present disclosure relates to samples derived from bodily fluids and the like.

[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.

[0004] International Publication No. 2022 / 024902 Patent Publication No. 2020-091121

[0005] When using chemical substances such as odorants as test markers, it is necessary to detect the chemical substances in body fluids such as urine. However, the inventors have conducted research and found that the sensitivity of detecting trace amounts of chemical substances added to body fluids is significantly lower than when a general-purpose biochemical buffer solution is used instead of body fluids.

[0006] Patent Document 2 describes a method for solving the same problem as above by diluting urine 10 to 100 times, but this method results in a significant decrease in the concentration of the chemical substance to be detected.

[0007] An object of the present disclosure is to provide a technique for detecting chemical substances in body fluids with higher sensitivity.

[0008] In view of the above-mentioned problems, the present inventors have conducted extensive research and found that the above-mentioned problems can be solved by using a sample derived from a body fluid, characterized in that the concentration of free divalent cations is higher than the concentration in the body fluid.

[0009] Item 1. A sample derived from a body fluid, characterized in that the concentration of free divalent cations is higher than the concentration in the body fluid.

[0010] Item 2. The sample according to Item 1, wherein the divalent cation is an ion of an alkaline earth metal of the fourth period or later.

[0011] Item 3. The sample according to Item 1, wherein the divalent cation is at least one selected from the group consisting of calcium ions and strontium ions.

[0012] Item 4. The sample according to Item 1, wherein the concentration is 0.2 mM or more higher than the concentration of free divalent cations in body fluids.

[0013] Item 5. 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.

[0014] Item 6. The sample according to Item 1, wherein the body fluid is urine.

[0015] Item 7. The sample according to Item 1, which is a sample for measuring the activity of a sensor protein, which is a protein involved in a reaction that detects the presence of a chemical substance and causes cations to flow into cells.

[0016] Item 8. The sample according to Item 7, wherein the sensor protein is an ion channel receptor and / or a G protein-coupled receptor.

[0017] Item 9. The sample according to Item 7, wherein the sensor protein is a sensor protein retained by a cell.

[0018] Item 10. A method for producing a sample according to any one of Items 1 to 9, which comprises subjecting a body fluid to a treatment for increasing the concentration of free divalent cations.

[0019] Item 11. A method for measuring the activity of a sensor protein, comprising adding the sample according to any one of Items 1 to 9 to a compartment containing the protein.

[0020] The present disclosure provides a technique for detecting chemical substances in body fluids with higher sensitivity, specifically, a sample derived from body fluid, a method for producing the sample, a method for measuring protein activity using the sample, etc.

[0021]

[0046] Figure 1 shows the results of measuring olfactory receptor activity when test substance a (a chemical substance recognized by insect olfactory receptor A), VUAA1, and citric acid were added in Test Example 1. The vertical axis shows the integrated value of luminescence obtained over time as measured using FlexStation 3. The horizontal axis shows the concentration of citric acid, and a "-" on the horizontal axis indicates that citric acid was not added.

[0047] Figure 1 shows the results of measuring olfactory receptor activity when test substance a (a chemical substance recognized by insect olfactory receptor A), VUAA1, and urea were added in Test Example 1. The vertical axis shows the integrated value of luminescence obtained over time as measured using FlexStation 3. The horizontal axis shows the concentration of urea, and a "-" on the horizontal axis indicates that urea was not added.

[0048] Figure 2 shows the results of measuring olfactory receptor activity when calcium ions were added and not added to human urine samples in Test Example 2. The vertical axis shows the integrated value of luminescence obtained over time as measured using FlexStation 3. The horizontal axis shows whether or not test substance b (a chemical substance recognized by insect olfactory receptor B) was added. In Test Example 3, the results of measuring olfactory receptor activity are shown for cases where strontium ions were not added and where they were added (concentrations in the urine sample: 2 mM and 5 mM). The vertical axis shows the integrated value of luminescence acquired over time, measured using FlexStation 3. The horizontal axis shows whether or not test substance b (a chemical substance recognized by insect olfactory receptor B) was added.

[0022] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0023] In this specification, any combination of upper and lower limits of a certain numerical range is also directly and unambiguously disclosed.

[0024] In one aspect, the present disclosure relates to a sample derived from a body fluid, characterized in that the concentration of free divalent cations is higher than the concentration in the body fluid (sometimes referred to herein as a "sample of the present disclosure"). This is described below.

[0025] The body fluid contains a chemical substance to be detected, and is not particularly limited.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 the addition of components, dilution with a solvent or solution, concentration, and purification. However, from the standpoint of simplicity and minimizing processing losses due to volatilization of the target chemical substance during the treatment process, the addition of components and / or dilution is preferred. When diluting with a solvent or solution, a low dilution ratio is desirable to minimize a decrease in the concentration of the target chemical substance. The dilution ratio (volume of the sample of the present disclosure / volume of the body fluid used to prepare the sample) is preferably less than 10x, 9.9x or less, 9.8x or less, 9.7x or less, 9.6x or less, 9.5x or less, 9x or less, 8x or less, 7x or less, 6x or less, 5x or less, 4x or less, 3x or less, 2x or less, 1.5x or less, 1.4x or less, 1.3x or less, 1.2x or less, or 1.1x or less. Furthermore, when concentrating, it is desirable that the concentration ratio be low from the viewpoint of minimizing processing losses due to volatilization of the chemical substance to be detected during the processing, and the concentration ratio (volume of body fluid used to prepare the sample of the present disclosure / volume of the sample) is preferably 2-fold or less, 1.9-fold or less, 1.8-fold or less, 1.7-fold or less, 1.6-fold or less, 1.5-fold or less, 1.4-fold or less, 1.3-fold or less, 1.2-fold or less, 1.1-fold or less, 1.09-fold or less, 1.08-fold or less, 1.07-fold or less, 1.06-fold or less, 1.05-fold or less, 1.04-fold or less, 1.03-fold or less, 1.02-fold or less, or 1.01-fold or less.

[0031] The sample of the present disclosure is characterized in that the concentration of free divalent cations is higher than that in the body fluid. That is, the sample of the present disclosure has a higher concentration of free divalent cations 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 higher concentration of free divalent cations 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) other than subject a has a higher concentration of free divalent cations than body fluid b (e.g., urine b).

[0032] The "free" divalent cation is a divalent cation in a free state that is not in the form of a complex with a ligand such as a chelating agent, and is not particularly limited insofar as it is so.

[0033] As the divalent cation, from the viewpoint of detectability of chemical substances, ions of alkaline earth metals are preferred, and ions of alkaline earth metals of the fourth period or later are particularly preferred. Specific examples include calcium ions, strontium ions, barium ions, etc. Among these, calcium ions and strontium ions are preferred, and calcium ions are particularly preferred, from the viewpoint of low toxicity and ease of handling, and from the viewpoint of detectability of chemical substances.

[0034] The free divalent cations can be one type alone or a combination of two or more types.

[0035] From the viewpoint of chemical substance detectability, the free divalent cation concentration in the sample of the present disclosure is preferably at least 0.2 mM higher, more preferably at least 0.5 mM higher, more preferably at least 1.0 mM higher, more preferably at least 1.5 mM higher, more preferably at least 1.7 mM higher, even more preferably at least 1.8 mM higher, even more preferably at least 1.9 mM higher, and particularly preferably at least 2.0 mM higher than the corresponding free divalent cation concentration in the body fluid used to prepare the sample. In one embodiment of the present invention, the free divalent cation concentration may be at least 2.5 mM, 3.0 mM, 4.0 mM, 4.5 mM, or 5.0 mM higher. The upper limit of the difference between the free divalent cation concentrations is not particularly limited and may be, for example, 1 M, 500 mM, 300 mM, 200 mM, 150 mM, 100 mM, 50 mM, 30 mM, 20 mM, 15 mM, or 10 mM.

[0036] The method for preparing the sample of the present disclosure is not particularly limited, but from the viewpoint of simplicity and detectability of chemical substances, a method in which a salt consisting of a divalent cation and an anion is added to the body fluid is preferred.

[0037] Examples of anions constituting the salt include halide ions (e.g., fluoride ion, chloride ion, bromide ion, iodide ion, etc.), perchlorate ion, chlorate ion, hypochlorite ion, sulfide ion, sulfate ion, hydrogen sulfate ion, thiosulfate ion, sulfite ion, selenide ion, selenate ion, selenite ion, nitrate ion, nitrite ion, phosphate ion, arsenate ion, carbonate ion, hydrogen carbonate ion, metasilicate ion, cyanide ion, tetrahydroxide borate ion, permanganate ion, chromate ion, dichromate ion, acetate ion, etc. Among these, from the viewpoints of ease of handling and detectability of chemical substances, halide ions are preferred, and chloride ions are particularly preferred.

[0038] The salts may be one type alone or a combination of two or more types.

[0039] The sample of the present disclosure can be produced by a method including treating a body fluid to increase the concentration of free divalent cations. The treatment is not particularly limited, and the treatments described above can be used, for example.

[0040] In one embodiment, the sample of the present disclosure may be a sample obtained by adding a salt consisting of divalent cations and anions to a body fluid. The concentration of the salt (added salt) in the sample of the present disclosure, in terms of divalent cations, may be the difference between the free divalent cation concentration in the sample of the present disclosure and the corresponding free divalent cation concentration in the body fluid. The concentration of the salt (added salt) in the sample of the present disclosure, in terms of divalent cations, is preferably at least 0.2 mM higher, preferably at least 0.5 mM higher, preferably at least 1.0 mM, preferably at least 1.5 mM, more preferably at least 1.7 mM, even more preferably at least 1.8 mM, even more preferably at least 1.9 mM, and particularly preferably at least 2.0 mM. In one embodiment of the present invention, the concentration may be at least 2.5 mM, at least 3.0 mM, at least 4.0 mM, at least 4.5 mM, or at least 5.0 mM. The upper limit of the concentration is not particularly limited, and is, for example, 1 M, 500 mM, 300 mM, 200 mM, 150 mM, 100 mM, 50 mM, 30 mM, 20 mM, 15 mM, or 10 mM.

[0041] In one embodiment, the sample of the present disclosure can be obtained by treating a body fluid with a calcium ion chelating agent (e.g., citric acid) to dissociate the complex between the calcium ion and the body fluid. Such treatments include, for example, pH adjustment and addition of a complexation inhibitor. Such treatments can increase the concentration of free divalent cations in the resulting body fluid-derived sample.

[0042] 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.

[0043] The sensor protein may be selected from proteins involved in a reaction that detects the presence of a chemical substance and induces the influx of cations into cells, such as a receptor protein that uses a 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 as an indicator after the detection of the chemical substance.

[0044] Although we do not wish to limit our interpretation, it is thought that the activity of proteins responsible for detecting chemical substances is weakened when chelating agents in body fluids form complexes with divalent cations. Specifically, the amount of divalent cations flowing into cells due to reactions that occur after chemical detection is reduced by the effect of the chelating agents, thereby weakening the chemical response activity measured by the influx of divalent cations.

[0045] The sensor protein is preferably an ionotropic receptor and / or a G protein-coupled receptor. The sensor protein is preferably an olfactory receptor protein, particularly preferably an insect olfactory receptor protein. The sensor protein preferably exhibits calcium ion channel activity.

[0046] Insect olfactory receptor proteins are membrane proteins with seven transmembrane domains that function as odor sensors in living organisms. From the amino terminus (hereafter sometimes referred to as the "N-terminus") to the carboxyl terminus (hereafter 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).

[0047] 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).

[0048] Specific examples of wild-type insect olfactory receptor proteins include AaOR1, AaOR2, AaOR3, AaOR4, AaOR5, AaOR6, AaOR8, AaOR9, AaOR10, AaOR11, AaOR13, AaOR14, AaOR15, AaOR16, AaOR17, AaOR19, AaOR20, AaOR21, AaOR22, AaOR23, AaOR24, AaOR25, AaOR26, AaOR27, AaOR28, AaOR30, AaOR31, AaOR33, AaOR34, AaOR36, AaOR38, AaOR40, AaOR41, AaOR42, AaOR43, AaOR44, AaOR45, AaOR46, AaOR47, AaOR48, AaOR49, AaOR50, AaOR51, AaOR52, AaOR53, AaOR54, AaOR55, AaOR56, AaOR57, AaOR58, AaOR59, AaOR60, AaOR61, AaOR62, AaOR63, AaOR64, AaOR65, AaOR66, AaOR67, AaOR68, AaOR69, AaOR70, AaOR71, AaOR72, AaOR73, AaOR74, AaOR75, AaOR76, AaOR77, AaOR78, AaOR79, AaOR80, AaOR81, AaOR82, AaOR83, AaOR84, AaOR85, AaOR86, AaOR87, AaOR88, AaOR89, AaOR90, AaOR91, AaOR92, R41, AaOR42, AaOR43, AaOR44, AaOR45, AaOR46, AaOR47, AaOR48, AaOR49, AaOR50, AaOR52, AaOR54, AaOR55, AaOR58, AaOR59, AaOR60, AaOR61, AaOR62, A aOR63, AaOR64, AaOR65, AaOR66, AaOR67, AaOR69, AaOR70, AaOR71, AaOR72, AaOR73, AaOR74, AaOR75, AaOR76, AaOR77, AaOR78, AaOR79, AaOR80, AaOR81, AaOR83, AaOR84, AaOR85, AaOR86, AaOR87, AaOR88, AaOR89, AaOR90, AaOR91, AaOR92, AaOR93, AaOR94, AaOR95, AaOR96, AaOR97, AaOR99, AaOR100, AaOR 101, AaOR102, AaOR103, AaOR104, AaOR105, AaOR106, AaOR107, AaOR108, AaOR109, AaOR110, AaOR112, AaOR113, AaOR114, AaOR115, AaOR116, AaOR117, A aOR118, AaOR119, AaOR121, AaOR122, AaOR123, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR8, AgOR9, AgOR10, AgOR11, AgOR12, A gOR13, AgOR14, AgOR15, AgOR16, AgOR17, AgOR18, AgOR20, AgOR21, AgOR22, AgOR23, AgOR24, AgOR25, AgOR26, AgOR27, AgOR28, AgOR29, AgOR30, AgOR31,AgOR32、AgOR33、AgOR34、AgOR35、AgOR36、AgOR37、AgOR38、AgOR39、AgOR40、AgOR41、AgOR42、AgOR43、AgOR44、AgOR45、AgOR46、AgOR47、AgOR48,AgOR49、AgOR50、AgOR51、AgOR53、AgOR54、AgOR55、AgOR56、AgOR57、AgOR58、AgOR59、AgOR60、AgOR61、AgOR62、AgOR63、AgOR64、AgOR65、AgOR66、AgOR68、AgOR69、AgOR70、AgOR71、AgOR72、AgOR73、AgOR74、AgOR75、AgOR76、AgOR77、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、BmOR6、BmOR8、BmOR9、BmOR10、BmOR12、BmOR13、BmOR17、BmOR18、BmOR19,BmOR20、BmOR23、BmOR24、BmOR25、BmOR26、BmOR29、BmOR33、BmOR35、BmOR36、BmOR41、BmOR42、BmOR44、BmOR45、BmOR49、BmOR51、BmOR52、BmOR54、BmOR55、BmOR56、BmOR60、BmOR61、BmOR63、DmOR1a、DmOR2a、DmOR7a、DmOR9a、DmOR10a、DmOR13a、DmOR19a、DmOR22a、DmOR22b、DmOR22c、DmOR23a、DmOR24a、DmOR30a、DmOR33a、DmOR33b、DmOR33c、DmOR35a、DmOR42a、DmOR42b、DmOR43a、DmOR43b、DmOR45a、DmOR45b、DmOR46a、DmOR47a、DmOR47b、DmOR49a、DmOR49b、DmOR56a、DmOR59b、DmOR59c、DmOR63a、DmOR65a、DmOR65b、DmOR65c、DmOR67a、DmOR67b、DmOR67c、DmOR67d、DmOR69a、DmOR71a、DmOR74a、DmOR82a、DmOR83a、DmOR83c、DmOR85a、DmOR85b、DmOR85c、DmOR85d、DmOR85e、DmOR85f、DmOR88a、DmOR92a、DmOR94a、DmOR94b、DmOR98a、DmOR98b、ClOR1、ClOR2、ClOR3、ClOR4、ClOR5、ClOR6、ClOR7、ClOR8、ClOR9、ClOR10、ClOR11、ClOR12、ClOR13、ClOR14、ClOR15、ClOR16、ClOR17、ClOR18A、ClOR19、ClOR20、ClOR21、ClOR22、ClOR23、ClOR24、ClOR25、ClOR26、ClOR27、ClOR28、ClOR29、ClOR30、ClOR31、ClOR32、ClOR33、ClOR34、ClOR35、ClOR36、ClOR37、ClOR38、ClOR39、ClOR40、ClOR41、ClOR42、ClOR43、ClOR44、ClOR45、ClOR46,ClOR47、LmOR1、LmOR2、LmOR3、LmOR5、LmOR6、LmOR7、LmOR8、LmOR10、LmOR11、LmOR16、LmOR17、LmOR20、LmOR22、LmOR24、LmOR27、LmOR31、LmOR33、LmOR34、LmOR35、LmOR36、LmOR37、LmOR38、LmOR39、LmOR41、LmOR42、LmOR44、LmOR45、LmOR46、LmOR47、LmOR48、LmOR49、LmOR50、LmOR51、LmOR52、LmOR55、LmOR56、LmOR57、LmOR58、LmOR60、LmOR61、LmOR62、LmOR64、LmOR67、LmOR68、LmOR70、LmOR71、LmOR72、LmOR73、LmOR75、LmOR76、LmOR77、LmOR80、LmOR83、LmOR84、LmOR86、LmOR88、LmOR90、LmOR93、LmOR94、LmOR95、LmOR97、LmOR99、LmOR102、LmOR103、LmOR104、LmOR105、LmOR107、LmOR108、LmOR110、LmOR112、LmOR118、LmOR119、LmOR120、Examples include LmOR122, LmOR123, LmOR124, LmOR126, LmOR127, LmOR129, LmOR130, LmOR131, LmOR133, and LmOR142.

[0049] As used herein, OR indicates odorant 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, Am indicates that it is derived from Apis mellifera, Cl indicates that it is derived from Cimex lectularius, and Lm indicates that it is derived from Locusta migratoria. 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 known sequences.

[0050] 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, relative to 100% of the chemical response activity of the wild-type sensor protein.

[0051] The amino acid mutation is, for example, an amino acid substitution, insertion, addition, or deletion, preferably a substitution, and particularly preferably a conservative substitution.

[0052] The sensor protein can include a wild-type amino acid sequence, or an amino acid sequence that has, for example, 70% or more, 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 to the wild-type amino acid sequence.

[0053] As used herein, chemical response activity refers to the activity of detecting the presence of a chemical substance and influxing cations into cells. When the sensor protein is an olfactory receptor, it may be an ionotropic receptor or a G protein-coupled receptor. In the case of an insect olfactory receptor, however, the term refers to the property that the olfactory receptor recognizes a chemical substance, and the olfactory receptor complex formed by the olfactory receptor and the olfactory receptor co-receptor is activated, resulting in ion channel activity. The chemical response activity of a sensor protein can be measured using the signal transduction activity of the sensor protein in contact with a chemical substance as an indicator (e.g., by quantifying and evaluating the amount of signal molecules). In the case of an insect olfactory receptor, the chemical response activity of a sensor protein can be measured using the ion channel activity of the olfactory receptor complex formed by the olfactory receptor and the olfactory receptor co-receptor in contact with a chemical substance as an indicator. For example, a chemical substance is contacted with cells expressing proteins that emit fluorescence or luminescence in response to ions (e.g., calcium ions) that flow into the cells when (a) the olfactory receptor, (b) the olfactory receptor co-receptor, and (c) the olfactory receptor complex respond, 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 chemical substances is determined to be. Specifically, the measurement can be performed according to the method described in Patent Document 1.

[0054] 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.

[0055] The cells are not particularly limited, but animal cells such as insect cells and mammalian cells are preferred from the viewpoint of suitability for detecting chemical substances.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] The exogenous polynucleotide preferably contains a coding sequence for a protein that emits fluorescence or luminescence in response to ions (such as calcium ions) that flow into the cell in response to a sensor protein (particularly an olfactory receptor protein). 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.).

[0060] 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.

[0061] 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.

[0062] The compartment can be, for example, a compartment within a cell chip. The cell chip is not particularly limited as long as it contains cells and an apparatus capable of holding the cells, and is, for example, a well plate or a dish equipped with an apparatus for holding cells.

[0063] 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.

[0064] The material of the compartment is not particularly limited as long as it can hold cells, and can be, for example, resin, metal, etc.

[0065] 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 , 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.

[0066] 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.

[0067] From the viewpoint of detection sensitivity, a compartment usually contains multiple cells. 2 The number of cells per 1000 mm is, for example, 50 to 20,000 cells / mm 2 From the viewpoint of detection sensitivity, cell viability, etc., the cell density 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 , particularly preferably 1000 to 5000 cells / mm 2 is.

[0068] 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.

[0069] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0070] Test Example 1. Search for inhibitors of olfactory receptor activity. HEK293FT cells were cultured in a 6 cm dish at 1.2 × 10 6 The cells were seeded at 1000 cells / dish and cultured in DMEM medium (Nacalai Tesque) containing penicillin-streptomycin (Nacalai Tesque) and 10% FBS (Corning) at 37°C under 5% CO2 for approximately 24 hours.

[0071] The insect olfactory receptor A expression plasmid, the insect olfactory receptor co-receptor expression plasmid, and the GFP-aequorin expression plasmid were mixed in Opti-MEM (Thermo Fisher Scientific), and the mixture was transfected into the above cells using Lipofectamine LTX and Plus reagent (both reagents are from Invitrogen). Four hours after transfection, 1 × 10 cells were plated onto a 96-well plate. 5 The cells were seeded at 1000 cells / well and cultured in DMEM medium containing penicillin-streptomycin and 10% FBS at 37°C under 5% CO2 conditions for approximately 16 hours.

[0072] The culture medium was then removed and replaced with 80 μL of Hanks' HEPES buffer (containing 0.5 μM coelenterazine h (Promega) and 0.1% BSA (Sigma-Aldrich)), and the cells were allowed to stand for 4 hours at room temperature in the dark. Next, using FlexStation 3 (Molecular Devices), test substance a corresponding to olfactory receptor A was added dropwise to the cell culture medium, and the amount of luminescence from the cells was measured.

[0073] In this test example, test substance a (a chemical recognized by insect olfactory receptor A) was added at 500 μM, and VUAA1 (AOBIOUS) was added at 50 μM. Furthermore, 20 μL of Hanks' HEPES buffer containing citric acid at 2%, 0.5%, or 0.125% or urea at 2%, 0.5%, or 0.125% was added to the 96-well plate, and the luminescence was measured. Test substance a, VUAA1, citric acid, or urea was diluted 5-fold by adding the solution to the 96-well plate.

[0074] The results are shown in Figures 1 and 2. It was shown that the activity of the olfactory receptor was inhibited in a citric acid concentration-dependent manner. This was thought to be due to the formation of a complex between citric acid, a calcium ion chelator, and calcium ions.

[0075] Test Example 2. Analysis of the effect of adding calcium ions when using body fluid samples. The expression plasmid was introduced into cells in the same manner as in Test Example 1, except that an insect olfactory receptor B expression plasmid was used, and the cells were seeded on a 96-well plate and luminescence measurements were performed.

[0076] In this example, VUAA1 (manufactured by AOBIOUS) was added to a human urine sample at a concentration of 50 μM. 2+For the group receiving VUAA1, CaCl2 was added to the urine sample at 2 mM, and for the group receiving test substance, test substance b (a chemical recognized by insect olfactory receptor B) was added to the urine sample at 3 μM. After preparation, 20 μL of urine sample was dropped onto the 96-well plate, and the luminescence was measured. By dropping the sample onto the 96-well plate, the urinary components, added test substance b, VUAA1, and added CaCl2 were finally diluted 5-fold.

[0077] The results are shown in Figure 3. As can be seen from the results of the Ca+-free group in Figure 3, no difference in olfactory receptor activity was observed when calcium was not added, despite the addition of the test substance. Based on the results of Test Example 1, this was thought to be due to the inhibition of olfactory receptor activity by the citric acid in urine forming a complex with calcium ions. As predicted, the addition of calcium ions confirmed a change in olfactory receptor activity due to the addition of the test substance (Ca+-added group in Figure 3).

[0078] Test Example 3. Analysis of the effect of adding strontium ions when using a body fluid sample. The insect olfactory receptor B expression plasmid was used, and the seeding density in the 96-well plate was 8.0 × 10 4 The expression plasmid was introduced into the cells in the same manner as in Test Example 1, except that the cells / well was used, and the cells were seeded on a 96-well plate, and luminescence measurement was carried out.

[0079] In this example, VUAA1 (manufactured by AOBIOUS) was added to a human urine sample at a concentration of 50 μM. 2+ For the groups receiving VUAA1, SrCl2 was added to the urine samples at 2 mM or 5 mM, and for the groups receiving test substance, test substance b (a chemical recognized by insect olfactory receptor B) was added to the urine samples at 3 μM. After preparation, 20 μL of urine sample was dropped onto the 96-well plate, and the luminescence was measured. By dropping the sample onto the 96-well plate, the urine components, added test substance b, VUAA1, and added SrCl2 were finally diluted 5-fold.

[0080] The results are shown in Figure 4. Even when strontium ions were added instead of calcium ions, a change in the activity of the olfactory receptor due to the addition of the test substance was confirmed.

Claims

1. A sample derived from a body fluid, characterized in that the concentration of free divalent cations is higher than said concentration in said body fluid.

2. The sample according to claim 1, wherein the divalent cation is an ion of an alkaline earth metal of the fourth period or later.

3. The sample according to claim 1, wherein the divalent cation is at least one selected from the group consisting of calcium ions and strontium ions.

4. The sample of claim 1, wherein said concentration is at least 0.2 mM higher than the concentration of free divalent cations in body fluids.

5. 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.

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

7. The sample according to claim 1, which is a sample for measuring the activity of a sensor protein, which is a protein involved in a reaction that detects the presence of a chemical substance and allows cations to flow into cells.

8. The sample according to claim 7, wherein the sensor protein is an ionotropic receptor and / or a G protein-coupled receptor.

9. The sample according to claim 7, wherein the sensor protein is a sensor protein retained by a cell.

10. A method for producing a sample according to any one of claims 1 to 9, which comprises subjecting a body fluid to a treatment for increasing the concentration of free divalent cations.

11. A method for measuring the activity of a sensor protein, comprising adding a sample according to any one of claims 1 to 9 to a compartment containing the protein.

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