TWI479152B - Methods of detecting allergic asthma by immunization with chitin - Google Patents

Description

Method for detecting allergic asthma patients by using chitin-induced immune response

The present invention relates to a method for diagnosing an allergic asthma patient, and more particularly to a method for diagnosing an allergic asthma patient by measuring the response of chitosan to peripheral blood immune cells taken from a patient.

To illustrate the related art of the present invention, the definitions and characteristics of hypersensitivity, asthma and chitin are described first.

An allergic reaction is an inflammatory reaction caused by the body fluids or cellular mediators of the immune system, which can cause tissue damage or even death. These reactions can be divided into four types depending on their induction mechanism, each of which has its specific action molecule and clinical manifestations. Clinically, most common allergic diseases, including asthma, allergic rhinitis and hay fever, are usually type 1 allergic reactions.

The first type of allergic reaction is induced by allergens, which may be proteins or small molecules of chemicals [Roitt et al., 1998]. The first type of allergic reaction occurs very quickly and can appear immediately within minutes of exposure to the allergen. In general, these reactions can be divided into two phases: the first phase is the beginning of sensitization, when the first exposure to an allergen, the plasma cells in the body will produce relative antibodies, especially antibody IgE, The antibody has a high affinity for tissue mast cells and FcεRI receptors on the surface of basophils in blood, and also has low affinity IgE receptors (FcεRII) on the surface of lymphocytes, eosinophils, platelets and macrophages. Or CD23) combined. When an IgE antibody binds to an Fc receptor on the surface of mast cells or basophils, the individual is sensitized. If the patient is exposed to the same allergen (the second stage of the first type of reaction), the allergen molecule will bind to the IgE on the cell receptor, causing mast cells or basophils to go. The granules act to release vasoactive substances [Lane and Lee, 1996; Boyce, 2004], in which the content of histamine is more and the effect is the fastest. Histamine is the main component in mast cell granules, which accounts for about 10% of the weight of the granules, which causes smooth muscle contraction, increased mucus secretion, and increased blood vessel expansion and permeability. In addition to histamine, there are many serotonins in the mast cell granules that cause smooth muscle contraction, increased respiratory rate, and further promote the release of histamine, causing allergic symptoms to worsen [Peters et al. , 1998].

Asthma, one of the most common allergic diseases, is a chronic respiratory inflammatory disease whose main symptoms include difficulty breathing, wheezing, chest tightness, slow cough, etc. These symptoms often last for a long time, but most patients can heal. It is also possible to control symptoms without hindering normal life. The main features of asthma are (1) inflammation of the respiratory tract, swelling of the mucous membrane above the respiratory tract; (2) narrowing of the respiratory tract, making breathing difficult; and (3) the respiratory tract becomes too sensitive due to inflammation, so the respiratory tract is sensitive to many irritants. It is easy to react strongly and cause asthma attacks. It is generally believed that the cause of asthma is quite complex, including the interaction of genetic and environmental stimuli, and for many patients, the disease may have taken root in infancy, so genetic and environmental factors have a beginning and development of the disease. Quite important impact. Clinically, it can be divided into two types: exogenous asthma and endogenous asthma: exogenous asthma is an allergic and immune asthma, and patients may be allergic to air or blood, like dust mites, pollen, perfume. Or viral antigen induces asthma; endogenous asthma is a primary, non-allergic asthma, caused by chronic, recurrent bronchial obstruction and has nothing to do with allergens [Kay, Trends Mol Med. 2005;11(4) :148-152].

Chitin is a natural polymer that is second only to cellulose in nature and is widely distributed in cell walls such as algae and mushrooms [Bulawa, 1993] and in exoskeletons such as shrimp, crab and insects [Neville et al .,1976]. Chitin is a high molecular polymer similar to cellulose chemical structure. N-acetylglucosamine and glucosamine are monomers, and the linear polysaccharides bonded by β-(1,4) glycosidic bonds are high. Molecules [Tharanathan and Kittur, 2003]. Since chitin itself is a highly crystalline solid, it forms internal and external hydrogen bonds in the molecular structure, so its water solubility is poor, and the hydrogen bond structure is very strong, making chitin in physical properties and chemical properties better than other polysaccharides. Classes have higher stability and are less prone to fracture and deformation under natural conditions [Merzendorfer and Zimoch, 2003].

In recent years, chitin has been widely used due to its lack of natural polymer sources, and it has also been found to have many biomedical properties, including (1) no toxicity and obvious side effects on animals [Minami Saburo et al., 2002 (2) It has biological activity and can enter the small intestine to absorb the fat of the milk formed by the food fat, to reduce the chance of digestive absorption of the milk globule, and to reduce the effect of triglyceride and cholesterol in the blood [Zacour et al., 1992], in order to improve obesity and prevent cardiovascular diseases. In addition, chitin can absorb and excrete negatively charged chloride ions in salt to prevent blood pressure rise [Freier et al., 2004]; (3) Academically, chitin is thought to protect the liver, inhibit tumor growth and growth, treat gastric ulcers, control blood pressure, and boost the function of the immune system [Esteban et al., 2000]; and (4) is highly biocompatible. Does not stimulate the production of antibodies. Therefore, chitin and its derivatives are widely used in medicine, chemical, agricultural, food processing, etc. [Tharanathan and Kittur, 2003; Synowiecki and A1-Khateeb, 2003; Khor and Lim, 2003].

Chitin has been found to enhance the immune activity of animals, and it can help inhibit the growth of tumors and protect the host against pathogen infections [Murata et al., 1991; Nishimura et al., 1985]. Confirmed. Including 30% or 70% of chitosan and carboxymethyl-chitin can effectively induce macrophage production of cytotoxicity and inhibit tumor growth [Nishimura et al., 1984; Nishimura et al., 1985]; At the same time, chitin was injected into mice and found to activate the complement system in vivo, including promoting the increase of C3 and C5 complement proteins, which is very similar to zymosan [Tokura] Et al., 1999]; In addition, related studies have further shown that chitin can effectively promote the secretion of nitric oxide by macrophages and enhance their resistance to bacteria and tumors [Peluso et al., 1994]. Therefore, chitin and chitosan are believed to enhance non-specific immune responses, such as promoting macrophage proliferation, secreting nitrogen compounds, and increasing bactericidal and anti-tumor capabilities.

In addition, Shibata et al. demonstrated that chitin enhances the immunity of TH1 against mycobacterial antigens [Shibata et al., 2001]. When mice were immunized with mycobacterial protein (MPB-59) alone, there would be The TH2 reaction is induced, which is characterized by an increase in the total amount of IgE and specific IgG1 in the serum. In addition, TH2 cells produce IL-4, IL-5 and IL-10, and spleen TH1 cells are not detected (spleen) TH1 cells) produced interferon-gamma (IFN-γ) and specific serum IgG2a (specific serum IgG2a). If chitin-conjugated mycobacterial protein (MPB-59) is used to immunize mice, the TH2 response is attenuated, and there is a clear tendency for the spleen TH1 response to increase, and IgG2a in serum will also increase. Therefore, chitin should enhance the specific immune response, promote antibody production and activate T cell responses.

Furthermore, Shibata et al. found that chitin can significantly stimulate the secretion of interferon-gamma by TH1 cells to increase macrophage activity [Shibata et al., 1997], while macrophages phagocytose these non-antigenic chitin particles, which will produce Cytokines associated with TH1 response such as IL-12, IL-18 and TNF-α [Shibata et al., 1997], and further activation of TH1 cells or production of IFN in natural killer cells (NK cells) -γ, in order to inhibit the response of TH2, redirect the entire immune response to TH1, and effectively reduce IgE in serum and eosinophilic white blood cells in the lung [Shibata et al., 2000], thereby achieving the effect of alleviating allergy symptoms. Similar findings have found that this treatment can improve the pathological response of the respiratory tract [Musumeci et al., 2008].

It is known that the inflammatory response of allergic asthma is highly correlated with the type 2 T helper cell (TH2) immune response induced by allergens. Therefore, it is known to use chitin to induce C. Interferon-type (IFN-γ) production inhibits the TH2 response of ovalbumin (OVA)-sensitized asthmatic mice to the pathological symptoms of asthmatic mice. When discussing the mechanism of chitin-protected ovalbumin-sensitized mice, we found that chitin can hardly stimulate the formation of interferon-gamma in the immune cells of sensitized or treated mice compared with normal mice. However, this feature has not been used to diagnose patients with allergic asthma.

It is an object of the present invention to provide a rapid and convenient method of diagnosing an allergic asthma patient.

Another object of the present invention is to provide a method for diagnosing allergic asthma by treating two peripheral blood immune cell samples taken from a subject.

In order to achieve the above object, the present invention provides a novel method for detecting an allergic asthma patient by using a chitin-induced immune response, which has the following steps: a first sample containing peripheral blood immune cells is collected from a subject and a second sample; adding chitin to the first sample; respectively measuring the amount of interferon-gamma in the first sample and the second sample to obtain a first value and a second value; And dividing the first value by the second value to obtain a ratio, wherein when the ratio is below a threshold, the subject is diagnosed as an allergic asthma patient.

The detailed description of the drawings and the preferred embodiments are set forth in the accompanying drawings.

In order to make the principles of the present invention more understandable, the properties of the TH1 and TH2 pathways, as well as the transcription factors associated with T cell responses, are described first.

TH1 and TH2 paths:

Under normal conditions, TH1 and TH2 cells maintain an equilibrium state, TH1 subpopulation mainly secretes IFN-γ, TNF-β and IL-2; while TH2 subpopulation produces IL-4, IL-5, IL. -6, IL-10 and IL-13 and other cytokines [Romagnani et al., 1997]. TH1 cells are mainly involved in cellular immune responses. The secreted cytokines activate cell cytotoxic, inflammatory and delayed allergic reactions. TH2 is involved in the production of antibodies, which can effectively stimulate B cell proliferation and produce antibodies (such as IgE). Against free microbes, the cytokines produced by TH2 cells are often associated with the regulation of allergic reactions [Romagnani, 1994]. In addition to the induction of different cytokines that specifically affect T-helper cell differentiation, gene transcription factors also have cell line specificity: it is currently known that TH1 and TH2 cell development require STAT-1 and STAT-6, respectively [Grogan et al Curr Opin Immunol, 2002; 14(3): 366-72]. In addition, transcription factors have also been found to be selectively expressed in TH1 and TH2 cells, for example, GATA-3 is well expressed in TH2 cells, whereas T-bet is well expressed in TH1 cells [Grogan et al., Curr Opin Immunol, 2002; 14 ( 3): 366-72].

It is generally believed that the allergic inflammatory response of asthma is related to the imbalance between TH1 and TH2 cells, especially the interleukin-4 (IL-4) and IL-13 secreted by TH2 cells are most valued [Dittrich et Al., 1997]. Because interferon-gamma has the ability to inhibit the synthesis of immunoglobulins and the differentiation of TH2 cells, in the absence of interferon-type C, it will promote allergic inflammatory reactions; and the cytokine IL-4 secreted by TH2 cells will Stimulates B cells to increase the secretion of IgE in a large amount [Gascan et al., 1991], and also inhibits macrophage activity, blocks the effect of IFN-γ on the activation of macrophages, and stimulates the growth and development of mast cells; as for IL-5 It can stimulate the growth and differentiation of eosinophils, and is therefore considered to be an important factor in causing allergic reactions [Robinson et al., N Engl J Med, 1992; 326(5): 298-304].

In addition, asthma is generally considered to be a chronic respiratory inflammatory disease associated with Th2 hyperimmune reaction, mainly by a large number of cytokines such as IL-4, IL-5 and IL-13, which drive eosinophils and mast cells to aggregate. And activation, leading to chronic lung inflammation, resulting in respiratory hyperreactivity (AHR), and can make recovery of airway block and mucus hyperplasia and respiratory inflammation [WillsKarp et al., Science, 1998; 282 (5397): 2258-2261]. Although excessive Th2 immunity caused by large amounts of IL-4, IL-5 and IL-13 should be associated with allergic diseases, there is increasing evidence that the low Th1 immune response is also responsible for the development of allergic diseases.

Transcription factors associated with T cell responses:

Immature helper T cells may affect their differentiation pathways due to different cytokine stimuli and different transcription factors, or the interaction of cytokines and transcription factors. For example, in the presence of interleukin-4 (IL-4), cells are stimulated to differentiate into type 2 helper T cells; in the presence of IL-12, cells are stimulated to differentiate into type 1 helper T cells [ Science, 295 (5553): 338-42]. In the context of molecular biological knowledge, the present invention selects the following transcription factors as experimental materials:

1. GATA-3: a specific transcription factor for type II helper T cells that initiates transcription of the IL-4 gene, thereby promoting the differentiation of unactivated helper T cells toward the second type of helper T cells. . In addition, GATA-3 also blocks the development of type 1 helper T cells by inhibiting IL-12 [Thomas et al., 2001].

2. T-bet: is a specific transcription factor of type 1 helper T cells, which promotes differentiation of unactivated helper T cells. The interferon-type G produced by IL-12 stimulation induces the expression of the T-bet gene, and T-bet also promotes the production of interferon-gamma, which is positive feedback relationship [Szabo et al., Science, 295 (5553): 338-42].

The present invention utilizes chitin to stimulate peripheral blood mononuclear cells of allergic asthmatic patients and normal healthy persons to secrete type C interferon, and thereby observe the difference in immune response. The experimental results showed that the concentration of interferon-type interferon produced by peripheral blood mononuclear cells in patients with allergic asthma was significantly lower than that of normal healthy people.

In the part of the relationship between the rate of C-type interferon production and the severity of allergens in patients with allergic asthma, the results showed that patients with hyperallergenic reactions to dust mite had lower IFN-γ production ratio. On the contrary, in patients with low allergic reactions to dust mites, the ratio of C-type interferon production is higher. Therefore, the immune response to chitin in asthma patients may be partially related to the patient's specific IgE response to dust mites. Among them, dust mites are rich in chitin, and have a specific concentration of IgE and IFN-γ concentration induced by chitin stimulation; although crab shells and shrimp shells also contain chitin, but It appears that there is no correlation between the degree of allergy to the patient's specimen and chitin stimulation.

Accordingly, the present invention discloses a method for detecting an allergic asthma patient by using a chitin-induced immune response, which has the following steps: a first sample containing a peripheral blood immune cell and a second test are taken from a subject a chitin to the first sample; respectively measuring the average amount of the interferon-gamma in the first sample and the second sample to obtain a first value and a second value; The first value is divided by the second value to obtain a ratio, wherein when the ratio is below a threshold, the subject is diagnosed as an allergic asthma patient.

The immune cells used in the present invention are preferably mononuclear cells; the ELISA system is used to quantify the type C interferon; and the ratio of the interferon-gamma production ratio is defined as the average amount of the interferon-type interferon in the chitin-stimulated sample. The average amount of interferon-type C was measured in a sample without chitin added, wherein the average amount of interferon-type C was obtained according to an ELISA standard curve.

The method of the present invention can also be used to diagnose healthy people without allergic asthma. When the C-type interferon production ratio is higher than 10, the subject is diagnosed as a healthy person without allergic asthma. When the C-type interferon production ratio was between 6-10, the subject was diagnosed as a suspected allergic asthma patient.

The method of the invention can also be used to diagnose patients who are severely allergic to dust mites. When the ratio of interferon-gamma production was less than 2, the subject was diagnosed as a patient with severe allergies to dust mites.

Example: Example 1:

The present invention collects 30 allergic asthma patients and 30 non-patients for research. The inclusion criteria for patients were: newly diagnosed allergic asthma patients (experimental group), and healthy people without history (control group). The exclusion criteria were: patients with a history of allergies. The following are the results of allergen tests for newly diagnosed allergic asthma patients.

Total IgE in patients with allergic asthma:

Allergic levels of allergens in patients with allergic asthma (Table I):

Allergic levels of allergens in allergic asthma patients (Table II):

Chitin preparation:

After the allergen test, chitin microparticle powder (about 4-5μm in diameter) was provided and tested by Dr. Chen Jinkun, a research and development unit of CNPC. After the micron chitin was confirmed by the endotoxin free test, it was prepared into a suspension of 10 mg/ml in phosphate buffered saline (PBS), and after high temperature sterilization, it was stored in a refrigerator at 4 ° C. When the experiment is carried out, mix it evenly and use it directly.

Isolation of peripheral blood mononuclear cells:

The blood collected from healthy people and asthma allergic patients was placed in a green tube (containing Na-Heparin anticoagulant). After centrifugation, the upper plasma was extracted and frozen to a refrigerator at -80 ° C, and stored for later use. Then, the phosphate buffer (Phosphate Buffered Saline---PBS) and the remaining blood (blood) were mixed and transferred to a centrifuge tube containing Ficoll. After centrifugation, it was found that the centrifuge tube was divided into three layers. (The principle is to use the concentration gradient of Ficoll), in which the white turbid color in the middle is layered into the desired Peripheral Blood Mononuclear Cell (PBMC), which is separated and washed with PBS 2~ 3 times, placed on ice.

Cell culture:

After separating PBMC, the cells were cultured in a 96 wells plate, reconstituted with RPMI 1640, and added with 10% fetal bovine serum (FBS) and 1% L-glutamic acid (L- Glutamine) and 2% Penicillin + Streptomycin into Complete medium, add 200 μl medium (for 96-well plate) or 2 ml medium (medium) to each well. (for 24-well plates) and control the number of cells in each well to 2 × 10 ⁵ (for 96-well plates) or 2 × 10 ⁶ (for 24-well plates), add to each sample Different stimuli, cultivate different appropriate time. The well plate was placed in an incubator and cultured at 37 ° C in a 5% carbon dioxide (CO ₂ ) environment.

Sample preparation (cell culture supernatant and cells):

After placing the wells of the cultured cells in the incubator, the wells were taken out on the fifth day, the cell liquid was slightly mixed, collected in a microcentrifuge (eppendorf) and centrifuged, and after centrifugation, it was seen at the bottom of the eppendorf. There will be a pellet of cells. Next, the cell culture supernatant (supernatant) will be collected into a new centrifuge tube, clearly labeled and frozen in a -80 ° C refrigerator for enzyme-bound immunosorbent assay (Enzyme-Linked). Immuno-sorbent Assay---ELISA).

Enzyme combined immunosorbent assay (ELISA):

The present invention detects IFN-γ by ELISA. First, the anti-human cytokine capture antibody was diluted 250-fold with a coating buffer, attached to a 96-well ELISA plate, and 100 μl was added to each well. 4°C one night (overnight). Then use washing buffer - 0.05% Tween-20 PBS - rinse several times (as indicated in the specification), then add the assay diluent to the well and fill it with non-specific At the position of the sex, 200 μl was added to each well and allowed to stand at room temperature for 1 hour. After that, it was washed several times with the washing buffer, and the standard for 2-fold serial dilution (up to a maximum initial concentration of 2000 pg/ml IFNγ) and the duplicated sample were added, and 100 μl was also added to each well and placed at room temperature. 2 hours. After washing several times with the washing buffer, 100 μl of a detection antibody (250-fold diluted with the sample diluent) was added to each well and allowed to stand at room temperature for 1 hour. Then, after washing several times with the washing buffer, avidin-horseradish peroxidase (Avidin-HRP) was added and allowed to stand at room temperature for 30 minutes. Finally, after washing several times through the washing buffer, 100 μl of the substrate (tetramethylbenzidine-TMB) was added to each well. After 15 minutes of reaction, 50 μl of 2N H 2 SO 4 was added to each well to terminate the reaction, and the ELISA analyzer was used. In the reader, the absorbance at a wavelength of 450 nm is measured.

Example 2: Micron chitin stimulates peripheral blood mononuclear cells to produce interferon-gamma:

In order to determine whether micron chitin can induce the production of IFN-γ, the present invention co-cultures different concentrations of chitin (5, 10, 20, 40, 80 μg/ml) with peripheral blood mononuclear cells of normal healthy persons, and The production of IFN-γ was detected at different times (4-7 days). PBMC was stimulated by 20 μg/ml chitin after being taken by a healthy person or a asthmatic patient. Those who do not have additional stimulation (blank) serve as a control group. After 5 days of culture, the culture supernatant was collected to measure the amount of IFN-γ produced by ELISA. The results of representative data of the experimental results are shown in Fig. 1. After peripheral blood mononuclear cells were stimulated with different concentrations of chitin, IFN-γ was produced. Among them, the optimal response was 20μg/ml, which was 1072.2pg/ml on the 4th day, 1897.4pg/ml on the 5th day, 2687.1pg/ml on the 6th day, and 4708.6pg on the 7th day. /ml. At the same concentration of chitin stimulation, the concentration of IFN-γ released increased with the increase of reaction time. As shown in Fig. 2, in order to compare the amount of IFN-γ produced by healthy people and asthmatic patients with PBMC, PBMC was obtained from healthy people and asthma patients, and then chitin and phytohemagglutinin ( Phytohemagglutinin; PHA), and anti-CD3 plus anti-CD28 stimulation. Those who do not have additional stimulation (blank) serve as a control group. After the required number of days of culture (2 days for PHA, 3 days for anti-CD3 plus anti-CD28, and 5 days for chitin), the culture supernatant was collected to measure IFN-γ production by ELISA. the amount.

Example 3: Differences in C-type interferon between peripheral blood mononuclear cells in asthmatic patients and healthy individuals:

In order to compare the difference of C-type interferon secreted by peripheral blood mononuclear cells between asthmatic patients and healthy people, peripheral blood mononuclear cells were cultured with micron chitin and ELISA was used to detect the type C in the cell culture supernatant. Interferon concentration, and the data is chitin-induced IFN-γ production ratio--using the ELISA standard curve to calculate the concentration of each sample, The mean IFN-γ concentration (double overlap) produced by the mass stimulation was divided by the mean IFN-γ concentration of the blank tester. The cell culture of the blank tester was not added to chitin.

In the healthy part, as shown in Figure 3, a total of 24 samples were collected. PBMC was stimulated by 20 μg/ml chitin after being taken by a healthy person or a asthmatic patient. Those who do not have additional stimulation (blank) serve as a control group. After 5 days of culture, the culture supernatant was collected to measure the amount of IFN-γ produced by ELISA. Most of the C-type interferon production ratios fall between 15 and 25, and the ratio of a few samples has reached more than 40. The ratio of most healthy people is above 10, and only one sample is below 10. . However, in the asthma patient group, there are 12 specimens. The ratio of the interferon-type production of most specimens is between 4 and 6, with an average of about 5, and the ratio of some specimens is less than 2. Next, the data of the two groups of asthmatic patients and healthy people were measured by the nonparametric Mann-Whitney U test or the student t-test. The alpha values obtained by the assay are all below 0.001, ie, p < 0.001 indicates a statistically significant difference, the average ratio of healthy people is about 20, and the average of asthma patients is about 4, and after calculation, the obtained p value is 0.0008, which is less than the set alpha value of 0.001, therefore, this result is statistically significant.

To understand the expression of the transcription factors GATA-3 and T-bet in chitin-stimulated PBMC, PBMC were stimulated by 20 μg/ml chitin after being taken from healthy or asthmatic patients. Those who do not have additional stimulation (blank) serve as a control group. After 5 days of culture, the cells were collected to analyze the expression of the transcription factors GATA-3 and T-bet via immediate RT-PCR. The data is shown in Figure 4.

From the above results, it can be seen that the PBMC of asthmatic patients and healthy people have significant differences in the production of interferon-type C when stimulated by micro-chitin.

Example 4: The association between asthmatic patients with low response to chitin and allergic diseases:

Next, it is the IgE part that is specific to a particular allergy. As shown in Figure 5, in order to understand the relationship between chitosan-induced interferon-gamma production and total serum IgE in asthmatic patients, PBMC was obtained from healthy people and asthma patients at a rate of 20 μg/ml. Stimulation stimulation. Those who do not have additional stimulation (blank) serve as a control group. After 5 days of culture, the culture supernatant was collected to measure the amount of IFN-γ produced by ELISA. The data herein is the ratio of interferon-gamma production of asthmatic patients (n=8) and the total amount of serum IgE associated therewith.

However, among allergens in Taiwan, dust mites are the first to cause children with allergic asthma attacks, and dust mites can be subdivided into house dust mites and dust mites. Therefore, the present invention firstly treats asthma patients to house dust. The degree of allergic reaction of cockroaches and dust mites (detecting the specific IgE concentration of dust mites) was compared with the ratio of C-type interferon production. The results are shown in Fig. 6. The invention observes that the patient with hyperallergenic reaction to dust mites has a lower ratio of C-type interferon production. Conversely, in patients with low allergic reaction to dust mites, the ratio of C-type interferon production is higher, and Part of the dust mites, especially statistically significant (p = 0.0106). The present invention contemplates that the immune response to chitin in asthma patients may be partially related to the patient's specific IgE to dust mites. In addition, the present invention also compares the test results of other allergens (crab, shrimp, dog fur, mites and proteins) with the ratio of interferon-gamma production. The results are shown in Fig. 7, but no correlation is found therein. .

In summary, the present invention provides a novel method of diagnosing an allergic asthma patient.

The disclosure of the present invention is a preferred embodiment. Any change or modification of the present invention originating from the technical idea of the present invention and being easily inferred by those skilled in the art will not deviate from the scope of patent rights of the present invention.

In summary, this case, regardless of its purpose, means and efficacy, is showing its technical characteristics that are different from the conventional ones, and its first invention is practical and practical, and it is also in compliance with the patent requirements of the invention. I will be granted a patent at an early date.

Figure 1 is a schematic diagram showing the interferon-type C produced by peripheral blood mononuclear cells (PBMC) obtained by a group of healthy subjects and a group of asthmatic subjects in a dose range of chitin Concentration profile.

Figure 2 is a schematic diagram for comparing the difference in the concentration of interferon-type C produced by peripheral blood mononuclear cells (PBMC) obtained from a group of healthy subjects and a group of asthmatic subjects under different stimuli. .

Figure 3 is a schematic diagram for comparing the difference in the concentration of interferon-type C produced by peripheral blood mononuclear cells (PBMC) obtained by a group of healthy subjects and a group of asthmatic subjects under chitin. . This ratio (chitin/blank) was determined by dividing the concentration of interferon-gamma produced by chitin stimulation by the concentration of interferon-gamma of the blank tester. Individual ratios and mean ratios measured by PBMCs from healthy subjects (n=24) and asthmatic subjects (n=12) were obtained under Student's t-test, where ** * represents p < 0.001.

Figure 4 is a schematic diagram showing the relative expression of the genes GTA-3 and T-bet in the chitin-stimulated PBMC, which were obtained by real-time PCR (polymerase chain reaction) analysis. The relative expression of this gene was obtained by dividing the average performance of transcription factors produced by chitin stimulation by the average expression of transcription factors without chitin (blank tester).

Figure 5 is a schematic diagram showing the association of chitin-induced interferon-gamma production with total serum IgE levels in asthmatic patients. The type C interferon (chitin/blank) is obtained by dividing the concentration of interferon-gamma produced by chitin stimulation by the concentration of interferon-gamma of the blank tester, and the total serum IgE of the patient with asthma The concentration concentration correlation was (n=8, R ² =0.1861).

Figure 6 is a schematic diagram showing the correlation between the production of interferon-gamma in asthmatic subjects and the degree of allergy to dust mites. The grade of allergic to dust mite in asthmatic subjects is classified as highly allergic (specific IgE concentration ≧ 1.0 mg/dl for dust mites) and low allergy (<10.0 mg/dl). The correlation result for house dust mites was p = 0.0815, and the correlation result for dust mites was p = 0.0106, and the results were obtained under Student's t-test.

Figure 7 is a schematic diagram showing the association of hepatitis C interferon production with the severity of allergic reactions to common allergens in asthmatic patients. Patients were classified as allergic (specific IgE concentration of allergens ≧1.0 mg/dl) and non-allergic (<1.0 mg/dl) according to the degree of allergic reaction to common allergens. The patient's interferon-gamma production ratio was correlated with allergic/non-allergic to common allergens and was obtained under Student's t-test and p=0.2344.

Claims (6)

A method for detecting an allergic asthma patient by using an immune reaction induced by chitin, comprising the steps of: taking a first sample and a second sample containing peripheral blood immune cells; and adding a particle size of 4-5 a micron-sized chitin to the first sample; measuring the average amount of the interferon-gamma in the first sample and the second sample on the fifth day to obtain a first value and a second value And dividing the first value by the second value to obtain a ratio, wherein when the ratio is below 10, the subject is diagnosed as an allergic asthma patient. A method for detecting an allergic asthma patient by using a chitin-induced immune response according to the first aspect of the patent application, wherein the peripheral blood immune cells are monocytes. A method for detecting an allergic asthma patient using a chitin-induced immune response according to the first aspect of the patent application, wherein the measurement of the amount of the interferon-gamma comprises performing an enzyme-binding immunosorbent assay (ELISA). A method for detecting an allergic asthma patient using a chitin-induced immune response according to claim 1, wherein the measurement of the amount of interferon-gamma comprises using an ELISA standard curve to obtain the average. A method for detecting an allergic asthma patient using a chitin-induced immune response according to the first aspect of the patent application, wherein when the ratio is higher than 10, the subject is diagnosed as a healthy person. A method for detecting an allergic asthma patient using a chitin-induced immune response according to the first aspect of the patent application, wherein when the ratio is less than 2, the subject is diagnosed as a patient who is severely allergic to dust mites.