KR20020075996A - Skin-test reagents and serum specific IgE, IgG subtypes detection kits, comprising crude extracts of spider mites such as citrus red mite, two-spotted spider mite and European red mite - Google Patents

Abstract

PURPOSE: Provided are skin test reagents containing crude extracts of spider mite(citrus mite, european red mite and two-spotted spider mite) and serum-specific IgE and IgG subtype detection kits, thereby easily and specifically diagnosing respiratory diseases caused by the spider mite. CONSTITUTION: The skin test reagent contains the crude extracts of spider mite which is any one selected from citrus red mite, european red mite and spotted spider mite in the amount of 1 mg/ml. A preserving agent is optionally added to the skin test reagent, wherein the preserving agent is glycerin. The serum-specific IgE and IgG subtype detection kit comprises a vessel containing a crude extract of citrus red mite, a detectable component-labeled antibody as a second antibody for spider mite specific IgE and IgG subtype, and a manual.

Description

Skin-test reagents and serum specific IgE, IgG subtypes detection kits, comprising crude extracts of spider mites containing leaf mite (mandarin mite, spotted mite, apple mite) such as citrus red mite, two-spotted spider mite and European red mite}

The present invention relates to techniques for diagnosing the cause of allergic diseases. Specifically, the present invention relates to a technique for diagnosing a case where leaf mite (mandarin mite, spotted mite, apple mite), which is one of the mites widely present in rural and urban areas including an orchard, is the cause of the allergic disease. It is about.

Chronic allergic diseases such as allergic rhinitis, bronchial asthma and atopic dermatitis are very important diseases in the national health, so that about 20-30% of the population of Korea is affected by allergic diseases. For the treatment of allergic diseases, the first and foremost task is to accurately diagnose the allergen causing the allergic disease.

In particular, allergens that cause allergic diseases vary greatly depending on the surrounding environment, and since the environment is different from country to country, allergen diagnosis techniques useful in one country are often not equally effective in other countries. Therefore, it is very important for the public health to develop a diagnosis technique based on the allergens that are important in our country.

The inventors have found that leaf mite is an important allergen in the Korean environment, and by developing a diagnosis technology for allergic diseases including the same, it is possible to expect innovative progress in diagnosing allergens of allergic diseases clinically.

Inhaling allergens that cause allergic diseases can be divided into indoor allergens released from house dust mites, pet hair, cockroaches, and indoor fungi, as well as outdoor allergens such as trees, grass, weed pollen, and outdoor fungi [Kim YY et al., Prevalence ofchildhood asthma based on questionnaire and methacholine bronchial provocation test. Clin Exp Allergy 1997; 27: 761-8.

Recently, the present inventors have newly discovered that leaf mite, tangerine mite, spot mite, and apple mite are allergens that cause allergic diseases commonly found in rural and urban areas of Korea. Citrus red mite ( Panonychus citri ), a spider mite (Phyder mite, Tetranychidae ) that inhabits citrus leaves and inhales chlorophyll, is the first to report an allergen causing bronchial asthma in citrus farmers (Kim YK) et al., New occupational allergen in citrus farmers: citrus red mite ( Panonychus citri ) .Ann Allergy Asthma Immunol 1999; 82: 223-8) Kim YK et al., Citrus red mite is the most common sensitizing allergen in citus farmers with asthma and rhinitis.Clin Exp Allergy 1999; 29: 1102-9; Kim YK et al., Citrus red mite ( Panonychus citri ) is a common sensitizing allergen among children living around citrus orchards.Anne Allergy Asthma Immunol 2000; 80: 200-4). The inventors also found that spotted mite and apple mite, which are widely distributed in the land area of Korea, are important as a cause of allergic diseases (Kim YK et al., Spider mite allergy in apple-cultivating farmers: european red mite ( Panonychus ulmi ) and two-spotted spider mite ( Tetranychus urticae ) may be important allergens in the development of work-related asthma and rhinitis symptoms.J Allergy Clin Immunol 1999; 104: 1285-92; Jee YK et al., Two-spotted spider mite ( Tetranychus urticae ): an important allergen in asthmatic non-farmers symptomatic in summer and fall months.Anne Allergy Asthma Immunol 2000; 84: 543-8).

Leaf mites are small animals that live on plant leaves and suck chlorophyll, and they are phylogenetic and belong to the same mites as house dust mites and storage mites. Spotted mite is widely distributed not only in apples, pears, grapes and other fruits but also in plants such as flowers, vegetables and weeds. In particular, the spotted mite has a high density of occurrence in crops that are frequently sprayed with pesticides and intensive cultivation management such as pruning. This is presumably due to the removal of natural enemies by pesticides, which do not play an important role in the control of the density of the natural enemies on the spotted mite, and the nutritional conditions of the host plants have improved, thus increasing the survival and reproduction rate of the spotted mite. Indeed, leaf mites, including spotted mites, did not matter until after World War II, although pests that caused damage to fruit trees were widely used after World War II. Its density has begun to increase, and recent studies have found it the most problematic pest in fruit farming.

Accordingly, the present inventors have completed the present invention by developing a skin test reagent, a serum specific IgE and IgG subtype antibody detection kit which can easily diagnose leaf mites as a cause of allergic diseases by standardizing these leaf mites qualitatively and quantitatively. Was done.

Accordingly, an object of the present invention is to provide a skin test reagent that can easily and specifically diagnose allergic diseases in which leaf mite (mandarin mite, spotted mite, apple mite), which has been previously determined to be unknown, is the causative agent. will be.

Still another object of the present invention is serum specific IgE, IgG, which can easily and specifically diagnose allergic diseases in which leaf mite (mandarin mite, spotted mite, apple mite) has been judged as the cause of the past. It is to provide a subtype antibody detection kit.

1 is an IgE immunoblot photograph showing the results of immunoblot analysis of Tangerine mite. M at the top of the picture is a molecular weight indicating protein, numbers 1 to 10 are serum of 10 allergic patients judged to be caused by leaf mite, C is a control serum, and B is a buffer solution.

Figure 2 is an IgE immunoblot photograph showing the results of immunoblot analysis of spotted mites. At the top of the photo, A is the serum of a patient who has sensitized to both spot mites and house dust mites, B is the serum of a patient who has only sensitized to spot mites, and from the left, M is the molecular weight marker and the number is the patient's serum. , C represents control serum and B represents buffer.

Methods for diagnosing allergens of allergic diseases include skin tests and detecting allergen-specific antibodies in the serum of patients. These two methods are complementary, and skin tests cannot be performed when acute dermatitis is taken when skin stunting or anti-allergic drugs are used. In this case, the serum of the patient is used to detect the presence of allergen-specific antibodies in serum. Diagnose the cause allergens.

The skin test reagent of the present invention contains the leaf mite clause as an active ingredient. The leaf mite is one or more selected from the group consisting of tangerine mite, spotted mite and apple mite. Leaf mite constituents used in the skin test reagents according to the present invention may be prepared by conventional methods known in the art, for example, protein extraction using Coca solution, but are not limited thereto. The content of the protein finally contained may vary according to the method of preparing the leaf mite clause, but in the present invention, a skin test reagent containing a suitable concentration of a clause effective as a skin test reagent is minimized by minimizing such variation. The concentration of the formula contained in the skin test reagent according to the present invention is suitably about 0.1 to 10 mg / ml, preferably 0.5 to 5 mg / ml, more preferably 1 mg / ml.

The skin test reagent according to the present invention may preferably contain a preservative for long term preservation of the protein of interest. Glycerin is suitable as a preservative, but is not limited thereto.

The skin test reagent according to the present invention is used as follows. Skin test reagent is dropped on the patient's back or forearm, and then terminal with a 23G or 26G needle to check for swelling and redness after 15 minutes to diagnose allergic symptoms caused by leaf malignant allergens.

The inventors have found that leaf marrow allergens cause IgE mediated inflammatory responses. Skin irritation test showed that asthma patients with leaf malignant allergens had IgE antibodies specific to leaf malignant antigen (Kim, YK et al., Sensitivity to citus red mite and the development of asthma. Ann Allergy Asthma Immunol 2000; 80; : See 483-8). In addition, the measurement of two IgG subtypes IgG1 and IgG4 antibodies in these patients confirmed the significant association between serum malignant specific IgG1 and G4 antibody values and IgE antibody values. An increase in specific IgE, IgG1 and G4 led to the conclusion that allergic diseases caused by leaf malignant allergens can be effectively diagnosed.

The allergen-specific IgE, IgG subtype detection kit refers to a kit for diagnosing allergic diseases caused by leaf mite by detecting the presence of leaf malignant allergen-specific IgE and IgG1, IgG4 antibodies in patient serum. The leaf mite allergen-specific IgE or IgG subtype antibody detection kit according to the present invention may be used for skin stunting or anti-allergic drugs, or for severe atopic dermatitis, i.e., when a skin test cannot be performed and as a skin test reagent. It is to diagnose the causative allergen when the diagnosis is inappropriate.

The leaf mite allergen-specific IgE, IgG subtype antibody detection kit of the present invention is (1) a container containing a leaf marrow clause, and (2) a detectable component as a secondary antibody against the leaf marrow allergen-specific IgE, IgG subtype antibody. Instructions for use of the labeled antibody and (3) kit.

According to the most preferred embodiment of the present invention, component (1) in the detection kit is well coated with 100 mcg (1 mg / ml, 100 μl) leaf mite. The content of leaf mite constituents included in the diagnostic kit may be in the range of 10 to 1000 mcg, and the preferred effective amount may be selected by those skilled in the art.

In another preferred embodiment of the present invention, the secondary antibody to (2) component mite allergen-specific IgE, IgG1 and IgG4 antibodies is an antibody to which biotin is attached. However, the present invention is not limited thereto, and any antibody labeled with other detectable components such as radioisotopes may be used. Biotinylated antibodies cause color reaction using streptavidin-peroxidase, H ₂ O ₂ and ABTS (2,2'-azido-bis-3 ethylbenthiazoline sulfonic acid) as substrate, The color reaction can be measured by absorbance at 405 nm. Thus, the diagnostic kit according to the invention may optionally further comprise ABTS as streptavidin-peroxidase, H ₂ O ₂ and a substrate.

Diagnosis using the kit according to the present invention is performed as follows. The patient's serum was placed in wells coated with 100 mcg of leaf mite, followed by an antigen antibody reaction, followed by washing to remove all serum containing antibodies not attached to the wells, to which anti-IgE with biotin was attached. (Or IgG1, IgG4) is added to react and the non-reactive anti-IgE antibodies are removed, followed by the addition of streptavidin-peroxidase and substrate to give a color reaction, which is measured by absorbance at 490 nm.

The detection kit of the skin test reagent and the leaf mite allergen-specific antibody according to the present invention was completed through a process of extracting the constituents from the collected leaf mite and standardizing them quantitatively and qualitatively.

In the present invention, as a qualitative standardization process of leaf mite constituents, immunoenzyme method, SDS-PAGE and immunoblot analysis are performed on 20 leaf malignant allergy patients, and the leaf mite is a quantitative standardization process. A skin terminal test is performed on 20 allergic patients to determine histamine equivalenet potency (HEP).

Hereinafter, the present invention will be described in more detail through, but not limited to, a standardized process for completing the present invention and preferred examples of skin test reagents and specific antibody detection kits. Those skilled in the art will appreciate that modifications and variations are possible without departing from the spirit of the invention within the scope of the invention.

Example 1 Preparation of Leaf Mites

Tangerine mites were obtained from the Jeju Citrus Research Center (Seogwipo City, Jeju Island), and spotted mites and apple mites were obtained from the Apple Research Center (Gunwi-gun, Gyeongsangbuk-do). Methods for isolating leaf mite protein from each leaf mite are described in Elliot Middleton et al. Allergy: Principles and Practice, 5th edition, Mosby.

After removing impurities from each leaf mite, the mixture was reacted with Coca solution (40 g / L sodium chloride, 4 g / L phenol, 2.5 g / L sodium bicarbonate) at 1:40 wt / vol for 24 hours at room temperature, followed by centrifugation. It was. The supernatant obtained after centrifugation was dialyzed at 4 ° C. in phosphate buffer solution (pH 8.0) for 2 days. It was lyophilized at minus 70 ℃ and weighed. The lyophilized article was diluted with the phosphate buffer solution at a concentration of 1 mg / ml, and used for the preparation of a specific antibody detection kit, and the skin test reagent was prepared by adding the same amount of phosphate buffer solution and glycerin.

Example 2 Standardization of Tangerine Mite

Standardization of tangerine mites was conducted in a qualitative and quantitative manner. Qualitative standardization was performed by enzyme-linked immunosorbent assay (ELISA) inhibition test, immunoblot analysis, and quantitative method to determine HEP (histamine equivalent potency).

The sera of patients used in each standardized test were caused by previously known allergen tests among farmers engaged in tangerine and apple farming in Jeju Island, and children and adolescents living in orchards and apple orchards in Jeju Island. Serum was separated from the peripheral blood of this unknown patient and used.

1. Determination of the Presence of Specific IgE and IgG Subtype Antibodies to Tangerine Antigen (ELISA)

After preliminary experiments determine the appropriate concentration of tangerine mite, the 1 mg / ml concentration of the source is dissolved in carbonate buffer (pH 9.6) and 100 per well in a 96-well ELISA plate (Immuno Dynatech, USA). 씩 was added thereto and reacted at 4 ° C. for 24 hours. This was washed three times with 0.05% Tween-PBS (hereinafter referred to as PBS-T) three times, and 10% newborn calf serum (Sigma Chemical Co., USA, hereinafter referred to as NCS) was added thereto for 1 hour. In each well, 100 μl of serum of the patient and 20 patients negative for allergic skin terminal test with tangerine malignant antigen were added to each well and allowed to stand at room temperature for 2 hours. After washing three times with PBS-T again, 100 μl of anti-IgE antibody (Sigma Chemical Co., USA) 1: 1000 v / v diluted biotin was added to each well and reacted for 2 hours. 100 μl of streptavidin-peroxidase (Sigma Chemical Co., USA) diluted 1: 500 v / v was added thereto, reacted for 30 minutes, and washed again three times. Here ABTS as H ₂ O ₂ and the substrate (2.2'- azido-solution in 100 ml of ethyl bis -3 Ben thiazol sleepy sulfonic porik 70 mM citrate phosphate buffer, Acid 55 mg; Sigma Chemical Co., USA ) 100 After the reaction was added for 5 minutes, the reaction was blocked with 2 mM NaN ₃ , and the absorbance was measured at 405 nM.

Measurement of specific IgG ₁ and IgG ₄ antibodies is carried out in the same manner as described above, but instead of anti-IgE antibodies, anti-IgG1 or anti-IgG4 antibodies (Sigma Chemical Co., USA) 1: 1000 v / v dilution 100 It was measured by adding the μL.

2.ELISA Inhibition Test

ELISA enzyme inhibition tests with these antigens were performed to observe cross-antigenicity and antigen specificity with antigens related to tangerine mites (house dust mites, storage mites) and other inhalable antigens. House dust mites and storage mites, which are related antigens, were purchased from Allergopharma (Allergopharma, Hamburg, Germany) and used in dilution at a concentration of 1 mg / ml in phosphate buffer solution (pH 8.0). Cockroach antigens were used as other inhalable antigens, which were also purchased from Allergo Pharma and diluted in the same manner as above. Each antigen (associated antigen and other antigens) to function as a serum and inhibitor of the patient was diluted to 0.01, 0.1, 1.0, 10, 100 μl / ml, respectively, and 100 μl were mixed and reacted at room temperature for 1 hour. 100 μl per well was added to the ELISA plate to which the antigen was attached, and ELISA was performed to measure IgE antibody against leaf malignant antigen (see ELA method for detecting IgE antibody). As a control, the same amount of PBS was added instead of the inhibitor, and then the degree of inhibition (%) was calculated. As a result, there was no cross antigenicity by other related antigens in patients with positive skin test only in tangerine mite, and cross antigenicity within 50% of other related antigens could be observed in patients who simultaneously tested positive for tangerine mite and house dust mite. there was. As a result, it was confirmed that the leaf mite antigen as an allergen is a unique allergen different from the related antigen known previously.

3. SDS-PAGE and Immunoblot Analysis

SDS-PAGE and immunoblot analysis were performed to confirm the presence of major allergens in the tangerine mite. SDS-PAGE was performed on a 12% gel using a 90 × 40 mm mini-gel instrument (Hoeffer Scientific, San Francisco, Calif., USA). Prepare a comb having two widths of 3.5 mm width and 80 mm, and add the label protein reagent (Pharmacia, Uppsala, Sweden) for molecular weight measurement from the left and the leaf marrow antigen extracted by the method described in Example 1 above. It was run. This was then replaced with a nitrocellulose membrane. Subsequently, the translocation sites of the labeled protein and the leaf mite protein were cut and stained with Ponceu S solution to confirm the position of the labeled protein and the antigenic protein. The antigen-transferred nitrocellulose membrane was cut at 3 mm intervals, and then reacted with 1% bovine serum albumin and PBS-T solution for 1 hour to block nonspecific protein binding. IgE, IgG4 as a serum stock solution) was added 500 μl and reacted at room temperature for 16 hours. After washing three times with PBS-T solution, it was reacted with biotin attached anti-human IgE antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 3 hours at room temperature. After washing three times with PBS-T solution, the mixture was added with 500 μl of streptavidin-peroxidase (Stremavidin-peroxidase) (Sigma Chemical Co., St. Louis, Mo.) 1: 500 and reacted for 1 hour. . Again washed 5 times with PBS-T solution, 3 ml of 0.3% 4-kilo-1-naphthol (Sigma Chemical Co., St. Louis, MO) dissolved in methanol, 50 ml of Tris buffered saline And 30% H₂O₂A solution of 25 µl of the solution was added 1 ml per segment and reacted for 15 minutes, followed by washing three times with distilled water to observe the reaction. From the results of the immunoblot analysis performed as described above, when the protein band reacted with the serum of 20 patients was 50% or more, it was determined as a major allergen. As a result, major allergens of protein bands of 24 kDa and 34 kDa were observed (see FIG. 1).

3. Determination of histamine equivalent potency (HEP)

As part of the quantitative standardization of tangerine mites, the antigenicity (allergenic potency) of the tangerine mites is expressed as HEP crystals. The skin terminal test was carried out using a diagnostic reagent diluted with 1 mg / ml concentration of tangerine mite, which was prepared by the method of manufacturing the method described in Example 1 above. Twenty patients with allergic symptoms from tangerine mite were subjected to a skin terminal test with the leaf mite diagnostic reagent prepared as described above and compared with the size of histamine-induced swelling. In case of the same size as the histamine-induced swelling, when defined as 1 HEP (= 1000 BU / ml), 1.02 HEP was shown for the skin test reagent prepared by the method described above. This means that, when prepared by the method described above, there is a substance that reacts to a degree almost equal to the concentration of 1 mg / ml of histamine at the concentration of 1 mg / ml of leaf mite.

Example 3 Standardization of Spotted Mite Clause

The standardization of spotted mite clauses was performed in the same way as the experiment on standardization of tangerine mite clauses.

In qualitative standardization, some cross-antigens with related antigens, house dust mites and storage mites, were observed but no cross-reactivity with other inhaled antigens, and immunoblot analysis identified three major allergens of 10, 29, and 60 kDa. Could be (see Figure 2).

In the quantitative normalization, the murine mites showed an antigenicity of 0.95 HEP by the above method.

Example 4 Standardization of Apple Mite Clause

The standardization of apple mite clauses was carried out in the same way as the experiment on the standardization of tangerine mite clauses.

Qualitative standardization showed some cross-antigens with related antigens but no cross-reactivity with other inhaled antigens, and immunoblot analysis identified three major allergens of 33, 41 and 51 kDa.

In the quantitative standardization, the apple mites showed an antigenicity of 0.01 HEP by the above method.

Summarizing the above experimental results, qualitative standardization results showed that the constituents extracted from leaf mite contained many proteins, two of which were tangerine mite and three major allergens of spotted mite and apple mite. It was found to contain. Quantitative standardization showed 0.9-1.1 HEP when the concentration of the above-mentioned constituents of each leaf mite was 1 mg / ml.

Example 5 Preparation of Skin Test Reagent

As in Example 1, the same amount of glycerin was added to the tangerine m allergens at a concentration of 1 mg / ml diluted in a phosphate buffer solution to prepare a tangerine m allergen diagnostic skin test reagent.

Similarly, as in Example 1, the same amount of glycerin was added to a spotted mite allergen at a concentration of 1 mg / ml diluted in a phosphate buffer solution to prepare a spotted malignant allergen diagnostic skin test reagent.

Similarly, the same amount of glycerin was added to apple mite allergens at a concentration of 1 mg / ml diluted in phosphate buffer solution, as in Example 1, to prepare an apple mite allergen diagnostic skin test reagent.

Example 6 Preparation of a Leaf Mite Specific IgE and IgG Subtype Antibody Detection Kit.

(1) 100 μl of the tangerine mite extract at a concentration of 1 mg / ml extracted in Example 1 was coated on 94-well (NUMC immunoplate, Roskilde, Denmark) to prepare a well coated tangerine mite. In a similar manner, wells coated with spotted mites, apple mites and mixtures thereof were prepared.

(2) Secondary antibodies against leaf malignant allergen-specific IgE and IgG subtype antibodies were biotin-labeled antibodies purchased from Vector Co. Berlingham, CA, USA.

(3) Streptavidin-peroxidase, H ₂ O ₂ and ABTS used as substrates were purchased from Sigma (St. Louis, MO, USA) and included in the kit.

The kit is used as follows.

The serum of the patient is placed in the well of (1), reacted and washed, and the secondary antibody of (2) is added thereto, reacted and washed, and the enzyme and the substrate of (3) are added thereto to develop a color reaction. Is measured by absorbance at 405 nm.

According to the present invention, among allergic diseases such as bronchial asthma and allergic rhinitis, which have been previously unknown, a leaf mite can be easily and specifically diagnosed.

In addition, according to the present invention, allergic diseases caused by leaf mites can be easily diagnosed by simply separating and testing the serum even in a case where the skin test is impossible or inappropriate.

Diagnosing leaf mites as a cause of allergic diseases is essential for establishing treatment policies such as avoidance therapy and immunotherapy.

Claims (10)

A skin test reagent for diagnosing allergic diseases caused by leaf mite allergens, which contains leaf mite clause as an active ingredient. The skin test reagent according to claim 1, wherein the leaf mite clause is at least one member selected from the group consisting of tangerine mite, spotted mite and apple mite. The skin test reagent according to claim 1 or 2, wherein the skin test reagent contains a manganese mite, spotted mite, apple mite or a mixture thereof as an active ingredient at a concentration of 1 mg / ml. The skin test reagent according to claim 1 or 2, further comprising a preservative for preserving the active ingredient. The skin test reagent according to claim 4, wherein the preservative is glycerin. (1) a container containing a leaf mite article, (2) an antibody labeled with a detectable component as a secondary antibody to a leaf mite specific IgE and IgG subtype antibody, and optionally (3) including instructions for use of the kit, Leaf mite serum specific IgE and IgG subtype antibody detection kits for the diagnosis of allergic diseases caused by leaf mite allergens. 7. The leaf mite serum specific IgE and IgG subtype antibody detection kit according to claim 6, wherein the leaf mite clause is at least one member selected from the group consisting of tangerine mite, spotted mite and apple mite. 8. The detection kit according to claim 6 or 7, wherein the leaf kit contains at least 1 mg / ml of a mandarin mite, spotted mite, apple mite, or a mixture thereof. 8. The detection kit according to claim 6 or 7, wherein the antibody labeled with a detectable component as a secondary antibody against IgE and IgG antibodies is biotin labeled. 10. The detection kit of claim 9, further optionally comprising streptavidin-peroxidase, H ₂ O _2, and ABTS as a substrate.