WO2023163240A1 - Stimulation composition for rapid test of natural killer cell activity and method for rapid test of natural killer cell activity using same - Google Patents

Abstract

The present invention relates to a technology for a rapid test of natural killer cell activity and, more specifically, to a stimulation composition for a rapid test of natural killer cell activity that can stimulate a natural killer cell and can test the activity of the natural killer cell within 48 hours at the latest after culturing, and to a rapid test of natural killer cell activity using same.

Description

Stimulating composition for rapid test of natural killer cell activity and rapid test method for natural killer cell activity using the same

The present invention relates to a technology for rapid testing of natural killer cell activity, and more specifically, a stimulating composition for rapid testing of natural killer cell activity capable of testing natural killer cell activity within 18 to 48 hours at the latest after culturing by stimulating natural killer cells, and It relates to a rapid test of natural killer cell activity using this.

As humans age, the number of immune cells decreases and the activity of immune cells gradually decreases. For this reason, immunity continues to fall in inverse proportion to age, and as life expectancy increases these days, living in the 100-year-old era, cancer patients due to reduced immunity are bound to increase.

The human body maintains immunity by various types of immune cells. That is, natural killer cells gradually lose their activity as humans age, and in the case of cancer patients, in particular, it can be confirmed through experiments that the activity of natural killer cells is significantly different from that of healthy people.

On the other hand, natural killer cells maintain almost no activity (cancer cell killing power) even in normal people who are said to have good immunity, and are immediately activated when they encounter infected cells or bacteria to show a killing effect. The activity of natural killer cells can be expressed by the activation time and the magnitude of cytotoxicity, which is how quickly the ability to remove abnormal cells is secured when stimulated.

According to these research results, it seems that the possibility of cancer can be predicted by measuring the activity of natural killer cells. In addition, if the activity of natural killer cells can be directly confirmed, the possibility of cancer occurrence can be more accurately predicted, providing accurate information to many semi-healthy people and cancer patients who are concerned about the possibility of cancer. may be able to help

However, no attempt has yet been made to predict the possibility of disease by measuring the activity of natural killer cells by a direct method rather than an indirect method.

The present inventors have completed the present invention by developing a technology capable of quickly examining the activity of natural killer cells, focusing on the fact that the possibility of disease onset can be predicted by measuring the activity of natural killer cells.

Therefore, an object of the present invention is to culture lymphocytes isolated from peripheral blood in order to measure immunity and predict the possibility of infection of infectious diseases, so that natural killer cell activity can be measured within 18 to 48 hours at the latest. To provide a stimulating composition for rapid testing of natural killer cell activity capable of stimulating cells and a rapid testing method for natural killer cell activity using the same.

Another object of the present invention is to provide a pharmaceutical composition for preventing or ameliorating symptoms caused by viruses, which is effective in preventing and/or improving various diseases caused by viruses by using a stimulating composition for rapid testing of natural killer cell activity.

Another object of the present invention is to provide a cosmetic composition effective for anti-inflammatory action and skin smoothness using a stimulating composition for rapid testing of natural killer cell activity.

The object of the present invention is not limited to the object mentioned above, and other objects not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the above object, the present invention provides a stimulating composition for a rapid test of natural killer cell activity comprising IL-2, IL-12, IL-18 and a basal medium as active ingredients.

In a preferred embodiment, the IL-12 and the IL-18 are included in a weight ratio of 1:3 to 1:10.

In a preferred embodiment, the IL-2 is 700 IU/mL to 5000 IU/mL, the IL-12 is 1 ng/mL to 4 ng/mL, and the IL-18 is 3 ng/mL to 40 ng/mL. do.

In addition, the present invention is a separation step of separating lymphocytes from the peripheral blood of the measurement subject; A culture step of culturing the separated lymphocytes with the stimulatory composition of any one of claims 1 to 3 for 18 to 48 hours or less; and a test step of examining the activity of the natural killer cells obtained in the culturing step.

In a preferred embodiment, the stimulatory composition rapidly increases the activity of natural killer cells obtained from the lymphocytes within 24 hours from the culturing step.

In a preferred embodiment, the inspection step includes fluorescent staining the target cells; reacting the fluorescently stained target cells with the natural killer cells; and measuring the intensity of the fluorescent substance released when the target cell attacked by the natural killer cells is destroyed.

In addition, the present invention provides a pharmaceutical composition for preventing or ameliorating symptoms caused by a virus, comprising any one of the above-described stimulating compositions for rapid testing as an active ingredient.

In a preferred embodiment, the pharmaceutical composition is formulated as a spray formulation.

In addition, the present invention is any one of the above-described stimulation composition for rapid testing; And it provides a cosmetic composition comprising a cosmetic base.

The present invention has the following excellent effects.

First, according to the present invention, a stimulating composition for rapid testing of natural killer cell activity capable of stimulating natural killer cells so that the activity of natural killer cells can be measured within 18 to 48 hours at the latest by culturing lymphocytes isolated from peripheral blood, and a stimulating composition for rapid testing of natural killer cell activity. Using the natural killer cell activity rapid test method, immunity can be measured and the possibility of infection of infectious diseases can be predicted.

In addition, according to the present invention, various diseases caused by viruses can be prevented and/or improved by using a stimulating composition for rapid testing of natural killer cell activity through a pharmaceutical composition for preventing or improving symptoms caused by viruses.

In addition, according to the present invention, it is effective in anti-inflammatory action and skin smoothness by using a stimulating composition for rapid testing of natural killer cell activity through a cosmetic composition.

These technical effects of the present invention are not limited to the above-mentioned range, and even if not explicitly mentioned, the effects of the invention that can be recognized by those skilled in the art from the description of specific contents for the practice of the invention described later included of course

1 is a graph showing the results of measuring the activity of a natural killer cell stimulating composition according to an embodiment of the present invention and cultured natural killer cells over time in normal people and cancer patients.

Figure 2 is a graph showing the average activity according to the culture time based on the results measured in Figure 1.

Terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "comprise" or "having" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features or It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present invention, they should not be interpreted in an ideal or excessively formal meaning. don't

The term "basal medium" as used herein refers to any medium capable of supporting the growth of cells. The basal medium supplies standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, energy sources, buffer systems and essential amino acids.

Characteristic parts of each of the various embodiments of the present invention can be partially or entirely combined or combined with each other, technically various interlocking and driving are possible, and each implementation can be implemented independently of each other or together in an association relationship may be carried out.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like reference numbers used to describe the invention throughout the specification indicate like elements.

The technical feature of the present invention is that by measuring the activity of natural killer cells, the possibility of cancer as well as immunity can be measured, and the possibility of various diseases such as infectious diseases can be predicted. it is confirmed In addition, in order to measure immunity and predict the possibility of disease by measuring the activity of natural killer cells, it is important how quickly natural killer cells can be activated and measured. The development of a natural killer cell stimulating composition that can increase the cytotoxicity of natural killer cells to the maximum, and the development of a rapid test method for natural killer cell activity using this composition.

Therefore, the stimulating composition for rapid testing of natural killer cell activity of the present invention includes IL-2, IL-12, IL-18 and a basal medium as active ingredients. When activating natural killer cells through numerous experiments, natural killer cells produced much more IFN-g when using a basal medium containing IL-12 and IL-18 at a certain ratio than IL-2 alone, and the titer This is because it was confirmed that

IL-2 is a glycoprotein with a molecular weight of 14 to 17 kDa that is produced when T cells recognize and activate antigens. Since it promotes cell growth, it is known as a substance that promotes growth by acting on NK cells, enhances the killing ability of NK cells, and promotes their growth by acting on B cells. As an embodiment, the stimulating composition of the present invention may contain 700 IU to 5000 IU of IL-2 per 1 mL of the basal medium.

IL-12 is produced in dendritic cells (DC), macrophages, and B cells, induces the production of IFN-γ and TNF-α in NK cells and T lymphocytes, and reduces the production of IL-4, which inhibits IFN-γ. As a substance that increases the cytotoxicity of NK cells and CD8+ cytotoxic T lymphocytes, it has a close relationship with the IL-2 signaling system in NK cells, as well as IL-12 receptor β1 and IL-12 in NK cells. It is known to induce the expression of the receptor β2 to express and activate proteins related to the IL-12 signaling system. This mechanism has been well demonstrated in the ability of NK cells to produce IFN-γ and to kill target cells. The IL-12 receptor β2 appears to play an important role in IL-12 function, which has been reported to inhibit Th2 generation while simultaneously advancing Th1 generation. Signaling of IL-12 in T cells and NK cells is included in the JAK-STAT signaling system, and the activity of the IL-12 receptor β2 plays an important role in inducing phosphorylation and activating STAT4, a transcription factor. As an embodiment, IL-12 may be included in the stimulating composition of the present invention in an amount of 1 ng to 4 ng per 1 mL of the basal medium.

IL-18 is produced by macrophages and is a proinflamatory cytokine. It binds to the IL-18 receptor and, together with IL-12, triggers an immune response to bacteria or virus-infected cells and increases the production of IFN-γ in NK cells and T cells. It is a substance that serves As an embodiment, the stimulating composition of the present invention may contain 3ng to 40ng of IL-18 per 1mL of the basal medium.

The basal medium is any medium capable of supporting the growth of cells, and examples of the basal medium include Dulbecco's Modified Eagle Medium (DMEM), DME/F12, Minimum Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (G-MEM), PF CHO (SAFC Biosciences) and Iscove ( Iscove's modified Dulbecco's medium. In the examples below, BME was used as a basal medium.

As described above, IL-12 and IL-18 included in the stimulatory composition of the present invention may be included in the basal medium at a constant ratio. In particular, the ratio exhibiting the optimal effect is experimentally in the range of 1:3 to 1:10. It was a case where it was included in the weight ratio.

Next, the natural killer cell activity rapid test method of the present invention includes a separation step of separating lymphocytes from the peripheral blood of the subject; A culture step of culturing the isolated lymphocytes with any one of the above-described stimulating compositions for 48 hours or less; and an inspection step of examining the activity of the natural killer cells obtained in the culturing step.

Here, the above-described stimulating composition in the culturing step can rapidly increase the activity of natural killer cells obtained from lymphocytes within 24 hours.

The inspection step includes fluorescent staining of target cells; reacting the fluorescently stained target cells with the natural killer cells; and measuring the intensity of the fluorescent substance released when the target cell attacked by the natural killer cells is destroyed. In this way, in the inspection step, cancer cells (Target cells) are fluorescently stained in vitro, reacted with self-activated lymphocytes, that is, natural killer cells (Effector cells), and the fluorescence intensity emitted from the Target cells destroyed by being attacked by Effector cells is measured. The killing ability of effector cells was evaluated. That is, the higher the cytotoxic activity, the higher the measured fluorescence intensity.

As an embodiment, K562 cell line was used as a target cell, and Calcein AM was used as a fluorescent dye. Calcein AM is a lipid-soluble diester fluorogenic esterase substrate and can cross cell membranes. Calcein AM (non-fluorescence) is converted to Calcein (fluorescence) by hydrolysis by intracellular esterase of undamaged cell membrane in living cells, resulting in fluorescence. The esterase activity is maintained by intact membranes of living cells, resulting in fluorescence. However, in the case of damaged cell membranes of dead cells, esterase activity is lost and fluorescence cannot be maintained (in the case of dead cells, fluorescence cannot be maintained).

In this way, when the target cell (K562 cell line) is fluorescently stained with Calcein AM and reacted with the effector cell (NK cell), the target cell attacked by the effector cell is destroyed and emits a fluorescent material. In other words, fluorescent material comes out only when living target cells are destroyed. The more damaged by the effector cell, the higher the fluorescence intensity emitted from the destroyed target cell. Fluorescence intensity emitted from the target cell can be measured with a device capable of measuring fluorescence intensity. According to this principle, the cytotoxic activity of effector cells can be confirmed.

Next, the pharmaceutical composition for preventing or improving symptoms caused by a virus of the present invention includes the above-described stimulating composition of the present invention as an active ingredient. When the pharmaceutical composition of the present invention includes a carrier or excipient, water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used at least one, but are limited thereto It is not, and all conventional carriers and excipients can be used.

In addition, when the pharmaceutical composition for preventing or improving symptoms caused by a virus of the present invention is formulated, a conventional filler, extender, binder, disintegrant, surfactant, anticoagulant, lubricant, wetting agent, flavoring agent, emulsifier or preservative is further included. and can be implemented in all known formulations. As an embodiment, it may be implemented in a spray form.

Next, the cosmetic composition of the present invention is a stimulation composition for rapid inspection; and a cosmetic base. In particular, the stimulation composition for rapid testing may be used in an amount of 0.1% to 50% by weight based on the total weight of the cosmetic composition. As described above, when the stimulating composition for rapid testing of natural killer cell activity is applied to the skin, the activity of NK cells in the skin is increased to promote the secretion of various cytokines, and various secreted cytokines cause allergic rhinitis, allergic dermatitis, and atopy. It was effective in improving inflammation, such as dermatitis, acne, and inflammation caused by insect bites, and had the effect of quickly removing itching. It was confirmed that the skin regeneration effect through the treatment of burns and wounds and the removal of deposited pigments were very good, and that it was also effective in skin whitening.

The cosmetic composition of the present invention differs only by including the stimulation composition for rapid test of the present invention in a certain weight, using a known cosmetic base using a cosmetic composition manufacturing method including emulsion, liposome, salt dispersion, etc. Since it can be implemented as a body lotion, skin lotion, essence, cream, etc., a detailed description thereof will be omitted.

Example 1

After adding 3600 IU/mL of IL-2, 2 ng/mL of IL-12, and 10 ng/mL of IL-18 to the basic medium Eagle (BME), mix them evenly to obtain a stimulating composition (Media D) for rapid testing of natural killer cell activity. prepared.

Comparative Examples 1 to 3

Comparative Example Compositions 1 to 3 (Media A, Media B, Media C) were prepared in the same manner as in Example 1, except for having the component ratios shown in Table 1 below.

Media	Ingredients
Media A	Add 3600 IU/mL of IL-2 to Basic Medium Eagle (BME)
Media B	Add 3600 IU/mL of IL-2 and 2 ng/mL of IL-12 to Basic Medium Eagle (BME)
Media C	Add 3600 IU/mL of IL-2 and 10 ng/mL of IL-18 to basic medium Eagle (BME)

Example 2 The prepared stimulant composition for rapid testing of natural killer cell activity was placed in a spray container and prepared as a spray formulation.

Experimental Example 1

Lymphocytes were isolated from peripheral blood and cultured for 3 days using the compositions obtained in Example 1 and Comparative Examples 1 to 3, respectively, with natural killer cells cultured for 14 days, and the titer of natural killer cells and the production of natural killer cells in the culture medium The amounts of interferon gamma, IL-8 and IL-10 were measured and the results are shown in Table 2 as follows.

The titer was measured using the K562 cell line, a cancer cell line, and the titer was evaluated by measuring the extent to which natural killer cells directly kill the K562 cell line. The activity test process was performed using a conventional method, and the result value was expressed as a percentage (%) of dead cancer cell lines.

1. The control group (K562 only control group) was prepared as follows.

(1)Media only

Fluorescence measurement of the culture medium itself

(2) Natural glass (Negative control)

Measures the amount of fluorescence emitted by the target cell self-destruction in the natural state (culture medium + target cell)

(3) Maximum glass (Positive control)

Measure the amount of fluorescence emitted from the target cell when the target cell is lysed to the maximum by treatment with 1% Triton-X 100 (culture medium + target cell + 1% Triton-X 100)

(4) Sample

Measure the amount of fluorescence emitted from the target cell destroyed by the effector cell by treating the target cell and the effector cell together (Target cell + Effector cell)

2. The test method is as follows.

(1) A target cell (K562) was prepared by fluorescent staining as follows.

(1-1) After culturing the K562 cells, collect them in a 15 ml tube and centrifuge at 1500 rpm for 5 minutes. The supernatant was discarded, and the pellet was released with 1 ml of culture medium (RPMI1640 + 10% FBS), followed by cell counting.

(1-2) After adjusting the cell count to 1×10 ⁶ /ml in a 1.5 ml tube, 10 μl of Calcein AM (1 mg/DMSO 1 ml) staining solution was added and 5% CO ₂ was maintained at 37° C. for 30 minutes. Incubation was carried out. (Cells were inverted at 15-minute intervals.)

(1-3) After incubation, centrifugation was performed at 1500 rpm for 5 minutes. After removing the supernatant and resuspension with 1 ml of the culture medium (RPMI1640 + 10% FBS), the cells were washed by centrifugation at 1500 rpm for 5 minutes. (It was repeated twice.)

(1-4) After discarding the supernatant, 1 ml of the culture solution was added, and the cells were counted with a hemocytometer to prepare a cell concentration ranging from 1×10 ⁴ /50 μl/well to 2×10 ⁵ /ml.

(2) Effector cells were prepared as follows.

(2-1) Immune cells (including NK cells) were centrifuged at 1500 rpm for 5 minutes and washed once with culture medium (RPMI1640 + 10% FBS).

(2-2) After discarding the supernatant, 1 ml of the culture solution was added, and cell counting was performed with a hemocytometer to prepare a cell concentration 10 times that of the target cell.

(3) Control and target cells / effector cells were prepared in a 96-well plate as follows and dispensed into wells. All tests were performed in triplicate.

(3-1) Natural glass Well (Negative control)

Culture medium (RPMI1640 + 10% FBS) 100 μl + Target cell 100 μl

(3-2) Maximum Glass Well (Positive control)

Culture medium (RPMI1640 + 10% FBS) 70 μl + Target cell 100 μl + 1% Triton X-100 30 μl

(3-3) Culture well

Culture medium (RPMI1640 + 10% FBS) 200 μl

(3-4) Sample Well

Target cell (K562) 100 μl + Effector cell (NK cell) 100 μl

(4) The culture medium and the sample were mixed for good reaction.

(5) Incubation was performed at 5% CO ₂ and 37°C for 4 hours.

(6) After the reaction, centrifugation was performed at 1500 rpm for 5 minutes.

(7) After centrifugation, 120 μl was transferred to a black plate and the fluorescence value was measured with a Fluoroskan ascent plate reader.

3. Cytotoxicity was calculated as follows using the cytotoxic activity formula.

Cytotoxicity (%) of effector cells

= (Fluorescence value of sample - natural free fluorescence value) / (maximum free fluorescence value - natural free fluorescence value) X 100

Media	IFN-g (ng/mL)	IL-8 (pg/mL)	IL-10 (pg/mL)	Potency (%, E/T=10:1)
Media A	8.82	11.15	29.61	40.57
Media B	14.3	9.15	18.9	69.12
Media C	11.55	21.65	18.5	62.15
Media D	122.53	20.6	13.76	79.49

From Table 2, Media D, the stimulating composition of the present invention, had the highest titer and the highest concentration of interferon gamma, so when cultured with natural killer cells containing IL-2, IL-12 and IL-18 at a certain ratio, natural killer cells It can be seen that the activity can be greatly increased. Experimental Example 2

Lymphocytes were isolated from the blood of terminal cancer patients and normal people and cultured with the stimulating composition (Media D) for rapid testing of natural killer cell activity obtained in Example 1, and after 24 hours and 48 hours, natural killer cell activity (K562 cell line killing power) was measured and the results are shown in Tables 3 and 4 and FIGS. 1 and 2, respectively.

incubation day		day 0	day 1	day 2
cancer patient 1	0.51	20.00	24.71
cancer patient 2	-9.18	6.23	24.71
cancer patient 3	-1.91	44.67	62.61
average cancer patients	-3.53	23.63	37.34

incubation day		day 0	day 1	day 2
normal person 1	1.26	98.59	83.10
normal person 2	1.58	89.31	92.15
normal person 3	1.21	82.99	91.09
normal person 4	1.65	71.06	108.80
normal person 5	-0.15	90.58	109.12
normal person 6	-1.30	102.60	77.60
normal person 7	2.12	87.10	91.67
normal person average	0.91	88.89	93.36

From the results of Tables 3 and 4 and FIGS. 1 and 2, in the case of normal people in a healthy state, stimulating natural killer cells to measure natural killer cell activity can be raised to the maximum in 24 hours of culture, but terminal cancer patients In the case of , natural killer cell activity increased very slowly, and even after 48 hours of culture, it was found that the average activity was less than 40%. The activity of natural killer cells can be measured within an hour, and if the activity value of the measured natural killer cells is high, the immunity is good, so there is no high probability of getting a disease even when exposed to a disease source such as a virus, and if the activity value is low, the immunity is not good. It clearly shows that exposure to a disease agent increases the probability of developing a disease.

Therefore, if the natural killer cell activity rapid test method of the present invention is used, countermeasures can be prepared in advance before getting sick. If a rapid test method for killer cell activity is performed and the degree of immunity of each subject is notified, it will be possible to provide a preventive method that can preemptively respond to diseases.

In addition, if the natural killer cell activity rapid test method of the present invention is used, the level of immunity can be grasped as described above, so that the recovery of immunity of cancer patients under treatment can be tracked and managed, which can be expected to be of great help in cancer treatment. .

Experimental Example 3

A spray formulation prepared with the stimulating composition (Media D) in Example 2 was sprayed once in the morning and evening during the winter and spring seasons when colds are prevalent for 30 minutes in a nursing home. After the change of season, it was sprayed once every 2-3 days. The results are shown in Table 5.

test subject	Number of people	before treatment	after treatment
Elderly in convalescence (age 70 and older)	30 people	About 6-7 cold cases occur each year, especially 2 elderly people die of influenza in the year before treatment.	1 cold patient
Nursing home staff and officials	20 people	3 patients	Cold symptoms disappear in 1-2 days

As shown in Table 5, as a result of spraying once in the morning and evening, there was one elderly person with a cold. As a result, it was confirmed that cold symptoms disappeared in 1-2 days. This shows that natural killer cells located in the mucous membrane can be directly stimulated and activated when the pharmaceutical composition containing the stimulatory composition of the present invention is sprayed on the skin and especially the mucous membrane.

In addition, although it was not presented as an experimental example, it was confirmed that it was effective for herpes virus infection, where the lips swollen when tired, and that it was cured in about 3 days on average if applied immediately when the lips swollen.

The above experimental results show that the pharmaceutical composition containing the stimulating composition (Media D) of the present invention as an active ingredient can prevent or improve cold symptoms caused by a cold virus, as well as prevent or improve diseases caused by viruses such as herpes virus. show what is

Therefore, it can be seen that the present invention can be used for the prevention and treatment of various viral infections such as cold virus infection, that is, corona virus, by the stimulating composition (Media D).

Although the present invention has been shown and described with preferred embodiments as described above, it is not limited to the above embodiments, and to those skilled in the art within the scope of not departing from the spirit of the present invention Various changes and modifications will be possible.

Claims (9)

1. A stimulating composition for rapid testing of natural killer cell activity comprising IL-2, IL-12, IL-18 and a basal medium as active ingredients.
2. According to claim 1,

   The IL-12 and the IL-18 are included in a weight ratio of 1: 3 to 1: 10, characterized in that the natural killer cell activity stimulating composition for rapid testing.
3. According to claim 2,

   The IL-2 is 700 IU / mL to 5000 IU / mL, the IL-12 is 1 ng / mL to 4 ng / mL, and the IL-18 is 3 ng / mL to 40 ng / mL. A stimulatory composition for a rapid test of killer cell activity.
4. Separation step of separating lymphocytes from the peripheral blood of the measurement subject;

   A culture step of culturing the separated lymphocytes with the stimulatory composition of any one of claims 1 to 3 for 18 to 48 hours or less; and

   A rapid test method for natural killer cell activity comprising: a test step of examining the activity of the natural killer cells obtained in the culturing step.
5. According to claim 4,

   A rapid test method for natural killer cell activity, characterized in that the stimulating composition rapidly increases the activity of natural killer cells obtained from the lymphocytes within 24 hours in the culturing step.
6. According to claim 4,

   The inspection step includes fluorescent staining of target cells; reacting the fluorescently stained target cells with the natural killer cells; and measuring the intensity of the fluorescent substance released when the target cell attacked by the natural killer cells is destroyed.
7. A pharmaceutical composition for preventing or improving symptoms caused by a virus, comprising the stimulating composition for rapid testing according to any one of claims 1 to 3 as an active ingredient.
8. According to claim 7,

   The pharmaceutical composition is a pharmaceutical composition for preventing or improving symptoms caused by a virus, characterized in that formed in a spray formulation.
9. The stimulation composition for rapid examination according to any one of claims 1 to 3; And a cosmetic composition comprising a cosmetic base.

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