JPH07133227A - Purified mite allergen - Google Patents

Abstract

PURPOSE:To provide the subject allergen, an antigen for desensitization therapies useful from the standpoint of effectiveness and safety, thus useful as a desensitization therapeutic agent/preventive agent for mite-allergic diseases. CONSTITUTION:The objective purified mite allergen consisting of (A) HM2-1, a glycoprotein 29000 in molecular weight containing 25wt.% of carbohydrate(s) or (B) HM2-2, a glycoprotein 32000 in molecular weight containing 37wt.% of carbohydrate(s), which can be obtained by separation, followed by purification, through dispreparative SDS-PAGE from an extract fluid derived from the excreta during mite culture. This allergen is useful in quick and accurate diagnoses of mite allergic diseases. Specifically, this purified mite allergen can be obtained by a process in which the excreta during mite culture is subjected to extraction with saturated saline solution and/or a buffer solution of medium ionic strength and the resultant extract fluid is fractionated using a combination of techniques such as gel filtration, SDS-PAGE and ELISA technique using mite patient serum IgG or IgE.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a purified mite allergen having allergen activity.

[0002]

2. Description of the Related Art Indoor dust mites are important as a major cause of allergic diseases such as atopic bronchial asthma. Conventionally, as a treatment method for allergic diseases, desensitization therapy in which allergen, which is a causative agent of allergy, is administered and desensitized is considered to be the most important fundamental therapy, and antigens such as hay fever and insect allergies are particularly important. In diseases that are easily identified,
It can be said that the evaluation is now established. However, this hyposensitization therapy involves the risk of anaphylaxis due to allergens, and thus requires safe administration of therapeutic antigens.

As for mite allergic diseases, Dermatop (Dermatop
hagoides pteronyssinus) and Dermatophagoides farinae (Derm
Atophagoides farinae) are reported to be important [J. Allergy: 42, 14-28, (1968)]. Conventionally, as the major mite allergen, a mite excrement, a glycoprotein with a molecular weight of 24-28 kD contained in a mite body (pI 4.6-7.2)
And protein having a molecular weight 14.5~20kD (pI 5~8.3), etc. have been reported [J. Immunol:. 125, 587-592 , (1980) / J.
Allergy Clin. Immunol .: 76, 753-761, (1985) / Immun
ology: 46 , 679-687, (1982) / Int. Arch. Allergy Ap
pl. Immunol .: 81, 214-223, (1986) / J. Allergy Cli
n. Immunol .: 75, 686-692, (1985)]].

On the other hand, the inventors of the present invention showed specific reactivity to a serum IgG of a mite asthma patient in a fraction having a higher molecular weight and a lower molecular weight than the above-mentioned major mite allergens, and leukocyte histamine of a mite asthma patient. It was reported that there is a component that induces release (Japan Society for Agricultural Chemistry, 62 : 411, 1988). However, except for a part, it has not been purified to the extent that it can be used as an antigen for treating desensitization.

As a method for diagnosing mite allergic disease,
Traditionally, most of the interviews have been conducted with an intradermal reaction test using an indoor dust (house dust) extract or a mite body extract, and rarely, the measured value of IgE antibody titer in serum by the RAST method. It was only to use (relative value) together, and it was quite difficult to directly diagnose a mite allergic disease.

[0006]

Conventionally, house dust extract has been used as a method for desensitizing bronchial asthma with indoor dust mite as a specific antigen. Its composition is extremely unclear, and since it contains many kinds of impurities and may induce anaphylaxis, its dose is extremely limited and the therapeutic effect is extremely low. Therefore, from the viewpoint of efficacy and safety, the emergence of a useful antigen for treating desensitization is expected.
In addition, rapid and accurate diagnosis of mite allergic disease is important for proper treatment of mite allergic disease,
The establishment of a diagnostic system is expected.

[0007] An object of the present invention is to provide exactly this point, and to provide a novel purified mite allergen which is extremely useful as a therapeutic agent or diagnostic agent for mite allergic diseases. That is, an object of the present invention is to provide a novel purified mite allergen having allergen activity and a method for producing the novel purified mite allergen, which can be extracted from excrement in mite culture, and a novel mite allergic disease treatment. To provide an agent and a novel diagnostic agent for mite allergic disease.

[0008]

[Means for Solving the Problems] The present inventors have conducted extensive studies on allergens in the extract of excrement of Dermatophagoides farinae for the purpose of developing therapeutic agents and diagnostic agents for mite allergic diseases. As a result, various gel filtration, S
DS-PAGE and IgG in serum of mite allergic patients,
By fractionating in combination with an ELISA method using IgE or the like, a strong allergen activity was found in glycoproteins contained in various fractions, and further studies were conducted to complete the present invention.

That is, the gist of the present invention relates to two types of purified mite allergens, HM2-1 and HM2-2, having the following physicochemical and biological properties.

(1) The physicochemical properties and biological properties of the purified mite allergen HM2-1 are contained in the excrement extract solution in the mite culture.
Included in the fraction (HM2) eluted near the ovalbumin peak by gel filtration with cA54 (saline),
Then, it is a component (HM2-1) obtained from a band having a molecular weight of about 29,000, which is separated by disc preparative SDS-PAGE under the following conditions. Gel composition: 12.5% gel (SDS-PAGE) running buffer : 0.025M Tris, 0.192M
Glycine, 0.1% SDS Running conditions: 100 Vc.v. It is a glycoprotein containing about 25% carbohydrate and has allergen activity.

(2) The physicochemical and biological properties of the purified mite allergen HM2-2 are contained in the excrement extract in mite culture, and Ultrogel A
Included in the fraction (HM2) eluted near the ovalbumin peak by gel filtration with cA54 (saline),
Then, it is a component (HM2-2) obtained from a band having a molecular weight of about 32,000, which is separated by disc preparative SDS-PAGE under the following conditions. Gel composition: 12.5% gel (SDS-PAGE) running buffer : 0.025M Tris, 0.192M
Glycine, 0.1% SDS Running conditions: 100 Vc.v. It is a glycoprotein containing about 37% carbohydrate and has allergen activity.

Further, according to the present invention, the excrement in the mite culture is subjected to extraction treatment with a saturated saline solution and / or a buffer having a medium ionic strength, and the obtained extract is subjected to gel filtration, SDS-P.
Methods such as AGE and serum IgG of mite allergic patients,
A method for producing the above-mentioned purified mite allergen, which comprises fractionating by combining the methods of an ELISA method using IgE.

Further, the present invention provides a therapeutic agent for mite allergic disease and a diagnostic agent for mite allergic disease, which comprises the purified mite allergen as an active ingredient.

In the present invention, each of the purified mite allergens comprises a single purified mite allergen and a plurality of mite allergens (that is, a mixture of the purified mite allergens), as long as the above-mentioned requirements are satisfied. Any of the above embodiments).

The purified mite allergen of the present invention will be described in more detail below. That is, the properties of the purified mite allergen of the present invention are as follows. (1) Purified mite allergen HM2-1 Color and properties: white (lyophilized product) Water solubility: readily soluble Molecular weight: detected as a band of about 29,000 by SDS-PAGE. Composition component: According to analysis from the composition component, it is a glycoprotein and the sugar content occupies about 25%. The amino acid composition is as shown in Table 5. Has allergen activity. Does not provoke an anaphylactic reaction.

(2) Purified mite allergen HM2-2 Color and properties: white (lyophilized product) Water solubility: readily soluble Molecular weight: detected by SDS-PAGE as a band of about 32,000. Composition component: According to the analysis from the composition component, it is a glycoprotein and the sugar content occupies about 37%. The amino acid composition is as shown in Table 5. Has allergen activity. Does not provoke an anaphylactic reaction.

The molecular weight of the purified mite allergen of the present invention is
It is measured by SDS-PAGE (12.5% gel).

The amino acid composition of the purified mite allergen of the present invention can be obtained by the following procedure. That is, 200 μl of 0.1% sample solution was mixed with 200 μl of 12N hydrochloric acid,
Hydrolyze at 110 ° C. for 24 hours in a sealed test tube with N ₂ substitution. After drying to dryness, add a small amount of water and dry again to dehydrochlorine by repeating 3 times, and add 1 ml of diluting buffer for amino acid analyzer.
And is quantified with an amino acid analyzer (amino acid analysis system; Hitachi, Ltd.).

The sugar content of the purified mite allergen of the present invention is
This is a result of quantifying total neutral sugars by the phenol-sulfuric acid method using Glucose as a standard.

The allergen activity is judged by an intradermal reaction activity against a mite allergic patient, a patient leukocyte histamine release test using HPLC, a dot blot test and the like. Regarding the anaphylactic reaction, guinea pigs are immunized by a conventional method, and the anaphylactic reaction at the time of booster immunization is observed.

Examples of the method for producing the purified mite allergen of the present invention include the following methods. a) Preparation of Crude Mite Excretion Antigen The raw material for producing the mite antigen is not particularly limited, and for example, either Dermatophagoides farinae or Dermatophagoides farinae may be used. These mites are cultured in a mite culture medium and subjected to extraction treatment. The extraction treatment was performed by adding saturated saline and / or a phosphate buffer, stirring at room temperature for about 1 hour, and allowing the mixture to stand at room temperature for 30 minutes, followed by centrifugation (3000 rpm).
, 30 minutes) and pool the supernatant. At this time, any other buffer may be used as the extraction solvent as long as it is a buffer solution having a medium ionic strength. For example, lactate buffer, acetate buffer,
Examples include citrate buffer, Tris-HCl buffer, borate buffer and the like. Mite bodies float on the surface of the supernatant, and are removed by filtration. The pooled supernatant is filtered and dialyzed against ion-exchanged water (crude mite excrement extract) as a starting material. Next, this crude mite excrement extract is passed through, for example, an ultrafiltration membrane (UF-20CS-10PS, manufactured by Tosoh Corp.) having a molecular weight of 10,000 cut, and passed through with a high molecular weight crude mite excretion antigen that does not pass through this membrane. Fractionated into low molecular weight mite excrement antigen
Further fractionation is carried out for high molecular weight crude mite excretion antigen.

B) Preparation of each purified mite allergen from high molecular weight crude mite excretion antigen The fraction of the high molecular weight mite excretion antigen is (i) IgE, IgG, specific to mite allergen serum of mite allergic patient,
Rabbit anti-mite excrement antiserum, rabbit anti-mite excrement antibody,
Measurement of the antigen activity of each fraction by measuring the reaction with a mouse monoclonal antibody specific for excrement antigen by enzyme-linked immunosorbent assay (ELISA) (Immunochemistr
y: 8, 871, (1971)), (ii) Measurement of antigen activity of each fraction by radioimmunoassay using rabbit anti-mite excrement antiserum, (iii) Allergen activity of each fraction by intradermal reaction activity , (Iv) measurement of allergen activity of each fraction by leukocyte histamine release activity of mite allergic patient, known purification methods such as gel filtration chromatography, ultrafiltration, ion exchange chromatography, affinity Purification can be carried out by appropriately combining chromatography, hydrophobic chromatography, focus electrophoresis method, gel electrophoresis method and the like.

For example, specifically, a high molecular weight crude mite excretion antigen is subjected to gel filtration with Ultrogel AcA54. The eluate at that time was used as IgG and IgE in serum of a mite allergic patient.
IgG in rabbit and rabbit anti-polymer crude mite excrement antigen serum
The reactivity with the is monitored by an ELISA method, and the protein content and the absorption at 280 nm are fractionated by a guide. Fractions showing strong intradermal reaction activity were further subjected to gel filtration and SD.
The active ingredient which is the purified mite allergen of the present invention can be purified by appropriately combining purification means such as S-PAGE, reverse phase chromatography, dual mode chromatography, reverse phase HPLC and the like.

For example, first, of the various fractions fractionated with Ultrogel AcA54, the fraction showing the strongest intradermal reaction activity is described, for example, in Disc Preparative S.
Fractionation is performed using DS-PAGE under the following conditions. Gel composition: 12.5% gel (SDS-PAGE) Running buffer: 0.025M Tris, 0.192M
Glycine, 0.1% SDS Electrophoresis conditions: 100 Vc.v. The purified mite allergens HM2-1 and HM2-2 thus obtained were white (lyophilized product) in color and were easily soluble in water. is there. In the anaphylactic reaction test using guinea pigs, it does not induce an anaphylactic reaction.

The purified mite allergen of the present invention can be applied as a therapeutic agent for mite allergic diseases, and is useful, for example, as a therapeutic agent for hyposensitization. Here, the mite allergic disease refers to any allergic disease caused by a mite specific antigen, such as atopic bronchial asthma, allergic rhinitis, allergic conjunctivitis, and atopic dermatitis.

The purified mite allergen purified by the above method is concentrated and collected as a solution or a syrup, or further dried and collected as a powder to be used as a therapeutic agent for hyposensitization against mite allergic disease. . This therapeutic agent for hyposensitization is used as it is, or as required, generally used adjuvants and various additives such as stabilizers, excipients, solubilizers, emulsifiers, buffers, soothing agents. , A preservative, a coloring agent and the like can be used as a compounding agent.

The therapeutic agent for hyposensitization can be administered by a usual administration route such as oral, intradermal, subcutaneous, intramuscular, intraperitoneal administration. Furthermore, it can be used as a transdermal or transmucosal drug such as troches, sublingual tablets, eye drops, nasal sprays, poultices, creams, lotions and the like. The dose and frequency of administration of the present therapeutic agent for hyposensitization are appropriately selected depending on the administration route, symptoms and the like so as to be within a range of about 20 μg or less per adult, and are administered about once a week.
Further, the therapeutic agent for hyposensitization is useful not only as a therapeutic agent for mite allergic disease but also as a preventive agent. The therapeutic agent for hyposensitization has no anaphylaxis-inducing effect and can be safely used for the human body.

The purified mite allergen of the present invention is useful as a diagnostic agent for mite allergic diseases. That is, the patient's blood, and a fixed amount of a blood cell suspension obtained by suspending the blood cell fraction obtained by centrifugation from this blood in a buffer solution, and titrated using purified mite allergen as a titration reagent, respectively, and stimulated by allergen stimulation. measuring the amount of histamine released from basophils (a type of white blood cell) [allergy: 33, 692, (198
4) / allergy: 33, 733, (1984)].

In this histamine release titration, the amount of histamine released from 50% of the maximum release amount (the inflection point of the titration curve) is determined. In this titration, (i) the allergen sensitivity of the patient is directly measured from the titration value of the blood cell suspension. (Ii) Usually, the titration value of blood is higher than that of blood cell suspension. This is because there is an IgG antibody (blocking antibody) having an allergen neutralizing ability in plasma.

Therefore, the blocking antibody titer can be obtained from the magnitude of the shift of the blood titration curve from the blood cell suspension titration curve. From the sensitivity and this blocking antibody titer, as shown in Table 1, accurate diagnosis of mite allergy is possible. It is also useful as a monitor of the effect of desensitization treatment.

[0031]

[Table 1]

[0032]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. The various analytical methods used in the following examples are collectively described at the end of Example 9 except for some.

Example 1 Preparation of high molecular weight crude mite excretion antigen: Rat, mouse, hamster feed M (Oriental Yeast Co.) at a temperature of 26
In the environment of ± 2 ° C. and humidity of 75% RH, Dermatophagoides farinae was bred so as to have a mite density of 20,000 / 30,000 mite / g medium.
After adding 10 times the amount of saturated saline solution in terms of mass to the frozen insecticidal mite culture and gently stirring at room temperature for 1 hour, 30
Centrifugation was performed at 00 rpm for 30 minutes. At this time, mite bodies float on the surface of the supernatant and were collected by suction filtration. Similarly, stirring and centrifugation were repeated twice. The supernatant obtained after three times of centrifugation and suction filtration is used as a crude mite excrement antigen (Dff). The crude mite excrement antigen extract is dialyzed against tap water overnight and centrifuged (6000 ppm, 10 minutes, 4 ° C.) to remove insoluble matter. Next, ultrafiltration with a molecular weight cut-off of 10,000 (membrane is UF-20CS-10
PS, manufactured by Tosoh Co., Ltd.) twice, and fractionated into a molecular weight of 10 kD and above and below and dialyzed for 3 days. Each fraction was concentrated and freeze-dried to obtain a high-molecular-weight crude mite excrement antigen and a low-molecular-weight crude mite excretion antigen, respectively. From 1 kg of mite culture, 5.5 g of high molecular weight crude mite excrement antigen (HM) was obtained. this is,
It contained about 0.2% of low molecular weight mite excrement antigen (LM).

Example 2 Ultrogel AcA of high molecular weight crude mite excrement antigen (HM)
Fractionation by 54: 30 ml of the high molecular weight crude mite excrement antigen (HM) solution obtained in Example 1 (HM / saline = 3.
Ultrogel AcA54 column (IBF, size 4) in which 3%) was equilibrated with physiological saline (0.9% NaCl solution) in advance.
X 85 cm), and gel filtration was performed at a flow rate of 2 ml / min to fractionate into 32 ml fractions. IgG in serum of a mite allergic patient for each fraction,
The reactivity to IgE and IgG in rabbit anti-polymer crude mite excrement antigen serum was measured, and the amount of neutral sugar was measured by the phenol-sulfuric acid method, the amount of protein was measured by the Folin-Lowry method, and the absorbance at 280 nm was measured. . From these results, these fractions were fractionated into HM1 to HM4 as shown in FIGS. From 5.5 g of HM antigen, 1.19 g of HM1 and 1.3 of HM2
5 g, HM3 1.37 g, and HM4 0.22 g
Was obtained.

Here, the histogram of FIG. 1 is ELIS.
The antigenicity of each fraction against IgG in rabbit anti-polymer crude mite excrement antigen serum measured using Method A is shown. In addition, the histogram of FIG. 2 shows IgG and I in serum of a mite allergy patient measured by the ELISA method.
The antigenicity of each fraction against gE is shown. The results of measuring the molecular weight of these four fractions by SDS-PAGE (12.5% gel) are shown in FIG. Also, this 4
Table 2 shows the results of examining the intradermal reaction activity of the fractions against a mite allergic patient.

[0036]

[Table 2]

As is clear from Table 2, HM1 and HM
2, HM3, and HM4 all showed strong intradermal reaction activity against mite-positive patients, but HM2, which showed strong reaction with both IgG and IgE in the serum of mite allergic patients, was further purified.

Example 3 Disc preparative SD of mite allergen HM2
Fractionation by S-PAGE: HM2, which had high activity in the fractionation with Ultrogel AcA54, was fractionated by disc preparative SDS-PAGE (NA-1800 type, manufactured by Nippon Eido Co., Ltd.) under the following conditions. . Gel composition: 12.5% gel (SDS-PAGE) Running buffer: 0.025M Tris, 0.192M
Glycine, 0.1% SDS Running conditions: 100 Vc.v.

First, 45 ml of the separating gel was poured into the column to be solidified, and then 10 ml of the concentrated gel was poured to solidify.
Next, the column cap at the bottom was slowly rotated and removed, and the bottom surface of the gel was washed with running buffer. A filter paper and a membrane filter, which had been attached to the migration buffer and deaerated beforehand, were attached thereto in that order. After removing air between the gel bottom surface, the filter paper and the membrane filter, a column cap from which the gel making plate was removed was attached. The degassed running buffer was added to the anode buffer layer. The column was put there, and the air entering the bottom of the column was removed. The degassed running buffer was added to the cathode buffer layer, and the sample (20 mg /
(10 ml) was applied, and then migration was started. Next, the eluate eluted from the capillary was collected under the condition of fraction / 10 minutes. As a result, 120 fractions were obtained.

The molecular weight of each fraction was determined by SDS-PAG.
It was determined by E (12.5% gel) (Fig. 4). From these results, 17.3 mg of a fraction (HM2-1) having a purified mite allergen having a molecular weight of about 29,000 and a fraction (HM2-2) 7. 7 having a purified mite allergen having a molecular weight of about 32,000. 0 mg was obtained as the main fraction.

Example 4 Mite allergen activity and anaphylaxis induction test for purified mite allergens HM2-1, HM2-2:
The purified mite allergen of the present invention was found by the following experiment to have allergen activity and not induce anaphylaxis.

Experimental Example 1 100 μg of each of the purified mite allergens HM2-1 and HM2-2 was injected intraperitoneally into three Balb / c mice (4 weeks old) mixed with Freund's complete adjuvant (Difco). Therefore, the first immunization was performed at week 0. Boosts were given at the 2nd and 4th week. Anti-HM2- in mouse serum
The 1 antibody titer and the anti-HM2-2 antibody titer were clearly increased, and sufficient immunogenicity was observed. Therefore, it was judged that they all have the ability to induce blocking antibodies and can be used as therapeutic antigens.

Experimental Example 2 The purified mite allergen HM2 was intraperitoneally injected into two guinea pigs.
-1 and 1 mg of each HM2-2 aqueous solution were added to Alum and injected to give a primary immunization at the 0th week, and booster immunizations were performed at the 3rd and 6th weeks. As a control, physiological saline containing Alum was similarly injected into the abdominal cavity of one guinea pig. The anti-HM2-1 antibody titer and anti-HM2-2 antibody titer in the serum of guinea pigs showed a clear increase, and sufficient immunogenicity was observed. However, anaphylactic symptoms were not observed at all in the observation immediately after the booster immunization at the third and sixth weeks.
From this, the purified mite allergens HM2-1 and H
It was judged that M2-2 did not induce any anaphylaxis.

Experimental Example 3 The allergen activities of the purified mite allergens HM2-1 and HM2-2 were assayed by an intradermal reaction test for mite allergic patients. The method of the intradermal reaction test is as described below. The results are shown in Table 3.

[0045]

[Table 3]

Example 5 The allergen activities of the purified mite allergens HM2-1 and HM2-2 were measured by the leukocyte histamine release test of mite allergic patients. The leukocyte histamine release test was performed according to a known method as described below [Allergy: 3
3 , 692, (1984)]. The result is shown in FIG. Histamine-releasing activity was observed in both the purified mite allergens HM2-1 and HM2-2.

Example 6 With respect to the purified mite allergens HM2-1 and HM2-2, the amount of neutral sugar was expressed by the phenol-sulfuric acid method, and the amount of protein was expressed by the Folin-Lowry method in terms of glucose or bovine serum albumin, respectively. Table 4
Shown in.

[0048]

[Table 4]

Example 7 Amino acid composition analysis of purified mite allergens HM2-1 and HM2-2: For each purified mite allergen, the constituent amino acids of the protein were analyzed by the method described below. The results are shown in Table 5.

[0050]

[Table 5]

Example 8 Preparation of Antigen Preparation for Desensitization Treatment: Purified mite allergen HM2-1 or HM2- obtained in Example 4 using 0.9% saline containing 0.5% phenol as a solvent. 2 was dissolved in each to a concentration of 1 mg / ml and used as a stock solution of the antigen for treating hyposensitization.

Example 9 Preparation of Titration Reagent for Tick Allergy Diagnosis: Purified mite allergen H obtained in Example 3 using Hanks buffer as a solvent
Each of M2-1 and HM2-2 was dissolved at a concentration of 1 mg / ml to prepare a stock solution of a histamine release titration reagent.

Various analytical methods and special materials used in the above examples will be described below. (1) Gel chromatography Ultrogel AcA54 (column size 4 × 85 cm) or Sephacryl S200 (column size 4 × 85 cm) equilibrated with 0.9% NaCl (physiological saline) solution
To this, 30 ml of a crude polymer mite excrement antigen solution (3.3%) from which insoluble matter was removed by centrifugation was added, and physiological saline was added to 2 m.
Flowing at a flow rate of 1 / min, fractionation was performed in 32 ml fractions. The eluate was monitored by UV 280 nm absorption.

(2) Disc preparative SDS-P
AGE disc preparative SDS-PAGE (NA-18
Fractionation was performed with a 00 type, manufactured by Nippon Eido Co., Ltd. under the following conditions. Gel composition: 12.5% gel (SDS-PAGE) Running buffer: 0.025M Tris, 0.192M
Glycine, 0.1% SDS Running conditions: 100 Vc.v.

First, 45 ml of the separating gel was poured into the column to be solidified, and then 10 ml of the concentrated gel was poured to solidify.
Next, the column cap at the bottom was slowly rotated and removed, and the bottom surface of the gel was washed with running buffer. A filter paper and a membrane filter, which had been attached to the migration buffer and deaerated beforehand, were attached thereto in that order. After removing air between the gel bottom surface, the filter paper and the membrane filter, a column cap from which the gel making plate was removed was attached. The degassed running buffer was added to the anode buffer layer. The column was put there, and the air entering the bottom of the column was removed. The same degassed migration buffer was added to the cathode buffer layer, and after applying the sample, migration was started. The eluate eluted from the capillary was collected under the condition of 10 minutes / fraction.

(3) ELISA method (enzyme immunoassay method) The antigen activity of each fraction during gel filtration and the antibody titer of mouse and rabbit were measured by this method. EL first
In an ISA microtiter plate (96 wells), 50 μl of each fraction at the time of gel filtration, or a solution of an immunogen in a bicarbonate buffer solution (pH 9.2) at the time of measuring the antibody titer of mouse or rabbit, Add 50 μl each,
Incubated at 37 ° C for 1 hour. The solution was removed by suction, and the well was washed 3 times with a washing buffer, and 200 μl of 1% bovine serum albumin (BSA) solution was added to the well,
Incubated at 37 ° C for 1 hour (blocking).

After washing the wells, 50 μl of serum (patient serum, rabbit antiserum) diluted to an appropriate concentration with an ELISA dilution buffer was added and incubated at room temperature for 1 hour. After washing the wells, add 50 μl of the peroxidase-labeled second antibody solution diluted with the dilution buffer,
Incubated for hours. After washing the wells, 0.1M citrate buffer containing 0.1% ABTS as substrate,
100 μl of 0.3% hydrogen peroxide was added and reacted at room temperature. The reaction time is 1 to 40 minutes (determined by the degree of color development).
Subsequently, the reaction was stopped by adding 100 μl of 10 mM sodium azide solution, and the absorption of the solution at 420 nm was measured.

(4) Intradermal reaction test A sample was dissolved in 0.9% sodium chloride and 0.5% phenol solution and 20 μl was injected into the forearm and flexor skin of a mite allergic patient with a tuberculin syringe. The major axis and minor axis of erythema and wheal appearing after 15 minutes were measured, and the average value was tested. Table 6 shows the standard of the test. However, if the wheal is 8 mm or more even if the erythema is 19 mm or less, it is determined to be positive.

[0059]

[Table 6]

(5) Leukocyte histamine release test The allergen activity of each fraction in disc preparative SDS-PAGE was measured by this method. 1. Preparation of washed whole blood cell suspension A 10 ml blood sample was centrifuged (1400 rpm, 1
0 minutes) and divide into blood cells and plasma. 50 ml of blood cells
Then, 40 ml of Hanks buffer is added and centrifugal washing (1400 rpm, 10 minutes) is performed. The supernatant is suctioned off with an aspirator, and the obtained blood cell portion is washed twice with Hanks buffer. 1 with this Hank's buffer
Make up the volume to 0 ml and use as washed whole blood cell suspension.

2. Histamine release test method The antigen fraction is dissolved in Hanks buffer to prepare an antigen solution having a concentration four times that of the antigen concentration used. To 100 μl of this antigen solution, 100 μl of Hank's buffer and 200 μl of the washed whole blood cell suspension prepared in the above 1 are added, and incubated at 37 ° C. for 30 minutes. The supernatant from which histamine was released was 1400 r
200 μl is collected by centrifugation for 10 minutes at pm, 10 μl of 60% perchloric acid is added to this supernatant, and after stirring, 140 μl of deproteinized supernatant is collected by centrifugation (3000 rpm, 10 minutes). This is subjected to histamine quantitative HPLC to quantify histamine contained in the supernatant.

To measure the total amount of histamine contained in blood cells, directly add 60% perchloric acid 4 to 200 μl of whole blood cell suspension.
Add 0 μl to destroy blood cells. This is 3000r
After centrifugation at pm for 10 minutes, histamine contained in the obtained supernatant is measured by histamine quantitative HPLC to obtain the total amount of histamine. In order to measure the amount of histamine released nonspecifically, a histamine release test is performed using Hanks buffer instead of the antigen solution and used as a control.

(6) Phenol Sulfuric Acid Method (Quantitative Method for Neutral Sugar) In a conventional method, 0.2 ml of a sample was placed in a test tube and 7%
After adding 0.1 ml of phenol water and incubating at room temperature for 10 minutes, add 1.2 ml of 85% concentrated sulfuric acid while cooling with ice, heat at 100 ° C. for 10 minutes, and then leave in cold water for 30 minutes. , OD 490 nm absorption is measured.

(7) Folin Lowry Method (Protein Quantification Method) In a conventional method, 0.2 ml of a sample is placed in a test tube, 1 ml of an alkaline copper salt solution is added, and the mixture is incubated at room temperature for 10 minutes. To this, 1 ml of 1N phenol reagent solution was added, stirred, and then incubated at room temperature for 30 minutes.
The absorption at D750 nm is measured.

(8) Amino acid analysis of protein 200 μl of 0.1% sample solution was added to 200 μl of 12N hydrochloric acid
In a sealed test tube mixed with 1 and replaced with N ₂ , 110
It was hydrolyzed at 24 ° C. for 24 hours. Dehydrochlorination was carried out by repeating the operation of drying to dryness and adding a small amount of water again to dryness three times, and dissolving this in 1 ml of a diluting buffer for amino acid analyzer, and amino acid analyzer (high performance liquid chromatograph; ( Hitachi, Ltd.).

(9) Rabbit antiserum: A 0.2% sample solution (500 μl) and Freund's complete adjuvant (500 μl) were mixed and emulsified, and then injected every week, in the vicinity of lymph nodes at the base of the forepaws of rabbits. Blood was collected weekly from the ear vein. After immunizing for 8 weeks and confirming the increase in antibody titer by ELISA,
Three days after the final immunization, whole blood was collected by suction from the ear.

[0067]

Industrial Applicability The purified mite allergen of the present invention is a useful therapeutic antigen for hyposensitization from the viewpoint of efficacy and safety,
It is useful as a therapeutic / preventive agent for hyposensitization against mite allergic diseases. Further, the purified mite allergen of the present invention,
It is useful in the rapid and accurate diagnosis of mite allergic disease.

[Brief description of drawings]

FIG. 1 shows the elution pattern of high molecular weight crude mite excretion antigen obtained in Example 2 by gel filtration on Ultrogel AcA54. For each fraction,
The antigen activity (histogram) against rabbit serum IgG, the neutral sugar, the protein, and the UV absorption value at 280 nm measured by the ELISA method are plotted.
In addition, peaks of BD (blue dextran), BSA, and OVA (ovalbumin) are also shown in the figure.

FIG. 2 is similar to FIG. 1, and is Ultrogel AcA5 of high molecular weight crude mite excretion antigen obtained in Example 2.
4 shows the elution pattern by gel filtration in 4. The antigen activity (histogram) against patient serum IgG and IgE for each fraction is shown together with the value of UV absorption at 280 nm. In addition, peaks of BD (blue dextran), BSA, and OVA (ovalbumin) are also shown in the figure.

FIG. 3 shows the results of measuring the molecular weight of the HM fraction and the HM1 to 4 fractions by SDS-PAGE (12.5% gel).

FIG. 4 shows HM2, HM2-1 and HM2-2.
It is a result of measuring the molecular weight of the fraction by SDS-PAGE (12.5% gel).

FIG. 5 shows the results of a mite allergic patient leukocyte histamine release test for the purified mite allergens HM2-1 and HM2-2 obtained in Example 5.

Claims (5)

[Claims] 1. A purified mite allergen having the following physicochemical and biological properties. Ultrogel A contained in the extract of excrement in mite culture
Included in the fraction (HM2) eluted near the ovalbumin peak by gel filtration with cA54 (saline),
Then, a component (HM2-1) obtained from a band having a molecular weight of about 29,000, which is separated by disc preparative SDS-PAGE under the following conditions: Gel composition: 12.5% gel (SDS-PAGE) running buffer : 0.025M Tris, 0.192M
Glycine, 0.1% SDS electrophoresis conditions: 100 Vc.v. It is a glycoprotein containing about 25% sugar, and has allergen activity. 2. A purified mite allergen having the following physicochemical and biological properties. Ultrogel A contained in the extract of excrement in mite culture
Included in the fraction (HM2) eluted near the ovalbumin peak by gel filtration with cA54 (saline),
Then, a component (HM2-2) obtained from a band having a molecular weight of about 32,000, which is separated by disc preparative SDS-PAGE under the following conditions: Gel composition: 12.5% gel (SDS-PAGE) running buffer : 0.025M Tris, 0.192M
Glycine, 0.1% SDS electrophoresis conditions: 100 Vc.v. It is a glycoprotein containing about 37% sugar, and has allergen activity. 3. The excrement in the mite culture is subjected to extraction treatment with saturated saline and / or a buffer having a medium ionic strength,
The obtained extract is subjected to gel filtration, SDS-PAGE, etc., and ELISA using tick patient serum IgG, IgE
The method for producing a purified mite allergen according to claim 1 or 2, wherein the method is combined and fractionated. 4. A therapeutic agent for mite allergic diseases, which comprises the purified mite allergen according to claim 1 or 2 as an active ingredient. 5. A tick allergic disease diagnostic agent comprising the purified mite allergen according to claim 1 or 2 as an active ingredient.