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WO2018235915A1 - Procédé de mesure, kit de mesure et dispositif de mesure - Google Patents

Procédé de mesure, kit de mesure et dispositif de mesure Download PDF

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Publication number
WO2018235915A1
WO2018235915A1 PCT/JP2018/023670 JP2018023670W WO2018235915A1 WO 2018235915 A1 WO2018235915 A1 WO 2018235915A1 JP 2018023670 W JP2018023670 W JP 2018023670W WO 2018235915 A1 WO2018235915 A1 WO 2018235915A1
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WIPO (PCT)
Prior art keywords
hair
antibody
active substance
physiologically active
cortisol
Prior art date
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PCT/JP2018/023670
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English (en)
Japanese (ja)
Inventor
雅也 上田
豪哉 川添
康彦 内海
研 平野
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Sharp Corp
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Sharp Corp
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Publication date
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Publication of WO2018235915A1 publication Critical patent/WO2018235915A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present disclosure relates to a method, a measurement kit and a measurement device for measuring the accumulation amount of a physiologically active substance in hair.
  • Non-Patent Document 1 reports that Cortisol levels were determined by ELISA (enzyme-linked immunosorbent assay) using neonatal hair as a sample.
  • One aspect of the present disclosure aims to provide a simple method of measuring the accumulation amount of a physiologically active substance in hair.
  • a measuring method concerning one mode of this indication is a method of measuring accumulation amount of physiologically active substance in hair, and the above-mentioned physiologically active substance is peculiar to a section of the above-mentioned hair. And a detection step of detecting the antibody bound to the physiologically active substance in the reaction step.
  • the accumulated amount of the physiologically active substance in the hair can be simply measured.
  • FIG. 6 is a schematic view showing an optical system used in a detection step of detecting an antibody bound to a physiologically active substance in the method according to Embodiment 1.
  • FIG. 1 In the method which concerns on Embodiment 2, it is a schematic diagram showing each step of the reaction process of making the antibody which specifically recognizes a physiologically active substance react with respect to the cross section of hair. It is a microscope image showing the result of having measured the accumulation amount of cortisol contained in hair by the method concerning Embodiment 2.
  • FIG. 3 In the method which concerns on Embodiment 3, it is a schematic diagram showing each step of the reaction process which makes the antibody which specifically recognizes a physiologically active substance react with respect to the cross section of hair. It is a microscope image showing the result of having measured the accumulation amount of cortisol contained in hair by the method concerning Embodiment 3.
  • FIG. 1 In the method which concerns on Embodiment 2, it is a schematic diagram showing each step of the reaction process of making the antibody which specifically recognizes a physiologically active substance react with respect to the cross section of hair. It is a microscope image showing the result of having measured the accumulation amount of cortisol contained in hair by the method concerning Embodiment 3.
  • FIG. 1 It is a figure showing the result of having measured accumulation amount of cortisol contained in hair by a method concerning Embodiment 5.
  • A is a microscope image
  • (b) is an image showing fluorescence intensity.
  • it is a schematic diagram showing each step of the reaction process which makes the antibody which specifically recognizes a physiologically active substance react with respect to the cross section of hair.
  • A is a method in which the first antibody and the second antibody are reacted simultaneously
  • (b) is a method in which the first antibody and the second antibody are reacted separately.
  • non-human mammals in addition to humans.
  • non-human mammals include primates (monkeys, apes, etc.), amphibians (bovines, boars, pigs, sheep, goats, etc.), metamorphosing animals (eg, horses), rodents (mouse, rats, hamsters) And squirrels), lagomorphs (such as rabbits) and carnivores (such as dogs, cats and ferrets).
  • the non-human mammal described above may be a domestic animal or a companion animal, and may be a wild animal.
  • hair intends "hair that grows on a subject.”
  • examples of hair include hair, eyebrows, eyebrows, eyebrows, nose hair, ear hair, eyebrows, pubic hair, body hair.
  • hair or a beard it is preferable to use hair or a beard, because the hair can be easily collected.
  • the eyebrow is thick hair, it is preferable in that it is easy to detect a physiologically active substance accumulated in the pulp.
  • using hair as a biological sample has the following advantages over using blood, saliva and the like as a biological sample.
  • Hair collection is non-invasive and easy. Therefore, even a person who is not a doctor or a medical person can take a hair sample.
  • the risk of being contaminated with other types of samples is low. For example, if blood contaminates when saliva is collected, the level of the physiologically active substance can not be accurately measured because the level of the physiologically active substance is usually different in saliva and blood. However, the collected hair can be easily prevented from being contaminated with other types of samples (blood, saliva, etc.) by washing.
  • bioactive substance intends a substance involved in maintenance, regulation, etc. of biological activity and physiological function.
  • physiologically active substances include hormones, neurotransmitters, cytokines, vitamins, enzymes, proteins, peptides and the like.
  • the measurement method of the present disclosure is a method of measuring the accumulation amount of a physiologically active substance on hair.
  • it may be a method of determining vital activity and physiological function of a subject from whom the hair is collected.
  • the stress-related physiologically active substance in the hair, it is possible to know the stress that the subject has experienced chronically.
  • stress-related physiologically active substances include cortisol (hydrocortisone; 11 ⁇ , 17 ⁇ , 21-trihydroxypregna-4-ene-3, 20-dione), DHEA (dehydroepiandrosterone; 3 ⁇ -hydroxyandrone St-5-en-17-one), DHEA-S (sulfate-bound DHEA; DHEA-sulfate; DHE-S), adrenocortical hormone such as testosterone, catecholamine (adrenaline, noradrenaline), neurotransmitter such as Neuropeptide Y And acidic proteins such as chromogranin A.
  • cortisol, adrenalin and chromogranin A are associated with an increase in accumulated amount and chronic stress.
  • DHEA and DHEA-S are associated with increased accumulation and decreased chronic stress.
  • a substance having a steroid skeleton is easily accumulated in the hair. For this reason, measuring the accumulated amount of the substance having a steroid skeleton in the hair is one of the preferred embodiments of the method of the present disclosure.
  • the substance having a steroid skeleton include cortisol, DHEA, DHEA-S, testosterone and the like.
  • antibody as used herein includes immunoglobulins. That is, IgA, IgD, IgE, IgG, IgM, and Fab fragments thereof, F (ab ') 2 fragments, Fc fragments and the like are included. More specifically, all classes of polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotypic antibodies, humanized antibodies, humanized antibodies by genetic recombination and the like can be mentioned, but are not limited thereto Absent.
  • the "antibody” in the present specification also includes antibody fragments and modified antibodies as long as they specifically recognize a specific physiologically active substance. Examples of antibody fragments include Fab fragments and F (ab ') 2 fragments.
  • Embodiment 1 is a method of measuring the accumulation amount of a physiologically active substance in hair, which comprises: (1) reacting the antibody that specifically recognizes the physiologically active substance to the cross section of the hair; (2) A measurement method including a detection step of detecting the antibody bound to the physiologically active substance in the reaction step will be described.
  • the method for measuring the accumulation amount of a physiologically active substance in hair comprises: (1) a reaction step of reacting an antibody that specifically recognizes the physiologically active substance with respect to the cross section of the hair And (2) a detection step of detecting an antibody bound to the physiologically active substance in the reaction step.
  • a reaction step of reacting an antibody that specifically recognizes the physiologically active substance with respect to the cross section of the hair comprises: (1) a detection step of detecting an antibody bound to the physiologically active substance in the reaction step.
  • the data regarding the accumulation amount of the physiologically active substance in the said hair can be acquired.
  • the tool used to collect the hair is not particularly limited, and known tools (scissors, shaving, etc.) may be used.
  • the hair collected from the subject in the collecting step is preferably hair or eyebrows. Hair or eyebrows can be easily collected without using a special tool.
  • the number of hairs collected from the subject in the collecting step is not particularly limited as long as it is one or more. From the viewpoint of measuring the accumulated amount of physiologically active substance more accurately, it is preferable to collect a plurality of hairs.
  • the number of hairs collected at this time is not particularly limited as long as it can be statistically interpreted. When using hair as hair, it is sufficient for the method of the present disclosure to be implemented if about 20 can be embedded to make a hair sample.
  • the collected hair 1 is embedded in an embedding agent 2 after passing through processes such as washing as appropriate ((a) of FIG. 1).
  • an embedding agent 2 paraffin, celloidin, gelatin, carbowax, acrylic resin, methacrylic resin, epoxy resin, cyanoacrylate, ice or the like can be used.
  • the embedded hair 1 is cut using a microtome, a cutter, a jig or the like to obtain a hair sample 10 (FIG. 1 (b)).
  • the thickness of the hair sample 10 is not particularly limited, but may be about 10 nm to 500 ⁇ m as a standard.
  • the shape of the hair sample 10 to be produced is not limited to thin pieces, and it is sufficient if the cross section of the hair 1 can be obtained.
  • the hair sample 10 may be prepared immediately before measuring the accumulated amount of the physiologically active substance, or may be prepared in advance. However, among the physiologically active substances contained in the hair, those whose amount is likely to change with the passage of time from hair collection, from the viewpoint of accurately measuring the physiologically active substance, the hair sample prepared immediately before the measurement It is preferred to use 10.
  • a plurality of hair samples 10 may be prepared by cutting a plurality of places.
  • the direction in which the hair 1 is cut is not particularly limited, and may be perpendicular, oblique or parallel to the growth axis of the hair. This will be described based on FIG. FIG. 2 is a diagram showing coordinates defining the cut surface of the hair 1.
  • the Y-axis is the growth axis of the hair 1 and the thick arrows are the normal vectors of the cutting plane (ie, the direction vectors perpendicular to the cutting plane).
  • the normal vector can be arbitrarily determined in the range of 0 ° ⁇ ⁇ 180 ° and 0 ° ⁇ ⁇ ⁇ 180 °.
  • reaction step an antibody that specifically recognizes a physiologically active substance is reacted with the cross section of the hair formed in the cutting step.
  • a method (direct method) of detecting a label bound to a primary antibody that recognizes a physiologically active substance is employed.
  • the label bound to the primary antibody is, for example, a dye, a fluorescent dye, a radioactive isotope, an enzyme, micro / nano particles (metal particles (gold particles, silver particles, etc.), quantum dots (composition is CdSe) Etc.), polystyrene beads, glass beads etc.).
  • a dye or a fluorescent dye is used as a label, light emitted from the dye or the fluorescent dye may be detected.
  • a radioactive isotope is used as a label, radiation emitted from the radioactive isotope may be detected.
  • an enzyme When using an enzyme as a label, it may be detected using a reaction involving the enzyme.
  • a labeling substance a dye, a fluorescent dye, a radioactive isotope, an enzyme, etc.
  • the detection may be performed by a method utilizing the physical properties of the above-mentioned fine particles (for example, light scattering by surface plasmon resonance or the like, absorbance, etc. can be detected).
  • a fluorescent dye as a label from the viewpoint that miniaturization of a detection device is possible.
  • These include light detection elements (photodiodes, phototransistors, complementary metal-oxide semiconductor (CMOS) imaging elements, charge-coupled device (CCD) imaging elements, etc.) and excitation light source elements (semiconductor lasers, This is because small-sized and inexpensive LEDs are widely used in industry.
  • CMOS complementary metal-oxide semiconductor
  • CCD charge-coupled device
  • excitation light source elements semiconductor lasers, This is because small-sized and inexpensive LEDs are widely used in industry.
  • Fluorescent dye labels are also preferred in that they differ from radioactive isotopes and do not require special facilities to handle.
  • fluorochrome labels are also preferred in that they can also be detected with a fluorescent microscope that is widespread at the laboratory level.
  • a method may be used in which a label (conjugate) in which a primary antibody and a labeled antibody are directly bound is produced to stain an antigen.
  • the primary antibody used for immunostaining may be appropriately determined depending on the antigen (physiologically active substance) recognized by the primary antibody.
  • an anti-cortisol antibody may be used.
  • anti-Cortisol antibodies include Anti-Cortisol antibody XM210 ab1949, abcam product and the like.
  • anti-DHEA antibodies include Anti-Dehydroepiandrosterone Antibody, Rabbit-Poly and Gene Tex.
  • the antibody and the fluorescent dye are linked by known techniques. From the viewpoint of light resistance, it is preferable that the fluorescent dye be contained in the fluorescent particle having a functional group capable of reacting with the antibody to form a bond. At this time, the fluorescent dye is encapsulated by the nanoparticles to form a fluorescent particle, and a bond is formed between the nanoparticle and the antibody.
  • the component of the above-mentioned nanoparticles include those capable of stably containing a fluorescent dye, such as polystyrene, polyamide, polylactic acid, polyacrylonitrile, polyglycidyl methacrylate, melamine resin and the like.
  • Examples of the fluorescent dye contained in the nanoparticles include rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, pyromecene-based dye molecules, etc. Can be mentioned.
  • Alexa Fluor registered trademark, manufactured by Invitrogen
  • BODIPY registered trademark, manufactured by Invitrogen
  • Cy registered trademark, manufactured by GE Healthcare
  • DyLight registered trademark, manufactured by Thermo Fisher Scientific
  • ATTO registered trademark, manufactured by ATTO-TEC
  • pigment molecules MFP (registered trademark, manufactured by Mobitec)
  • Dye molecules may be used.
  • a known method is used for binding of the fluorescent particle to the antibody. For example, amidation by reaction of amine and carboxylic acid, sulfidation by reaction of maleimide and thiol, imination by reaction of aldehyde and amine, amination by reaction of epoxy and amine, and the like can be used.
  • the hair sample 10 includes, in addition to the hair cross section 11 (having the medullary cross section 11a inside), a location 12 where the antibody may bind nonspecifically and a location 13 where the fluorescent dye may bind nonspecifically. (S1).
  • the antibody blocking agent 12a is dropped on the hair sample 10 to block the portion 12 where the antibody may nonspecifically bind. This prevents nonspecific binding between the primary antibody 14a and the site 12 where the antibody may bind nonspecifically.
  • the fluorescent dye blocking agent 13a is dropped on the hair sample 10 to block the portion 13 where the fluorescent dye can nonspecifically bind. This prevents nonspecific binding between the fluorescent dye 15 and the site 13 where the fluorescent dye can bind nonspecifically.
  • Examples of the blocking agent for antibody 12a include artificially synthesized polymers, normal serum, animal serum (rabbit serum, goat serum, rat serum etc.), bovine serum albumin, gelatin, casein and the like.
  • Examples of the fluorescent dye blocking agent 13a include reagents that reduce background staining such as FX signal enhancer (manufactured by Thermo Fisher Scientific). Further, if it can be judged that there is no problem even if the treatment with the blocking agent for fluorescent dye is omitted depending on the sample, the treatment with the blocking agent for fluorescent dye may be omitted.
  • a primary antibody 14a that specifically recognizes a specific physiologically active substance is dropped on the hair sample 10 and allowed to bind to the physiologically active substance.
  • the primary antibody 14a is previously bound to the fluorescent dye 15, the fluorescent dye 15 is bound to the existing location of the physiologically active substance via the primary antibody 14a.
  • the hair sample 10 after the reaction step is appropriately subjected to a treatment (for example, a sealing step) necessary for the detection step.
  • a treatment for example, a sealing step
  • PBS phosphate buffered saline
  • the hair sample 10 is placed on a slide glass and the mounting agent is dropped. 3. Place a coverslip on the hair sample.
  • the encapsulating agent used in the encapsulating step is not particularly limited as long as the fluorescent dye 15 is not photobleached.
  • the cover glass and the slide glass used in the sealing step are also not particularly limited.
  • the antibody bound to the physiologically active substance is detected in the reaction step.
  • an example of an optical system used in the detection step will be described based on FIG.
  • the hair sample 10 enclosed and enclosed between the cover glass 21 and the slide glass 22 is placed on the stage 23.
  • fluorescence 32 is emitted from the fluorescent dye 15 contained therein.
  • the excitation light 31 is reflected by the dichroic mirror 25 and irradiated to the hair sample 10 via the optical lens 24. Further, the fluorescence 32 passes through the dichroic mirror 25, and only a specific wavelength component is extracted by the filter 26.
  • the fluorescence 32 emitted from the fluorescent dye 15 can be detected with less noise. Since the fluorescent dye 15 is bound to the primary antibody 14a and the primary antibody 14a is bound to the physiologically active substance, detecting the fluorescence 32 detects the primary antibody 14a bound to the physiologically active substance It will be.
  • the fluorescence 32 may be observed from the column of a fluorescence microscope, or the result detected by a CCD camera or the like may be displayed on a display means such as a monitor.
  • image analysis may be performed on an image captured by a CCD camera (image representing the distribution of the primary antibody 14 a in the cross section of the hair by fluorescence) to obtain numerical data indicating the fluorescence distribution.
  • a method of analyzing the image will be described in a fifth embodiment.
  • FIG. 4 what is described in FIG. 4 is only an example of an optical system that can be used in the detection process. Since the detection step in the method of the present disclosure is sufficient if the antibody bound to the physiologically active substance is detected in the reaction step, detection of the fluorescence 32 is sufficient as the detection step.
  • a hair sample 10 which is collected from the same subject and reacted with an antibody that specifically recognizes a physiologically active substance in cross sections having different growth timings.
  • this evaluation method can be used to evaluate changes in the degree of stress of a subject.
  • the cutting step one hair is cut every 1 cm, and in the reaction step, an antibody that specifically recognizes cortisol in each cut cross section is reacted. Furthermore, the change in the degree of stress of the subject can be evaluated by plotting the intensity (detection result) of the fluorescence 32 of each cross section detected in the detection step in order from the root to the tip of the hair.
  • the growth rate of hair varies from subject to subject, but in the case of a human being, it is said that hair grows up to 1 cm in one month. Therefore, in the above example, the change in the degree of stress of the subject can be evaluated at intervals of about one month.
  • the intensity of the fluorescence 32 may be an average value of one cross section of the hair sample 10.
  • the intensity of the fluorescence 32 may be an average value of the cross sections of the plurality of hair samples 10 at the same growth time.
  • the plunger of one syringe was removed and the hair was inserted into the syringe. 3. After the resin for embedding was filled inside the above-mentioned syringe, the plunger was inserted. 4. The plunger of the syringe containing the hair was pushed to push extra bubbles and resin in the syringe to the other connected syringe. 5. The embedding resin was allowed to cure for approximately 1 hour while immediately cooling into ice water.
  • the embedded hair was exfoliated using a microtome (or jig (self-made) and a cutter knife).
  • the thickness of the flakes was about 300 ⁇ m.
  • the obtained slice was placed in a microtube, and 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, poured into the above-mentioned microtube, and tapped. The PBS was then removed by aspiration. This process was repeated three times to wash the hair sample.
  • the hair sample is placed in a microtube, and 10% gelatin-PBS (w / v) (from Sigma Aldrich gelatin (from cold waterfish)) as a blocking agent for primary antibody is further added to Wako Pure Chemical Industries 10 ⁇ PBS 1 mL of the solution (diluted with ( ⁇ )) was injected into the above-mentioned microtube. After tapping the microtube, the blocking agent for primary antibody was reacted for about 1 hour. Then, 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, injected into the above-mentioned microtube, and tapped. The PBS was then removed by aspiration. This process was repeated three times to wash the hair sample.
  • FX signal enhancer manufactured by Thermo Fisher Scientific
  • a fluorescent dye blocking agent as a fluorescent dye blocking agent
  • 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, injected into the above-mentioned microtube, and tapped.
  • the PBS was then removed by aspiration. This process was repeated three times to wash the hair sample.
  • a solution containing the conjugate of the anti-cortisol antibody and the fluorescent dye described above was injected into a microtube for about 200 ⁇ L (the amount in which the hair sample is immersed) and reacted for about 1 hour.
  • a hair sample was immersed in 10% gelatin-PBS instead of the above solution.
  • 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, injected into the above-mentioned microtube, and tapped.
  • the PBS was then removed by aspiration. This process was repeated three times to wash the hair sample.
  • the same washing operation was performed once with about 1.0 mL of pure water in order to prevent precipitation of the salt contained in PBS at the time of drying and to reduce the background at the time of fluorescence detection.
  • the hair sample was then transferred onto a glass slide and allowed to air dry (approximately 30 to 60 minutes) until the hair sample was dry. Finally, an antifading agent (ProLong® Diamond, manufactured by Thermo Fisher Scientific) was dropped onto the hair sample, and a cover glass was covered.
  • an antifading agent ProLong® Diamond, manufactured by Thermo Fisher Scientific
  • ECLIPSE TE2000-U manufactured by Nikon
  • AURA Light Engine manufactured by Lumencor
  • the dichroic mirror Chroma, 41008
  • a filter Chroma, 41008 was used to block wavelengths below 662 nm.
  • EM-CCD manufactured by Hamamatsu Photonics
  • a measurement method further including a secondary reaction step of causing a secondary antibody that specifically recognizes the antibody to react with an antibody bound to a physiologically active substance is described.
  • the physiologically active substance can be detected with higher sensitivity.
  • an indirect method is used as a method of immunostaining.
  • the indirect method is a method of performing a first antigen-antibody reaction using an unlabeled primary antibody, and then reacting a labeled secondary antibody that specifically recognizes the primary antibody.
  • detection sensitivity is higher in the indirect method than in the direct method described in the first embodiment.
  • the measurement method according to the second embodiment will be described below based on FIG.
  • the step of collecting hair from a subject (collecting step), the step of forming a cut surface on the hair (cutting step), and the step of detecting an antibody bound to a physiologically active substance (detection step) are described in the first embodiment.
  • the description on the antibody and the fluorescent dye according to the step (reaction step) of reacting the antibody that specifically recognizes the physiologically active substance with respect to the cross section of the hair is also as described in the first embodiment.
  • the state before reacting the antibody (S1a), the step of allowing the antibody blocking agent to act (S2a), and the step of allowing the fluorescent dye blocking agent to act (S3a) are respectively S1 and S2 in Embodiment 1 (FIG. 3). , Same as S3.
  • a primary antibody 14a that specifically recognizes a specific physiologically active substance is dropped on the hair sample 10 and allowed to bind to the physiologically active substance.
  • the fluorescent dye is not bound to the primary antibody 14a.
  • the secondary antibody 14b that specifically recognizes the primary antibody 14a is dropped onto the hair sample 10 and allowed to bind to the primary antibody 14a.
  • the fluorescent dye 15 is bound to the existing site of the physiologically active substance through the primary antibody 14a and the secondary antibody 14b. Become.
  • the hair sample is placed in a microtube, and 10% gelatin-PBS (w / v) (from Sigma Aldrich gelatin (from cold water fish)) is added as a blocking agent for the primary antibody, 10 ⁇ PBS (Wako Pure Chemical Industries, Ltd.) 1) was diluted into the above micro tube. After tapping the microtube, the blocking agent for primary antibody was reacted for about 1 hour. Then, 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, injected into the above-mentioned microtube, and tapped. The PBS was then removed by aspiration. This process was repeated three times to wash the hair sample.
  • FX signal enhancer manufactured by Thermo Fisher Scientific
  • a fluorescent dye blocking agent as a fluorescent dye blocking agent
  • 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, injected into the above-mentioned microtube, and tapped.
  • the PBS was then removed by aspiration. This process was repeated three times to wash the hair sample.
  • an anti-Cortisol antibody (Anti-Cortisol antibody XM210 ab1949, manufactured by abcam) diluted 200-fold with 10% gelatin-PBS is injected into a microtube for about 200 ⁇ L (the amount in which the hair sample is immersed), and about 1 hour It was made to react.
  • a hair sample was immersed in 10% gelatin-PBS instead of the above antibody.
  • 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, injected into the above-mentioned microtube, and tapped. The PBS was then removed by aspiration. This process was repeated three times to wash the hair sample.
  • a fluorescently labeled secondary antibody (goat anti-mouse antibody, attached to Alexa Fluor 488 Goat Anti-Mouse SFX kit (manufactured by Thermo Fisher Scientific)) diluted 100-fold with 10% gelatin-PBS.
  • a fluorescently labeled secondary antibody (goat anti-mouse antibody, attached to Alexa Fluor 488 Goat Anti-Mouse SFX kit (manufactured by Thermo Fisher Scientific)) diluted 100-fold with 10% gelatin-PBS.
  • 1.0 mL of 1 ⁇ PBS was weighed using a precision pipette and a disposable pipette tip, injected into the above-mentioned microtube, and tapped.
  • the PBS was then removed by aspiration. This process was repeated three times to wash the hair sample. Furthermore, the same washing operation was performed once with about 1.0 mL of pure water.
  • the hair sample was then transferred onto a glass slide and allowed to air dry (approximately 30 to 60 minutes) until the hair sample was dry. Finally, an antifading agent (ProLong® Diamond, manufactured by Thermo Fisher Scientific) was dropped onto the hair sample, and a cover glass was covered.
  • an antifading agent ProLong® Diamond, manufactured by Thermo Fisher Scientific
  • the hair sample prepared for speculum observation was observed under a fluorescence microscope to confirm the presence of cortisol.
  • BZ-X710 manufactured by Keyence
  • ECLIPSE TE2000-U manufactured by Nikon
  • a metal halide lamp with a built-in main body was used as an excitation light source.
  • the above excitation light source was irradiated to the hair sample through an excitation filter (OP-87763 manufactured by Keyence) having a center wavelength of 470 nm (full width at half maximum 40 nm).
  • the dichroic mirror manufactured by Keyence, OP-87763
  • a filter manufactured by Keyence, OP-87763
  • a cooled CCD incorporated in the main unit was used for fluorescence detection.
  • ECLIPSE TE2000-U a light having a center wavelength of 475 nm (full width at half maximum 28 nm) was irradiated using an AURA Light Engine (manufactured by Lumencor) as an excitation light source.
  • the dichroic mirror manufactured by Nikon, B-1A
  • a filter was used to block wavelengths below 535 nm.
  • EM-CCD manufactured by Hamamatsu Photonics
  • FIG. 6 A microscopic image representing the results is shown in FIG.
  • the upper part of FIG. 6 is a hair sample reacted with the primary antibody, and the lower part is a negative control.
  • the left column is a microscope image in bright field
  • the right column is a fluorescence microscope image. As shown in FIG. 6, the strongest fluorescence was observed in the medulla. That is, it was shown that cortisol is localized in the medulla.
  • Third Embodiment Embodiment 3 describes a measurement method including the reaction with other compounds for the primary antibody and / or the secondary antibody. According to the configuration of the third embodiment, the physiologically active substance can be detected with high sensitivity.
  • Examples of measurement methods including reactions with other compounds include ABC (Avidin-Biotin Complex) sensitization and TSA (Thyramide signal amplification) sensitization.
  • ABC sensitization is a method of improving the sensitivity of immunostaining by utilizing the formation of a complex between avidin and biotin.
  • the TSA sensitization method is a method of improving the sensitivity of immunostaining utilizing the property of tyramide that it is bound to a nearby aromatic molecule by the catalytic action of HRP in the presence of hydrogen peroxide.
  • a primary antibody 14a that specifically recognizes a specific physiologically active substance is dropped on the hair sample 10 and allowed to bind to the physiologically active substance.
  • the fluorescent dye is not bound to the primary antibody 14a.
  • the secondary antibody 14b that specifically recognizes the primary antibody 14a is dropped onto the hair sample 10 and allowed to bind to the primary antibody 14a.
  • the secondary antibody 14 b is previously bound to biotin 16.
  • biotin 16 that specifically binds to biotin 16 is dropped onto the hair sample 10 and allowed to bind to biotin 16.
  • the fluorescent dye 15 is attached to the existing location of the physiologically active substance via the primary antibody 14a, the secondary antibody 14b, the biotin 16 and the avidin 17 It will be combined.
  • avidin 17 is a series of proteins (avidins) functionally defined by binding to biotin 16 with high affinity and specificity.
  • avidins include avidin, streptavidin, neutravidin.
  • an anti-Cortisol antibody (Anti-Cortisol antibody XM210 ab1949, manufactured by abcam) diluted 200-fold with 10% gelatin-PBS was used as a primary antibody, reacted and washed in the same manner as in Experimental Example 2.
  • 10% gelatin-PBS was used in the same manner except for using.
  • the hair sample was then transferred onto a glass slide and allowed to air dry (approximately 30 to 60 minutes) until the hair sample was dry. Finally, an antifading agent (ProLong® Diamond, manufactured by Thermo Fisher Scientific) was dropped onto the hair sample, and a cover glass was covered.
  • an antifading agent ProLong® Diamond, manufactured by Thermo Fisher Scientific
  • the hair sample prepared for speculum observation was observed under a fluorescence microscope to confirm the presence of cortisol.
  • ECLIPSE TE2000-U manufactured by Nikon
  • the excitation light source light with a center wavelength of 475 nm (full width at half maximum 28 nm) was irradiated using AURA Light Engine (manufactured by Lumencor).
  • the dichroic mirror manufactured by Nikon, B-1A
  • a filter was used to block wavelengths below 535 nm.
  • EM-CCD manufactured by Hamamatsu Photonics
  • FIG. 8 A microscopic image representing the results is shown in FIG.
  • the upper part of FIG. 8 is a hair sample reacted with the primary antibody, and the lower part is a negative control.
  • the strongest fluorescence was observed in the medulla. That is, it was shown that cortisol is localized in the medulla.
  • Embodiment 4 a measurement method including a quantification step of quantifying the accumulated amount of the physiologically active substance will be described. According to the configuration of the fourth embodiment, the accumulated amount of the physiologically active substance can be quantified according to the detected fluorescence intensity.
  • the quantification step is a step of correlating the fluorescence intensity obtained from the immunostained hair sample with the absolute amount of the accumulated amount of the physiologically active substance contained in the hair.
  • concentration of the physiologically active substance in another hair (comparative hair) similar to the hair to be measured (the target hair) is previously determined by another method (for example, ELISA) than fluorescent staining using an antibody Measure quantitatively by the method).
  • the comparative hair is a hair which is presumed to have accumulated approximately the same amount of physiologically active substance as the target hair, and is, for example, a hair growing near the target hair.
  • the fluorescence intensity by the immunostaining is obtained by performing processing of correlating the concentration of the physiologically active substance at the specific site of the comparative hair with the fluorescence intensity at the site corresponding to the specific site of the target hair for a plurality of fluorescent intensities.
  • a calibration curve can be created that corresponds to the actual concentration of physiologically active substance.
  • an artificial sample containing a physiologically active substance of a known concentration is prepared, and the sample is subjected to the same immunostaining as the immunostaining to be applied to the subject hair.
  • a calibration curve showing the relationship between the fluorescence intensity and the concentration of the physiologically active substance can be obtained.
  • the dot plot method is mentioned as a method of producing the said artificial sample.
  • the calibration curve derived in this manner can also be diverted to the measurement of the amount of physiologically active substance accumulated in the hair thereafter.
  • ELISA is a general method that can measure a specific physiologically active substance contained in a sample, and measurement results can be quantified by using a dilution series of known concentration. Specific operating procedures can follow, for example, the protocol attached to the kit for ELISA.
  • Embodiment 4 in order to derive a calibration curve representing the relationship between the fluorescence intensity and the accumulated amount (concentration) of the physiologically active substance by ELISA, (1) measuring the accumulated amount of physiologically active substance in the hair by ELISA Then, (2) the correlation with the fluorescence intensity may be determined.
  • a physiologically active substance as cortisol.
  • Hair is collected from the subject for ELISA (for a more specific method, see Experimental Example 1).
  • the hair is preferably grown in the same part as the hair collected from the subject for immunostaining.
  • the collected ELISA hair is cut into an appropriate length.
  • the portion cut out from the hair for ELISA corresponds to the portion for producing a hair sample from the hair for immunostaining.
  • a portion of 0 to 1 cm is cut out from the root side of hair for immunostaining to prepare a hair sample
  • a portion of 0 to 1 cm is cut out from the root side of hair for ELISA and measurement by ELISA is performed .
  • the hair milled in 4 is placed in a microtube, and 1.5 mL of methanol (manufactured by Wako Pure Chemical Industries, methanol (for LC / MS), product number 138-14521) is added.
  • the extract is cortisol extracted by stirring with end-over-end stirring at 10 rpm for 16 to 24 hours at room temperature.
  • the resultant is centrifuged at 6500 G for 5 minutes using a centrifuge (Himac K-15, manufactured by Hitachi Koki Co., Ltd.). The supernatant after solid-liquid separation is taken out, and methanol is evaporated using a centrifugal concentrator (manufactured by Tomy Seiko, MV-100). This solidifies cortisol-containing solids.
  • a saliva ELISA assay kit (Salimetrics, Cortisol EIA Kit) is diverted and used. Add 25 ⁇ L per well of the following samples, 2 wells each. -Solution obtained by diluting the solid obtained in 6 with 200 ⁇ L of ASSAY diluent-Control solution (cortisol solution of known concentration) ⁇ Cortisol standard solution (kit attachment, 6 series, for preparation of standard curve): • Assay diluent (kit supplied, for zero control and nonspecific binding).
  • TMB tetramethyl benzidine
  • the fluorescence intensity measured by the method of Experimental Examples 1 to 3 can be associated with the cortisol contained in the hair. That is, since the fluorescence intensity (known amount) measured from the hair sample and the cortisol concentration (known amount, quantified by ELISA) in the portion of the hair corresponding to the hair sample are known, the fluorescence intensity and the hair sample An equation representing the correlation with the cortisol concentration contained in Therefore, once an equation representing such a correlation is prepared, the cortisol concentration can be calculated from the result of immunostaining without the subsequent ELISA.
  • the dilution series (the concentration is known) of the target physiologically active substance is dot-plotted on the membrane.
  • the dot plot on the membrane is immunostained in the same conditions as the immunostaining applied to the hair sample (eg, in the conditions described in Embodiments 1-3).
  • the fluorescence intensity (fluorescence intensity per unit area) of the dot-plotted film is measured.
  • the dot plot is a physiologically active substance solution whose concentration is known
  • a calibration curve showing the correlation between the fluorescence intensity and the physiologically active substance concentration is obtained. Based on the calibration curve obtained in 4.3, the concentration of the physiologically active substance contained in the hair sample is calculated from the fluorescence intensity emitted from the hair sample.
  • membrane known membranes such as PVDF membrane and nitrocellulose membrane can be used. Further, the concentration range of the dilution series of the target physiologically active substance can be determined appropriately.
  • the concentration of the physiologically active substance is calculated from the result of the immunostaining without using the dot plot thereafter.
  • the distribution of a physiologically active substance in a portion where the fluorescence intensity is weak, which is difficult to distinguish by visual recognition of a fluorescently labeled image, can also be obtained as quantified data.
  • the fluorescence intensity per unit area can be calculated, data easy to compare between samples can be obtained.
  • the accumulation amount of a physiologically active substance (such as cortisol) localized to the medulla can be quantified without being affected by the difference in the size of the medulla from the subject.
  • a plot of fluorescence intensity is generated using an image processing software that can quantify the fluorescence intensity of the image with an image including a fluorescence-labeled image.
  • image processing software is not particularly limited, and known image processing software such as ImageJ (free software) and Image Pro Plus (manufactured by Media Cybernetics, registered trademark) may be used.
  • the data representing the distribution of the physiologically active substance is preferably in the form of Surface Plot (three-dimensional plot).
  • a hair cross section is represented by the X axis and the Y axis
  • a fluorescence intensity is represented by the Z axis.
  • the data representing the distribution of the physiologically active substance by the above-mentioned Surface Plot can be rotated, tilted and colored as required, and can also be output to the outside.
  • Such data have, for example, the advantage that they can be explained in an easy-to-understand manner in the context of explaining the amount of accumulation of physiologically active substances to medical staff.
  • a fluorescence intensity distribution represented by a heat map may be mentioned.
  • a fluorescence intensity distribution represented by a heat map may be mentioned.
  • FIG. (A) of FIG. 9 is a microscopic image showing the result of immunostaining, and is the same as FIG. 6 showing the result of Experimental Example 2.
  • (B) of FIG. 9 is a surface plot image representing the luminance in the right column (fluorescent microscope image) of (a) of FIG. The Z axis represents luminance.
  • Sixth Embodiment Embodiment 6 is a method of measuring the accumulation amount of a physiologically active substance in hair, which specifically recognizes a second physiologically active substance different from the physiologically active substance with respect to the cross section of the hair.
  • the measuring method further includes a second reaction step of reacting the antibody of According to the configuration of Embodiment 6, it is possible to simultaneously measure the accumulation amount of two or more kinds of physiologically active substances.
  • the measurement method according to the fifth embodiment will be described below based on FIG.
  • the step of collecting hair from a subject (collecting step), the step of forming a cut surface on the hair (cutting step), and the step of detecting an antibody bound to a physiologically active substance (detection step) are described in the first embodiment.
  • the description on the antibody and the fluorescent dye according to the step (reaction step) of reacting the antibody that specifically recognizes the physiologically active substance with respect to the cross section of the hair is also as described in the first embodiment. Since the state before reacting the antibody, the step of allowing the antibody blocking agent to act, and the step of allowing the fluorescent dye blocking agent to act are the same as S1 to S3 in the first embodiment (FIG. 3), they are omitted from FIG. Do.
  • the step of reacting the antibody includes (1) a method in which two or more types of antibodies are mixed and reacted, and (2) a method in which two types of antibodies are reacted one by one.
  • the method (1) is preferable from the viewpoint of reducing the number of work processes.
  • the method (2) may be adopted.
  • the reaction of the primary antibody is performed by the method of (1)
  • the reaction of the secondary antibody is performed by the method of (2), such as (1) and (2) You may use together the method of.
  • methods (1) and (2) will be described in order.
  • FIG. 10 is a schematic view showing a method of mixing and reacting (1) two or more types of antibodies.
  • the primary antibody 14a that specifically recognizes a physiologically active substance and the second primary antibody 14c that specifically recognizes a second physiologically active substance are dropped onto the hair sample 10 in a mixed state, The substance and the second physiologically active substance are bound respectively.
  • the secondary antibody 14b that specifically recognizes the primary antibody 14a and the second secondary antibody 14d that specifically recognizes the second primary antibody 14c are dropped onto the hair sample 10, and the primary antibody 14a and the primary antibody 14a are dropped. It is bound to the second primary antibody 14c.
  • the fluorescent dye 15 is bound to the location of the physiologically active substance via the primary antibody 14a and the secondary antibody 14b. Become.
  • the second secondary antibody 14 is previously bound to the second fluorescent dye 15a, the second primary antibody 14c and the second The second fluorescent dye 15a is bound via the secondary antibody 14d.
  • FIG. 10 is a schematic view showing a method of reacting (2) two types of antibodies one by one.
  • the primary antibody 14a that specifically recognizes a physiologically active substance is dropped on the hair sample 10 and allowed to bind to the physiologically active substance.
  • the second primary antibody 14c that specifically recognizes the second physiologically active substance is dropped on the hair sample 10 and allowed to bind to the second physiologically active substance.
  • the secondary antibody 14b that specifically recognizes the primary antibody 14a is dropped onto the hair sample 10 and allowed to bind to the primary antibody 14a.
  • the fluorescent dye 15 is bound to the existing site of the physiologically active substance through the primary antibody 14a and the secondary antibody 14b. Become.
  • the second secondary antibody 14d that specifically recognizes the second primary antibody 14c is dropped on the hair sample 10 and allowed to bind to the second primary antibody 14c.
  • the second secondary antibody 14d is previously bound to the second fluorescent dye 15a, the second primary antibody 14c and the second The second fluorescent dye 15a is bound via the secondary antibody 14d.
  • the same hair sample 10 can be obtained by using fluorescent dyes exhibiting different wavelengths of fluorescence as the fluorescent dye 15 and the second fluorescent dye 15a.
  • the accumulated amount of the physiologically active substance and the second physiologically active substance contained in the above can be measured.
  • a goat anti-mouse antibody Alexa Fluor (registered trademark) 488 Goat obtained by diluting an anti-Cortisol antibody (Anti-Cortisol antibody XM210 ab1949, abcam) diluted 200-fold as a primary antibody and diluted 100-fold as a secondary antibody.
  • Anti-Mouse Antibody manufactured by Thermo Fisher Scientific was used.
  • a goat anti-rabbit antibody Anti-Dehydroepiandrosterone Antibody, Rabbit-Poly, Gene Tex, catalog number: GTX10999
  • diluted 200-fold as a primary antibody and diluted 100-fold as a secondary antibody Alexa Fluor (registered trademark) 647 Goat Anti-rabbit Antibody (manufactured by Thermo Fisher Scientific) was used.
  • NC represents a negative control (Negative Control).
  • ECLIPSE TE2000-U (manufactured by Nikon) was used for the speculum.
  • AURA Light Engine manufactured by Lumencor
  • a ray of central wavelength 475 nm full width at half maximum 28 nm
  • a ray of central wavelength 640 nm full width at half maximum 30 nm
  • a dichroic mirror (Nikon, B-1A) that reflects a wavelength shorter than 505 nm is used, and for detection of DHEA, a dichroic mirror (a light from Chroma, 41008) that reflects a wavelength shorter than 660 nm is used.
  • a filter (Nikon, B-1A) for blocking a wavelength of less than 535 nm was disposed on the detection side of cortisol, and a filter (41008, manufactured by Chroma) for blocking a wavelength of less than 662 nm was disposed on the detection side of DHEA.
  • the configuration of the other optical systems is the same as that of Experimental Example 1.
  • FIG. 11 is an image showing the results of double immunostaining of cortisol and DHEA.
  • the upper row corresponds to the sample reacted with the primary antibody, and the lower row corresponds to the negative control.
  • the left column shows the result of detection of 520 nm fluorescence (peak wavelength of the fluorescence emitted by the secondary antibody for cortisol) and the right column shows the fluorescence of 669 nm (peak wavelength of the fluorescence emitted by the secondary antibody for DHEA).
  • the hair can be more easily obtained using a known hair removal device.
  • the medulla is also expected to be thick, and there is also an advantage that it is easy to detect a physiologically active substance (for example, cortisol) which tends to accumulate in the medulla.
  • the washing solution is removed, and after washing with a buffer solution (such as PBS) suitable for immunostaining, embedding is carried out in the same manner as in Embodiment 1 (FIG. 12: ⁇ 1a is embedded in embedding agent 2) ).
  • a buffer solution such as PBS
  • embedding is carried out in the same manner as in Embodiment 1 (FIG. 12: ⁇ 1a is embedded in embedding agent 2) ).
  • the hair sample can be prepared by cutting, and the accumulated amount of the physiologically active substance can be measured in the same manner as in the first embodiment.
  • Embodiment 8 describes a kit used in measuring the accumulation amount of the physiologically active substance in the hair in Embodiments 1 to 7.
  • a kit according to Embodiment 8 comprises (1) an embedding agent for embedding hair, and (2) an antibody reactive agent containing an antibody that specifically recognizes a physiologically active substance contained in the hair. Have. The following will be described in order.
  • An embedding agent is used in cutting the hair to make a hair sample (see FIGS. 1, 12).
  • the main component of the embedding agent paraffin, celloidin, gelatin, carbowax, acrylic resin, methacrylic resin, epoxy resin, cyanoacrylate, ice (ice of purified water suitable for freezing and fixing), etc. can be used.
  • the antibody reactive agent includes an antibody that specifically recognizes a physiologically active substance (eg, cortisol) contained in hair.
  • a physiologically active substance contained in hair can be detected (direct method).
  • a labeled antibody as a secondary antibody that specifically recognizes the antibody contained in the above-mentioned antibody reactive agent, the physiologically active substance contained in the hair is higher. It can be detected by sensitivity (indirect method).
  • the physiologically active substance contained in the hair can be detected with higher sensitivity also by combining it with the ABC sensitization method, TSA sensitization method or the like.
  • the kit according to Embodiment 8 includes, in addition to the embedding agent and the antibody reaction agent, drugs (reagents, solvents, buffers, cleaning agents, etc.) required for use of the kit, devices, containers (bottles, plates, tubes, etc.) Dish etc.), a manual etc. may be provided.
  • drugs reagents, solvents, buffers, cleaning agents, etc.
  • devices devices, containers (bottles, plates, tubes, etc.) Dish etc.)
  • a manual etc. may be provided.
  • the medicines, devices, containers, instructions and the like described above may be separately obtained by the user through the market or communication lines.
  • Embodiment 9 describes an apparatus used when measuring the accumulation amount of the physiologically active substance in the hair in Embodiments 1 to 7.
  • the apparatus according to Embodiment 9 detects the fluorescence generated depending on the antibody bound to the physiologically active substance in the reaction step.
  • the above-mentioned device is provided with a member for detecting fluorescence (CCD imaging device, CMOS imaging device, photomultiplier tube, photodiode, phototransistor, etc.).
  • the said apparatus may be equipped with the member (Laser, LED, a high pressure mercury lamp, a metal halide lamp etc.) which excites fluorescence as an arbitrary structure.
  • FIG. 4 An example of an optical system combining other optical members and elements is shown in FIG. The description of the optical system shown in FIG. 4 is as described in the first embodiment.
  • a reaction involving the enzyme for example, a color reaction
  • Such devices may be suitably assembled by techniques known in the art.
  • a labeling substance a dye, a fluorescent dye, a radioactive isotope, an enzyme or the like
  • detection may be performed using physical properties (such as surface plasmon resonance) of the fine particles.
  • Such devices may be suitably assembled by techniques known in the art.
  • Embodiments 1 to 3 can be arbitrarily combined with Embodiments 4 to 7. That is, the measurement methods described herein can be performed by direct methods or by indirect methods. More specifically, the fluorescent dye may be linked to the primary antibody, linked to the secondary antibody, or linked to another compound such as avidin.
  • an antibody to be biotinylated may be a primary antibody or a secondary antibody.
  • an antibody to be biotinylated may be a primary antibody or a secondary antibody.
  • TSA sensitization may be any of the first and second embodiments.
  • the methods of the present disclosure can also be incorporated into radiolabeled antibodies, enzyme labeled antibodies, microparticulate labeled antibodies.
  • a radiolabeled antibody When a radiolabeled antibody is used, the radiation emitted by the antibody may be detected in the detection step.
  • an enzyme-labeled antibody When an enzyme-labeled antibody is used, a reaction involving the enzyme may be used to detect the antibody bound to the physiologically active substance.
  • detection using a phenomenon involving the physical properties of the microparticle, or a labeling substance (a dye, a fluorescent dye, a radioactive isotope, an enzyme, etc.) contained in the microparticle It is sufficient to detect in response.
  • the negative control is a cross-section of the hair which has been subjected to treatment in the reaction step with the primary antibody 14a without using the primary antibody 14a (for example, only in the diluted solution of the primary antibody 14a (such as 10% gelatin-PBS)).
  • the fluorescence intensity in the hair sample 10 and the background value be measured and processed under the same conditions (for example, the average value of the fluorescence intensity in the pulp of the hair etc. is calculated).
  • the method of the present disclosure is easier to align the conditions of the negative control with the conditions of the experimental group than the conventional method.
  • the conditions of the negative control and the experimental group can be aligned. 1.
  • the hair being embedded is cut along one plane. At this time, two cross sections of the hair are generated, so one is used as an experimental group and the other as a negative control. 2.
  • the hair sample is allowed to react under negative control conditions and a first observation is made. The same hair sample is then reacted with the primary antibody (and optionally with the secondary antibody) and subjected to a second observation.
  • the method of the present disclosure is characterized by simplicity as compared to the prior art.
  • the method of the present disclosure can be analyzed by making a cross-section of the hair, and there is no need to apply a destructive treatment to the hair.
  • the method of the present disclosure is different from and superior to methods using mass spectrometry or liquid chromatography.

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Abstract

L'invention concerne un procédé pratique pour mesurer la quantité accumulée d'une substance biologiquement active dans les poils/cheveux. Le procédé de mesure comprend : une étape de mise en réaction pour faire réagir une coupe transversale de poils/cheveux avec un anticorps qui reconnaît spécifiquement la substance biologiquement active ; et une étape de détection pour détecter un anticorps qui s'est lié à la substance biologiquement active pendant l'étape de mise en réaction.
PCT/JP2018/023670 2017-06-23 2018-06-21 Procédé de mesure, kit de mesure et dispositif de mesure Ceased WO2018235915A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
JP2011133371A (ja) * 2009-12-24 2011-07-07 Shiseido Co Ltd 毛髪の糖化度の特定方法、及び、これを用いる評価方法
WO2013032905A1 (fr) * 2011-08-26 2013-03-07 University Of Rochester Modulation de l'activité de wnt par ciblage de gpr177
WO2016037016A1 (fr) * 2014-09-03 2016-03-10 The Brigham And Women's Hospital, Inc. Compositions, systèmes et procédés pour la production de cellules ciliées de l'oreille interne pour le traitement de la perte auditive

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Publication number Priority date Publication date Assignee Title
JP2011133371A (ja) * 2009-12-24 2011-07-07 Shiseido Co Ltd 毛髪の糖化度の特定方法、及び、これを用いる評価方法
WO2013032905A1 (fr) * 2011-08-26 2013-03-07 University Of Rochester Modulation de l'activité de wnt par ciblage de gpr177
WO2016037016A1 (fr) * 2014-09-03 2016-03-10 The Brigham And Women's Hospital, Inc. Compositions, systèmes et procédés pour la production de cellules ciliées de l'oreille interne pour le traitement de la perte auditive

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NISSIMOV, J. ET AL.: "Method for chronological recording of antigen appearance in human head-hair shafts and its use for monitoring glycation products in diabetes", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 320, 27 November 2006 (2006-11-27), pages 1 - 17, XP055568973 *

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