Body fluid-derived test sample

Abstract

Provided is a technique for detecting a chemical in a body fluid with higher sensitivity. A sample of a body fluid source, characterized in that the concentration of free divalent cations is higher than the concentration in the body fluid.

Description

Technical Field

[0001] This disclosure relates to samples derived from bodily fluids, etc. Background Technology

[0002] A group of odor substances characterizing specific human diseases and mental states has been identified. Due to their high value as diagnostic markers, the development of various odor sensors targeting these substances has become prevalent. Because biological olfactory receptors possess superior characteristics in terms of diversity, sensitivity, and selectivity compared to previous odor sensor elements such as semiconductors, there is a promising prospect for developing new odor sensors that utilize olfactory receptors as sensor elements.

[0003] Patent document 1 discloses the use of cells expressing modified olfactory receptors and lipid bilayer membranes with modified olfactory receptors as odor sensors.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: International Publication No. 2022 / 024902

[0007] Patent Document 2: Japanese Patent Application Publication No. 2020-091121 Summary of the Invention

[0008] The problem that the invention aims to solve

[0009] When using odorants and other chemical substances as markers, it is necessary to detect chemicals in bodily fluids such as urine. However, during their research, the inventors discovered that the detection sensitivity for trace amounts of chemicals added to bodily fluids is significantly reduced compared to using a common biochemical buffer solution instead of bodily fluids.

[0010] Patent Document 2 describes a method to solve a problem similar to the one described above by diluting urine 10 to 100 times before use. However, in this method, the concentration of the chemical substance being tested is significantly reduced.

[0011] The subject of this disclosure is to provide a technique for detecting chemical substances in bodily fluids with higher sensitivity.

[0012] Methods for solving problems

[0013] In view of the above-mentioned problems, the inventors conducted in-depth research and found that the above-mentioned problems can be solved by using a sample of a body fluid source characterized by a concentration of free divalent cations higher than the concentration in the body fluid. That is, this disclosure includes the following methods.

[0014] Project 1. A sample derived from body fluids, characterized in that the concentration of free divalent cations is higher than the concentration in the body fluid.

[0015] Project 2. The sample described in Project 1, wherein the divalent cation is an ion of an alkaline earth metal from the fourth period onwards.

[0016] Item 3. The sample described in Item 1, wherein the divalent cation is selected from at least one of calcium ions and strontium ions.

[0017] Item 4. The sample described in Item 1, wherein the concentration is at least 0.2 mM higher than the concentration of free divalent cations in body fluids.

[0018] Item 5. The sample described in Item 1, wherein the body fluid is selected from at least one of urine, blood, saliva, sweat, tears, tissue fluid, joint fluid, follicular fluid, cerebrospinal fluid, semen, breast milk, and vaginal fluid.

[0019] Item 6. The sample described in Item 1, wherein the body fluid is urine.

[0020] Item 7. The sample described in Item 1 is a sample for measuring the activity of a sensor protein that senses the presence of a chemical substance and participates in a reaction that causes cations to flow into the cell.

[0021] Item 8. The sample described in Item 7, wherein the sensor protein is an ion channel receptor and / or a G protein-coupled receptor.

[0022] Item 9. The sample described in Item 7, wherein the sensor protein is a sensor protein maintained by cells.

[0023] Item 10. A method for preparing a sample as described in any one of Items 1 to 9, comprising treating a body fluid to increase the concentration of free divalent cations.

[0024] Item 11. A method for determining the activity of a sensor protein, comprising adding a sample described in any one of Items 1 to 9 to a region containing the sensor protein.

[0025] Invention Effects

[0026] According to this disclosure, a technique for detecting chemical substances in bodily fluids with higher sensitivity can be provided. Specifically, a sample derived from bodily fluids, a method for preparing the sample, and a method for determining the activity of proteins using the sample can be provided. Attached Figure Description

[0027] [ Figure 1This section shows the results of olfactory receptor activity measurements in Experiment 1 with the addition of test substance a (a chemical substance recognized by insect olfactory receptor A), VUAA1, and citric acid. The vertical axis shows the cumulative value of luminescence obtained over time using FlexStation3. The horizontal axis shows the concentration of citric acid; a "-" on the horizontal axis indicates that no citric acid was added.

[0028] [ Figure 2 This section shows the results of olfactory receptor activity measurements in Experiment 1 with the addition of test substance a (a chemical substance recognized by insect olfactory receptor A), VUAA1, and urea. The vertical axis shows the cumulative value of luminescence obtained over time using FlexStation3. The horizontal axis shows the concentration of urea; a "-" on the horizontal axis indicates that no urea was added.

[0029] [ Figure 3 This section shows the results of olfactory receptor activity measurements in Experiment 2, with and without the addition of calcium ions to human urine samples. The vertical axis shows the cumulative value of luminescence over time, measured using FlexStation3. The horizontal axis shows the presence or absence of the tested substance b (a chemical substance recognized by insect olfactory receptor B).

[0030] [ Figure 4 This section shows the results of olfactory receptor activity measurements in Experiment 3, with and without the addition of strontium ions to human urine samples (concentrations of 2 mM and 5 mM in the urine samples). The vertical axis shows the cumulative value of luminescence obtained over time using FlexStation3. The horizontal axis shows the presence or absence of the test substance b (a chemical substance recognized by insect olfactory receptor B). Detailed Implementation

[0031] In this specification, the expressions “containing” and “comprising” include the concepts of “containing”, “comprising”, “substantially composed of” and “composed of only”.

[0032] Furthermore, in this specification, the range of values ​​formed by any combination of the upper and lower limits is also directly and uniquely disclosed.

[0033] In one embodiment, this disclosure relates to a sample (sometimes referred to herein as "the sample of this disclosure") that is a sample derived from bodily fluids, characterized in that the concentration of free divalent cations is higher than the concentration in the bodily fluids. This will be described below.

[0034] There are no particular restrictions on the chemical substances that can be tested in bodily fluids.

[0035] There are no particular restrictions on the biological source of the bodily fluids (the subject of the test). For example, various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, horses, cattle, and pigs can be listed, with humans being the preferred example.

[0036] There are no particular restrictions on who can be the subject of the examination. For example, samples can be submitted where it is unclear whether the subject has the disease, samples where the subject is determined to have the disease, samples where the subject is determined not to have the disease, or samples where information related to the disease or condition is unclear. When the subject is a person, regardless of past medical history, including those considered healthy, anyone can be a subject. Examples include patients undergoing health diagnoses or comprehensive physical examinations, and individuals undergoing examinations for the subject's disease using methods such as medical history taking, questionnaires, scoring, pathological diagnosis, imaging diagnosis, and blood biochemistry tests.

[0037] Examples of bodily fluids include, for example, urine, blood, saliva, sweat, tears, tissue fluid, synovial fluid, follicular fluid, cerebrospinal fluid, semen, breast milk, and vaginal fluid. Of these bodily fluids, urine is preferred from the viewpoint of being the most suitable source of the sample for this disclosure.

[0038] Body fluids can be collected from a biological source by means of methods known or based on known methods. The collected body fluids can be used immediately in the preparation of samples disclosed herein or after storage (e.g., after storage under refrigeration or freezing) in the preparation of samples disclosed herein.

[0039] The term "sample derived from bodily fluids" does not refer to the bodily fluids themselves, but rather to a sample obtained by subjecting the bodily fluids to a certain treatment that affects their composition. This definition is not particularly limited. Such treatment may include various processes such as adding components, dilution with solvents or solutions, concentration, and purification. However, from the viewpoints of simplicity and minimizing treatment losses caused by the volatilization of the chemical substance being tested during the treatment process, adding components and / or dilution is preferred. In the case of dilution with solvents or solutions, from the viewpoint of minimizing the decrease in the concentration of the chemical substance being tested, a low dilution ratio is desirable. The dilution ratio (volume of the sample disclosed herein / volume of the bodily fluid used to prepare the sample) is preferably less than 10 times, 9.9 times or less, 9.8 times or less, 9.7 times or less, 9.6 times or less, 9.5 times or less, 9 times or less, 8 times or less, 7 times or less, 6 times or less, 5 times or less, 4 times or less, 3 times or less, 2 times or less, 1.5 times or less, 1.4 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less. Furthermore, in the case of concentration, from the viewpoint of suppressing processing losses caused by the volatilization of chemical substances of the target substance during the processing, a low concentration ratio is desirable. The concentration ratio (volume of body fluid supplied to the sample of this disclosure / volume of the sample) is preferably 2 times or less, 1.9 times or less, 1.8 times or less, 1.7 times or less, 1.6 times or less, 1.5 times or less, 1.4 times or less, 1.3 times or less, 1.2 times or less, 1.1 times or less, 1.09 times or less, 1.08 times or less, 1.07 times or less, 1.06 times or less, 1.05 times or less, 1.04 times or less, 1.03 times or less, 1.02 times or less, or 1.01 times or less.

[0040] The sample of this disclosure is characterized in that the concentration of free divalent cations is higher than that in the body fluid. That is, the sample of this disclosure has a higher concentration of free divalent cations compared to a prepared body fluid, which is not a typical body fluid. To be more specific, for example, a sample a of this disclosure derived from body fluid a (e.g., urine a) taken from a subject (subject a) has a higher concentration of free divalent cations compared to body fluid a (e.g., urine a), and a sample b of this disclosure derived from body fluid b (e.g., urine b) taken from a subject different from subject a has a higher concentration of free divalent cations compared to body fluid b (e.g., urine b).

[0041] "Free" divalent cations are divalent cations in a free state that do not form complexes with ligands such as chelating agents, and there are no particular restrictions in this definition.

[0042] From the viewpoint of detectability of chemical substances, alkaline earth metal ions are preferred as divalent cations, especially those from period 4 and later. Specifically, examples include calcium ions, strontium ions, and barium ions. Among these, calcium ions and strontium ions are preferred, and calcium ions are particularly preferred, from the viewpoints of low toxicity, ease of operation, and detectability of chemical substances.

[0043] Free divalent cations can be a single type or a combination of two or more types.

[0044] From the viewpoint of chemical substance detection, the concentration of free divalent cations in the sample of this disclosure is preferably 0.2 mM higher than the corresponding concentration of free divalent cations in the prepared body fluid supplied with the sample, preferably 0.5 mM higher, preferably 1.0 mM higher, preferably 1.5 mM higher, more preferably 1.7 mM higher, further preferably 1.8 mM higher, even more preferably 1.9 mM higher, and particularly preferably 2.0 mM higher. Furthermore, in one aspect of the invention, this concentration may be 2.5 mM higher, 3.0 mM higher, 4.0 mM higher, 4.5 mM higher, or 5.0 mM higher. There is no particular upper limit to the difference between the two, 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.

[0045] The method for preparing the sample disclosed herein is not particularly limited, but from the viewpoint of simplicity and detectability of chemical substances, the method of adding a salt composed of divalent cations and anions to body fluids is preferred.

[0046] Examples of anions that constitute salts include, for example, halide ions (such as fluoride, chloride, bromide, iodide, etc.), perchlorate ions, chlorate ions, hypochlorite ions, sulfide ions, sulfate ions, bisulfate ions, thiosulfate ions, sulfite ions, selenide ions, selenate ions, selenite ions, nitrate ions, nitrite ions, phosphate ions, arsenate ions, carbonate ions, bicarbonate ions, metasilicate ions, cyanide ions, tetrahydroxyborate ions, permanganate ions, chromate ions, dichromate ions, and acetate ions. Among these, halide ions are preferred, and chloride ions are particularly preferred, from the viewpoint of ease of operation and detectability of chemical substances.

[0047] Salt can be a single type or a combination of two or more types.

[0048] The sample disclosed herein can be prepared by a method including treating the body fluid to increase the concentration of free divalent cations. There are no particular limitations on this treatment; for example, the treatment described above can be applied.

[0049] In one embodiment, the sample of this disclosure may be a sample prepared by adding a salt composed of divalent cations and anions to body fluids. The concentration of the salt (added salt) in the sample of this disclosure, converted to divalent cations, can be the difference between the concentration of free divalent cations in the sample of this disclosure and the corresponding concentration of free divalent cations in the body fluids. The concentration of the salt (added salt) in the sample of this disclosure, converted to divalent cations, is preferably 0.2 mM or higher, preferably 0.5 mM or higher, preferably 1.0 mM or higher, preferably 1.5 mM or higher, more preferably 1.7 mM or higher, further preferably 1.8 mM or higher, even more preferably 1.9 mM or higher, and particularly preferably 2.0 mM or higher. Furthermore, in one embodiment of the invention, the concentration may be 2.5 mM or higher, 3.0 mM or higher, 4.0 mM or higher, 4.5 mM or higher, or 5.0 mM or higher. There is no particular upper limit to this concentration, such as 1M, 500mM, 300mM, 200mM, 150mM, 100mM, 50mM, 30mM, 20mM, 15mM, or 10mM.

[0050] In one embodiment, the sample of this disclosure can be obtained by treating a body fluid to dissociate a chelating agent (e.g., citric acid) from calcium ions in the body fluid and its complex. Examples of such treatment include pH adjustment and the addition of a complexation inhibitor. This treatment increases the concentration of free divalent cations in the resulting body fluid-derived sample.

[0051] The samples disclosed herein can be samples for sensor protein activity assays (=sample compositions). Sensor protein activity assays can detect chemical substances recognizable by the sensor protein. The samples disclosed herein can be used in contact with sensor proteins.

[0052] Sensor proteins can be selected from those that detect the presence of chemical substances and participate in the reaction that causes cations to flow into the cell; for example, they could be receptor proteins that use chemical substances as ligands. The chemical response activity of the sensor protein can be evaluated by using the amount of cations that flow into the cell after the chemical substance is detected as an indicator.

[0053] While not wishing to be limited in interpretation, it is believed that the activity of proteins responsible for detecting chemical substances is reduced by the formation of complexes between chelating agents and divalent cations in body fluids. Specifically, the amount of divalent cations flowing into cells after chemical substance detection is reduced due to the influence of chelating agents, thus weakening the chemical substance response activity indicated by the influx of divalent cations.

[0054] The sensor protein is preferably an ion channel receptor and / or a G protein-coupled receptor. Furthermore, the sensor protein is preferably an olfactory receptor protein, particularly an insect olfactory receptor protein. The sensor protein preferably functions as a calcium ion channel.

[0055] Insect olfactory receptor proteins are membrane proteins with a seven-transmembrane structure that function as odor sensors in organisms. They are formed by the following sequence from the amino terminus (hereinafter sometimes called the "N-terminus") to the carboxyl terminus (hereinafter sometimes called the "C-terminus"): N-terminal region (NT), first transmembrane domain (TM1), first extracellular loop (EC1), second transmembrane domain (TM2), first intracellular loop (IC1), third transmembrane domain (TM3), second extracellular loop (EC2), fourth transmembrane domain (TM4), second intracellular loop (IC2), fifth transmembrane domain (TM5), third extracellular loop (EC3), sixth transmembrane domain (TM6), third intracellular loop (IC3), seventh transmembrane domain (TM7), and C-terminal region (CT). In this disclosure, each region was determined using structural prediction (with default conditions) using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membranespanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html).

[0056] Preferred insects that serve as sources of olfactory receptor proteins include: Diptera such as mosquitoes and fruit flies; Lepidoptera such as silkworm moths; Hymenoptera such as honeybees; Orthoptera such as grasshoppers; and Hemiptera such as bedbugs. Further preferred examples include: Diptera such as mosquitoes and fruit flies; Orthoptera such as grasshoppers; and Hemiptera such as bedbugs. Examples of mosquitoes include *Anopheles gambiae*, *Aedes aegypti*, and *Culex quinquefasciatus*. Examples of fruit flies include *Drosophila melanogaster*, *Drosophila pseudoobscura*, and *Drosophila virillis*. Examples of insects belonging to the family Bombyx mori include the domestic silkworm (Bombyx mori), the wild mulberry silkworm (Bombyx mandarina), and the trilocha varians. Examples of insects belonging to the family Apidae include the Western honeybee (Apis mellifera), the honey honeybee (Apis florea), the giant honeybee (Apis dorsata), and the ground bumblebee (Bombus terrestris). Examples of insects belonging to the family Locustae include the Asian migratory locust (Locusta migratoria). Examples of insects belonging to the family Cimexidae include the temperate bedbug (Cimex lectularius).

[0057] As wild-type insect olfactory receptor proteins, specifically, for example, 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, Aa OR42, AaOR43, AaOR44, AaOR45, AaOR46, AaOR47, AaOR48, AaOR49, AaOR50, AaOR52, AaOR54, AaOR55, AaOR58, AaOR59, AaOR60, AaOR61, AaOR62, AaOR63, A aOR64, 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, AaOR101, AaO R102, AaOR103, AaOR104, AaOR105, AaOR106, AaOR107, AaOR108, AaOR109, AaOR110, AaOR112, AaOR113, AaOR114, AaOR115, AaOR116, AaOR117, AaOR118, AaOR119, AaOR121, AaOR122, AaOR123, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR8, AgOR9, AgOR10, AgOR11, AgOR12, AgOR13, A gOR14, 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、LmOR122、LmOR123, LmOR124, LmOR126, LmOR127, LmOR129, LmOR130, LmOR131, LmOR133, LmOR142, etc. ,

[0058] In this specification, OR represents the olfactory receptor, Dm represents the Drosophila melanogaster source, Bm represents the silkworm source, Ag represents the Anopheles gambiae source, Aa represents the Aedes aegypti source, Am represents the Western honeybee source, Cl represents the temperate bedbug source, and Lm represents the Asian migratory locust source. The amino acid sequences and coding sequences of the various olfactory receptor proteins containing them are well known or can be easily identified by sequence identity searches based on well-known sequences.

[0059] Sensor proteins may contain amino acid mutations in the wild-type amino acid sequence, provided that 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, further 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.

[0060] Amino acid mutations, such as substitutions, insertions, additions, or deletions of amino acids, are preferred, with substitutions being particularly preferred, especially conservative substitutions.

[0061] The sensor protein may contain: a wild-type amino acid sequence, and an amino acid sequence having, for example, 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more of the same amino acid sequence as the wild-type amino acid sequence.

[0062] In this specification, chemical response activity refers to the activity of sensing the presence of a chemical substance and causing cations to flow into the cell. In the case of an olfactory receptor, the sensor protein can be an ion channel receptor or a G protein-coupled receptor. However, in the case of an insect olfactory receptor, it refers to the property of the olfactory receptor recognizing a chemical substance and the activation of the olfactory receptor complex formed by the olfactory receptor and its co-receptor, thus exhibiting ion channel activity. The chemical response activity of a sensor protein can be measured as an indicator (e.g., quantifying / evaluating the amount of signaling molecules) of the sensor protein in contact with the chemical substance. In the case of an insect olfactory receptor, the chemical response activity of the olfactory receptor can be measured as an indicator of the ion channel activity of the olfactory receptor complex formed by the olfactory receptor and its co-receptor in contact with the chemical substance. For example, a cell expressing a protein that fluoresces or emits light through ions (such as calcium ions) flowing into the cell in response to (a) the olfactory receptor, (b) the co-receptor, and (c) the olfactory receptor complex is contacted with a chemical substance, and the amount of light emitted by the cell is measured. The higher the measured amount of light emitted, the higher the chemical response activity of the olfactory receptor. Specifically, it can be determined according to the method described in Patent Document 1.

[0063] The sensor protein is preferably in a cellular state. The sample disclosed herein can be used to contact the cell that holds the sensor protein.

[0064] There are no particular restrictions on the cells used. From the perspective of suitability for detecting chemical substances, animal cells such as insect cells and mammalian cells are preferred.

[0065] The cells preferably contain exogenous polynucleotides encoding sequences of sensor proteins. This allows for the expression of any sensor protein, and further enables the increase of expression levels of target sensor proteins and the improvement of detection sensitivity for target chemicals.

[0066] Exogenous polynucleotides are polynucleotides that contain a base sequence not derived from cellular genomic DNA (specifically, chromosomal genomic DNA), and there are no particular restrictions in this definition.

[0067] When the sensor protein is an insect olfactory receptor, the exogenous polynucleotide preferably contains the coding sequence of the insect olfactory receptor co-receptor. The insect olfactory receptor co-receptor is a membrane protein with a seven-transmembrane structure, similar to the olfactory receptor, and functions by forming a heterogeneous complex with the olfactory receptor. The olfactory receptor complex, a heterogeneous complex composed of the olfactory receptor and the olfactory receptor co-receptor, possesses ion channel activity activated by odor substances, releasing sodium ions (Na+) upon activation. + ), calcium ions (Ca 2+ Cations such as ions flow into the cells.

[0068] The exogenous polynucleotide preferably contains the coding sequence of a protein that emits fluorescence or light through ions (such as calcium ions) flowing into the cell in response to a sensor protein (particularly, an olfactory receptor protein). Examples of such proteins include Aequorin, Yellow Cameleon, and GCaMP. Alternatively, the cells disclosed herein preferably contain ion-dependent fluorescent dyes such as calcium ion-dependent fluorescent dyes (e.g., Fura-2, Fluo-3, Fluo-4, etc.).

[0069] The coding sequences for insect olfactory receptor co-receptors, fluorescent or luminescent proteins, and antibiotic resistance genes are preferably contained in exogenous polynucleotides in the form of expression cassettes. The structure of the expression cassette is the same as that of sensor protein expression cassettes. The promoter of the expression cassette can be shared among multiple coding sequences.

[0070] In one embodiment, this disclosure relates to a method for determining the activity of a sensor protein, comprising adding a sample of this disclosure to a partition containing the sensor protein.

[0071] This partition could be, for example, a partition within a cell chip. There are no particular restrictions on a cell chip as long as it contains cells and instruments that can hold those cells, such as dishes or well plates for holding cells.

[0072] The form of the partitions is not particularly limited as long as it preserves the cells. From the viewpoints of cell preservation, preparation efficiency, or chemical detection, pore-shaped partitions are preferred.

[0073] There are no particular restrictions on the material used for partitioning, as long as it can preserve the cells. Materials can include, for example, resin, metal, etc.

[0074] From the perspective of detection sensitivity or preparation efficiency, the area of ​​the partition is preferably 0.5–100 mm². 2 More preferably 1 to 50 mm 2 Further optimization is preferred, with a diameter of 1.5–40 mm. 2 Further preferred size: 2-40mm 2 In a preferred embodiment of this disclosure, the area is 50 mm². 2 Below, 35mm 2 Below, or 15mm 2 the following.

[0075] From the viewpoint of detection sensitivity or preparation efficiency, the number of partitions is preferably 1 to 2000, more preferably 4 to 1600, and even more preferably 8 to 400. In a preferred embodiment of this disclosure, the number is 10 or more, 20 or more, or 50 or more.

[0076] From a detection sensitivity perspective, a partition typically contains multiple cells. The area per mm² of a partition... 2 The cell count (cells) is, for example, 50 to 20,000 cells / mm². 2 From the perspectives of detection sensitivity and cell viability, a concentration of 100–15,000 cells / mm² is preferred. 2 More preferably, 100 to 10,000 cells / mm 2 Further optimization is needed to select cells ranging from 200 to 10,000 per mm. 2 Further optimization yields 500–7000 cells / mm². 2 Especially preferred is 1000-5000 cells / mm². 2 .

[0077] Cell chips may contain only one type of sensor protein, but preferably contain two or more (more preferably three or more, further preferably four or more, even more preferably five or more, ten or more, fifteen or more, or twenty or more) different types of sensor proteins.

[0078] Example

[0079] The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0080] Experimental Example 1. Exploration of Substances that Inhibit Olfactory Receptor Activity

[0081] HEK293FT cells were loaded at 1.2 × 10⁻⁶. 6 One cell / culture dish was seeded into a 6 cm culture dish and cultured in DMEM medium (made by Nacalai Tesque) containing penicillin-streptomycin (made by Nacalai Tesque) and 10% FBS (made by Corning) for about 24 hours at 37°C and 5% CO2.

[0082] In Opti-MEM (Thermo Fisher Scientific), a mixture of insect olfactory receptor A expression plasmid, insect olfactory receptor co-receptor expression plasmid, and GFP-jellyfish luminescent protein expression plasmid was transfected into the aforementioned cells using Lipofectamine LTX and Plus reagents (both manufactured by Invitrogen). Four hours after transfection, cells were inoculated at a dose of 1×10⁻⁶. 5 Cells / well were seeded into 96-well plates and cultured in DMEM medium containing penicillin-streptomycin and 10% FBS at 37°C and 5% CO2 for approximately 16 hours.

[0083] Subsequently, the culture medium was removed and replaced with 80 μL of Hanks' HEPES buffer (containing 0.5 μM coelenterate h (Promega) and 0.1% BSA (Sigma-Aldrich)), and the cells were incubated at room temperature in the dark for 4 hours. Then, using a FlexStation 3 (Molecular Devices), the test substance a, corresponding to olfactory receptor A, was dropped into the cell culture medium, and the luminescence of the cells was measured simultaneously.

[0084] In this experimental example, 20 μL of Hanks' HEPES buffer, containing 500 μM of test substance a (a chemical substance recognized by insect olfactory receptor A) and 50 μM of VUAA1 (manufactured by AOBIOUS), and further containing 2%, 0.5%, or 0.125% citric acid, or 2%, 0.5%, or 0.125% urea, was added to the above-mentioned 96-well plate, and the luminescence was measured. By adding the test substance a, VUAA1, citric acid, or urea to the 96-well plate, the final concentration was diluted 5-fold.

[0085] The results are shown in Figure 1 and Figure 2 The activity of olfactory receptors was inhibited in a concentration-dependent manner by citric acid. This is thought to be due to the complexation of citric acid and calcium ions, which act as chelators for calcium ions.

[0086] Experimental Example 2. Analysis of the effect of calcium ion addition when using body fluid samples

[0087] Except for using an insect olfactory receptor B expression plasmid, the expression plasmid was introduced into cells in the same manner as in Experiment 1, seeded in 96-well plates, and subjected to luminescence assay.

[0088] In this embodiment, VUAA1 (manufactured by AOBIOUS) was prepared to be added to a human urine sample to a concentration of 50 μM, before adding Ca. 2+ In the group where CaCl2 was added to the urine sample to a concentration of 2 mM, and in the group where the test substance was added, test substance b (a chemical substance recognized by insect olfactory receptor B) was added to the urine sample to a concentration of 3 μM. 20 μL of the prepared urine sample was dropped into the 96-well plate, and the luminescence intensity was measured. By dropping the sample into the 96-well plate, the urine components, added test substance b, VUAA1, and added CaCl2 were ultimately diluted 5-fold.

[0089] The results are shown in Figure 3 .like Figure 3In the group without added Ca+, no difference in olfactory receptor activity was detected despite the addition of the test substance in the absence of calcium. Based on the results of Experiment 1, this is believed to be due to the inhibition of olfactory receptor activity by the formation of a complex between citric acid in urine and calcium ions. Furthermore, as this is hypothesized, the addition of calcium ions could confirm the change in olfactory receptor activity caused by the addition of the test substance. Figure 3 (Ca+ addition group).

[0090] Experimental Example 3. Analysis of the effect of strontium ion addition when using body fluid samples

[0091] In addition to using an insect olfactory receptor B expression plasmid and an inoculation density of 8.0 × 10⁶ in 96-well plates. 4 In addition to the single cell / well method, the expression plasmid was introduced into cells and seeded in a 96-well plate, as in Experiment 1, for luminescence assay.

[0092] In this embodiment, VUAA1 (manufactured by AOBIOUS) was prepared to be added to a human urine sample to a concentration of 50 μM, before adding Sr. 2+ In the control group, SrCl2 was added to the urine sample to make 2 mM or 5 mM, while in the control group, test substance b (a chemical substance recognized by insect olfactory receptor B) was added to the urine sample to make 3 μM. 20 μL of the prepared urine sample was dropped into the 96-well plate, and the luminescence intensity was measured. By dropping the sample into the 96-well plate, the urine components, added test substance b, VUAA1, and added SrCl2 were ultimately diluted 5-fold.

[0093] The results are shown in Figure 4 When strontium ions are added instead of calcium ions, changes in the activity of olfactory receptors caused by the addition of the tested substance can also be confirmed.

Claims

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

2. The sample according to claim 1, wherein, The divalent cations are ions of alkaline earth metals from the fourth period onwards.

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

4. The sample according to claim 1, wherein, The 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 bodily fluid is selected from at least one of the following: urine, blood, saliva, sweat, tears, tissue fluid, synovial fluid, follicular fluid, cerebrospinal fluid, semen, breast milk, and vaginal fluid.

6. The sample according to claim 1, wherein, The fluid in question is urine.

7. The sample of claim 1, which is a sample for determining the activity of a sensor protein, wherein the sensor protein is a protein that senses the presence of a chemical substance and participates in a reaction that causes cations to flow into cells.

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

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

10. A method for preparing a sample according to any one of claims 1 to 9, comprising treating a body fluid to increase the concentration of free divalent cations.

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

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