Method for analyzing cortisol using hair to measure stress in companion animals

Abstract

The present invention relates to a method for analyzing cortisol using hair to measure stress in companion animals. More specifically, the present invention relates to a method for analyzing cortisol using hair of a companion animal, which performs a pretreatment process of sampling, washing, grinding, and extracting a hair sample, followed by cortisol analysis on a microplate reader using a cortisol enzyme immunoassay kit. The analysis method of the present invention enables simple and accurate assessment of stress levels in companion animals.

Description

A method for analyzing cortisol using hair to measure stress in companion animals.

[0001] The present invention relates to a method for analyzing cortisol using hair for measuring stress in companion animals. More specifically, the present invention relates to a method for analyzing cortisol using companion animal hair, comprising preprocessing hair samples for sampling, washing, grinding, and extraction, followed by cortisol analysis using a microplate reader using a cortisol enzyme immunoassay kit.

[0002] Cortisol (hydrocortisone), or cortisol, is a glucocorticoid hormone produced by the adrenal cortex, with smaller amounts produced by other tissues. It is often called the "stress hormone" because it plays a role in the stress response and influences blood pressure, blood sugar levels, and other stress-adaptive processes. The adrenal cortex secretes cortisol in response to stress or low blood glucocorticoid levels. This hormone increases blood sugar, suppresses the immune system, and aids in the metabolism of carbohydrates, proteins, and fats. Cortisol also inhibits bone formation. Various synthetic forms of cortisol are used to treat a variety of conditions. As an anti-inflammatory, it is also used for various inflammatory and allergic conditions. Metabolically, cortisol promotes gluconeogenesis, decreases hepatic glycogen deposition, and reduces glucose utilization. Cortisol production follows a circadian rhythm dependent on ACTH. Furthermore, cortisol levels peak in the morning and decline throughout the day. Most serum cortisol, with the exception of about 4%, is bound to proteins, including corticosteroid-binding globulin and serum albumin. Only free cortisol is available to most receptors, through which physiological processes are regulated. Abnormal cortisol levels have been linked to various conditions, including prostate cancer, depression, and schizophrenia, and abnormal cortisol levels are known to be associated with Cushing's syndrome and Addison's disease.

[0003] Meanwhile, "stress," the root of all illness, is also very detrimental to pet health. Unlike people who can manage stress on their own through various methods, pets require their owners to help them manage it. When people are under a lot of stress, their health deteriorates. They become depressed, lose their appetite, and suffer from insomnia. This disrupts their daily routine, weakening their immune system and exposing them to various diseases. Pets are no different. If they're constantly stressed, they can develop various diseases. Therefore, guardians need to recognize their pets' signals and help them manage their stress, but this is not an easy task. Diseases caused by stress in pets include periodontal disease and depression. Periodontal disease occurs when a pet licks itself, causing hair to become lodged between its teeth. This hair then allows bacteria to enter the gums, leading to periodontal disease. Periodontal disease causes red or swollen gums. Meanwhile, a pet with depression may not feel any physical symptoms, but may not eat or play well. They may also be unwelcome when their owner sees them. These symptoms could be early signs of depression, so it's recommended to take your pet to the hospital for a checkup. In addition, you may develop ulcerative colitis (chronic diarrhea), thyroid disease, or diabetes.

[0004] That is, the present invention was developed to easily and accurately identify the stress of companion animals as described above, and specifically relates to a cortisol analysis method using hair for measuring stress in companion animals. More specifically, the present invention relates to a method for analyzing cortisol using companion animal hair, which comprises a preprocessing process of sampling, washing, grinding, and extracting a hair sample, and then analyzing cortisol using a microplate reader using a cortisol enzyme immunoassay kit.

[0005] The purpose of the present invention is to provide a method for analyzing cortisol using hair to measure stress in companion animals.

[0006] Another object of the present invention is to provide a method for pretreating a hair sample for cortisol analysis of the companion animal.

[0007] To achieve the above purpose, the present invention provides a method for analyzing cortisol using hair to measure stress in companion animals, according to the following steps:

[0008] (1) Step of sampling and washing a pet hair sample;

[0009] (2) A step of extracting cortisol from a washed hair sample; and

[0010] (3) Step of analyzing the extracted cortisol.

[0011] In one embodiment of the present invention, the "step (1)" includes (1-1) a step of cutting and sampling the pet's hair from the back of the pet's neck as a diagnosis target; and (1-2) a step of taking 200 to 300 mg of the sampled hair sample, adding 1 to 10 mL of isopropyl alcohol, and reacting for 1 to 10 minutes. The steps may be repeated 1 to 3 times to wash the hair sample and then dry it, but the present invention is not limited thereto.

[0012] In one embodiment of the present invention, the "step (2)" may be, but is not limited to, the steps of (2-1) freeze-pulverizing a washed and dried sample; (2-2) adding 0.5 to 2 mL of an organic solvent to the freeze-pulverized sample and extracting the sample at room temperature for 12 to 24 hours using a digital rotator; (2-3) separating the organic solvent from the sample after the extraction is complete; and (2-4) concentrating the organic solvent of the separated sample with nitrogen. At this time, the organic solvent may preferably be methanol or ethanol, and more preferably may be methanol.

[0013] In one embodiment of the present invention, the "nitrogen enrichment" may be performed for 10 to 50 minutes at a nitrogen supply device pressure of 60 to 100 psi during nitrogen enrichment, but is not limited thereto.

[0014] In one embodiment of the present invention, the "step (2)" may be, but is not limited to, the steps of (2-1) freeze-pulverizing a washed and dried sample; (2-2) adding 0.8 to 1.2 mL of an organic solvent to the freeze-pulverized sample and extracting the sample at room temperature for 14 to 18 hours using a digital rotator; (2-3) separating the organic solvent from the sample after the extraction is complete; and (2-4) concentrating the organic solvent of the separated sample with nitrogen. At this time, the organic solvent may preferably be methanol or ethanol, and more preferably may be methanol.

[0015] The present invention relates to a method for analyzing cortisol using hair to measure stress in companion animals. More specifically, the method involves analyzing cortisol using companion animal hair, comprising preprocessing hair samples, including sampling, washing, grinding, and extraction, followed by cortisol analysis using a microplate reader using a cortisol enzyme immunoassay kit. The analysis method of the present invention enables simple and accurate assessment of companion animal stress.

[0016] FIG. 1 is a photographic diagram illustrating a process for analyzing cortisol using pet hair in one embodiment of the present invention.

[0017] FIG. 2 is a diagram showing a standard curve using 4PLC software by measuring cortisol in a pet hair sample according to one embodiment of the present invention.

[0018] FIG. 3 is a diagram showing the results of cortisol measurement after different pretreatment equipment was applied to a pet hair sample in one embodiment of the present invention.

[0019] FIG. 4 is a diagram showing the results of cortisol measurement after different pretreatment equipment was applied to a pet hair sample in one embodiment of the present invention.

[0020] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0021] In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0022] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise stated.

[0023] Hereinafter, the present invention will be described in more detail through examples. However, the above examples and experimental examples are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0024]

[0025] <Experimental Method> Cortisol Analysis Method for Pet Hair Samples (See Figure 1)

[0026] 1-1. Hair sample sampling and washing

[0027] (1) Cut 3 to 5 cm of hair from the back of the pet's neck using surgical precision scissors, place in aluminum foil, and store at room temperature to prepare for use as a sample.

[0028] (2) Take 250 mg of the sample, place it in a 15 mL conical PP centrifuge tube, and seal it with a stopper.

[0029] (3) Add 5 mL of isopropyl alcohol (IPA) to each sample, mix gently, and incubate for 3 minutes.

[0030] (4) Pour the IPA gently, repeat this twice, and dry the washed sample.

[0031]

[0032] 1-2. Extraction

[0033] (1) Freeze-pulverize the dried sample after washing.

[0034] (2) Weigh 50 mg of the freeze-crushed sample into 1.5 mL conical PP centrifuge tubes, add 1 mL of organic solvent (preferably methanol or ethanol, more preferably methanol) to each sample, and extract overnight (16 h) at room temperature using a designed digital rotator.

[0035] (3) After extraction is complete, take out the sample and place it in a centrifuge. When performing the process once at 4000 RCF, the sample remains in the form of a pellet at the bottom and the organic solvent is separated. If extraction step (4) is not performed immediately, store the sample at -20℃.

[0036] (4) Take 0.5 mL of organic solvent from each sample and place it in a 1.5 mL conical PP centrifuge tube. Place the sample in a thermo shaker preheated to 38°C and concentrate it with nitrogen using an automatic nitrogen concentrator. The pressure of the nitrogen supply device is set to 60 to 100 psi and the process is carried out for 10 to 50 minutes.

[0037]

[0038] 1-3. Cortisol Assay Kit Usage and Analysis

[0039] (1) Reagent Preparation: Allow the kit reagents to sit at room temperature for 30 minutes and ensure that all samples have reached room temperature and are appropriately diluted before running the kit.

[0040] (2) Assay Buffer: Dilute the concentrate (Assay Buffer Concentrate) 1:5 with 3rd-stage purified water (deionized water). Once diluted, it is stable for 3 months at 4°C.

[0041] (3) Wash Buffer: If the concentrate (Wash Buffer Concentrate) is diluted 1:20 with 3rd-stage purified water (deionized water), it is stable for 3 months at room temperature.

[0042] (4) Standard Preparation: Label test tubes #1 through #7. Pipette 450 μL of Assay Buffer into tube #1 and 250 μL into tubes #2 through #7. Because the cortisol stock solution contains an organic solvent, pre-rinse the pipette tip several times to ensure accurate transfer. Carefully add 50 μL of the cortisol stock solution to tube #1 and mix thoroughly. Take 250 μL of the cortisol solution from tube #1 and add it to tube #2 and mix thoroughly. Repeat the serial dilution in tubes #3 through #7. The cortisol concentrations in the tubes are 3,200, 1,600, 800, 400, 200, 100, and 50 pg / mL (50 to 3,200 pg / mL). Use the standard solutions within 2 hours of preparation (Table 1).

[0043]

[0044] Std1(#1)Std2(#2)Std3(#3)Std4(#4)Std5(#5)Std6(#7)Std7(#7)Assay Buffer Volume (μL)450250250250250250250AdditionStockStd1(#1)Std2(#2)Std3(#3)Std4(#4)Std5(#5)Std6(#7)Volume of Addition (μL)50250250250250250250Final Conc. (pg / mL)3200160080040020010050

[0045] (5) Assay protocol

[0046] The tester should run all standard solutions and samples in duplicate to ensure accurate calculation of cortisol concentrations.

[0047] 1) Add 150 μL of Diluted Assay Buffer (DAB (1x5)) to each nitrogen-concentrated sample and mix thoroughly.

[0048] 2) Add 50 μL of DAB (1x5) to the sample or standard solution into the plate well using a pipette (duplicate the sample or standard solution).

[0049] 3) Add 75 μL of DAB (1x5) buffer to the non-specific binding (NSB) well (Blank).

[0050] 4) Add 50 μL of DAB (1x5) buffer to the maximum binding (B0 or ​​Zero standard) well.

[0051] 5) Use a pipette to add 50 μL of DetectX cortisol Conjugate to each well.

[0052] 6) Add 25 μL of DetectX Cortisol Antibody to each well except NSB.

[0053] 7) Gently tap the side of the plate to ensure proper mixing of the reagents, cover the plate with a plate sealer, and shake using a thermo shaker at 700 to 900 rpm at room temperature for 1 hour.

[0054] 8) Afterwards, aspirate each solution from the plate using a pipette, and wash each well four times with 300 μL DWB (1x20). Pat the plate dry with a clean absorbent towel.

[0055] 9) Add 100 μL of TMB Substrate to each well.

[0056] 10) Incubate the plate at room temperature for 30 minutes without shaking.

[0057] 11) Add 50 μL of stop solution to each well.

[0058] 12) Read the optical density (OD) generated in each well at 450 nm using a microplate reader.

[0059] 13) Calculate the cortisol concentration of each sample using the built-in 4PLC software of the Microplate Reader.

[0060]

[0061] <Experimental Results>

[0062] Following the sampling, washing, grinding, and extraction processes described above, cortisol results were obtained using a microplate reader using a Cortisol Enzyme immunoassay kit. Table 2 below shows the results calculated using 4PLC software (plotting). Figure 1 also shows the typical standard curves of 4PLC software (plotting).

[0063]

[0064] WellContentNet OD4-Parameter fit - Net OD in pg / mL4-Parameter fit - %B / B0 in pg / mL%B / B0 in % (450)A01Sample X20.534880.971977.95648.80702B01Sample X50.648552.376635.83759.26502C02Sample X60.518936.5091035.15347.35351d01blank bd01positive control P1.094n.094n.ana100e02positive control P1.094n.ana100

[0065] Additional Experiments

[0066] In order to optimize the cortisol analysis method using pet hair confirmed through the above experimental method and experimental results, the preprocessing equipment was changed and the sampling, freeze-pulverization, sample extraction, and cortisol kit processing processes of the above experimental method were compared. The results are shown in Figs. 3 and 4. In the case of the preprocessing equipment, the cortisol extraction efficiency was better with a digital rotator than with a shaker, and the reproducibility was confirmed through three replicate tests under the digital rotator conditions. In addition, the recovery rate was confirmed to be 124% when spiking a cortisol standard. This is a common recovery rate (%) in the micropipette process, and it can be said that reproducibility and accuracy were secured.

[0067]

[0068] As described above, specific embodiments of the present invention have been described in detail. However, those skilled in the art who understand the spirit of the present invention will be able to easily suggest other inventions that are backwards or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the scope of the same spirit. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description described above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Cortisol analysis method using hair to measure stress in pets according to the following steps: (1) Step of sampling and washing a pet hair sample; (2) A step of extracting cortisol from a washed hair sample; and (3) Step of analyzing the extracted cortisol.

2. In paragraph 1, The above step (1) is, (1-1) A step of cutting and sampling the pet's hair from the back of the pet's neck to be diagnosed; and (1-2) The process proceeds by taking 200 to 300 mg of the sampling hair sample, adding 1 to 10 mL of isopropyl alcohol, and reacting for 1 to 10 minutes. Characterized in that the hair sample is washed and dried by repeating the above steps 1 to 3 times. A cortisol analysis method using hair to measure stress in companion animals.

3. In paragraph 1, The above step (2) is, (2-1) Step of freeze-pulverizing the dried sample after washing; (2-2) Step of adding 0.5 to 2 mL of organic solvent to the freeze-pulverized sample and extracting at room temperature for 12 to 24 hours using a digital rotator; (2-3) A step of separating the organic solvent from the sample after extraction is completed; and (2-4) Characterized in that it proceeds in a step of concentrating nitrogen in the organic solvent of the separated sample. A cortisol analysis method using hair to measure stress in companion animals.

4. In paragraph 3, The nitrogen supply device pressure during the above nitrogen enrichment is characterized by being 60 to 100 psi and proceeding for 10 to 50 minutes. A cortisol analysis method using hair to measure stress in companion animals.

5. In paragraph 3, The above step (2) is, (2-1) Step of freeze-pulverizing the dried sample after washing; (2-2) A step of adding 0.8 to 1.2 mL of organic solvent to the freeze-pulverized sample and extracting at room temperature for 14 to 18 hours using a digital rotator; (2-3) A step of separating the organic solvent from the sample after extraction is completed; and (2-4) Characterized in that it proceeds in a step of concentrating nitrogen in the organic solvent of the separated sample. A cortisol analysis method using hair to measure stress in companion animals.

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