JP2019007834A - Method for measurement, measurement kit, and measurement device - Google Patents

Abstract

To provide an easy method for measuring the time-dependent change of the amount of the stored physiologically active substance of hairs.SOLUTION: The method for measurement includes; a cutting step of cutting hairs to form more than one cross section of the hairs or of cutting out pieces of hairs; a reaction step of causing an antibody which specifically recognizes the physiologically active substance to react with the cross sections generated by the cutting step; and a detection step of detecting the antibody combined with the physiologically active substance in the reaction step.SELECTED DRAWING: Figure 3

Description

  The present disclosure relates to a measurement method, a measurement kit, and a measurement apparatus that measure changes over time in the amount of physiologically active substance accumulated in hair.

  There is a technique for measuring a substance contained in hair collected from a subject and determining the physiological state of the subject. For example, Non-Patent Document 1 reports that cortisol levels were determined by ELISA (enzyme-linked immunosorbent assay) using neonatal hair as a sample.

Yamada J et al (2007) "Hair Cortisol as a Potential Biologic Marker of Chronic Stress in Hospitalized Neonates", Neonatology, Vol.92 (No.1), pp.42-49

  However, the conventional techniques as described above tend to increase the size of the apparatus because the number of steps required for measuring physiologically active substances in hair increases.

  An object of one embodiment of the present disclosure is to provide a simple method for measuring a temporal change in the amount of accumulated physiologically active substance in hair.

  In order to solve the above-described problem, a measurement method according to one embodiment of the present disclosure is a method of measuring a change over time in the accumulated amount of a physiologically active substance in hair, wherein at least a part of the hair is A cutting step for cutting along the growth axis direction, a reaction step for reacting an antibody specifically recognizing the physiologically active substance to the cross section produced by the cutting, and the physiologically active substance in the reaction step Detecting the bound antibody.

  In addition, a measurement method according to another aspect of the present disclosure is a method for measuring a temporal change in the accumulated amount of a physiologically active substance in hair, and the hair is cut so that a plurality of cross sections are formed in the hair. Or a cutting step of notching the hair, a reaction step of reacting an antibody that specifically recognizes the physiologically active substance to a plurality of cross-sections generated by the cutting step, and the reaction step described above Detecting the antibody bound to the physiologically active substance.

  According to one embodiment of the present disclosure, it is possible to measure a change with time in the accumulation amount of a physiologically active substance in hair, which is simpler than the conventional method.

In the method which concerns on Embodiment 1, it is a schematic diagram showing the cutting process which cut | disconnects hair. (A) represents the hair 1 embedded in the embedding agent 2. (B) represents a plurality of hair samples 10 obtained by cutting (a) at a plurality of locations. In the said cutting process, it is a figure showing the coordinate which prescribes | regulates the cut surface of hair. In the method which concerns on Embodiment 1, it is a schematic diagram showing each step of the reaction process which makes the antibody which recognizes a bioactive substance specifically react with the cross section of hair. In the method which concerns on Embodiment 1, it is a schematic diagram showing the optical system used for the detection process which detects the antibody couple | bonded with the bioactive substance. 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 recognizes a bioactive substance specifically react with the cross section of hair. It is a figure showing the experimental result which actually measured the time-dependent change of the accumulation amount of a physiologically active substance (cortisol) with the method concerning Embodiment 2. (A) is the schematic diagram showing having set the some site | part in the hair 1 embedded by the embedding agent 2. FIG. (B) is an antibody-stained image showing the accumulated amount of cortisol at each site. (C) is a graph showing the fluorescence intensity in each part. It is a figure showing the other experimental result which actually measured the time-dependent change of the accumulation amount of a physiologically active substance (cortisol) with the method concerning Embodiment 2. (A) is a schematic diagram showing a cut part in hair 1 embedded in embedding agent 2. (B) is an antibody-stained image representing the accumulated amount of cortisol at each cut site. In the method which concerns on Embodiment 3, it is a schematic diagram showing each step of the reaction process of making the antibody which recognizes a bioactive substance specifically react with the cross section of hair. It is an antibody dyeing | staining image showing the experimental result which measured the accumulation amount of the physiologically active substance (cortisol) actually by the method which concerns on Embodiment 3. FIG. (A) is the schematic diagram showing an example of the cutting method which cut | disconnects hair in a growth axis direction in the method which concerns on Embodiment 5. FIG. (B) is the schematic diagram showing the other example of the cutting method which cut | disconnects hair in a growth axis direction in the method which concerns on Embodiment 4. FIG. It is a figure showing the experimental result which actually measured the time-dependent change of the accumulation amount of a physiologically active substance (cortisol) with the method concerning Embodiment 5. (A) is a microscope image showing the hair cross section cut | disconnected in the growth axis direction. (B) is the graph which analyzed the fluorescence intensity distribution in the hair cross section cut | disconnected in the growth axis direction along the growth axis. (C) is the figure which represented the said fluorescence intensity distribution with the heat map. (A) is the schematic diagram showing an example of the cutting method which notches hair 1 and exposes hair cross-section 11 in the method which concerns on Embodiment 6. FIG. (B) is the schematic diagram showing the other example of the cutting method which notches hair 1 and exposes hair cross-section 11 in the method which concerns on Embodiment 6. FIG. (C) is the schematic diagram showing the further another example of the cutting method which notches the hair 1 and exposes the hair cross section 11 in the method which concerns on Embodiment 6. FIG. In the method which concerns on Embodiment 7, it is a schematic diagram showing each step of the reaction process of making the antibody which recognizes a bioactive substance specifically react with the cross section of hair. It is a figure showing the experimental result which measured the accumulation amount of the physiologically active substance (cortisol and DHEA) actually by the method which concerns on Embodiment 7. In the method which concerns on Embodiment 8, it is a schematic diagram showing the cutting process which cut | disconnects hair.

  Hereinafter, an example of an embodiment of the present disclosure will be described in detail. However, the present disclosure is not limited to these.

  Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more and B or less”.

  Prior to the description of the embodiments, terms widely used in this specification will be described.

  The range of “subject” herein includes non-human mammals in addition to humans. Examples of non-human mammals include primates (monkeys, apes, etc.), cloven-hoofers (cattle, wild boar, pigs, sheep, goats, etc.), odd-hoofed animals (such as horses), rodents (mouse, rats, hamsters). , Squirrel, etc.), rabbit eyes (rabbit, etc.), and meat (dogs, cats, ferrets, etc.). The non-human mammal described above may be a domestic animal, a pet animal, or a wild animal.

  In this specification, “hair” intends “hair growing on a subject”. When the subject is a human, examples of hair include head hair, eyelashes, eyebrows, eyelashes, nasal hair, ear hair, eyelashes, pubic hair, and body hair. Among these, in the method of the present disclosure, it is preferable to use hair or wrinkles because collection is easy. Furthermore, since hair has a long growth period, it is possible to measure changes over time in the amount of accumulated physiologically active substance over a long period (several months (eg, 1 to 12 months) to several years (eg, 1 to 9 years)). There is sex. In addition, since wrinkles are thick hair, it is easy to detect physiologically active substances accumulated in the medulla.

Here, the use of hair as the biological sample has the following advantages over the use of blood, saliva, or the like as the biological sample.
(1) Hair collection is non-invasive and easy. For this reason, even a person who is not a doctor or a medical person can collect a hair sample.
(2) Low risk of contamination by other types of samples. For example, when blood is mixed when collecting saliva, the level of the physiologically active substance is usually different between the saliva and the blood, so the level of the physiologically active substance cannot be measured accurately. However, the collected hair can be easily prevented from being mixed with other types of samples (blood, saliva, etc.) by washing.
(3) Suitable for measuring the “accumulated amount” of a physiologically active substance. That is, the level of the physiologically active substance measured from blood, saliva, etc. tends to be influenced by biological conditions and environmental conditions at the time of sample collection due to circadian rhythm, acute biological reaction, and the like. On the other hand, since the growth rate of hair is about 1 cm / month for hair, a measurement value reflecting “accumulated amount of physiologically active substance secreted during a specific period” can be obtained by using hair as a sample. Can do.

  In this specification, the term “physiologically active substance” intends a substance involved in the maintenance and regulation of life activity and physiological function. Specific examples of the physiologically active substance include hormones, neurotransmitters, cytokines, vitamins, enzymes, proteins, peptides and the like.

  The method of the present disclosure is a method for measuring a change with time of the accumulation amount of a physiologically active substance in hair. Therefore, it can be a method for judging the change in the life activity and physiological function of the subject from which the hair has been collected.

  For example, by measuring the time-dependent change in the amount of stress-related physiologically active substance accumulated in the hair, it is possible to know the time-dependent change in stress experienced chronically by the subject. Examples of such stress-related physiologically active substances include cortisol (hydrocortisone; 11β, 17α, 21-trihydroxypregna-4-ene-3,20-dione), DHEA (dehydroepiandrosterone; 3β-hydroxyandro Sta-5-en-17-one), DHEA-S (sulfate-binding DHEA; DHEA-sulfate; DHE-S), testosterone, catecholamine (adrenaline, noradrenaline), chromogranin A, Neuropeptide Y, and the like. Among these, cortisol, adrenaline, and chromogranin A are associated with an increase in accumulated amount and an increase in chronic stress. On the other hand, DHEA and DHEA-S are associated with an increase in accumulated amount and a decrease in chronic stress.

  In addition, for example, it is known that in patients with depression, which is a stress-related disease, the amount of cortisol accumulated is high and the amount of accumulated DHEA is low. In such a case, it is also within the scope of the present disclosure to obtain a change over time in the ratio of the accumulated amounts of a plurality of physiologically active substances (in the above example, the value of “cortisol accumulated amount / DHEA accumulated amount”). included.

  In addition to those exemplified above, the relationship between changes in the accumulated amount of various physiologically active substances and changes in the corresponding life activities and physiological functions over time is interpreted using common technical knowledge in the medical and physiological fields. be able to.

  It is known that substances having a steroid skeleton are easily accumulated in the hair. For this reason, it is one of the preferable aspects of the method of this indication to measure the time-dependent change of the accumulation amount of the substance which has a steroid skeleton in hair. Examples of the substance having a steroid skeleton include cortisol, DHEA, DHEA-S, testosterone and the like.

As used herein, “antibody” includes immunoglobulins. That is, IgA, IgD, IgE, IgG, IgM, and these Fab fragments, F (ab ′) ₂ fragments, Fc fragments and the like are included. More specifically, examples include, but are not limited to, all classes of polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotype antibodies, humanized antibodies, and humanized antibodies by gene recombination. Absent. The “antibody” in the present specification includes antibody fragments and modified antibodies as long as a specific physiologically active substance is specifically recognized. Examples of antibody fragments include Fab fragments and F (ab ′) ₂ fragments.

  A method for producing an antibody that specifically recognizes a specific physiologically active substance is a well-known technique. For example, see [Harlow (Ed.), “Antibodies: a laboratory manual”, New York: Cold Spring Harbor Laboratory, 1988], [Iwasaki Ikuo et al., “Monoclonal antibodies: hybridomas and ELISA”, Kodansha, 1991]. can do. Of course, you may purchase and use the antibody currently marketed.

Embodiment 1
In the first embodiment, (1) a cutting step of cutting the hair so that a plurality of cross sections are formed in the hair, and (2) a physiologically active substance is specific to the plurality of cross sections generated by the cutting step. A measurement method including a reaction step of reacting an antibody to be recognized in (3) and a detection step of detecting an antibody bound to the physiologically active substance in the reaction step will be described. According to the structure which concerns on Embodiment 1, the data regarding the time-dependent change of the accumulation amount of the physiologically active substance in the said hair can be acquired. Hereinafter, the preparation from the hair sample to the detection of the antibody will be described in order based on FIGS.

  First, prior to the reaction step, hair is collected from the subject (collecting step), and a cut surface is formed on the hair (cutting step). Therefore, the collection and cutting of hair will be briefly described.

  The tool used for collecting hair is not particularly limited, and a known tool (scissors, shaving, etc.) can be used.

  The hair collected from the subject in the collecting step is preferably scalp hair or wrinkles. This is because, as described above, the hair or wrinkle can be easily collected without using a special instrument.

  The number of hairs collected from the subject in the collection process 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 is a number that can be statistically interpreted. When using head hair as hair, if about 20 hairs can be embedded to make a hair sample, it is sufficient to carry out the method of the present disclosure.

  From the viewpoint of preventing contamination of skin, blood, etc., it is preferable to collect a portion that does not include the hair root when collecting hair. On the other hand, if long hair can be collected, it is possible to measure changes with time in the amount of accumulated physiologically active substance over a longer period. From this viewpoint, it is preferable to collect hair from the vicinity of the root.

  The collected hair 1 is subjected to a process such as washing as appropriate, and then embedded in an embedding agent 2 ((a) in FIG. 1). As the main component of the embedding agent, paraffin, celloidin, gelatin, carbowax, acrylic resin, methacrylic resin, epoxy resin, cyanoacrylate, ice and the like can be used.

  Next, the embedded hair 1 is cut using a microtome, a cutter, a jig or the like to obtain a plurality of hair samples 10 ((b) in FIG. 1). The thickness of the hair sample 10 is not particularly limited, but may be about 10 nm to 500 μm. Moreover, the shape of the hair sample 10 produced is not limited to a thin piece, and it is sufficient if a cross section obtained by cutting the hair 1 at a plurality of locations is obtained.

  What is necessary is just to set suitably the space | interval (l) which obtains the hair sample 10 according to required time resolution. For example, when the hair is scalp hair, it stretches about 1 cm in one month. For this reason, if the hair sample 10 is cut out with l = 1 cm, data representing a change with time of the physiologically active substance having a time resolution of one month can be obtained.

  Here, the hair sample 10 may be prepared immediately before the accumulated amount of the physiologically active substance is measured, 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 since hair collection, a hair sample prepared immediately before measurement from the viewpoint of accurately measuring the physiologically active substance 10 is preferably used.

  The direction when cutting the hair 1 is not particularly limited, and may be perpendicular to the hair growth axis, may be crossed, or may be parallel. This will be described with reference to FIG. FIG. 2 is a diagram illustrating coordinates that define a cut surface of the hair 1. In the above coordinates, the Y axis is the growth axis of the hair 1, and the thick arrow is the normal vector of the cut surface (that is, the direction vector perpendicular to the cut surface). At this time, the normal vector can be arbitrarily determined in the range of 0 ° ≦ θ ≦ 180 ° and 0 ° ≦ φ ≦ 180 °.

  Further, in the cutting step, instead of completely cutting the hair, a part of the hair may be cut out to expose the hair cross section. The description regarding the cutting process of notching a part of hair will be described later in Embodiment 6.

  In the 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. Regarding the above steps, first, a general description of the antibody to be used will be given, and then described with reference to FIG.

  In the reaction step, a known technique relating to immunostaining can be used. Such a method can be referred to, for example, [Kuniaki Takada, Naosuke Saito, Hayato Kawakami, “Dyeing and Bioimaging Experiment Handbook” Yodosha, 2006 (separate volume of experimental medicine)].

  In the first embodiment, a method (direct method) for detecting a label bound to a primary antibody that recognizes a physiologically active substance is employed. The label bound to the primary antibody includes, for example, a dye, a fluorescent dye, a radioisotope, an enzyme, a micro / nano fine particle (metal fine particle (gold fine particle, silver fine particle, etc.), a quantum dot (a quantum dot whose composition is CdSe). Etc.), polystyrene beads, glass beads, etc.). When a dye or a fluorescent dye is used as a label, light emitted from the dye or the fluorescent dye may be detected. When a radioisotope is used as a label, the radiation emitted from the radioisotope may be detected. When an enzyme is used as the label, detection using a reaction involving the enzyme may be performed. When micro / nanoparticles are used as the label, detection is performed according to the labeling substance (dye, fluorescent dye, radioisotope, enzyme, etc.) bound to the surface of the microparticle or contained in the microparticle. Just do it. Alternatively, detection may be performed by a method using the physical properties of the fine particles (for example, light scattering or absorbance due to surface plasmon resonance or the like can be detected).

  From the viewpoint that the detection device can be miniaturized, it is preferable to use a fluorescent dye as a label. This is because the photodetection elements (photodiodes, phototransistors, complementary metal-oxide semiconductor (CMOS) image sensors, charge-coupled device (CCD) image sensors, etc.) and excitation light source elements (semiconductor lasers, This is because a small and inexpensive LED is widely used industrially. Further, unlike the radioisotope, the fluorescent dye label is preferable in that a special facility for handling is not required. Furthermore, the fluorescent dye label is also preferable in that it can be detected with a fluorescent microscope that is widely used at the laboratory level.

  In the first embodiment, a method may be used in which a label (conjugate) obtained by directly binding a primary antibody and a labeled antibody is prepared and the antigen is stained.

  The primary antibody used for immunostaining may be appropriately determined according to the antigen (bioactive substance) recognized by the primary antibody. For example, when cortisol is used as an antigen, an anti-cortisol antibody may be used.

  The antibody and the fluorescent dye are bound by a known technique. From the viewpoint of light resistance, a state in which a fluorescent dye is included in a fluorescent particle having a functional group capable of reacting with an antibody to form a bond is preferable. At this time, the fluorescent dye is encapsulated by the nanoparticles to form fluorescent particles, and a bond is formed between the nanoparticles and the antibody. Examples of the components of the nanoparticles include those that can stably include a fluorescent dye, such as polystyrene, polyamide, polylactic acid, polyacrylonitrile, polyglycidyl methacrylate, and melamine resin.

  Examples of fluorescent dyes encapsulated in the nanoparticles include rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromesene dye molecules. Is mentioned. Or Alexa Fluor (registered trademark, manufactured by Invitrogen) dye molecule, BODIPY (registered trademark, manufactured by Invitrogen) dye molecule, Cy (registered trademark, manufactured by GE Healthcare) dye molecule, DY dye molecule (registered trademark, manufactured by DYOMICS) ), HiLyte (registered trademark, manufactured by AnaSpec) dye molecule, DyLight (registered trademark, manufactured by Thermo Fisher Scientific) dye molecule, ATTO (registered trademark, manufactured by ATTO-TEC) dye molecule, MFP (registered trademark, manufactured by Mobitec) ) Dye molecules may be used.

  A well-known method is used for the binding between the fluorescent particles and 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 can be used.

  Hereinafter, based on FIG. 3, a reaction process is divided and demonstrated to the step of S1-S4. S <b> 1 to S <b> 4 are schematic diagrams in which the vicinity of the cross section of one hair in the hair sample 10 is enlarged. The hair sample 10 includes a hair cross section 11 (having a medullary cross section 11a inside), a portion 12 where an antibody can bind nonspecifically, and a portion 13 where a fluorescent dye can bind nonspecifically. (S1).

  In S2, the antibody blocking agent 12a is dropped onto the hair sample 10 to block the portion 12 where the antibody can bind nonspecifically. This prevents non-specific binding between the primary antibody 14a and the portion 12 where the antibody can bind non-specifically.

  In S3, the fluorescent dye blocking agent 13a is dropped on the hair sample 10 to block the portion 13 where the fluorescent dye can bind nonspecifically. This prevents nonspecific binding between the fluorescent dye 15 and the portion 13 where the fluorescent dye can bind nonspecifically.

  Examples of the antibody blocking agent 12a include artificial synthetic 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). Moreover, when it can be judged that there is no problem even if the treatment with the fluorescent dye blocking agent is omitted depending on the sample, the treatment with the fluorescent dye blocking agent may be omitted.

  In S4, a primary antibody 14a that specifically recognizes a specific physiologically active substance is dropped on the hair sample 10 and is bound to the physiologically active substance. Here, since the primary antibody 14a is previously bound to the fluorescent dye 15, the fluorescent dye 15 is bound to the location where the physiologically active substance is present via the primary antibody 14a.

The hair sample 10 after the reaction process is appropriately subjected to a treatment (for example, an encapsulation process) necessary for the detection process. A specific example of the encapsulation process is as follows.
1. Wash with an aqueous wash such as PBS (phosphate buffered saline) and allow to air dry.
2. The hair sample 10 is placed on a slide glass, and an encapsulant is dropped.
3. A cover glass is placed on the hair sample.

  The encapsulant used in the encapsulating step is not particularly limited as long as the fluorescent dye 15 is not photobleached. The cover glass and slide glass used in the enclosing process are not particularly limited.

  In the detection step, the antibody bound to the physiologically active substance in the reaction step is detected. Hereinafter, an example of the optical system used in the detection step will be described with reference to FIG.

  In FIG. 4, the hair sample 10 sandwiched and enclosed between the cover glass 21 and the slide glass 22 is placed on the stage 23. When the hair sample 10 is irradiated with the excitation light 31, fluorescence 32 is emitted from the fluorescent dye 15 contained therein.

  At this time, the excitation light 31 is reflected by the dichroic mirror 25 and is applied to the hair sample 10 via the optical lens 24. Further, the fluorescence 32 passes through the dichroic mirror 25 and only the specific wavelength component is extracted by the filter 26.

  By adopting the above configuration, 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 a tube of a fluorescence microscope, or the result detected by a CCD (charge-coupled device) camera or the like may be displayed on a display means such as a monitor. Further, image analysis may be performed on an image captured by a CCD camera (an image in which the distribution of the primary antibody 14a in the cross section of the hair is expressed by fluorescence) to obtain numerical data indicating the fluorescence distribution. A method for analyzing the image will be described in a fifth embodiment.

  However, what is illustrated in FIG. 4 is merely 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 only needs to detect the antibody bound to the physiologically active substance in the reaction step, if the fluorescence 32 is detected, it is sufficient as the detection step.

  In addition, said description is comprised about the case where a fluorescent pigment | dye is used as a label | marker. When other types of labels are used, the above description may be read as appropriate according to common general technical knowledge regarding microscopic observation.

[Embodiment 2]
In the second embodiment, a measurement method that further includes a secondary reaction step of reacting a secondary antibody that specifically recognizes the antibody with an antibody bound to a physiologically active substance will be described. According to the configuration according to the second embodiment, a physiologically active substance can be detected with higher sensitivity.

  In Embodiment 2, an indirect method is used as a method of immunostaining. The indirect method is a method in which a first antigen-antibody reaction is performed using an unlabeled primary antibody, and then a labeled secondary antibody that specifically recognizes the primary antibody is reacted. In general, the indirect method has higher detection sensitivity than the direct method described in the first embodiment.

  Hereinafter, the measurement method according to the second embodiment will be described with reference to FIG. In addition, 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 Embodiment 1. As described in. Further, the description of the antibody and the fluorescent dye relating to the step (reaction step) of reacting the antibody specifically recognizing the physiologically active substance with the hair cross section is as described in the first embodiment.

  The state before reacting the antibody (S1a), the step of causing the antibody blocking agent to act (S2a), and the step of causing the fluorescent dye blocking agent to act (S3a) are respectively S1 and S2 in Embodiment 1 (FIG. 3). , S3.

  In S4a, a primary antibody 14a that specifically recognizes a specific physiologically active substance is dropped onto the hair sample 10 and is bound to the physiologically active substance. Unlike Embodiment 1, in Embodiment 2, a fluorescent dye is not bound to the primary antibody 14a.

  In S5a, the secondary antibody 14b that specifically recognizes the primary antibody 14a is dropped on the hair sample 10 and is bound to the primary antibody 14a. Here, since the secondary antibody 14b is preliminarily bound to the fluorescent dye 15, the fluorescent dye 15 is bound to the location where the physiologically active substance is present via the primary antibody 14a and the secondary antibody 14b. Become.

<Experimental example 1>
With the measurement method according to the second embodiment, the change over time in the amount of accumulated cortisol contained in the hair was measured.

  First, protective equipment (goggles and gloves) was worn, and non-infectious treatment was performed on instruments used for hair collection (laboratory tables, medical scissors, clips, mirrors, etc.). For the non-infectious treatment, 70% (v / v) ethanol (self-prepared) was used.

  Thereafter, the subject himself collected hair from the vicinity of the root (about 50 to 150 hairs).

  About 20 of the obtained hairs were put into a 50 mL centrifuge tube, and about 40 mL of 2-propanol was injected into the centrifuge tube. Next, the centrifuge tube was overturned and stirred for 3 minutes with a stirrer (manufactured by Thermo Fisher Scientific) at 10 rpm. After stirring, 2-propanol was removed. Thereafter, the hair was washed again under the same conditions as described above. The hair thus treated was placed on a paper towel and allowed to air dry for 30-60 minutes.

  Next, the embedding / cutting process will be described. On the day before the experiment, 19.8 g of LR White Acrylic Resin (Sigma Aldrich) and 8.0 g of benzoyl peroxide (Sigma Aldrich) were mixed and allowed to stir overnight at room temperature. Hereinafter, the resin prepared by the above operation is referred to as an embedding resin.

5 mL of the embedding resin was injected into a 25 mL tube, about 100 μL of LR accelerator (manufactured by Sigma Aldrich) was added, and the mixture was immediately stirred by inversion. The hair was set in a syringe, and the embedding resin was poured into the syringe and embedded. Specifically, the following procedure was used.
Two 1.2 mL syringes (manufactured by Terumo, Terumo Syringe) were prepared, and their tips were connected by a resin tube (manufactured by Saint-Gobain, TYGON (registered trademark) tube).
2. The plunger of one syringe was removed and the hair was inserted into the syringe.
3. After filling the syringe with an embedding resin, a plunger was inserted.
4). The plunger of the syringe containing the hair was pushed, and excess air bubbles and resin in the syringe were pushed out to the other connected syringe.
5. The embedding resin was cured for about 1 hour while being immediately cooled in ice water.

In the case where bubbles are mixed when the embedding resin is injected, the following measures may be taken.
1. The syringe is erected in the vertical direction so that the tip of the syringe into which the embedding resin is injected is up. Thereby, a bubble is moved to a syringe front-end | tip.
In the state of 2.1, the air bubbles are moved to the other syringe side by further pushing the plunger.

  The embedded hair obtained in this way is represented schematically in FIG. Seven parts were set with respect to the embedded hair at intervals of 5 mm from the side close to the root (first to seventh parts in FIG. 6A). Thereafter, flaky hair samples were cut out from each of the set seven sites using a microtome (or jig (self-made) and cutter knife). The thickness of the flake was about 300 μm.

  The obtained thin piece was put into a microtube, 1.0 mL of 1 × PBS was weighed using a precision pipette and a disposable pipette tip, injected into the microtube, and tapped. Thereafter, PBS was removed by aspiration. This process was repeated three times to wash the hair sample.

  A hair sample is put into a microtube, and 10% gelatin-PBS (w / v) (from Sigma Aldrich gelatin (from cold water fish) is used as a blocking agent for a primary antibody, and 10 × PBS (− 1 ml) was injected into the 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 microtube, and tapped. Thereafter, PBS was removed by aspiration. This process was repeated three times to wash the hair sample.

  Next, as a fluorescent dye blocking agent, about 3 drops of FX signal enhancer (manufactured by Thermo Fisher Scientific) (amount in which the hair sample is immersed) was injected into the microtube and allowed to react 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 microtube, and tapped. Thereafter, PBS was removed by aspiration. This process was repeated three times to wash the hair sample.

  Next, about 200 μL of anti-cortisol antibody (Anti-Cortisol antibody XM210 ab1949, manufactured by abcam) diluted 200 times with 10% gelatin-PBS is injected into the microtube for about 1 hour. Reaction. Then, 1.0 mL of 1 × PBS was weighed using a precision pipette and a disposable pipette tip, injected into the microtube, and tapped. Thereafter, PBS was removed by aspiration. This process was repeated three times to wash the hair sample.

  Next, 200 μL of fluorescently labeled secondary antibody (goat anti-mouse antibody, attached to Alexa Fluor (registered trademark) 488 Goat Anti-Mouse SFX kit (Thermo Fisher Scientific)) diluted 100 times with 10% gelatin-PBS About (the amount that the hair sample is immersed in) was injected into the microtube and allowed to react 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 microtube, and tapped. Thereafter, PBS was removed by aspiration. This process was repeated three times to wash the hair sample. Furthermore, in order to prevent the salt contained in PBS from precipitating during drying and to reduce the background during fluorescence detection, the same washing operation was performed once with about 1.0 mL of pure water.

  Next, the hair sample was transferred onto a slide glass and air-dried until the hair sample was dried (about 30 to 60 minutes). Finally, an anti-fading agent (ProLong (registered trademark) Diamond, manufactured by Thermo Fisher Scientific) was dropped onto the hair sample and covered with a cover glass.

  The hair sample prepared for speculum was observed under a fluorescence microscope, and the change with time of the accumulated amount of cortisol was measured. At this time, ECLIPSE TE2000-U (made by Nikon) was used as a fluorescence microscope. An AURA Light Engine (manufactured by Lumencor) was used as an excitation light source, and a light beam having a central wavelength of 475 nm (full width at half maximum of 28 nm) was irradiated. The dichroic mirror (Nikon, B-1A) was designed to reflect wavelengths shorter than 505 nm. On the detection side, a filter (manufactured by Nikon, B-1A) was used to block wavelengths of less than 535 nm. EM-CCD (manufactured by Hamamatsu Photonics) was used for fluorescence detection.

  The results of microscopic observation are shown in FIGS. 6B and 6C. FIG. 6B is a fluorescence microscopic image of a hair sample cut out from each of 1 to 7 (a part of the sample is imaged). In each of the microscopic images, a bright part was observed at the center of the black hair cross section, indicating that cortisol was localized in the medulla.

  (C) of FIG. 6 is a graph showing the fluorescence intensity (luminance) in the hair samples cut out from the first to seventh parts, respectively. For each of a plurality of hair cross sections contained in each hair sample, the peak value of the fluorescence intensity of the medulla is measured, and the average value of the fluorescence intensity per hair fragment is determined for each of the first to seventh parts. Calculated. From the graph, it was found that the cortisol concentration at the fifth site was high. This suggests that the chronic stress experienced by the subject was high at the time corresponding to the fifth site (ie, 2.5 months before hair collection).

<Experimental example 2>
By using the same procedure as in Experimental Example 1, the amount of cortisol accumulated in the hair over time was measured using “white hair that was black-dyed from the middle” as the hair. For the embedded hair, as in Experimental Example 1, first to seventh regions were set at intervals of 5 mm from the side close to the root. Among these, the 1st-4th site | part was gray hair, and the 5th-7th site | part was dyed black.

  The result of microscopic observation is shown in FIG. In each microscopic image, a bright part was observed at the center of the hair cross section, indicating that cortisol was localized in the medulla. In addition, despite the intrinsic change in white hair and the exogenous change in hair coloring, the presence of cortisol could be confirmed by the method according to Embodiment 2.

[Embodiment 3]
In the third embodiment, a measurement method including a reaction with a primary antibody and / or a secondary antibody with another compound will be described. According to the configuration according to the third embodiment, the physiologically active substance can be detected with higher sensitivity when acquiring data related to the temporal change in the accumulated amount of the physiologically active substance in the hair.

  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 for improving the sensitivity of immunostaining by utilizing the phenomenon that avidin and biotin form a complex. The TSA sensitization method is a method for improving the sensitivity of immunostaining by utilizing the property of tyramide that binds to a nearby aromatic molecule by the catalytic action of HRP in the presence of hydrogen peroxide. Hereinafter, as an example, the step of immunostaining by ABC sensitization will be described with reference to FIG.

  The steps up to S4b (blocking for antibody and blocking for fluorescent dye) are the same as in the first embodiment.

  In S4b, first, a primary antibody 14a that specifically recognizes a specific physiologically active substance is dropped on the hair sample 10 and is bound to the physiologically active substance. At this time, as in the second embodiment, no fluorescent dye is bound to the primary antibody 14a.

  In S5b, the secondary antibody 14b that specifically recognizes the primary antibody 14a is dropped on the hair sample 10 and is bound to the primary antibody 14a. Here, the secondary antibody 14b is previously bound to biotin 16.

  In S <b> 6 b, avidin 17 that specifically binds to biotin 16 is dropped onto hair sample 10 to bind to biotin 16. Here, since the biotin 16 is preliminarily bonded to the fluorescent dye 15, the fluorescent dye 15 is passed through the primary antibody 14a, the secondary antibody 14b, the biotin 16 and the avidin 17 at the location where the physiologically active substance is present. Will be combined.

  Here, avidin 17 is a series of proteins (avidins) that are functionally defined by binding to biotin 16 with a high degree of affinity and specificity. Examples of avidins include avidin, streptavidin, and neutravidin.

<Experimental example 3>
The amount of cortisol accumulated in hair was measured by the measurement method (ABC sensitization method) according to Embodiment 3.

  The procedure for collecting, embedding and cutting hair to produce a hair sample is as described in Experimental Example 1.

  Further, an anti-cortisol antibody (Anti-Cortisol antibody XM210 ab1949, manufactured by abcam) diluted 200-fold with 10% gelatin-PBS as a primary antibody was used for reaction and washing in the same procedure as in Experimental Example 2. As a negative control, the same treatment was performed except that 10% gelatin-PBS was used instead of the antibody.

  Next, about 150 μL of biotinylated antibody (manufactured by Thermo Fisher Scientific, Biotin-XX goat anti-mouse IgG (H + L)) diluted 200 times with 10% gelatin-PBS (amount in which the hair sample is immersed), The mixture was injected into a microtube and allowed to react 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 microtube, and tapped. Thereafter, PBS was removed by aspiration. This process was repeated three times to wash the hair sample.

  Next, about 100 μL of fluorescently labeled streptavidin (manufactured by Thermo Fisher Scientific, Streptavidin, Alexa Fluor (registered trademark) 488 conjugate) diluted to 10 μg / mL with 10% gelatin-PBS (amount in which the hair sample is immersed), micro The solution was poured into a tube and allowed to react 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 microtube, and tapped. Thereafter, PBS was removed by aspiration. This process was repeated three times to wash the hair sample. Further, the same washing operation was performed with about 1.0 mL of pure water.

  Next, the hair sample was transferred onto a slide glass and air-dried until the hair sample was dried (about 30 to 60 minutes). Finally, an anti-fading agent (ProLong (registered trademark) Diamond, manufactured by Thermo Fisher Scientific) was dropped onto the hair sample and covered with a cover glass.

  A hair sample prepared for microscopic examination was observed under a fluorescence microscope to confirm the presence of cortisol. At this time, ECLIPSE TE2000-U (made by Nikon) was used as a fluorescence microscope. An AURA Light Engine (manufactured by Lumencor) was used as an excitation light source, and a light beam having a central wavelength of 475 nm (full width at half maximum of 28 nm) was irradiated. The dichroic mirror (Nikon, B-1A) was designed to reflect wavelengths shorter than 505 nm. On the detection side, a filter (manufactured by Nikon, B-1A) was used to block wavelengths of less than 535 nm. EM-CCD (manufactured by Hamamatsu Photonics) was used for fluorescence detection.

  A microscope image representing the result is shown in FIG. The upper part of FIG. 9 is a hair sample subjected to the reaction with the primary antibody, and the lower part is a negative control. As shown in FIG. 9, the strongest fluorescence was observed in the medulla. That is, it was shown that cortisol was localized in the hair medulla. By using this method, it is possible to acquire data relating to changes over time in the amount of accumulated physiologically active substance in hair.

[Embodiment 4]
In the fourth embodiment, a measurement method including a quantitative process for quantifying the amount of accumulated physiologically active substance will be described. According to the configuration according to the fourth embodiment, the amount of accumulated physiologically active substance can be quantified according to the detected fluorescence intensity when acquiring data related to the temporal change in the amount of accumulated physiologically active substance in hair. .

  The quantification step is a step of associating the fluorescence intensity obtained from the immunostained hair sample with the absolute amount of the physiologically active substance accumulated in the hair. For this association, the concentration of the physiologically active substance in another hair (comparative hair) similar to the hair to be measured (target hair) is previously determined by a method different from fluorescent staining using an antibody (for example, ELISA). Method) quantitatively. The comparative hair is hair presumed to accumulate substantially the same amount of physiologically active substance as the target hair, for example, hair that grows in the vicinity of the target hair.

  By performing the process of associating the concentration of the physiologically active substance in the specific part of the comparative hair with the fluorescent intensity in the part corresponding to the specific part of the target hair for a plurality of fluorescent intensities, A calibration curve that correlates the actual physiologically active substance concentration can be created.

  Alternatively, instead of comparative hair, an artificial sample containing a physiologically active substance at a known concentration is prepared, and immunostaining similar to the immunostaining applied to the target hair is performed on the sample. A calibration curve showing the relationship between the fluorescence intensity and the concentration of the physiologically active substance can be obtained. An example of a method for producing the artificial sample is a dot plot method.

  The calibration curve derived in this way can also be used for the measurement of changes over time in the accumulated amount of physiologically active substance in the subsequent hair.

  Hereinafter, the ELISA method and the dot plot method will be described in order.

  ELISA is a general technique for measuring a specific physiologically active substance contained in a sample, and the measurement result can be quantified by using a dilution series having a known concentration. The specific operation procedure can follow the protocol attached to the kit for ELISA, for example.

  In Embodiment 4, in order to derive a calibration curve representing the relationship between the fluorescence intensity and the accumulated amount (concentration) of a physiologically active substance by ELISA, (1) a specific portion in hair (a hair sample for immunostaining) The accumulated amount of the physiologically active substance in the portion corresponding to the cut-out portion) is measured by ELISA, and then (2) the correlation between the accumulated amount of the physiologically active substance and the fluorescence intensity is obtained. Hereinafter, an example of a specific operation procedure will be described using cortisol as a physiologically active substance.

  1. Hair is collected from the subject for ELISA (see Experimental Example 1 for a more specific method). It is preferable that the hair grows in the same part as the hair collected from the subject for immunostaining.

  2. The collected ELISA hair is cut into an appropriate length. At this time, the part cut out from the hair for ELISA corresponds to the part for producing a hair sample from the hair for immunostaining. For example, when preparing a hair sample by cutting out the 0-1 cm portion from the root side of the hair for immunostaining, cut out the 0-1 cm portion from the base side of the hair for ELISA, and perform measurement by ELISA. .

  3. 3 mg of the cut hair (Tomy Seiko, TM-625S) is put in a microtube, and 1 mL of isopropyl alcohol (manufactured by Kishida Chemical Co., Ltd., 2-propanol for high performance liquid chromatography, product number 140-64781) is added. Set on a stirrer (manufactured by ASONE, ACR-100), and after stirring for 3 minutes at 10 rpm, isopropyl alcohol is removed. The above process is repeated about 3 times to wash the hair.

  4). Using a bead-type cell crusher (Tomy Seiko, MS-100R) and zirconia beads (Tomy Seiko, ZB-20, diameter 2.0 mm), the hair is crushed under the conditions of a rotation speed of 4000 rpm and a crushing time of 60 seconds. To do. The cooling time is left for 1 minute or more, and the pulverization under the above conditions is repeated until the hair is sufficiently pulverized.

  5. The hair crushed in 4 is put into a microtube, and 1.5 mL of methanol (manufactured by Wako Pure Chemical Industries, methanol (for LC / MS), product number 138-14521) is added. From this, cortisol is extracted while stirring by inversion for 16 to 24 hours at 10 rpm at room temperature.

  6). After completion of the cortisol extraction step, the mixture is centrifuged at 6500 G for 5 minutes using a centrifuge (Hitachi Koki-15RX, manufactured by Hitachi Koki Co., Ltd.). The supernatant liquid after solid-liquid separation is taken out, and methanol is evaporated using a centrifugal concentrator (manufactured by Tommy Seiko, MV-100). Thereby, the solid substance containing cortisol is dried.

7). To determine the cortisol concentration, a saliva ELISA measurement kit (Salimetrics, Cortisol EIA Kit) is used after diversion. Add 25 μL per well of the following samples per well.
・ Solution obtained by diluting the solid material obtained in 6 in 200 μL ASSAY diluent ・ Control solution (cortisol solution of known concentration)
・ Cortisol standard solution (kit attachment, 6 series, for creating calibration curve)
・ Assay diluent (Attached to kit, for zero control and non-specific binding)
8). Add 200 μL of enzyme conjugate (HRP and cortisol complex) solution diluted to final concentration to each well. Stir for 5 minutes and allow to react for 1 hour at room temperature. At this time, cortisol in the sample solution, the control solution, and the standard solution binds to the anti-cortisol antibody competitively with the enzyme conjugate.

  9. Wash wells 4 times with 300 μL wash buffer (based on PBS). Cortisol that is not bound to the anti-cortisol antibody is washed away at this stage.

  10. Add 200 μL of tetramethylbenzidine (TMB) to each well. TMB reacts with HRP contained in the enzyme conjugate and develops color. Thereafter, 50 μL of STOP solution (methanesulfonic acid attached to the kit) is dropped to stop the enzyme reaction.

  11. The absorbance at 450 nm and 490 nm is measured using a plate reader (PerkinElmer, ARVO-MX).

12 The cortisol concentration in the hair is calculated according to the following procedure.
Calculate the average light intensity of two wells containing the same sample.
Subtract the light intensity of non-specific binding from the optical intensity of the zero control, standard solution, control solution, and hair sample.
Divide each well optical intensity (B) by the zero control average optical intensity (B0). Thereby, the binding rate (B / B0) of the enzyme conjugate to the normalized anti-cortisol antibody is calculated.
Create a calibration curve from the absorbance of the standard solution obtained. Since a 4-parameter non-linear regression curve is recommended, fit with an inverse sigmoid function (4-parameter).
-Quantify the cortisol concentration of the hair sample from the obtained calibration curve. Quantify the concentration of the control solution to confirm the accuracy of the measurement.

  By passing through the above process, the fluorescence intensity measured by the methods of Experimental Examples 1 to 3 can be correlated with the concentration of cortisol contained in the hair. That is, since the fluorescence intensity (known amount) measured from the hair sample and the cortisol concentration (known amount, determined by ELISA) in the portion of the hair corresponding to the hair sample are known, the fluorescence intensity and the hair sample An expression expressing the correlation with the cortisol concentration contained in is obtained. Therefore, once an expression representing such a correlation is created, the cortisol concentration can be calculated from the result of immunostaining without subsequent ELISA.

Next, an example of a method for quantifying the accumulated amount (concentration) of a physiologically active substance contained in a hair sample from the fluorescence intensity emitted from the hair sample by a dot plot method is shown below.
1. Dot plot the dilution series (concentration known) of the target physiologically active substance on the membrane.
2. The dot plot on the membrane is immunostained under the same conditions as the immunostaining applied to the hair sample (for example, under the conditions described in Embodiments 1 to 3).
3. Measure the fluorescence intensity (fluorescence intensity per unit area) of the dot-plotted film. Since the dot plot is a physiologically active substance solution with a known concentration, 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 by the hair sample.

  As the membrane, a known one such as a PVDF membrane or a nitrocellulose membrane can be used. Moreover, the concentration range of the dilution series of the target physiologically active substance can be determined as appropriate.

  Similar to the method using ELISA, once a calibration curve showing the correlation between fluorescence intensity and physiologically active substance concentration is obtained, the physiologically active substance concentration can be calculated from the result of immunostaining without subsequent dot plots. Can do.

[Embodiment 5]
Embodiment 5 is a method for measuring a change over time in the accumulated amount of a physiologically active substance in hair, (1) a cutting step of cutting at least a part of the hair along the growth axis direction of the hair; (2) a reaction step of reacting an antibody specifically recognizing the physiologically active substance to the cross section produced by the cleavage, and (3) detecting an antibody bound to the physiologically active substance in the reaction step. A measurement method including the detection step will be described. According to the measurement method according to the fifth embodiment, the change over time of the accumulated amount of the physiologically active substance can be obtained as continuous data.

  Hereinafter, based on FIG. 10, the cutting process of the measuring method according to the fifth embodiment will be described. In addition, the step of collecting hair from the subject (collection step), the step of reacting an antibody that specifically recognizes the physiologically active substance to the hair cross section (reaction step), and the detection of the antibody bound to the physiologically active substance The process (detection process) to be performed is as described in the first embodiment.

  FIG. 10A is a schematic diagram illustrating an example of a cutting method for cutting hair in the growth axis direction. The figure is a view of the hair 1 cut in the growth axis direction as viewed from the side (from the front of the cut surface). Since the hair 1 is cut in the growth axis direction, the hair cross section 11 is formed along the growth axis of the hair 1, and the medullary cross section 11 a is also formed along the growth axis of the hair 1.

  For this reason, from the hair cross section close to A, the accumulated amount of the physiologically active substance accumulated at a later time is measured. Conversely, from the hair cross section close to B, the accumulated amount of the physiologically active substance accumulated at an older time is measured. Such a hair cross section 11 is obtained when, for example, φ = 0 ° in FIG.

  FIG. 10B is a schematic diagram illustrating an example of a cutting method for cutting hair in the growth axis direction. In the figure, the hair 1 is cut along a plane oblique to the growth axis, and an elliptical hair section 11 and an elliptical medullary section 11a are formed.

  For this reason, from the hair cross section close to C, the accumulated amount of the physiologically active substance accumulated at a later time is measured. Conversely, from the hair cross section close to D, the accumulated amount of the physiologically active substance accumulated at an older time is measured. Such a hair cross section 11 is obtained, for example, when (φ = 90 °, θ = 70 to 85 °) in FIG.

  In addition to the above description, such as θ = 45 °, the values of φ and θ are appropriately set according to the measurement system (hair shape, optical arrangement of the detection system, etc.) and the desired time resolution. Can be set.

  In the method according to the fifth embodiment, the size of the cross section to be formed may be appropriately set according to a required measurement period of change with time. For example, since the hair grows about 1 cm per month, if the distance between AB (between CDs) is 1 cm, the change with time of the accumulated amount of physiologically active substance over one month can be measured. When the physiologically active substance is a substance that easily accumulates in the medulla (for example, cortisol), the size of the medullary cross section 11a may be set in the same manner as described above.

<Experimental example 4>
With the measurement method according to Embodiment 5, the change over time in the amount of accumulated cortisol contained in the hair was measured. Except for cutting the hair along the growth axis direction, the hair cross section was immunostained by the same procedure as in Experimental Example 1, and observed with a fluorescence microscope.

  The result of microscopic observation is shown in FIG. (A) of FIG. 11 is a microscope image showing the cross section of the observed hair. A hair cross-section along the growth axis was obtained over about 100 μm (corresponding to a stretch of 0.1 months). (B) of FIG. 11 is a graph (lower stage) showing the transition of the fluorescence intensity for the portion of about 70 μm in the hair cross section along the growth axis. The transition of the fluorescence intensity reflects the transition of the amount of cortisol accumulated in the hair, and is considered to be correlated with the transition of the degree of stress experienced by the subject. FIG. 11C is a diagram showing the fluorescence intensity distribution as a heat map. Also from this figure, it can be seen that the fluorescence intensity changes along the growth axis (the left side of the screen is generally high and the right side of the screen is generally low).

  Thus, it was shown that the change with time of the accumulated amount of cortisol contained in the hair can be continuously measured by the measurement method according to the fifth embodiment.

[Embodiment 6]
In the sixth embodiment, an example different from the first and fifth embodiments will be described with respect to the hair cutting method in the step of forming a cut surface on the hair (cutting step).

  In FIG. 12A, a plurality of portions of the hair 1 are notched in a V shape. Further, the embedding agent 2 in which the hair 1 is embedded is not completely cut (the resin below the V-shaped valley portion is connected without being cut). For this reason, a plurality of hair cross sections 11 derived from the same hair 1 are exposed on one section.

Thus, in handling a plurality of hair cross-sections 11 derived from the same hair 1, each flaky section can be treated as a single section instead of being reacted in different tubes as in the first to fifth embodiments. it can. As a result, the following advantages arise.
1. Since the accumulated amount of the physiologically active substance in the hair 1 can be observed by one scan, it becomes easy to measure with a desired time resolution.
2. Since there is no need to operate in parallel with multiple tubes, work can be speeded up, labor-saving, and speeded up.
3. Since one section is to be manipulated, it is easy to prepare the conditions for immunostaining, and the accuracy and precision of the measurement are improved (error between tubes can be eliminated).
4). The operability of the section is improved. On the other hand, in the cutting methods as in the first to fifth embodiments, a flaky section may be crushed during operation (eg, grasping with tweezers, reacting in a tube).

  In addition, you may combine the cutting process which not cuts the embedding agent 2 completely, but the cutting process which cuts the embedding agent 2 completely. In this case, a plurality of samples “a plurality of hair cross-sections 11 derived from the same hair 1 are exposed on one section” can be prepared from one or a plurality of hairs 1.

  FIG. 12B is a modification of the cutting method shown in FIG. 12 (b) is different from FIG. 12 (a) in that the hair 1 is not cut completely and the hair cross section 11 is exposed. By forming such a hair cross-section 11, the same effect as in FIG. 12A can be obtained.

  (C) of FIG. 12 has cut | disconnected the short site | part of the hair 1 further in several places. Such a hair cross-section 11 can be obtained, for example, by setting φ = 90 ° and θ = 80 ° in FIG. By cutting in this way, a plurality of hair cross sections 11 corresponding to the same growth period are obtained. For this reason, since the accumulation amount of the physiologically active substance in the said site | part of hair 1 can be measured in multiple times, it can measure more correctly and reliability improves.

  This will be described more specifically with reference to FIG. In the same figure, the point A and the point B can be regarded as portions corresponding to the same extension period T (represented by a broken line). However, the point A and the point B are located on different hair cross sections 11. For this reason, the accumulated amount of the physiologically active substance at the site corresponding to the extension period T can be measured at two points, point A and point B, and the number of samples of the measured value increases.

  Furthermore, the thickness of each thin piece can be set so that the plurality of hair cross-sections 11 obtained by cutting correspond to the same extension period (T ± t) in the hair 1. For example, all the flakes can be obtained from a portion corresponding to an extension period of one month (in the case of hair, a portion included in a length of 1 cm). Thereby, it is possible to secure a large number of samples of the measured value of the accumulated amount of the physiologically active substance from the single hair 1 during the predetermined extension period (T ± t).

  When the physiologically active substance to be measured tends to accumulate in the hair medulla, the hair 1 may be cut so that the points A and B occur on the hair medulla cross section 11a. In addition, the parts that can be considered to correspond to the same extension period T can be set in addition to the two points A and B.

[Embodiment 7]
Embodiment 7 is a method for measuring a temporal change in the accumulated amount of a physiologically active substance in hair, wherein a second physiologically active substance different from the physiologically active substance is specifically applied to the hair cross section. A measurement method that further includes a second reaction step of reacting the second antibody to be recognized will be described. According to the configuration of the seventh embodiment, it is possible to simultaneously measure the accumulated amounts of two or more types of physiologically active substances.

  Hereinafter, the measurement method according to the seventh embodiment will be described with reference to FIG. In addition, 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 Embodiment 1. As described in. Further, the description of the antibody and the fluorescent dye relating to the step (reaction step) of reacting the antibody specifically recognizing the physiologically active substance with the hair cross section is as described in the first embodiment. Since the state before reacting the antibody, the step of applying the blocking agent for the antibody, and the step of applying the blocking agent for the fluorescent dye are the same as S1 to S3 in Embodiment 1 (FIG. 3), they are omitted from FIG. To do.

  The step of reacting an 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. From the viewpoint of reducing the number of work steps, the method (1) is preferable. On the other hand, in the case (1), when the dyeing is weak or the dyeing is uneven, the method (2) may be adopted. For example, when immunostaining is performed by the indirect method, the reaction of the primary antibody is performed by the method (1), and the reaction of the secondary antibody is performed by the method (2). These methods may be used in combination. Hereinafter, the methods (1) and (2) will be described in order.

  (A) of FIG. 13 is a schematic diagram showing (1) a method of mixing and reacting two or more kinds of antibodies. In S4c, the primary antibody 14a that specifically recognizes the physiologically active substance and the second primary antibody 14c that specifically recognizes the second physiologically active substance are dropped into the hair sample 10 in a mixed state, and the physiological activity The substance and the second physiologically active substance are combined with each other.

  In S5c, a secondary antibody 14b that specifically recognizes the primary antibody 14a and a second secondary antibody 14d that specifically recognizes the second primary antibody 14c are dropped on the hair sample 10, and the primary antibody 14a and Binding to the second primary antibody 14c. Here, since the secondary antibody 14b is preliminarily bound to the fluorescent dye 15, the fluorescent dye 15 is bound to the location where the above-mentioned active substance is present via the primary antibody 14a and the secondary antibody 14b. Become. Similarly, since the second secondary antibody 14d is preliminarily bound to the second fluorescent dye 15a, the second primary antibody 14c and the second secondary antibody 14d are located at the location where the second physiologically active substance is present. The second fluorescent dye 15a is bound via the next antibody 14d.

  (B) of FIG. 13 is a schematic diagram showing (2) a method of reacting two types of antibodies one by one. In S4d1, the primary antibody 14a that specifically recognizes the physiologically active substance is dropped onto the hair sample 10 and is bound to the physiologically active substance.

  In S4d2, the second primary antibody 14c that specifically recognizes the second physiologically active substance is dropped on the hair sample 10 and is bound to the second physiologically active substance.

  In S5d1, a secondary antibody 14b that specifically recognizes the primary antibody 14a is dropped onto the hair sample 10 and is bound to the primary antibody 14a. Here, since the secondary antibody 14b is preliminarily bound to the fluorescent dye 15, the fluorescent dye 15 is bound to the location where the physiologically active substance is present via the primary antibody 14a and the secondary antibody 14b. Become.

  In S5d2, the second secondary antibody 14d that specifically recognizes the second primary antibody 14c is dropped onto the hair sample 10 and is bound to the second primary antibody 14c. Here, since the second secondary antibody 14d is preliminarily bound to the second fluorescent dye 15a, the second primary antibody 14c and the second secondary antibody 14d are located at the location where the second physiologically active substance is present. The second fluorescent dye 15a is bound via the next antibody 14d.

  When different physiologically active substances are immunostained with two kinds of fluorescent dyes in this way, the same hair sample 10 is obtained by using fluorescent dyes exhibiting different fluorescence wavelengths 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 can be measured.

<Experimental example 5>
With the measurement method according to Embodiment 7, the accumulated amounts of cortisol and DHEA contained in the hair were measured.

  For staining of cortisol, a 200-fold diluted anti-cortisol antibody (Anti-Dehydroepiandrosterone Antibody, Rabbit-Polyclonal (ab35062), manufactured by abcam) was used as a secondary antibody and a goat anti-mouse antibody (Alexa Fluor (100 ml) diluted 100-fold as a secondary antibody. (Registered trademark) 488 Goat Anti-Mouse Antibody, manufactured by Thermo Fisher Scientific).

  For staining of DHEA, a 200-fold diluted anti-DHEA antibody (Anti-Dehydroepiandrosterone Antibody, Rabbit-Poly, Gene Tex, catalog number: GTX10999) was used as a secondary antibody and a goat anti-rabbit antibody diluted 100-fold as a secondary antibody ( Alexa Fluor (registered trademark) 647 GoatAnti-rabbit Antibody (manufactured by Thermo Fisher Scientific) was used.

  Other reagents used were in accordance with Experimental Example 1 and are as shown in Table 1 below. In the table, “NC” represents negative control.

  For the speculum, ECLIPSE TE2000-U (manufactured by Nikon) was used. An AURA Light Engine (manufactured by Lumencor) was used as an excitation light source, and a light beam having a central wavelength of 475 nm (full width at half maximum of 28 nm) was irradiated for detection of cortisol, and a light beam having a central wavelength of 640 nm (full width at half maximum of 30 nm) was irradiated for detection of DHEA. A dichroic mirror (manufactured by Nikon, B-1A) that reflects wavelengths shorter than 505 nm was used for detection of cortisol, and a dichroic mirror (manufactured by Chroma, 41008) that reflects wavelengths shorter than 660 nm was used for detection of DHEA. . In addition, a filter that blocks wavelengths less than 535 nm (Nikon, B-1A) was disposed on the detection side of Cortisol, and a filter (Chroma, 41008) that blocks wavelengths below 662 nm was disposed on the detection side of DHEA. Other configurations of the optical system are the same as those in Experimental Example 1.

  The results of immunostaining are shown in FIG. FIG. 14 is an image showing the results of double immunostaining for 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 detecting fluorescence at 520 nm (peak wavelength of fluorescence emitted by the secondary antibody for cortisol), and the right column is the result of detection of fluorescence at 669 nm (peak wavelength of fluorescence emitted by the secondary antibody for DHEA).

  From the upper microscope image, the fluorescence caused by cortisol and the fluorescence caused by DHEA were confirmed. That is, it was shown that many types of physiologically active substances can be detected simultaneously from the same sample. By using this method, it is possible to acquire data relating to changes over time in the accumulated amount of various types of physiologically active substances in hair.

[Embodiment 8]
In the eighth embodiment, a measurement method in the case where wrinkles are used particularly as hair when measuring the accumulated amount of physiologically active substance accumulated in hair will be described. According to the structure which concerns on Embodiment 8, hair can be obtained more easily using a well-known hair removal apparatus. In addition, since wrinkles are generally thick hair, the hair medulla is expected to be thick, and there is an advantage that it is easy to detect a physiologically active substance (for example, cortisol) that tends to accumulate in the hair medulla.

  When using a scissors as hair, an electric shaving, a manual shaving (razor), etc. can be used for collection of scissors. There is an electric shaving that cleans the blade of the electric shaving after the hair removal treatment. In this case, a powder containing scissors is obtained by washing the blade of the electric shaving with a commercially available cleaning solution for electric shaving after the hair removal treatment.

  Thereafter, the washing solution is removed, and after washing with a buffer solution (PBS or the like) suitable for immunostaining, embedding is performed in the same manner as in Embodiment 1 (FIG. 15. ). From the embedded wrinkle shown in FIG. 15, a hair sample can be prepared by cutting, and the amount of accumulated physiologically active substance over time can be measured in the same manner as in the first embodiment.

[Embodiment 9]
In Embodiment 9, a kit used in Embodiments 1 to 8 for measuring a change over time in the amount of accumulated physiologically active substance in hair will be described.

  The kit according to Embodiment 9 includes (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. I have. Hereinafter, it demonstrates in order.

  The embedding agent is used when hair is cut to prepare a hair sample (see FIGS. 1 and 15). As 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 freeze fixation), and the like can be used.

  The antibody reactive agent contains an antibody that specifically recognizes a physiologically active substance (for example, cortisol) contained in hair. For example, as in Embodiment 1, a physiologically active substance contained in hair can be detected by using an antibody labeled as the antibody (direct method). Further, as in Embodiment 2, by using an antibody labeled as a secondary antibody that specifically recognizes the antibody contained in the antibody reactive agent, the physiologically active substance contained in the hair is made higher. Sensitivity can be detected (indirect method). Furthermore, the bioactive substance contained in the hair can be detected with higher sensitivity also by combining with the ABC sensitization method or the TSA sensitization method as in the third embodiment.

  In addition to the embedding agent and the antibody reactive agent, the kit according to Embodiment 9 includes drugs (reagents, solvents, buffers, cleaning agents, etc.), instruments, containers (bottles, plates, tubes, Etc.) and instructions may be provided. The above-described medicines, instruments, containers, instructions, and the like may be separately obtained by the user through the market or a communication line.

[Embodiment 10]
In the tenth embodiment, a device used in the first to eighth embodiments when measuring a change with time of the accumulation amount of the physiologically active substance in the hair will be described.

  The apparatus according to the tenth embodiment detects fluorescence generated depending on the antibody bound to the physiologically active substance in the reaction step. For this reason, the apparatus includes a member for detecting fluorescence (CCD image sensor, CMOS image sensor, photomultiplier tube, photodiode, phototransistor, etc.). Moreover, the said apparatus may be equipped with the member (a laser, LED, a high pressure mercury lamp, a metal halide lamp etc.) which excites fluorescence as arbitrary structures.

  An example of an optical system combining other optical members / elements is shown in FIG. The description about the optical system shown in FIG. 4 is as described in the first embodiment.

  When immunostaining is performed using a radiolabeled antibody, the radiation generated depending on the antibody bound to the physiologically active substance in the reaction step may be detected. Such an apparatus can be appropriately assembled by a known technique in this field.

  In addition, when immunostaining is performed using an enzyme-labeled antibody, the reaction involving the enzyme (for example, a color reaction) may be detected. Such an apparatus can be appropriately assembled by a known technique in this field.

  Further, when immunostaining is performed using antibodies labeled with microparticles, a labeling substance (a dye, a fluorescent dye, a radioisotope, an enzyme, etc.) contained in the microparticles may be detected. Alternatively, the detection may be performed using physical properties (surface plasmon resonance, etc.) of the fine particles. Such an apparatus can be appropriately assembled by a known technique in this field.

[Modification 1]
Embodiments 1 to 3 can be arbitrarily combined with Embodiments 4 to 7. That is, the measurement method described in this specification can be carried out by either a direct method or an indirect method. More specifically, the fluorescent dye may be linked to the primary antibody, may be linked to the secondary antibody, or may be linked to other compounds such as avidin.

  The third embodiment can be combined with any of the first and second embodiments. That is, for example, in the ABC sensitization method, an antibody that is biotinylated (an antibody that binds to biotin) may be a primary antibody or a secondary antibody. The same applies to other methods such as TSA sensitization.

  Furthermore, the method of the present disclosure can also be applied to radiolabeled antibodies, enzyme-labeled antibodies, and microparticle-labeled antibodies. When using a radiolabeled antibody, the radiation emitted by the antibody may be detected in the detection step. When an enzyme-labeled antibody is used, an antibody bound to a physiologically active substance may be detected using a reaction involving the enzyme. When using antibodies labeled with microparticles, detection using a phenomenon involving physical properties of the microparticles, or labeling substances (dyes, fluorescent dyes, radioisotopes, enzymes, etc.) contained in the microparticles What is necessary is just to perform the detection according to.

[Modification 2]
The hair cutting method in Embodiments 1, 5, and 6 can be arbitrarily combined with other embodiments. That is, in 2 to 4, 7, and 8, the cut surface formed on the hair is either perpendicular to the growth axis as in the first embodiment or in the growth axis direction as in the fifth embodiment. A special shape may be used as in the sixth embodiment.

[Modification 3]
For more precise measurement, prepare a negative control for each hair sample 10 and use the value obtained by subtracting the fluorescence intensity (background value) in the negative control from the fluorescence intensity in the hair sample 10 as the measurement value. Can do. Here, the negative control is a cross-section of the hair that has undergone the cutting step in the reaction step with the primary antibody 14a without using the primary antibody 14a (for example, only a diluted solution of the primary antibody 14a (10% gelatin-PBS or the like)). The hair sample 10 is produced by the same process as that described above except that the hair sample 10 is dipped). The fluorescence intensity in the hair sample 10 and the background value are preferably measured and processed under the same conditions (for example, both calculate the average value of the fluorescence intensity in the hair medulla).

The method of the present disclosure is easier to align the negative control conditions with those of the experimental group than the conventional method. For example, according to the following method, the conditions of the negative control and the experimental group can be made uniform.
1. Cut the embedded hair along one plane. At this time, since two cross sections of the hair are generated, one is used as an experimental group and the other is used as a negative control.
2. The hair sample is reacted under negative control conditions, and the first observation is performed. The same hair sample is then subjected to a reaction with the primary antibody (and optionally a reaction with the secondary antibody) and a second observation is made.

  The measurement using the negative control described above can be combined with any of the above-described embodiments and modifications.

[Comparison with conventional technology]
As described above, the method of the present disclosure is characterized by being simpler than the prior art. As an example, the method of the present disclosure can be analyzed by creating a cross section of the hair, and does not require destructive treatment on the hair. In this respect, the method of the present disclosure is different from and superior to the method using mass spectrometry or liquid chromatography.

  The present disclosure is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the embodiments can be obtained by appropriately combining technical means disclosed in different embodiments. Are also included in the technical scope of the present disclosure. Furthermore, a new technical feature can be formed by combining the technical means disclosed in each embodiment.

1: embedded hair 1a: embedded wrinkle 10: hair sample 11: hair cross section 11a: medullary cross section 12: place where antibody can bind nonspecifically 12a: blocking agent for antibody 13: fluorescent dye Non-specific binding site 13a: fluorescent dye blocking agent 14a: primary antibody 14b: secondary antibody 14c: second primary antibody 14d: second secondary antibody 15: fluorescent dye 15a: second fluorescent dye 16: Biotin 17: Avidin 21: Cover glass 22: Slide glass 23: Stage 24: Optical lens 25: Dichroic mirror 26: Filter 31: Excitation light 32: Fluorescence

Claims (10)

A method for measuring a change in accumulated amount of a physiologically active substance in hair over time,
A cutting step of cutting at least a part of the hair along the growth axis direction of the hair;
A reaction step of reacting an antibody specifically recognizing the physiologically active substance to the cross section produced by the cleavage,
And a detection step of detecting an antibody bound to the physiologically active substance in the reaction step. A method for measuring a change in accumulated amount of a physiologically active substance in hair over time,
Cutting the hair so as to form a plurality of cross-sections in the hair, or cutting the hair,
A reaction step of reacting an antibody that specifically recognizes the physiologically active substance to a plurality of cross-sections generated by the cutting step;
And a detection step of detecting an antibody bound to the physiologically active substance in the reaction step.   The measurement according to claim 2, wherein, in the cutting step, a plurality of cross sections derived from the hair are exposed on one piece without completely cutting the embedding agent in which the hair is embedded. Method.   4. The method according to claim 1, further comprising a second reaction step of reacting a second antibody that specifically recognizes a second physiologically active substance different from the physiologically active substance with respect to the cross section. The measuring method as described in.   The measurement method according to claim 1, further comprising a secondary reaction step of reacting the antibody bound to the physiologically active substance with a secondary antibody that specifically recognizes the antibody. .   The measuring method according to claim 1, wherein the hair is hair or wrinkles.   The measurement method according to any one of claims 1 to 6, wherein the antibody specifically recognizes cortisol. A measurement kit used in the measurement method according to any one of claims 1 to 7,
An embedding agent for embedding hair;
An antibody reactive agent comprising an antibody that specifically recognizes a physiologically active substance contained in the hair;
Measuring kit equipped with.   The kit according to claim 8, wherein the antibody specifically recognizes cortisol. A measurement device used in the measurement method according to any one of claims 1 to 7,
A measuring device for detecting fluorescence generated depending on an antibody bound to the physiologically active substance in the reaction step.