WO2002076477A1 - 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

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 Download PDF

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WO2002076477A1
WO2002076477A1 PCT/KR2002/000534 KR0200534W WO02076477A1 WO 2002076477 A1 WO2002076477 A1 WO 2002076477A1 KR 0200534 W KR0200534 W KR 0200534W WO 02076477 A1 WO02076477 A1 WO 02076477A1
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mite
spider
spider mite
crude extract
skin
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PCT/KR2002/000534
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French (fr)
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You-Young Kim
Kyung-Up Min
Hae-Sim Park
Sang-Heon Cho
Yoon-Keun Kim
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Bioalletech Co., Ltd.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43582—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from mites

Definitions

  • the present invention relates to kits for diagnosing allergic diseases caused by spider mites (citrus red mites, two-spotted mites and European red mites).
  • the causative allergens responsible for an allergic disease varies depending on the surrounding environment. countries around the world have different surrounding environments. Thus, many allergen-diagnosing techniques proved to be useful in one country are not effective in other countries. Therefore, developing a diagnosis technique specific to a population is very important for protection of that people's health.
  • the inventors of the present invention found for the first time that spider mites are a new important allergen in the Korean environment and developed a new diagnosing technology comprising the new allergen. Therefore, there is expected a great improvement in diagnosing allergic diseases in the clinical field.
  • Inhalant allergen which is the cause of allergic diseases can be divided into two groups, (1) indoor allergens such as home dust mite, pet hair, cockroach allergens, indoor mold, and (2) outdoor allergens such as wood allergens, weed, outdoor mold (Kim Y. et al., Prevalence of childhood asthma based on questionnaire and methacholine bronchial provocation test, Clin. Exp. Allergy 27:761-8, 1997).
  • indoor allergens such as home dust mite, pet hair, cockroach allergens, indoor mold
  • outdoor allergens such as wood allergens, weed, outdoor mold
  • Citrus red mite (Panonychus citri) is a common sensitizing allergen among children living around citrus orchards, Ann Allergy Asthma Immunol., 80:200-4, 2000).
  • a spider mite is a small organism which is parasitic on leaves by sucking chlorophyll. Taxonomically, it belongs to the order Acari with house dust mites and storage mites. TSM is parasitic to leaves of flowering plants, vegetables, weed as well as on leaves of fruit trees, i.e. the host range of the TSM parasite is very broad. TSM is also distributed in a very high density throughout the world including Korea. The density of TSM is characteristically high on plants of intensive cultivation which use high doses of pesticides. This is because a possible predator for the mites was eliminated with the use of high doses of pesticide. Further, the survival and breeding of TSM increases because nutrients for TSM are increased in intensively cultivated plants.
  • the present disclosure presents the spider mite, standardized quantitatively and qualitatively, based on which skin-test reagents and serum specific IgE, IgG subtype detection kits, which can diagnose spider mite as a cause of allergic diseases in a simple and easy manner, are revealed.
  • the object of the present invention is to provide a skin-test reagent capable of easy and specific diagnosis of allergic diseases caused by spider mite allergens (CRM, TSM and ERM).
  • Another object of the present invention is to provide a serum specific IgE, Ig-G subtype detection kit that can diagnose spider mites as a cause of allergic diseases in a simple and easy manner.
  • the present invention is for a skin test reagent for diagnosing allergic diseases caused by spider mites comprising spider mite crude extract as the effective component.
  • spider mite crude extract as the effective component.
  • One embodiment targets the following types of spider mites, the citrus red mite, the two-spotted mite and the European red mite.
  • the crude extract of spider mite is at a concentration of 1 mg/ml.
  • the skin test reagent may further comprise an agent to preserve the effective component.
  • the preservative agent is glycerin.
  • Another embodiment is a spider mite-specific IgE and IgG subtype detection kit for diagnosis of allergic diseases caused by spider mite allergen, comprising,
  • spider mite-specific IgE and IgG subtype detection kit embodiment include a spider mite which is one or more selected from the group consisting of citrus red mite, two-spotted mite and European red mite. Of course, these are exemplary and in no way limit the invention.
  • the detection kit embodiment is also preferably one in which the crude extract of spider mite is at a concentration of 1 mg/ml.
  • the preferable secondary antibody is biotin-labeled antibody.
  • the detection kit may have an additional component (3) further comprising streptavidine-peroxidase, H 2 O 2 and ABTS substrate.
  • Fig. 1 is an IgE immunoblot showing results from analysis of crude CRM extract.
  • M is the molecular weight marker
  • numbers 1 to 10 indicate serum of 10 patients
  • C is the control serum
  • B represents buffer for the control.
  • Fig. 2 is an IgE immonoblot showing results from analysis of crude TSM extract.
  • Lanes labeled A represent serum of patients who are sensitive to both TSM and house dust mite, while lanes labeled B represent serum of patients who are sensitive only to TSM. From the left, M is the molecular weight marker, the numbers indicate serum of patients, C is control serum, and B represents buffer for the control.
  • Skin test reagents according to the present invention comprise crude extract of spider mites as an effective ingredient.
  • the spider mite is one or more selected from the group consisting of CRM, TSM and ERM.
  • Crude extract of spider mite, used in the skin test reagent according to the present invention can be prepared by conventional methods known in the art. For example, protein extraction process using a Coca solution may be used. This invention is, of course, not restricted to this specific method. One skilled in the art can select any other known methods for obtaining crude extracts depending on the need, and tailor them to the present invention, after having the benefit of this disclosure.
  • the content of protein contained in the final product may be different.
  • the protein content variations within final products are minimized and skin test reagent comprising appropriate concentration of crude extract suitable for skin test reagent is provided.
  • the concentration of crude extracts within skin test reagent according to the present invention is 0.1 to 10 mg/ml, preferably 0.5 to 5 mg/ml and more preferably 1 mg /ml.
  • the skin test reagent according to the present invention can contain preservatives.
  • the preservative of choice is glycerin, but one skilled in the art can select any other known preservatives compatible with the present invention, after having the benefit of the present disclosure.
  • Skin test reagents according to the present invention are used as follows.
  • the skin test reagent according to the present invention are placed on the epidermis of the back, upper or lower arm of patients. The site is then pricked with a needle of 23G or 26G. After about 15 minutes, examination is made on whether a wheal or redness has occurred, and conclude whether or not the allergic symptom is caused by spider mite allergen.
  • the present inventors discovered that spider mite allergen triggers IgE- mediated inflammatory reaction. It was shown that asthma caused by spider mite allergen when tested by skin prick test, have IgE antibody specific to the spider mite allergen (Kim Y. et al, Sensitivity to citus red mite and the development of asthma. Ann Allergy Asthma Immunol, 80:483-8, 2000). Further, the present inventors studied the levels of two IgG subtypes, namely IgGl and IgG4, among allergic reactions caused by spider mite allergen, and identified reliable correlation between the levels of IgGl and G4 antibody and the level of IgE antibody. Based on these results, the present inventors concluded that the allergic diseases due to spider mite allergen can be diagnosed effectively by detecting an increase in the levels of spider mite-specific IgE, IgGl and IgG4 antibody within sera of patients.
  • allergen-specific IgE, IgG subtype detection kit refers to a product for diagnosis of allergic diseases due to spider mite allergen by detecting the presence of spider mite allergen specific IgE and IgGl and G4 antibody within sera of patients. Spider mite allergen specific IgE , Ig-G subtype detection kits according to the present invention are especially useful when skin tests are not practical or inappropriate, such as for the patients who suffer skin trouble, severe atopy, or have taken anti-allergic drugs.
  • the detection kit according to the present invention can comprise (1) a vial containing spider mite crude extract, (2) a secondary antibody labeled with detectable marker and specific to spider mite specific IgE and IgG subtype antibody, and (3) a manual for the kit user.
  • component (1) is a well coated with 100 meg (1 mg/ml, 100 ⁇ l) of spider mite crude extract.
  • the content of crude extract used in the detection kit of the present invention may range between 10 to 1000 ⁇ g. A person having ordinary skill in the art can select appropriate contents of crude extract for effective detection.
  • component (2) is biotin-labeled secondary antibody that has a specificity to IgE, IgGl and IgG4 antibody for spider mite allergen.
  • the present invention is not limited to the above embodiments, and any secondary antibody labeled with any detectable marker which one skilled in the art finds suitable after having the benefit of this disclosure, can be used.
  • Biotin-labeled secondary antibody will show a color reaction in the presence of streptavidine-peroxidase, H 2 O 2 , and ABTS (2,2'-azido-bis-3-ethylbenthiazoline sulfornic acid) substrate.
  • the color reaction can be detected by absorption spectroscopy at 405 nm.
  • the detection kit of the present invention may optionally further comprise streptavidine-peroxidase, H 2 O 2 , and ABTS substrate.
  • the diagnosis using the detection kit of the present invention is performed as follows. Serum sample obtained from test patients is added to the well coated with 100 meg of spider mite crude extract. After antigen-antibody reaction, the well is washed to remove serum containing unbound antibody. To the well, biotinylated anti-IgE (or IgGl, IgG4) antibody is added, unbound anti-IgE antibody is removed, and then, streptavidine-peroxidase and substrate are added to permit coloring reaction, which is detected by absorption at 490 nm.
  • biotinylated anti-IgE (or IgGl, IgG4) antibody is added, unbound anti-IgE antibody is removed, and then, streptavidine-peroxidase and substrate are added to permit coloring reaction, which is detected by absorption at 490 nm.
  • Skin test reagent and spider mite allergen specific antibody detection kit have been accomplished through the process of obtaining crude extract from a collected spider mite and standardizing the crude extract quantitatively and qualitatively.
  • Citrus red mite resource was provided from Citrus Research Center (Seoguipo-shi, Cheju Island, Republic of Korea) and two-spotted mite and European red mite were provided from Apple Research Center (Kunwee-kun, Kyungsang- bukdo province, Republic of Korea). Separation of crude extract proteins from each spider mite was performed according to the general method disclosed in the reference Elliot M. et al., Allergy: Principles and Practice, 5 edition, Mosby.
  • Impurities were removed from each spider mite resource, to which 1:40 wt/vol Coca solution (sodium chloride 5 g/L, phenol 4 g/L and sodium bicarbonate 2.5 g/L) was added. The solution was allowed for reaction for 24 hours at room temperature. After centrifugation, the supernatant was dialyzed against phosphate- buffer solution (pH 8.0) at 4 ° C for 2 days. The extract was lyophyilized at -70 ° C and weighed. The lyophilized crude extract was diluted with phosphate buffered solution to a 1 mg/ml concentration and used for the preparation of detection kit of the present invention. For skin test reagent, the extract in phosphate buttered solution was mixed with an equal amount of sterile glycerin.
  • Example 2 Standardization of Citrus Red Mite Crude Extract
  • the serum samples used for standardization are obtained from farmer patients who cultivate citrus trees or apple trees and from children and adolescent patients who live nearby the citrus or apple orchard. Those patients did not show positive response to any common inhalant allergen.
  • Optimal concentration of CRM crude extract was determined by preliminary testing. Crude extract was dissolved in carbonate buffer (pH 9.6) to a concentration of 1 mg/ml, which was added into 96 well ELISA plate (Immuno Dynatech, USA) with 100 ⁇ l per well. The plate was placed for reaction at 4 ° C for 24 hours. After the reaction, each well was washed three times with 0.05% Tween-PBS ("PBS-T"). Then, 250 ⁇ l of 10% newborn calf serum (NCS) (Sigma Chemical Co., USA) was added and incubated for one hour. The wells were then incubated for two hours at room temperature with 100 ⁇ l of patient's serum or control serum. Control serum was prepared from 20 patients showing negative response to skin prick test with
  • CRM allergen After washing three times with PBS-T, 100 ⁇ l of the 1 : 1000 vol vol diluted biotin labeled anti-human IgE antibody (Sigma Chemical Co., USA) were added to the wells and incubated for 2 hours. Then, 100 ⁇ l of 1 :500 vol/vol diluted streptavidin-peroxidase (Sigma Co, USA) were added to the wells and incubated for 30 minutes. The wells were then washed three times.
  • H 2 O 2 and ABTS 55 mg of 2,2'-azinobis-3-ethyl-benzthiazoline sulfuric acid in 100 ml solution of 70 mM citrate phosphate buffer; Sigma Chemical Co., USA
  • ABTS 55 mg of 2,2'-azinobis-3-ethyl-benzthiazoline sulfuric acid in 100 ml solution of 70 mM citrate phosphate buffer; Sigma Chemical Co., USA
  • the reaction was stopped by the addition of 2 mM NaN 3 and the absorbance was read at 405 nM.
  • serum-specific IgGl and IgG4 antibody was determined similarly as the method described above except adding 100 ⁇ l of the 1:1000 vol/vol diluted anti-human IgGl or IgG4 antibody (Sigma Chemical Co., USA) instead of anti-human IgE antibody.
  • the sera of the patients and each antigen were diluted to the concentration of 0.01, 0.1 , 1.0, 10 and 100 ⁇ l/ml. After mixing 100 ⁇ £/ml of each sample with different dilutions, the pooled sera were incubated for 1 hour at room temperature. 100 ⁇ l of the pooled samples were then incubated with the microtiter plate coated with spider mite antigen for 2 hours. ELISA was followed in order to detect the IgE antibody specific to spider mite antigen. (As to ELISA steps for detection of IgE antibody, please refer to section 1 hereinabove).
  • the antigenic potency of CRM crude extract was standardized with HEP determination.
  • the CRM crude extracts prepared according to the method described in example 1 hereinabove was diluted to the concentration of 1 mg/ml, which is then used for the preparation of skin test reagent.
  • skin test reagent skin prick test was performed for 20 patients showing allergic symptoms due to CRM.
  • the skin test reagent prepared according to method described above showed 1.02 HEP. This means that the skin test reagent comprising 1 mg/ml of CRM extract as prepared above contains the same allergenic materials as lmg/ml of histamine.
  • Example 3 Standardization of Two Spotted Mite Crude Extract
  • Standardization of TSM crude extract was done according to the same method used for the standardization of CRM crude extract.
  • the allergenic potential of skin test reagent prepared according to the method was 0.95 HEP.
  • Standardization of ERM crude extract was done according to the same method used for the standardization of CRM crude extract.
  • the allergenic potential of skin test reagent prepared according to the method was 1.01 HEP.
  • spider mite crude extracts contain many proteins, among which 2 major allergens for CRM and three major allergens for TSM and ERM, respectively, were identified. Further, with qualitative standardization, skin test reagents containing 1 mg/ml of crude extracts according to the present invention showed 0.9 to 1.1 HEP.
  • Skin test reagent for diagnosing CRM causative allergy was prepared by using the CRM crude extract prepared in example 1.
  • the CRM extract in phosphate-buffered solution diluted to the concentration of 1 mg/ml was mixed with an equal amount of sterile glycerin.
  • skin test reagent for diagnosing TSM causative allergy was prepared by using the TSM crude extract prepared in example 1.
  • the TSM extract in phosphate-buffered solution diluted to the concentration of 1 mg/ml was mixed with an equal amount of sterile glycerin.
  • skin test reagent for diagnosing ERM causative allergy was prepared by using the ERM crude extract prepared in example 1.
  • Example 6 Spider Mite specific IgE and IgG Subtype Antibody Detection Kit
  • IgG subtype antibody was purchased from Vector Co. (Berlingham, CA, USA).
  • Streptavidine-peroxidase, H 2 O 2 and ABTS substrate were purchased from Sigma (St. Louis, MO, USA) and included into the kit.
  • the detection kit comprising said components (1) to (3) are used as follows: Patients' sera are added to the well (1) and allowed for reaction. After washing the well, secondary antibody (2) is added into the well (1) and allowed for reaction. After washing again and adding enzyme and substrate (3) into the well, color reaction is detected by absorbance at 405 nm.
  • allergic diseases such as bronchial asthma and allergic rhinitis, which are caused by spider mites
  • the causative allergen for certain manifestations of such allergic diseases has been a mystery prior to this disclosure.
  • the present invention allows the detection of allergic diseases caused by spider mite antigen in those patients who are not susceptible to skin tests, by simple isolation of serum from the patients. Detection of the spider mites as causative allergens of allergic diseases is prerequisite for the diagnosis and treatment plan including the avoidance of the allergens and immunotherapy.

Abstract

Disclosed are skin test reagents and serum-specific IgE and IgG subtype detection kits comprising crude extracts of spider mites that which can diagnose specific allergic diseases caused by spider mites (Citrus Red Mite, Two-Spotted Mite, European Red Mite).

Description

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
TECHNICAL FIELD
The present invention relates to kits for diagnosing allergic diseases caused by spider mites (citrus red mites, two-spotted mites and European red mites).
BACKGROUND ART
Up to about 20 to 30% of the population suffers from allergic diseases such as rhinitis, bronchial asthma and atopy. Thus, dealing with these chronic allergic diseases is important for protection of the public health. The most important prerequisite to treat allergic diseases properly is to diagnose accurately the causative allergens responsible for these allergic diseases.
The causative allergens responsible for an allergic disease varies depending on the surrounding environment. Countries around the world have different surrounding environments. Thus, many allergen-diagnosing techniques proved to be useful in one country are not effective in other countries. Therefore, developing a diagnosis technique specific to a population is very important for protection of that people's health.
The inventors of the present invention found for the first time that spider mites are a new important allergen in the Korean environment and developed a new diagnosing technology comprising the new allergen. Therefore, there is expected a great improvement in diagnosing allergic diseases in the clinical field.
Inhalant allergen which is the cause of allergic diseases can be divided into two groups, (1) indoor allergens such as home dust mite, pet hair, cockroach allergens, indoor mold, and (2) outdoor allergens such as wood allergens, weed, outdoor mold (Kim Y. et al., Prevalence of childhood asthma based on questionnaire and methacholine bronchial provocation test, Clin. Exp. Allergy 27:761-8, 1997). Recently, the present inventors discovered that citrus red mite (CRM), two- spotted mite (TSM) and European red mite (ERM) are the culprit allergens affecting allergic diseases prevalent in the Korean rural and urban areas. It was also discovered for the first time that citrus red mite (CRM), belonging to the family of spider mites and infecting/damaging citrus leaves by absorbing chlorophyll, is a causative allergen responsible for asthmatic reactions in cultivating farmers (Kim Y. et al., New occupational allergen in citrus farmers: citrus red mite (Panonychus citri), Ann Allergy Asthma Immunol., 82:223-8, 1999). Further, it was revealed that spider mite is an important causative allergen affecting not only citrus-cultivating farmers but also ordinary peoples living nearby and not working on citrus cultivation
(Kim Y. et al., Citrus red mite is the most common sensitizing allergen in citrus farmers with asthma and rhinitis, Clin Exp Allergy 29:1102-9, 1999; (Kim Y. et al.,
Citrus red mite (Panonychus citri) is a common sensitizing allergen among children living around citrus orchards, Ann Allergy Asthma Immunol., 80:200-4, 2000).
Additionally, the present inventors discovered that two-spotted mite and European red mite, distributed widely in the Korean landscape, are important causative allergens for allergic diseases (Kim Y. 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., 104:1285-92, 1999; Jee Y. et al., Two-spotted spider mite (Tetranychus urticae): an important allergen in asthmatic non-farmers symptomatic in summer and fall months, Ann Allergy Asthma Immunol., 84: 543-8, 2000).
A spider mite is a small organism which is parasitic on leaves by sucking chlorophyll. Taxonomically, it belongs to the order Acari with house dust mites and storage mites. TSM is parasitic to leaves of flowering plants, vegetables, weed as well as on leaves of fruit trees, i.e. the host range of the TSM parasite is very broad. TSM is also distributed in a very high density throughout the world including Korea. The density of TSM is characteristically high on plants of intensive cultivation which use high doses of pesticides. This is because a possible predator for the mites was eliminated with the use of high doses of pesticide. Further, the survival and breeding of TSM increases because nutrients for TSM are increased in intensively cultivated plants. Compared to other harmful insects, the damage caused by the spider mite including TSM was not serious prior to World War π. However, with the wide-spread use of pesticides after World War π, many harmful insects were eliminated. And, the density of spider mites was increased. According to a recent report, spider mites ranked as the most notorious insect that damages fruit tree cultivation.
Accordingly, the present disclosure presents the spider mite, standardized quantitatively and qualitatively, based on which skin-test reagents and serum specific IgE, IgG subtype detection kits, which can diagnose spider mite as a cause of allergic diseases in a simple and easy manner, are revealed.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide a skin-test reagent capable of easy and specific diagnosis of allergic diseases caused by spider mite allergens (CRM, TSM and ERM).
Another object of the present invention is to provide a serum specific IgE, Ig-G subtype detection kit that can diagnose spider mites as a cause of allergic diseases in a simple and easy manner.
The present invention is for a skin test reagent for diagnosing allergic diseases caused by spider mites comprising spider mite crude extract as the effective component. One embodiment targets the following types of spider mites, the citrus red mite, the two-spotted mite and the European red mite. Preferably, the crude extract of spider mite is at a concentration of 1 mg/ml. The skin test reagent may further comprise an agent to preserve the effective component. Preferably, the preservative agent is glycerin.
Another embodiment is a spider mite-specific IgE and IgG subtype detection kit for diagnosis of allergic diseases caused by spider mite allergen, comprising,
(1) a container with spider mite crude extract; and
(2) a secondary antibody specific to spider mite-specific IgE and IgG subtype antibody which is labeled with detectable marker.
Specific targets for the spider mite-specific IgE and IgG subtype detection kit embodiment described above, include a spider mite which is one or more selected from the group consisting of citrus red mite, two-spotted mite and European red mite. Of course, these are exemplary and in no way limit the invention. The detection kit embodiment is also preferably one in which the crude extract of spider mite is at a concentration of 1 mg/ml. The preferable secondary antibody is biotin-labeled antibody. The detection kit may have an additional component (3) further comprising streptavidine-peroxidase, H2O2 and ABTS substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is an IgE immunoblot showing results from analysis of crude CRM extract. M is the molecular weight marker, numbers 1 to 10 indicate serum of 10 patients, C is the control serum, and B represents buffer for the control.
Fig. 2 is an IgE immonoblot showing results from analysis of crude TSM extract. Lanes labeled A represent serum of patients who are sensitive to both TSM and house dust mite, while lanes labeled B represent serum of patients who are sensitive only to TSM. From the left, M is the molecular weight marker, the numbers indicate serum of patients, C is control serum, and B represents buffer for the control.
DETAILED DESCRIPTION OF INVENTION
Generally, there are two methods used to diagnose causative allergens of unknown allergic diseases. One is the skin prick test and the other is to detect the existence of causative allergen-specific antibody in serum of patients. Both methods are complementary to each other. In cases where patients have skin trouble, have taken anti-allergic medication, or suffer severe atopy, the skin test method is not feasible. In such cases, diagnosis of causative allergen is performed by detection of specific antibody in their serum.
Skin test reagents according to the present invention comprise crude extract of spider mites as an effective ingredient. Preferably, the spider mite is one or more selected from the group consisting of CRM, TSM and ERM. Crude extract of spider mite, used in the skin test reagent according to the present invention, can be prepared by conventional methods known in the art. For example, protein extraction process using a Coca solution may be used. This invention is, of course, not restricted to this specific method. One skilled in the art can select any other known methods for obtaining crude extracts depending on the need, and tailor them to the present invention, after having the benefit of this disclosure.
Depending on the preparation process for crude extract of spider mite, the content of protein contained in the final product may be different. According to the present invention, the protein content variations within final products are minimized and skin test reagent comprising appropriate concentration of crude extract suitable for skin test reagent is provided. The concentration of crude extracts within skin test reagent according to the present invention, is 0.1 to 10 mg/ml, preferably 0.5 to 5 mg/ml and more preferably 1 mg /ml.
Preferably, in order to store the crude extract of protein for an extended duration, the skin test reagent according to the present invention can contain preservatives. Preferable the preservative of choice is glycerin, but one skilled in the art can select any other known preservatives compatible with the present invention, after having the benefit of the present disclosure.
Skin test reagents according to the present invention are used as follows.
The skin test reagent according to the present invention are placed on the epidermis of the back, upper or lower arm of patients. The site is then pricked with a needle of 23G or 26G. After about 15 minutes, examination is made on whether a wheal or redness has occurred, and conclude whether or not the allergic symptom is caused by spider mite allergen.
The present inventors discovered that spider mite allergen triggers IgE- mediated inflammatory reaction. It was shown that asthma caused by spider mite allergen when tested by skin prick test, have IgE antibody specific to the spider mite allergen (Kim Y. et al, Sensitivity to citus red mite and the development of asthma. Ann Allergy Asthma Immunol, 80:483-8, 2000). Further, the present inventors studied the levels of two IgG subtypes, namely IgGl and IgG4, among allergic reactions caused by spider mite allergen, and identified reliable correlation between the levels of IgGl and G4 antibody and the level of IgE antibody. Based on these results, the present inventors concluded that the allergic diseases due to spider mite allergen can be diagnosed effectively by detecting an increase in the levels of spider mite-specific IgE, IgGl and IgG4 antibody within sera of patients.
The term "allergen-specific IgE, IgG subtype detection kit" as used herein, refers to a product for diagnosis of allergic diseases due to spider mite allergen by detecting the presence of spider mite allergen specific IgE and IgGl and G4 antibody within sera of patients. Spider mite allergen specific IgE , Ig-G subtype detection kits according to the present invention are especially useful when skin tests are not practical or inappropriate, such as for the patients who suffer skin trouble, severe atopy, or have taken anti-allergic drugs.
The detection kit according to the present invention can comprise (1) a vial containing spider mite crude extract, (2) a secondary antibody labeled with detectable marker and specific to spider mite specific IgE and IgG subtype antibody, and (3) a manual for the kit user.
According to one preferred embodiment of the present invention, component (1) is a well coated with 100 meg (1 mg/ml, 100 μl) of spider mite crude extract. The content of crude extract used in the detection kit of the present invention may range between 10 to 1000 μg. A person having ordinary skill in the art can select appropriate contents of crude extract for effective detection.
According to another preferred embodiment of the present invention, component (2) is biotin-labeled secondary antibody that has a specificity to IgE, IgGl and IgG4 antibody for spider mite allergen. However, the present invention is not limited to the above embodiments, and any secondary antibody labeled with any detectable marker which one skilled in the art finds suitable after having the benefit of this disclosure, can be used.
Biotin-labeled secondary antibody will show a color reaction in the presence of streptavidine-peroxidase, H2O2, and ABTS (2,2'-azido-bis-3-ethylbenthiazoline sulfornic acid) substrate. The color reaction can be detected by absorption spectroscopy at 405 nm. Accordingly, the detection kit of the present invention may optionally further comprise streptavidine-peroxidase, H2O2, and ABTS substrate.
The diagnosis using the detection kit of the present invention is performed as follows. Serum sample obtained from test patients is added to the well coated with 100 meg of spider mite crude extract. After antigen-antibody reaction, the well is washed to remove serum containing unbound antibody. To the well, biotinylated anti-IgE (or IgGl, IgG4) antibody is added, unbound anti-IgE antibody is removed, and then, streptavidine-peroxidase and substrate are added to permit coloring reaction, which is detected by absorption at 490 nm.
Skin test reagent and spider mite allergen specific antibody detection kit according to the present invention have been accomplished through the process of obtaining crude extract from a collected spider mite and standardizing the crude extract quantitatively and qualitatively.
For the qualitative standardization, enzyme linked immuno assay, SDS- PAGE and immunoblot analysis were performed for 20 patients who suffer allergy symptom caused by spider mite allergen. For the quantitative standardization, skin prick test was performed for 20 patients and HEP (histamine equivalent potency) is determined.
EXAMPLES
Example 1 : Preparation of spider mite crude extract
Citrus red mite resource was provided from Citrus Research Center (Seoguipo-shi, Cheju Island, Republic of Korea) and two-spotted mite and European red mite were provided from Apple Research Center (Kunwee-kun, Kyungsang- bukdo province, Republic of Korea). Separation of crude extract proteins from each spider mite was performed according to the general method disclosed in the reference Elliot M. et al., Allergy: Principles and Practice, 5 edition, Mosby.
Impurities were removed from each spider mite resource, to which 1:40 wt/vol Coca solution (sodium chloride 5 g/L, phenol 4 g/L and sodium bicarbonate 2.5 g/L) was added. The solution was allowed for reaction for 24 hours at room temperature. After centrifugation, the supernatant was dialyzed against phosphate- buffer solution (pH 8.0) at 4 °C for 2 days. The extract was lyophyilized at -70 °C and weighed. The lyophilized crude extract was diluted with phosphate buffered solution to a 1 mg/ml concentration and used for the preparation of detection kit of the present invention. For skin test reagent, the extract in phosphate buttered solution was mixed with an equal amount of sterile glycerin. Example 2 : Standardization of Citrus Red Mite Crude Extract
Standardization of CRM crude extract was done through quantitatively and qualitatively. For qualitative standardization, ELIS A, inhibition test, and immunoblot analysis were performed. For quantitative standardization, HEP (histamine equivalent potency) was determined.
The serum samples used for standardization are obtained from farmer patients who cultivate citrus trees or apple trees and from children and adolescent patients who live nearby the citrus or apple orchard. Those patients did not show positive response to any common inhalant allergen.
1. Detection of the presence of IgE and IgG subtype antibody specific to the CRM antigen (ELIS A)
Optimal concentration of CRM crude extract was determined by preliminary testing. Crude extract was dissolved in carbonate buffer (pH 9.6) to a concentration of 1 mg/ml, which was added into 96 well ELISA plate (Immuno Dynatech, USA) with 100 μl per well. The plate was placed for reaction at 4 °C for 24 hours. After the reaction, each well was washed three times with 0.05% Tween-PBS ("PBS-T"). Then, 250 μl of 10% newborn calf serum (NCS) (Sigma Chemical Co., USA) was added and incubated for one hour. The wells were then incubated for two hours at room temperature with 100 μl of patient's serum or control serum. Control serum was prepared from 20 patients showing negative response to skin prick test with
CRM allergen. After washing three times with PBS-T, 100 μl of the 1 : 1000 vol vol diluted biotin labeled anti-human IgE antibody (Sigma Chemical Co., USA) were added to the wells and incubated for 2 hours. Then, 100 μl of 1 :500 vol/vol diluted streptavidin-peroxidase (Sigma Co, USA) were added to the wells and incubated for 30 minutes. The wells were then washed three times. One hundred microliters of H2O2 and ABTS (55 mg of 2,2'-azinobis-3-ethyl-benzthiazoline sulfuric acid in 100 ml solution of 70 mM citrate phosphate buffer; Sigma Chemical Co., USA) were added and incubated for 5 minutes. The reaction was stopped by the addition of 2 mM NaN3 and the absorbance was read at 405 nM.
The presence of serum-specific IgGl and IgG4 antibody was determined similarly as the method described above except adding 100 μl of the 1:1000 vol/vol diluted anti-human IgGl or IgG4 antibody (Sigma Chemical Co., USA) instead of anti-human IgE antibody.
2. ELISA Inhibition Test
Competitive ELISA inhibition tests were performed to determine the specificity of the spider mite antibody and the allergenic cross-reactivity to the spider mite-related antigen (such as house dust mites, storage mites) and other inhalant antigen. Related antigen, house dust mites and storage mites were purchased from Allergopharma (Hamburg, Germany) and diluted to 1 mg/ml concentration with phosphate buffered saline (pH 8.0). Cockroach antigen, purchased from Allergopharma and diluted as described above, was used as other inhalant antigen. The sera of the patients and each antigen (i.e., spider mite-related antigen and other inhalant antigen that function as inhibitor) were diluted to the concentration of 0.01, 0.1 , 1.0, 10 and 100 μl/ml. After mixing 100 μ£/ml of each sample with different dilutions, the pooled sera were incubated for 1 hour at room temperature. 100 μl of the pooled samples were then incubated with the microtiter plate coated with spider mite antigen for 2 hours. ELISA was followed in order to detect the IgE antibody specific to spider mite antigen. (As to ELISA steps for detection of IgE antibody, please refer to section 1 hereinabove). After studying the control samples in which equal volumes of PBS were incubated instead of inhibitors, we calculated the inhibition. According to the result, the positive responders to CRM antigen only showed no cross reactivity with other related antigens. But, the positive responders both to CRM and house dust mite showed a little cross-reactivity less than 50% with other related antigens. These results reveal that the CRM antigen is novel allergen that has no relation with known related allergen.
3. SDS-PAGE and Immunoblot analysis
SDS-PAGE and Immunoblot analysis were performed to determine the presence of major allergen within CRM crude extract. 12% gel SDS-PAGE was performed using 90 X 40 mm minigel apparatus (Hoeffer Scientific, San Francisco, CA, USA). Two kinds of comb with 3.5 mm width and 80 mm width were used. From left line, marker protein for assessment of molecular weight (Pharmacia, Uppsala, Sweden), and CRM crude extract antigen, prepared by the method described in example 1 hereinabove, were loaded and started electrophoresis. After transferring to nitrocellulose membrane, the nitrocellulose membrane was stained with Ponceu S solution to determine the positions of marker protein and antigen. The nitrocellulose position, to which CRM antigen is transferred, was excised out with 3 mm intervals. Then, each slice was incubated within 1% BSA and PBS-T solution for 1 hour to block non-specific binding of proteins, to which 500 μl of patients sera (un-diluted sera for IgE and IgG4) were added and incubated for 16 hours at room temperature. After washing with PBS-T three times, the samples were incubated with biotinylated anti-human IgE antibody (Vector Laboratories, Inc, Burlingame, CA, USA) for 3 hours at the room temperature. After washing again with PBS-T three times, 500 μl of streptavidin-peroxidase (Sigma Chemical Co., St. Louis, MO) 1:500 dilution was added to each slice sample, which was then incubated for 1 hour. After washing again with PBS-T solution for 5 times, to each slice sample, 1 ml of mixture solution consisting of 3 ml of 0.3% 4-kiro-l-naphtol (Sigma Chemical Co., St. Louis, MO) dissolved in methanol, 50 ml of Tris buffered saline, and 25 μl of 30% H2O2 solution, was added, and allowed reaction for 15 minutes. After washing with distilled water three times, results were monitered. The protein bands that react with more than half of 20 patients sera, was identified as major allergen. As a result, 24 kDa and 34 kDa of protein bands are identified as major allergen (See Figure 1).
4. Determination of HEP (histamine equivalent potency) for CRM
For quantitative standardization, the antigenic potency of CRM crude extract was standardized with HEP determination. The CRM crude extracts prepared according to the method described in example 1 hereinabove, was diluted to the concentration of 1 mg/ml, which is then used for the preparation of skin test reagent. Using the skin test reagent, skin prick test was performed for 20 patients showing allergic symptoms due to CRM. The mean diameter of the wheal formed by CRM was measured and compared with the size of the wheal formed by histamine (1 mg/ml). If the compared sizes are identical, the tested antigen is defined as having 1 HEP (=1000 BU/ml). The skin test reagent prepared according to method described above showed 1.02 HEP. This means that the skin test reagent comprising 1 mg/ml of CRM extract as prepared above contains the same allergenic materials as lmg/ml of histamine. Example 3 : Standardization of Two Spotted Mite Crude Extract
Standardization of TSM crude extract was done according to the same method used for the standardization of CRM crude extract.
The result of qualitative standardization indicated that the positive responders to TSM antigen showed partial cross reactivity with related antigens such as house dust mites and storage mites, but not with other inhalant antigens.
As a result of immunoblot analysis, three bands of 10, 29 and 60 kD were identified as major allergens (see Figure 2).
According to the quantitative standardization performed by the method described above, the allergenic potential of skin test reagent prepared according to the method was 0.95 HEP.
Example 4 : Standardization of European Red Mite Crude Extract
Standardization of ERM crude extract was done according to the same method used for the standardization of CRM crude extract.
The result of qualitative standardization indicated that the positive responders to ERM antigen showed partial cross reactivity with related antigens such as house dust mite and storage mite, but not with other inhalant antigens.
As a result of immunoblot analysis, three bands of 33, 41 and 51 kD were identified as major allergens.
According to the quantitative standardization performed by the method described above, the allergenic potential of skin test reagent prepared according to the method was 1.01 HEP.
In summary of the above results, with quantitative standardization, spider mite crude extracts contain many proteins, among which 2 major allergens for CRM and three major allergens for TSM and ERM, respectively, were identified. Further, with qualitative standardization, skin test reagents containing 1 mg/ml of crude extracts according to the present invention showed 0.9 to 1.1 HEP.
Example 5 : Production of Skin Test Reagent
Skin test reagent for diagnosing CRM causative allergy was prepared by using the CRM crude extract prepared in example 1. The CRM extract in phosphate-buffered solution diluted to the concentration of 1 mg/ml was mixed with an equal amount of sterile glycerin.
Similarly, skin test reagent for diagnosing TSM causative allergy was prepared by using the TSM crude extract prepared in example 1. The TSM extract in phosphate-buffered solution diluted to the concentration of 1 mg/ml was mixed with an equal amount of sterile glycerin.
Similarly, skin test reagent for diagnosing ERM causative allergy was prepared by using the ERM crude extract prepared in example 1.
Example 6 : Spider Mite specific IgE and IgG Subtype Antibody Detection Kit
(1) 100 μl of 1 mg/ml CRM crude extract prepared according to the method of example 1 was coated upon the wall of 94- well (NUMC, immunoplate, Roskilde, Denmark), to provide 94-well coated with CRM crude extract. TSM- and ERM- coated well were prepared similarly.
(2) Biotinylated secondary antibody specific to the CRM-specific IgE and
IgG subtype antibody was purchased from Vector Co. (Berlingham, CA, USA).
(3) Streptavidine-peroxidase, H2O2 and ABTS substrate were purchased from Sigma (St. Louis, MO, USA) and included into the kit.
The detection kit comprising said components (1) to (3) are used as follows: Patients' sera are added to the well (1) and allowed for reaction. After washing the well, secondary antibody (2) is added into the well (1) and allowed for reaction. After washing again and adding enzyme and substrate (3) into the well, color reaction is detected by absorbance at 405 nm. According to the present invention, allergic diseases such as bronchial asthma and allergic rhinitis, which are caused by spider mites, can be diagnosed easily and specifically. The causative allergen for certain manifestations of such allergic diseases has been a mystery prior to this disclosure. The present invention allows the detection of allergic diseases caused by spider mite antigen in those patients who are not susceptible to skin tests, by simple isolation of serum from the patients. Detection of the spider mites as causative allergens of allergic diseases is prerequisite for the diagnosis and treatment plan including the avoidance of the allergens and immunotherapy.
While the present invention has been described with reference to some preferred embodiments, it will become apparent to those skilled in the art that variations and modifications are possible without deviating from the broad principles of the present invention, which is defined by the following claims.

Claims

1. A skin test reagent for the diagnosis of allergic diseases caused by spider mites, comprising spider mite crude extract as the effective component.
2. The skin test reagent according to claim 1, wherein said spider mites is one or more selected from the group consisting of citrus red mite, two-spotted mite and European red mite.
3. The skin test reagent according to claim 1 or claim 2, wherein the crude extract of spider mite is at a concentration of 1 mg/ml.
4. The skin test reagent according to claim 1 or claim 2, further comprising an agent to preserve the effective component
5. The skin test reagent according to claim 4, wherein the agent is glycerin.
6. A spider mite-specific IgE and IgG subtype detection kit for diagnosis of allergic diseases caused by spider mite allergen, comprising,
(1) a container with spider mite crude extract; and
(2) a secondary antibody specific to spider mite-specific IgE and IgG subtype antibody which is labeled with detectable marker.
7. The spider mite-specific IgE and IgG subtype detection kit according to claim 6, wherein the spider mite is one or more selected from the group consisting of citrus red mite, two-spotted mite and European red mite.
8. The detection kit according to claim 6 or claim 7, wherein the crude extract of spider mite is at a concentration of 1 mg/ml.
9. The detection kit according to claim 6 or claim 7, wherein the secondary antibody is biotin-labeled antibody.
10. The detection kit according to claim 9, additionally comprising a component (3) further comprising streptavidine-peroxidase, H2O2 and ABTS substrate.
PCT/KR2002/000534 2001-03-27 2002-03-27 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 WO2002076477A1 (en)

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