KR101242797B1 - Composition for diagnosis of allergic rhinitis comprising agents determining lactoferrin expression level - Google Patents

Abstract

The present invention relates to a composition for diagnosing allergic rhinitis comprising a lactoferrin expression level measuring agent and a method for providing information for diagnosing allergic rhinitis comprising the same. The composition of the present invention is a composition having anti-inflammatory and anti-allergic activity, and may be usefully used for the prophylaxis and treatment of diseases caused by allergens.

Description

Composition for diagnosis of allergic rhinitis comprising agents determining lactoferrin expression level}

The present invention relates to a composition for diagnosing allergic rhinitis comprising a lactoferrin expression level measuring agent and a method for providing information for diagnosing allergic rhinitis comprising the same.

Allergic rhinitis is a typical allergic disease caused by the combination of genetic and environmental factors along with allergic asthma. Allergy refers to hypersensitivity reactions from normal and does not cause symptoms in normal people, but hypersensitivity reactions in allergy patients. Typical deteriorating factors for rhinitis are climate change, cold, air pollution, and stress.

Family history is one of the causes of allergic rhinitis. It is estimated that sensitization of antigens occurs in childhood, as 75% of patients begin symptoms before age 25. Infants born in households with allergy sufferers are at the highest risk of 10 years of life. If you are allergic to one of your parents, your child will be allergic to 50% of the time, and if both parents have allergies, your chances will increase to 75%. Atopic dermatitis, bronchial asthma, and allergic rhinitis are the three major allergic diseases, and these series of outbreaks are called allergic marches because they occur sequentially from a young age.

Symptoms of allergic rhinitis include seizure sneezing, clear runny nose, and itching and nasal congestion in the eyes and nose. Symptoms of sneezing and runny nose are usually severe during the morning waking up to decrease in the afternoon and continue to show nasal congestion. Itching occurs not only in the nose, but also in the eyes, neck, and ears, so it should be considered during treatment.

Congestion symptoms are the most common symptom, accounting for more than half, chronic and poor quality of life. It is followed by runny nose and sneezing, and other symptoms include tears, headache, olfactory deterioration, and obstructive nasal secretion. Complications may include otitis media, sinusitis, and sore throat.

Treatment of allergic rhinitis includes environmental therapy (avoidance therapy), avoiding allergens (antigens), the causative agent, drug therapy, and immunotherapy. Avoiding allergens is the most important treatment, but fundamentally avoiding them is practically difficult, so avoiding a single treatment alone is not enough to achieve a sufficient therapeutic effect, and it is necessary to control symptoms with proper medication.

Once allergic rhinitis develops, about 20% of the symptoms disappear naturally as they enter puberty or adulthood, but many cases last a lifetime, so proper prevention and treatment is important. Long-term allergic rhinitis can cause chronic rhinitis-like changes, which can lead to otitis media, nasal polyps, sinusitis olfactory, and chronic cough.

There are many ways to diagnose rhinitis, which can be applied in various ways depending on the patient and the treatment environment, and not all tests are performed at all times.

First, listening to your medical history will help you diagnose rhinitis. It is important to know in detail the age, occupation, type and severity of symptoms, age of onset, causes, residential environment, exposure to allergens, complications, allergic history, family history, treatment history and course. About 40% of allergic rhinitis patients have allergic diseases in their close family within uncles. Allergic rhinitis may be suspected if the family history of allergic disease and symptoms appear from childhood, seasonal changes, persistent and repeated symptoms over time, and symptoms associated with changes in living conditions.

People with allergies also have a nonspecific hypersensitivity reaction to nonspecific stimuli such as sudden temperature changes, cold air, tobacco smoke, and pollutants.If the symptoms worsen when cleaning the house, you may suspect hypersensitivity to house dust mites. If you have symptoms since moving to a new home, you should look closely at the changed environment to find the cause.

As a laboratory test, serum total Ig E test, specific Ig E test, blood eosinophil and eosinophil cationic protein, nasal smear test and the like can be performed. Biopsies include skin test and antigen-induced test. Among these, skin test is a simple, economical and diagnostic value test to identify the causative agent with or without reactivity.

The physical examination is mainly used to examine the inside of the nasal cavity using the nasal passages. The pale and swollen nasal mucosa is characteristic of allergic rhinitis. In children, long-term rhinitis causes a disorder of blood circulation in the nasal cavity. This may look bluish and your nose may be itchy, rubbing your nose frequently with your hands, or having horizontal wrinkles on your nose.

Simple sinus X-rays can detect thickening of the nasal mucosa, the presence of septal curvature, and general turbidity in the sinuses.

Nasal smear is a test to determine the distribution of epithelial and inflammatory cells in the nasal mucosa.

The skin test is the simplest and primary test for determining the cause of allergic disease. However, allergens that are positive in the skin test cannot be interpreted as the cause of allergic rhinitis (antigens), because even if there are no symptoms, the skin test may be positive. Therefore, the patient's medical history, examination findings, and other examination findings are combined.

However, these methods of diagnosis and test may be burdensome or painful to the subject, and the sensitivity and specificity of the allergen reaction may be inferior.

Against this background, the present inventors confirmed that the concentration of serum lactoferrin was significantly low when exposed to allergens caused by dust mites in patients with allergic rhinitis. Therefore, the present inventors confirmed that lactoferrin concentration may be a criterion for diagnosis of allergic rhinitis. The invention was completed.

One object of the invention to provide an allergic rhinitis diagnostic composition comprising a lactoferrin expression level measurement agent.

Another object of the present invention to provide an information providing method for diagnosing allergic rhinitis comprising the composition.

As one aspect for achieving the above object, the present invention provides a composition for diagnosing allergic rhinitis comprising a lactoferrin expression level measuring agent and an information providing method for diagnosing allergic rhinitis using the same.

As used herein, the term "lactoferrin" is a strong antiviral antimicrobial substance most commonly found in colostrum of humans and dairy cows. Lactoferrin has the important properties of binding and liberating iron, which is colorless when not combined with iron, but is red when combined with iron. When it was first discovered, it was called red protein. Soluble in water but insoluble in ethanol and weak in heat. Milk is rich in milk, and among them, colostrums are the most abundant. Human colostrum contains 6 ~ 8mg / l and breast milk secreted during lactation contains about 2mg / l. In addition, colostrum of milk contains 0.1 ~ 0.2mg / ℓ in 1.2mg / ℓ. Lactoferrin binds iron 260 times more strongly than transferrin (iron transport protein), and its action with iron is the center of various functions of lactoferrin. Lactoferrin requires iron for bacterial growth, and lactoferrin blocks this proliferation of iron before bacteria are supplied, thereby inhibiting the growth of bacteria and killing them. It also has a synergistic effect on neutralizing and agglomerating the surface charges of cells with basic (-) charges and purifying bacteria with immunoglobulins. In addition, bacteria that are useful to the human body, such as bifidus and Lactobacillus bacteria do not cause antibacterial activity, but in general, harmful bacteria function not only growth inhibition through the absorption of iron, but also directly through the cell membrane to kill and kill. Lactoferrin affinity receptors can be found in most immune cells. In addition, the combination with iron and glass to control the amount of iron in the body to prevent anemia. Lactoferrin has antiviral properties such as HIV (acquired immune deficiency) virus, hepatitis C (HCV), and herpes virus (HHV). Pure natural lactoferrin contained in colostrum of humans or cows has the functions of combating the herpes (HHV) virus and suppressing the reproduction of AIDS virus, and also has antifungal capacity. In relation to the regulation of immune function, the combination of various immune cells increases immunity and increases macrophage activity. Synthesis with various proteins may help strengthen immune secretion organs. As iron combined with lactoferrin is released, it acts as a catalyst for oxygen supply to cells and at the same time induces the binding of iron to antioxidant proteins. In addition, lactoferrin itself is a powerful antioxidant that helps the body's defense role. In the present invention, it was used as a marker protein for diagnosing allergic rhinitis.

As used herein, the term "agent for measuring expression level of lactoferrin" is an agent used to confirm the presence and degree of expression of lactoferrin in a biological sample in order to predict and diagnose an allergic rhinitis disease. Materials can be used. For example, the amount of lactoferrin may be confirmed using antibodies, proteins, compounds, pigments, radioactivity, and the like that specifically bind to lactoferrin, but is not limited thereto. Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunity Electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto. According to a specific example of the present invention, lactoferrin expression level was confirmed by ELISA using an antibody.

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody means an antibody that specifically binds to a marker of the present invention, which antibody is cloned into an expression vector according to a conventional method to obtain a protein encoded by the marker gene. From the obtained protein, it can be prepared by a conventional method.

The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

Antibodies used as lactoferrin expression level measurement agents of the present invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.

As used herein, the term "allergic rhinitis" means that the nasal mucosa is hypersensitivity to a specific substance and that the allergic agent (antigen) is exposed to the nasal mucosa. Inflammatory reactions are caused by various mediators secreted by inflammatory cells mediated by IgE antibodies. It is characterized by three main symptoms: seizure sneezing, clear runny nose, and stuffy nose, and allergic rhinitis can be suspected if you have two or more of these symptoms. In addition to the characteristic symptoms, it may be accompanied by itching around the nose, headache, and olfactory deterioration. Complications may include otitis media, sinusitis, and pharyngitis. Allergic rhinitis is classified into intermittent allergic rhinitis, which occurs only in a short period of time depending on the duration of symptoms, and persistent allergic rhinitis, which occurs over a month or more, and is mild according to the severity of symptoms. ) And moderate severe. It is also divided into seasonality, which occurs only in one particular season, and perennial seizures throughout the year. Seasonal allergic rhinitis is often associated with the season in which pollen is flying, but it is chronic, continues throughout the year, and occurs seasonally regardless of season.

As used herein, the term "allergen" refers to an allergen causing allergens, and house dust mites, house dust, pollen, animal hair, mold, milk, eggs, nuts, fish, peaches, buckwheat and the like are generally problematic. The four representative allergens in Korea are house dust mite, wormwood pollen, cat hair and alternaria fungus. For the purposes of the present invention, allergens may mean pathogenic substances that cause allergies derived from dermatophagoides pteronyssinus.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the development of allergic rhinitis.

 In a preferred embodiment of the present invention, it was confirmed that allergic rhinitis can be diagnosed by measuring the expression level of immunoglobulin and the concentration of lactoferrin expressed in serum by an allergen derived from a tick mite (FIG. 3 and Table 2). .

Therefore, in order to increase the specificity of the diagnosis of allergic rhinitis using the composition for the purpose of the present invention, the composition may further include an agent for measuring the expression level of allergen specific-IgE.

As used herein, the term "IgE" is an antibody that is involved in type 1 hypersensitivity in a mammalian immune response. As a monomeric antibody having four domains, a protein that plays an important role in allergic reactions, the present invention diagnoses allergic rhinitis. Used as a marker protein for.

The term "allergen specific-IgE" is an immunoglobulin that is expressed against a specific antigen during type 1 hypersensitivity, in which an IgE-class antibody specific for that antigen is bound to mast cells or basophils. This induction response can occur locally or systemically, depending on the type and amount of mediator these cells produce. This reaction is also called immediate hypersensitivity because it occurs within an hour of the antigen's arrival. This immediate hypersensitivity reaction is called systemically strong anaphylaxis, and can even cause asphyxiation and circulatory death. However, even when exposed to the same allergen, this reaction may or may not appear in some people, so this reaction is called atopy, which means a specific constitution.

As used herein, the term "agent for measuring the expression level of allergen-specific -IgE" is used to confirm the presence and expression of allergen-specific-IgE in a biological sample in order to predict and diagnose allergic rhinitis diseases. As the material to be used, various materials known in the art may be used. For example, the amount of protein can be determined using an antibody that specifically binds to allergen specific-IgE. The measurement method for this is the same as the case of measuring the expression level of the lactoferrin.

The present invention also relates to a method for providing information for diagnosing allergic rhinitis, including a composition for diagnosing allergic rhinitis, including an agent for measuring the expression level of lactoferrin.

More specifically, the present invention preferably comprises the steps of obtaining a sample from a subject suspected of allergic rhinitis, measuring the level of lactoferrin expression in the sample obtained in the step, the lactoferrin concentration is 307 ng / If less than ml provides a method of providing information for diagnosing allergic rhinitis comprising the step of determining the individual as allergic rhinitis. The allergic rhinitis may be a disease caused by a tick. An information providing method for diagnosing allergic rhinitis of the present invention may include obtaining a sample from an individual, and measuring the level of lactoferrin expression in the serum obtained in the step.

Further allergic rhinitis can be diagnosed by measuring allergen specific-IgE expression level measurements in a sample obtained from an individual.

As used herein, the term "individual" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine with different levels of expression of lactoferrin or / and IgE. . In a specific embodiment of the present invention, the sera of an allergic rhinitis patient were targeted.

Assays for measuring lactoferrin and IgE expression levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay , FACS, protein chips, and the like, but are not limited thereto. Through the above assay methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in allergic rhinitis suspects can be compared, and allergenicity is determined by measuring the significant expression concentrations of IgE and lactoferrin. It can be used for diagnosis and treatment of suspected disease, preferably allergic rhinitis suspect.

As used herein, the term “antigen-antibody complex” refers to a combination of a marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be measured quantitatively through the magnitude of a signal of a detection label. .

In a specific embodiment of the present invention, ELISA was used to measure protein expression levels. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. For example, by attaching an antibody to a solid support and reacting a sample, a labeled antibody that recognizes an antigen of the antigen-antibody complex is enzymatically developed or labeled for an antibody that recognizes an antigen of the antigen-antibody complex. It can be detected by the sandwich ELISA method which attaches the secondary antibody thus enzymatically and colors it. The degree of complexation of the marker protein with the antibody can be confirmed to diagnose an allergic disease, preferably an allergic rhinitis suspect.

Through the above detection methods, the lactoferrin expression level in the normal control group is compared with the lactoferrin expression level in the allergic disease, preferably in the allergic rhinitis suspect, and the specificity of the actual onset and diagnosis of the allergic rhinitis suspect and You can increase the sensitivity. Additionally, by comparing the expression levels of IgE, the sensitivity and specificity of allergic rhinitis diagnosis can be further increased. That is, by measuring the expression level of the markers (lactoferrin and IgE) of the present invention from a sample of an individual suspected of allergic rhinitis, and comparing the two by measuring the expression level of the marker of the present invention from a sample of a normal individual, Rhinitis can be determined.

In the specific experimental example of the present invention, despite the increased production of lactoferrin in nasal secretion after NPT treatment, the value of lactoferrin in the Dpt-specific AR group was compared with that of the asymptomatic Dpt-specific control group and the healthy control group without atopy. It was confirmed that the decrease significantly. Immunohistochemical analysis also showed that lactoferrin expression was reduced in the nasal mucosa of the Dpt-specific AR group compared to asymptomatic Dpt-specific controls (FIG. 4).

In addition, serum lactoferrin values were evaluated during the diagnostic evaluation process to prove specific AR patients in the control group. ROC curves were used to calculate optimal values and to select ranges with sensitivity above 60% and specificity below 50%. As a result of the ROC curve, the optimal concentration level of lactoferrin for allergic rhinitis diagnosis was less than 307 ng / mL. In addition, a combination of lactoferrin values and serum Dpt-specific IgE values were considered to improve specificity and sensitivity. The combination result (less than 307 ng / mL lactoferrin and greater than Dpt-specific IgE 00.35 kU / L) significantly improved the specificity to 79.2%. These results suggested that a combination of serum lactoferrin values and serum Dpt-specific IgE could be used as serological markers for early detection of Dpt-specific AR.

The composition of the present invention is a composition having anti-inflammatory and anti-allergic activity, and may be usefully used for the prophylaxis and treatment of diseases caused by allergens.

FIG. 1 is a result of collecting two-dimensional electrophoresis (2-DE) separation from nasal lavage fluid (NLFs) before (a) and after (b) nasal challenge test (NPT) including a Dpt allergen. Spot doubling or decreasing compared to the spot intensity of 2-DE of 209 protein spots in two gels was analyzed using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Significantly highly regulated (ratio 5: 5) Spot 1 after NTP treatment with Dpt demonstrated lactoferrin.
FIG. 2 shows (a) the change in lactoferrin value with Dpt allergen in nasal lavage fluid (NLFs) during nasal challenge test (NPT). Closed circle, group 1 (Dpt-specific AR); Open circle, group 2 (asymptomatic Dpt-specific control); Open triangle, group 3 (healthy control without atopic symptoms). Lactoferrin values after NPT treatment with Dpt are notable in Group 1 but in Groups 2 and 3. Statistical significant validation was assessed by Wilcoxon's signed-rank test. (b) Comparison of serum lactoferrin values in the control group was measured by ELISA, and statistically significant differences were assessed by ANOVA between the groups. The line represents the value of each group. * P <0.05, AR, allergic rhinitis
FIG. 3 shows the Receptive Response Characteristic (ROC) curve, which combines the serum values of lactoferrin (a) and the values of lactoferrin and serum Dpt-specific IgE (b), is used as an optimal separation to distinguish group 1 from other groups. It was. Arrows indicate the optimal values of the combination with lactoferrin (less than 307 ng / mL lactoferrin and greater than 0.35 kU / L Dpt-specific IgE).
Figure 4 shows the results of immunohistochemical analysis of lactoferrin expression in the nasal mucosa of the control group of groups 1 (a) and 2 (b). Lactoferrin expression was lower in Group 1 (Dpt-specific AR) than in Group 2 (Dpt-specific control). Arrangement: right panel, x 100; Left panel, x 400. AR, allergic rhinitis

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Research Project

Of the total patients, 117 patients who visited the subject were included in this study. Dpt-sensitive AR (Group I; n = 43); Three groups were asymptomatic Dpt-sensitive controls (Group II; n = 22) and healthy controls without atopy (Group III; n = 52). All groups underwent NPT with Dpt allergen and observed symptoms of rhinitis (eg nasal, nasal itching and runny nose). Group I is a group of allergic rhinitis patients with a history of rhinitis and positive reaction to NPT containing Dpt. Group III showed healthy volunteers without atopic disease that had a negative response to NPT.

Example 2 Skin Pric Test and Analysis of Total Ig E and Dpt-Specific Ig E

Nine representative inhalable allergens, the American dust mite (Dermatophagoides pteronyssinus), the American dust mite (Dermatophagoidesfarina), cat hair, dog hair, wood mixtures, grass mixtures, mugwort, ragweed, and Alternaria allergens (UK, Brad The skin test was performed using Ford Bend). Each test result is represented by A / H ratio. Serum levels of total IgE and Dpt-specific IgE were measured by fluorescence immunoassay using the Pharmacia cap system (Sweden, Uppsala, Pharmacia-Upjohn). Atopy was defined as an A / H ratio> 1 in a skin test or a common inhalation allergen of positive Dpt-specific IgE.

Example 3: Nasal mucosal challenge test and nasal wash

After prenasal examination, nasal mucosal challenge (NPT) and nasal wash were performed as described above. In summary: The test was performed after minimizing the effects of daily irritation by keeping them in the same place for 30 minutes in the morning. To confirm the presence of nonspecific hypersensitivity reactions, a non-mucosal induction test was performed with saline, except for patients who showed a positive response. . In non-specific hyperresponsive groups, 8 mm filter paper immersed in tick allergen solution (5000 BU /; manufactured by Allergopharma, Germany) until the allergic reaction occurs in the anterior portion of the lower nasal concha, or up to 10 minutes The test was performed. Thereafter, the symptoms of rhinitis such as nasal itch, nasal itching, nasal congestion, and runny nose were observed. Each symptom was expressed as a score of (Asymptomatic), 1, 2, and 3 (Severe symptoms that make life difficult). A total score of 3 or more was defined as positive for the nasal mucosal challenge test.

In addition, nasal lavage tests were performed before, 10, 30, 60, 180 and 360 minutes after the nasal mucosa challenge test. After the head was tilted back, 5 ml of saline was injected into one nostril by using a syringe, and the breath was stopped so that the saline did not fall behind the neck. After 10 seconds, he bowed his head to receive the nasal washing solution in the polypropylene tube. It was. This process is repeated for the opposite nose. In order to maximize cell viability, samples were centrifuged at 300 X g for 10 minutes and then the supernatant was prepared for slide morphological analysis, and the supernatant was stored at -80 ° C for later analysis.

Example 4 Measurement of Inflammatory Markers in Nasal Wash

The slide analysis confirmed the number of inflammatory cells, including eosinophils, neutrophils, and lymphocytes, and eosinophil cationic protein levels (ECP) in nasal washes were measured using a Pharmacia CAP system.

Example 5 Two-Dimensional Electrophoresis and Image Analysis

To identify the protein that causes AR, proteomic analysis was performed using the nasal wash solution before and after the nasal mucosa challenge test using Dpt in AR patients. Each sample was immersed with trichloroacetic acid / acetone for desalting and concentrating and subjected to two-dimensional electrophoresis analysis as described above. One-dimensional (equipotential electrophoresis) involves placing 1 mg of protein per sample on the negative side of a fixed pH gradient strip and applying a nonlinear pH gradient from 3 to 10 (Sweden, Uppsala, Amersham Pharmacia Biotechnology), etc. Point electrophoresis was performed at 80 kVh. SDS-PAGE was performed using a polyacrylamide gel at a concentration of 9% to 17% by two-dimensional separation, and the dye was carried out to reach the bottom end of the gel. At the end of each run, the gels were stained with Coomassie Brilliant Blue G-250 (US, CA, Hercules, Bio-Rad) for relative quantification of proteins. Stained gels were analyzed using GS-710 Image Densitometer (Bio-Rad) and Image Master Platinum 5 (USA, NJ, Piscataway, GE healthcare).

Example 6: Intra-gel Decomposition and Mass Spectrum Analysis

Mass spectra analysis was performed to confirm the protein name. The spots of 2-DE images obtained with nasal cleaning solution before and after nasal mucosal induction test using Dpt were analyzed and the protein names of the spots where the spot intensity increased or decreased more than two times were examined. Spots showing more than twofold sensitization were separated from the gel, cut into small pieces, and then treated with trypsin as in the previous paper (US, WI, Madison, Promega). Protein mass spectrometry was performed using a MALDI TOF / TOF analyzer equipped with a 355-nm laser and postponed the extraction and operation of the reflector mode. Data processing of the spectra was performed using Data Explorer version 4.4 software (US, MA, Framingham, PerSeptive Biosystems). Protein Matching and Protein Name Search for Swiss-Plot and Matrix Science Search This was done through a database search of the National Center for Bioinformatics Database (NCBI) via the engine.

Example 7: Determination of Lactoferrin

In each group, lactoferrin levels were analyzed by ELISA using Bioxytech kit (USA, OR, Portland, Oxis international) using nasal lavage fluid obtained sequentially during Dpt nasal mucosal challenge test and serum obtained prior to challenge test. Diluted standard samples as directed by the manufacturer were not reused. Serum and nasal wash samples were diluted 1:50 and 1: 100. The minimum detection level of lactoferrin is 1.0 ng / ml.

Example 8 Immunochemical Detection of Lactoferrin

Immunohistochemical staining was performed in nasal tissues to identify differences in the location and expression of lactoferrin in the nasal mucosa of AR and asymptomatic sensitive control samples. Primary antibody to lactoferrin (1: 500; Oxis international) was reacted for 1 hour at room temperature. Ultravision detection system (USA, CA, Fremont, Thermo Scientific) was used, Novared substrate was used as chromosome, and sections were mounted with hematoxylin.

Example 9: Statistical Analysis

Results were given as mean ± SD values. Significant differences between the groups were measured by ANOVA. Wilcoxon's signed-rank test was used to compare lactoferrin obtained in stages during NPT treatment between groups. The obtained operational characteristic curve (ROC) was used to measure the diagnostic value of serum lactoferrin and Dpt-specific IgE for AR diagnosis and satisfied a 95% confidence interval. Sensitivity and specificity were calculated as optimized cut-off values. All analyzes were performed using SPSS13.0 software (US, IL, Chicago, SPSS Corporation). The p-value <0.05 was considered a statistically significant value.

Experiment result

Experimental Example 1: Characteristics of Research Projects

Dpt-specific AR (Group I; n = 43) based on sensitivity status, rhinitis history, NPT results; Three groups were asymptomatic Dpt-specific controls (group II; n = 22) and healthy controls without atopy (group III; n = 52). Clinical characteristics of the controls are summarized in Table 1. There were no significant differences across age or gender in the three groups. (P> 0.05, respectively) Dpt, Dpt-specific IgE and total IgE, which are skin response factors between groups 1, 2, and 3, although there was no significant difference in factors between groups 1 and 2 (P> 0.05, respectively) Showed a significant difference.

Experimental Example 2: Proteomic analysis of nasal washes (NLFs)

To confirm that the protein is a diagnostic marker of AR, proteomic analysis was performed on each sample prior to obtaining NLFs in AR patients and after treatment with NTP including Dpt. Two-dimensional electrophoresis (2-DE) results are shown in FIG. 1. In each gel, 311 and 308 spots were identified, of which 209 grouped protein spots were compared and analyzed. Protein spots showing more than two times increase or decrease in spot intensity comparison of 2-DE were identified by MALDI-TOF-MS. Spot 1 was significantly increased after the challenge test (ratio, 5.5, Figure 1) compared with the spot strength before the challenge test after NPT using Dpt, was confirmed as lactoferrin.

Experimental Example 3: Lactoferrin Level Change in Nasal Wash During Nasal Induction Test

To assess the relevance of lactoferrin, we measured successive changes in lactoferrin levels in NLFs after NPT treatment with Dpt. There was no significant difference between the three groups at the baseline level of lactoferrin before the NPT. The level of lactoferrin in group 1 was significantly increased 10 min after NPT treatment with Dpt (P = 0.035) and increased again after 3 hours. In comparison, Groups 2 and 3 did not change significantly. There was no significant correlation between lactoferrin levels and ECP levels, and inflammatory cells also did not show significant changes in each group.

Experimental Example 4: Serum Lactoferrin Levels

Lactoferrin values were measured in three groups of serum using ELISA to confirm the lactoferrin results obtained by MALDI-TOF MS (FIG. 2). Lactoferrin values were found to be significantly lower in Group 1 (235.3 ± 91.9 ng / mL) compared to Groups 2 and 3 (374.7 ± 70.1 ng / mL and 355.9 ± 159.3 ng / mL; P <0.5, respectively).

Experimental Example 5: Diagnostic Evaluation of Serum Lactoferrin

For the diagnosis of AR, fluorinated lactoferrin values were measured using the ROC curve method (FIG. 3). In order to calculate the best sensitivity and specificity, ROC curve was used to find the optimal separation value, and lactoferrin concentration was less than 307 ng / mL (<307 ng / mL). To distinguish group 1 from groups 2 and 3, the odds ratio of lactoferrin in AR was 7.29 (95% CI: 2.36-22.53), with sensitivity of 81.4% and specificity between 58% and 62.5%. The isolated serum Dpt-specific IgE value was greater than 0.35 kU / L (> 0.35 kU / L) (considering the general range of 0--0.35 kU / L), and the highest values of sensitivity and specificity were group 3 and group 1 It was used as a significant value to distinguish between them. In contrast, only 17% of specificity was used to distinguish group 1 from group 2 (Table 2).

Lactoferrin values were combined with serum Dpt-specific IgE values and measured again using ROC curves to improve the intensity of the diagnosis (FIG. 3). The results obtained were 76.7%, 79.2% and 94% of sensitivity and specificity, respectively, to distinguish Group 1 from Groups 2 and 3 (Table 2).

Claims (10)

(1) measuring the level of lactoferrin expression in a sample obtained from the individual; And
(2) an information providing method comprising determining the individual as allergic rhinitis if the concentration of lactoferrin is less than 307 ng / ml as a result of measuring the expression level of the lactoferrin.

The method of claim 1, further comprising measuring allergen specific-IgE expression levels in the sample of step (1).

The method of claim 2, wherein when the allergen-specific-IgE expression level is measured, when the amount of allergen-specific-IgE expression is greater than 0.35 kU / L, the method includes determining the individual as allergic rhinitis. .

The method of claim 1 or 2, wherein the allergic rhinitis is caused by Dermatophagoides pteronyssinus (Dpt).

The method of claim 1 or 2, wherein the sample comprises tissue, cells, whole blood, plasma, serum, saliva, sputum, cerebrospinal fluid or urine.

The method according to claim 1 or 2, wherein the level of lactoferrin expression in step (1) is determined by using an antibody specific for lactoferrin.

delete A composition for diagnosing allergic rhinitis comprising a lactoferrin expression level measuring agent, wherein the measuring agent is an antibody specific for lactoferrin.

The allergic rhinitis diagnostic composition according to claim 8, further comprising an antibody specific for allergen specific-IgE.
The composition for diagnosing allergic rhinitis of claim 8, wherein the allergic rhinitis is caused by Dermatophagoides pteronyssinus (Dpt).

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