JP2869531B2 - Refined mite allergen - Google Patents

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 House dust mites are important as a major cause of allergic diseases such as atopic bronchial asthma. Conventionally, as a method of treating allergic diseases, desensitization therapy in which allergens that are allergens are administered and desensitized has been regarded as the most important fundamental therapy, and in particular, antigens such as hay fever and insect allergy are used. In diseases that are easily identified,
The evaluation is now well established. However, since this desensitization therapy involves the risk of anaphylaxis due to allergens, it is necessary to administer a safe therapeutic antigen.

[0003] As for mite allergic disease, Dermatop mite (Dermatop) is used as mite allergen in indoor dust.
hagoides pteronyssinus) and Dermatophagoides farinae (Derm)
atophagoides farinae) have been reported to be important [J. Allergy: 42, 14-28, (1968)]. Conventionally, as major mite allergens, glycoproteins having a molecular weight of 24-28 kD (pI 4.6-
7.2) and / or a protein with a molecular weight of 14.5-20 kD (pI 5-8.3)
[J. Immunol.: 125,587-592, (1980)
/ J. Allergy Clin. Immunol.: 76,753-761, (1985) /
Immunology: 46,679-687, (1982) / Int. Arch. Aller
gy Appl. Immunol.: 81,214-223, (1986) / J. Allergy
Clin. Immunol .: 75,686-692, (1985), etc.].

[0004] On the other hand, a fraction having a higher molecular weight and a lower molecular weight than the main mite allergen described above are added to the serum of a mite asthma patient.
The present inventors have reported that there is a component that shows specific reactivity to IgG and induces leukocyte histamine release in mite asthma patients (Japanese Society of Agricultural Chemistry, 62: 411, 1988). However, purification to the extent that it can be used as a desensitizing therapeutic antigen has not yet been performed.

[0005] As a method of diagnosing mite allergic disease,
Conventionally, most of the intracutaneous reaction tests using an indoor dust (house dust) extract and / or mite body extract have been conducted mainly by interviews, and in rare cases, measured values of serum IgE antibody titers (relative Value), it was quite difficult to directly determine the mite allergic disease.

[0006]

Conventionally, indoor dust (house dust) extract has been used for the desensitization treatment of bronchial asthma using a house dust mite as a specific antigen. At present, its composition is extremely unclear, it contains many kinds of impurities, and its dose is extremely limited due to the possibility of inducing anaphylaxis, and its therapeutic effect is extremely low at present. Therefore, from the viewpoints of efficacy and safety, the emergence of useful antigens for the treatment of 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.

The object of the present invention is exactly at this point, and it is an object of the present invention to provide a novel purified mite allergen which is extremely useful as a therapeutic or diagnostic agent for mite allergic diseases. That is, a first object of the present invention is to provide a novel purified mite allergen having allergen activity, which can be extracted from an excrement extract in mite culture. A second object of the present invention is to provide a method for producing the novel purified mite allergen. A third object of the present invention is to provide a novel agent for treating mite allergic diseases. A fourth object of the present invention is to provide a novel diagnostic agent for mite allergic disease.

[0008]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies on allergens in a cultured extract of Dermatophagoides farinae from which mite bodies have been removed with the aim of developing therapeutic agents and diagnostic agents for mite allergic diseases. Stacked. as a result,
The present inventors have found that glycoproteins having a molecular weight of 70,000 to 80,000 and glycoproteins having a molecular weight of 1,500 to 5,000 have strong allergenic activity, and further studied to complete the present invention.

That is, the present invention is a purified mite allergen having the following physicochemical and biological properties. It is included in the excrement extract during mite culture. It is a glycoprotein containing about 40% or more carbohydrates. Molecular weight: 1,500 to 5,000 (Sephadex G25 gel filtration method) Has allergen activity. In addition, the present invention relates to a method for excreting exudates in a mite culture using a saturated saline and / or
Alternatively, the method for producing a mite allergen is characterized in that the extract is subjected to an extraction treatment with a buffer having a moderate ionic strength, and the obtained extract is fractionated using a technique such as gel filtration. Further, the present invention is a therapeutic agent for mite allergic disease and a diagnostic agent for mite allergic disease, comprising the mite allergen as an active ingredient.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In this specification, amino acids and the like may be represented by abbreviations based on IUPAC-IUB and conventional abbreviations in the art, and examples thereof are as follows.

The abbreviations for amino acid residues are as follows.

Abbreviations for sugar are as follows.

In the present invention, the purified mite allergen comprises a single mite allergen or a plurality of mite allergens (that is, an embodiment of a mixture of mite allergens) as long as the above conditions are satisfied. Either may be used.

Hereinafter, representative examples of the purified mite allergen of the present invention (low molecular weight purified mite allergen (LM-ch)) will be described in more detail. (1) Color and properties: white or light brown (lyophilized product) (2) Water solubility: easily soluble (3) Molecular weight: 1,500 to 5,000 by gel filtration using Sephadex G25. (4) Composition: According to analysis from the composition, the sugar content was 55%.
Close%. In the phenol-sulfuric acid method, the sugar content is about 41%.

[Amino acid composition] 0.2% sample solution 200
The [mu] l is mixed with 12N HC1 200 [mu] l, in a sealed test tube with N ₂ substitution was 24hr hydrolyzed at 110 ° C.. After dehydration, the operation of adding a small amount of water and re-drying was repeated three times to remove hydrochloric acid. This was dissolved in 1 ml of a dilution buffer for an amino acid analyzer, and the solution was dissolved with an amino acid analyzer (Beckman). Quantified.

[Sugar composition] One sugar in the whole was determined by a phenol-sulfuric acid method using Glucose as a standard. Individual neutral sugars and amino sugars were analyzed in 4N TFA (trifluoroacetic acid) in 10
The mixture was hydrolyzed at 0 ° C. for 4 hours, reduced with NaBH ₄ , heated at 100 ° C. for 4 hours using acetic anhydride, acetylated, and quantified by GLC.

Asx 67.2 Thr 28.2 Ser 32.0 Glx 113.1 Gly 85.3 Ala 32.3 Cys 2.2 Val 17.2 Ile 9.9 Leu 11.9 Tyr 0.9 Phe 4.7 His 5.4 Arg 16.6 Lys 20.8 HexNAc 64.3 dHex 17.2 Pen 252.2 Hex 216.4 (μg / mg)

(5) It has allergen activity. Intracutaneous reaction activity in patients with mite allergy, and histamine release test on patients using HPLC were determined. (6) It does not induce an anaphylactic reaction. Guinea pigs are immunized by a conventional method, and observed for an anaphylactic reaction at the time of booster immunization.

[Production Method] As a method for producing the purified mite allergen of the present invention, for example, the following method is exemplified.

A) Preparation of Crude Mite Excretion Antigen As a raw material for producing mite antigens, either Dermatophagoides farinae or Dermatophagoides farinae may be used. These mites are cultured in a mite culture medium and subjected to extraction treatment.

In the extraction treatment, a saturated saline solution and / or a phosphate buffer solution are added, and the mixture is stirred, left at room temperature for 30 minutes, centrifuged (3000 rpm, 30 minutes), and the supernatant is pooled. At this time, any other extraction solvent may be used as long as it is a buffer having a moderate ionic strength. For example, lactate buffer,
Examples include an acetate buffer, a citrate buffer, a Tris-HCl buffer, a borate buffer, and the like. The 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 excretion extract) as a starting material.

Next, the crude mite excretion extract is passed through, for example, an ultrafiltration membrane (UF-20CS-10PS) having a molecular weight of 10,000 cuts, and is passed through a high molecular crude mite excretion antigen which does not pass through the membrane. Fractionated into low molecular weight mite excretion antigens.

B) Purification of crude mite excretion antigen The crude mite excretion antigen was purified by the following steps: (i) mite asthma patient serum-specific IgE, IgG, rabbit anti-polymer mite excretion antiserum, rabbit anti-polymer mite excretion antibody,
Measurement of the antigen activity of each fraction by measuring the reaction with a mouse monoclonal antibody specific for the excretion antigen by enzyme immunoassay (ELISA) (Immunochemistr
y: 8, 871, (1971)). (ii) Measurement of antigen activity of each fraction by radioimmunoassay using rabbit anti-polymeric mite excrement antiserum. (iii) Measurement of allergen activity of each fraction by intradermal reaction activity. (iv) Measurement of allergen activity of each fraction by mite allergy patient leukocyte histamine release activity. Purification using known purification methods, such as gel filtration chromatography, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focal electrophoresis, gel electrophoresis, etc., alone or in combination under a monitor such as can do. For example, the following method is exemplified.

[0024] The low molecular weight mite excretion antigen is separated by Sephadex.
Gel filtration is performed using G25 (Pharmacia Fine Chemical). The elution pattern at that time is monitored by the ability of mite asthma patients to release leukocyte histamine, the reactivity to patient serum-specific IgE by ELISA and the sera of rabbit anti-polymeric mite excreta, and the protein content and absorption at 280 nm are divided into fractions using a guide. Fractions with strong intradermal reaction activity were further added to Ultrogel AcA 54
The antigen-active fraction can be purified by gel filtration using (LKB). Alternatively, it is also effective to first fractionate with Ultrogel AcA54 and then gel-filter the low molecular fraction with Sephadex G25.

[Application as a therapeutic agent for mite allergic disease] The purified mite allergen of the present invention is useful as a therapeutic agent for hyposensitization to mite allergic disease. Here, the mite allergic disease refers to any allergic disease caused by a specific antigen of a tick, such as atopic bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and the like.

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

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

[Application as mite allergic disease diagnostic agent] The purified mite allergen of the present invention is useful as a diagnostic agent for mite allergic disease. That is, the blood of the patient, and a fixed amount of a blood cell suspension obtained by suspending a blood cell fraction obtained by centrifugation from this blood in a buffer solution were titrated using purified mite allergen as a titration reagent, and allergen stimulation was performed. The amount of histamine released from basophils (a type of leukocyte) is measured [allergy: 33,692, (1984) / allergy: 33,733, (1984)].

In this histamine release titration, the amount of histamine released from 50% of the maximum release (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) The titration value of the blood is usually higher than the value of the blood cell suspension. This is due to the presence of IgG antibodies (blocking antibodies) having allergen neutralizing ability in plasma. Therefore, the blocking antibody titer is obtained from the magnitude of the shift of the blood titration curve from the blood cell suspension titration curve. From the sensitivity and the blocking antibody titer, an accurate diagnosis of mite allergy is possible as shown in Table 1. It is also useful as a monitor of the desensitizing treatment effect.

[0030]

[Table 1]

[Allergen activity and anaphylaxis induction test] The purified mite allergen of the present invention was found to have allergen activity and not to induce anaphylaxis by the following experiment.

Experimental Example 1 Dff-1-2-0.1A was injected intraperitoneally into three Balb / C mice (4 weeks old).
A first immunization was performed at week 0 by injecting 100 μg of the mixture with Freund complete adjuvant (Difco) and injecting the mixture. Booster immunizations were performed on the second and fourth weeks. Anti-Df in mouse serum
As shown in FIG. 1, the f-1-2-0.1A antibody titer was clearly increased, and sufficient immunogenicity was observed. Therefore, it was determined that the antibody had the ability to induce a blocking antibody and could be used as a therapeutic antigen.

Experimental Example 2 1 mg of Dff-1-2-0.1A eluate was intraperitoneally injected into two guinea pigs.
A primary immunization was performed at week 0 and a boost was performed at week 3 by injection in addition to Alum. Control is guinea pig 1
Physiological saline containing Alum was similarly injected intraperitoneally into the animals. As shown in FIG. 2, the anti-Dff-1-2-0.1A antibody titer in the guinea pig serum was clearly increased, and sufficient immunogenicity was recognized. However, no anaphylactic symptoms were observed immediately after the third week of boosting. From this, it was determined that Dff-1-2-0.1A had no anaphylaxis-inducing property.

Experimental Example 3 The allergen activity of Dff-1-2-0.1A was assayed by an intradermal reaction test for mite allergic patients. The intradermal reaction test followed the following method. Sample 0.9% NaCl, 0.5% pheno
l Dilute to 0.0005% with solution. Using a syringe for tuberculin, 20 μl of this solution was injected into the forearm and flexor skin of the mite allergic patient, and the major and minor axes of red rash and wheal that appeared about 15 minutes later were measured, and the average was used for the test. Was. The test criteria are as shown in Table 2.

[0035]

[Table 2]

(However, even if the erythema is 19 mm or less, the wheal is 8 mm
If it is above, it is determined as positive (+). As a result, as shown in Table 3, allergen activity was recognized in Dff-1-2-0.1A. However, no anaphylactic induction was observed.

[0037]

[Table 3]

Experimental Example 4 The allergen activity of LM-1A, LM-1B, LM-2A and LM-2B was assayed by an intradermal reaction test for allergic mite patients. The intradermal reaction test was performed in the same manner as in Experimental Example 3. As a result, as shown in Table 4, allergen activity was observed in LM-1A, LM-1B and LM-2B. However, anaphylaxis induction was not recognized similarly to Experimental Examples 2 and 3.

[0039]

[Table 4]

Experimental Example 5 The allergen activity of LM-1A, LM-1B, LM-2A and LM-2B was measured by a histamine release test on leukocytes from mite allergic patients. The leukocyte histamine release test was performed according to a known method [allergy: 33,692, (1984)]. That is, the reaction between each fraction sample and the leukocyte surface IgE in the mite allergic patient serum was determined by HPLC (High Performance Liquid Chr
omatography). As shown in FIG. 3, histamine releasing activity was observed in the LM-1A, LM-1B and LM-2B fractions.

[0041]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of high molecular weight mite excretion antigen and low molecular weight mite excretion antigen: Rat Mice, Hamster Feed M (Oriental Yeast Co.), temperature 26 ± 2 ° C., humidity 75% RH In the environment described above, Dermatophagoides farinae are reared so that the density of the mites becomes 2 to 30,000 / g, and 1 L of a saturated saline solution is added to 100 g of the mite culture medium, followed by sufficient stirring. After allowing to stand at room temperature for 30 minutes, centrifugation is performed at 3000 rpm for 30 minutes. At this time, the mite bodies float on the surface of the supernatant, and are removed by filtration. The same operation is repeated by adding saturated saline to the precipitate again. Further, 1 L of 10 mM phosphate buffer is added to the precipitate, and the same operation as in the case of saturated saline is repeated (twice). The obtained extract was dialyzed overnight against tap water, and subjected to ultrafiltration with a molecular weight cut off of 10,000 (membrane: UF-20CS-10PS, Tosoh) to obtain a molecular weight of 1
Fractionated into more than 10,000 and less. Each fraction was concentrated and lyophilized to obtain a high molecular weight crude mite excretion antigen and a low molecular weight crude mite excretion antigen. From 3.52 kg of the mite culture medium, 93 g of high molecular crude mite excretion antigen and 70 g of low molecular crude mite excretory antigen were obtained.

Example 2 Purification of high molecular weight crude mite excretion antigen: (a) Fractionation by gel filtration on Ultrogel AcA54:
Ultrogel AcA54 equilibrated with 9% NaCl solution (LKB)
Then, 30 ml of a high molecular crude mite excrement antigen solution (3.3%) from which insoluble matter was removed by centrifugation was added and fractionated. At that time, UV28
Monitored at 0 nm absorbance. Flow rate is 2ml / min, 1 frc
Sampling was performed at 32 ml.

Reactivity with IgG in serum of mite allergic patient, IgG in serum of rabbit anti-polymer crude mite excretion antigen,
V absorption, neutral sugar content by phenol-sulfuric acid method, Folin-Lowr
The protein amount by the y method was measured for each fraction, and as shown in FIG. 4, fractionation was performed into four fractions Dff-1 to Dff-4. As a result of examining the intracutaneous reaction activity of the four fractions against mite allergic patients, Dff-1, Dff-2 and Dff-3 showed high activity on average ++, and Dff-4 showed almost no activity. Reactivity with IgG in serum of mite allergic patients and IgG in serum of rabbit anti-polymer crude mite excretion antigen was Dff-
1 was strong. In addition, the anti-polymer crude mite excretion antigen monoclonal antibody (MoAb T4) reacted with all fractions. The yield was 228.3 mg for Dff-1 from 1 g of crude high mite excrement antigen.

(B) Fractionation by gel filtration on Sepharose 6B Df having high activity by fractionation on Ultrogel AcA54 (LKB)
f-1 was fractionated on Sepharose 6B (Pharmacia Fine Chemical). As a result of assaying the reactivity with patient serum IgG and IgE by ELISA, fraction Nos. 18 to 26 showed high activity. Here, radioimmunoassay (RIA) was performed. First, ¹²⁵ I-Dff-1 was purified by affinity chromatography using rabbit anti-polymer crude mite excretion antigen serum-Sepharose CL-4B. As a result of RIA using the Gly-HCl eluted fraction at this time, high activity was detected in fraction Nos. 18 to 26 as in ELISA. From these results, as shown in FIG. 5, it was fractionated into three fractions Dff-1-1 to Dff-1-3. Dff-1 1g to Dff-1-1 130.2mg, Df
379.1 mg of f-1-2 and 158.5 mg of Dff-1-3 were obtained. These three
As a result of examining the intradermal reaction activity of the fractions with respect to the patient, all fractions showed high activities. Where ELISA, RI
A and Dff-1-2 showing high activity in the intradermal reaction were further purified. MoAb T4 reacted with all fractions.

(C) Fractionation by ion exchange chromatography on DEAE-Toyopearl (1) For Dff-1-2, DEAE-Toyopearl (Tosoh) and CM-
Preliminary experiments were conducted to determine which of these compounds was adsorbed at pH 6.0 with Toyopearl (Tosoh), and it was confirmed that they were adsorbed to DEAE-Toyopearl. Ion-exchange chromatography was performed using this. As a result of the previous preliminary experiment, elution of the active ingredient was observed in the elution of 0.3 M NaCl.
Eluted with NaCl. The result is shown in FIG. 0.3M NaCl
Activity was detected in both elution and 1M NaCl elution, and almost no activity was observed. These active fractions
The Dff-1-2 0.3M elution fraction (Dff-1-2-0.3M) and the Dff-1-2 1M elution fraction (Dff-1-2-1.0M) were pooled. Dff-1-2
From 100mg, Dff-1-2-0.3M is 34.6mg, Dff-1-2-1.0M is 4.
79 mg, 7.32 mg, were obtained. When the intradermal reaction activity was examined, Dff-1-2-0.3M showed high activity, and Dff-1-2-1.
0M showed little activity.

(D) Fractionation by ion exchange chromatography with DEAE-Toyopearl (2) In order to further fractionate Dff-1-2-0.3M, the NaCl concentration was reduced to 0 during ion exchange chromatography. Eluted with a ~ 0.5M gradient. Antigen activity of IgG and MoAb in patient serum
As a result of monitoring with T4, it was found that the peak of the reaction with the patient's serum IgG did not match, and therefore, at least two components were contained immunologically. Then, in order to obtain a peak portion for MoAb T4, elution was performed with NaCl 0.1M and 0.3M stepwise. At this time, patient serum IgG and Mo
As a result of monitoring with Ab T4, MoAb T
4 showed high reactivity of patient serum IgG to the 0.3M elution fraction. In addition, the same fraction was assayed for reactivity with patient serum IgE, and as shown in FIG.
High reactivity was shown in the 1M elution fraction. Therefore, the four peaks in the ELISA were identified as Dff-1-2-0.1A and Dff-1-2-0.1A, respectively.
B, pooled as Dff-1-2-0.3A, Dff-1-2-0.3B. Dff-1-2 100mg to Dff-1-2-0.1A 7.7mg, Dff-1-2-0.
3.5 mg of 1B, 7.0 mg of Dff-1-2-0.3A, and 2.5 mg of Dff-1-2-0.3B were obtained. These four fractions were examined for intradermal reaction activity. 0.1M
Most activity was recovered in the peak on the front side of NaCl elution, Dff-1-2-0.1A.

Here, as a result of composition analysis of these four fractions (Table 5), Dff-1-2-0.1A of the active fraction has a sugar content of 77%.
It was found to account for a considerable amount of%.

[0049]

[Table 5]

The SDS-PAGE of the four fractions using 7.5% acrylamide gel revealed that the active fraction Dff-1-2-0.1A did not stain at all with Coomassie staining. As a result of the dyeing, red was dyed in a relatively wide range, not as a band in a considerably high molecular weight region. This was judged to be difficult to migrate due to the high sugar content. In other words, it was impossible to estimate the molecular weight from the results, so the molecular weight was determined by ultracentrifugation. Specific volume is 0.
At 6076, one major peak and one
A minor peak was detected, confirming the presence of at least two components. The sedimentation coefficient of the major component is S _{20, W} = 2.1495, mino
The r component was S _{20, W} = 5.4044, and the average molecular weight was calculated to be 74,238 by the sedimentation equilibrium method.

Example 3 Purification of low molecular weight crude mite excretion antigen: (a) Fractionation by gel filtration on Sephadex G25 260 g of low molecular weight crude mite excretion antigen (LM) obtained in Example 1
g was fractionated by gel filtration chromatography on Sephadex G25 (Pharmacia Fine Chemical). FIG. 8 shows the result. In the vicinity of the void volume, a reaction with rabbit anti-excretion serum and patient serum IgE was observed, followed by elution of protein, neutral sugar, and UV activity in this order. Therefore, the ELISA active region near the void volume is LM-1, the protein peak is LM-2, and the neutral sugar peak is LM-1.
Pooled as LM-3 and the rest as LM-4 and fractionated into 4 fractions. The yield after lyophilization was 47 mg for LM-1,
LM-2 68 mg, LM-3 35 mg and LM-430 mg. LM-1 ~ L
When the allergen activity of M-4 was assayed by an intradermal reaction test for mite allergy patients, LM-1 and LM-2 showed allergen activity.

(B) Gel Filtration Chromatography of LM-1 with Ultrogel AcA54 LM-1 (360 mg) was refractionated by gel filtration chromatography with Ultrogel AcA54 (LKB). For gel filtration, sample (500 mg / 20 ml), Buffer 0.9% NaCl flow rate 120 ml / h
r. Performed with a column volume of 1600 ml and monitored for leukocyte histamine release activity along with response to antiserum. The result is shown in FIG.

Leukocyte histamine releasing activity was determined by the ELIS of the first half.
Although the activity is considerable in the A activity part, the largest activity peak is observed near the total volume. First half
The portion having high ELISA activity is considered to be a response due to the slightly mixed high molecular crude antigen (HM) component during the ultrafiltration in Example 1. LM-1A is the high-molecular-weight part showing ELISA activity.
The portion having the highest histamine releasing activity was fractionated as LM-1B and lyophilized. The yield is 18% for LM-1A.
% (66 mg), and LM-1B was 42% (150 mg) of the whole.
When LM-1A and LM-1B were subjected to an intradermal reaction test for mite allergic patients, as shown in Table 4, LM-1A
Allergen activity was observed in LM-1B.

(C) Ultrogel AcA-54 of LM-2 (390 mg)
Fractionation by Gel Filtration on HPLC LM-2 was subjected to gel filtration with Ultrogel AcA-54 (LKB) and refractionated. The conditions for gel filtration are the same as for LM-1. Patient leukocyte histamine release activity was monitored along with the response to antisera. The result is shown in FIG. The ELISA active part was pooled as LM-2A and the histamine releasing part was pooled as LM-2B. The yield was 130 mg for LM-2A and 150 mg for LM-2B. When an intradermal reaction test was performed on LM-2A and LM-2B against mite allergic patients, only LM-2B showed allergen activity (Table 2).

Example 4 Purification and composition analysis of LM-ch The composition analysis of the low molecular allergens LM-1B and LM-2B which showed a strong intradermal reaction was performed on mite allergic patients. As a result, since the values of both the amino acid composition and the sugar composition were very similar, it was determined that they were the same component. Therefore, both components were purified at once. That is, low-molecule crude mite excretion antigen (LM) is
First fractionation with AcA54, ELISA using rabbit anti-Dff serum
The antigen activity was measured in each fraction. The obtained low molecular weight fraction (LM-c) was further fractionated into a high molecular weight fraction (LM-ch) and a low molecular weight fraction (LM-cl) using Sephadex G25.
These results are shown in FIGS. The conditions for gel filtration are:
This is the same as the third embodiment. LM-c corresponding to LM-1B and LM-2B
h was analyzed for amino acid composition and sugar composition. Table 6 shows the results. The sugar content analyzed from the composition was 55%. However, the sugar content measured by the phenol-sulfuric acid method is about
41%.

[0056]

[Table 6]

Example 5 Estimation of Molecular Weight of LM-2B by Sephadex G25 Gel Filtration In order to estimate the molecular weight of LM-2B, LM-2B was subjected to gel filtration using a Sephadex G25 column, and at the same time, blue dextran (BD ), Vitamin B ₁₂ and K _{2 CrO 4} were calibrated. The result is shown in FIG. The histogram in the figure shows the histamine releasing activity on leukocytes in the mite allergic patient serum. VV is the elution position of BD, also the B ₁₂ is the elution position of vitamin B _12, TV
Indicates the elution position of K _{2 CrO 4} . It seems that the peak of leukocyte histamine releasing activity tends to slightly deviate from the peak of void volume to the lower molecular side.
The molecular weight of the void of Sephadex G25 used as a gel filtration carrier is 5K. Therefore, it is estimated that the molecular weight of the component exhibiting leukocyte histamine releasing activity of LM-2B is 1,500 to 5,000. Since LM-ch and LM-2B are considered to be the same component, the molecular weight of LM-ch is estimated to be 1,500 to 5,000.

Example 6 Preparation of antigen preparation for therapeutic treatment for desensitization: Dff-1-2-0.1A at 1 mg / ml in 0.9% saline containing 0.5% phenol as a solvent
To obtain a stock solution of the antigen for desensitization treatment.

Example 7 Preparation of a titration reagent for mite allergy diagnosis: Dff-1-2-0.1A was dissolved at a concentration of 1 mg / ml using Hanks buffer as a solvent.
Use as stock solution of histamine free titration reagent.

[0060]

Industrial Applicability According to the present invention, a purified mite allergen useful as a therapeutic or diagnostic agent for mite allergic diseases is provided.

[Brief description of the drawings]

FIG. 1 is a diagram showing changes in antibody titer when mice were immunized with Dff-1-2-0.1A.

FIG. 2 is a graph showing changes in antibody titer when guinea pigs were immunized with Dff-1-2-0.1A.

FIG. 3 is a diagram showing the results of gel filtration of LM-1 and LM-2 with Ultrogel AcA54.

[Fig. 4] Fig. 4 is a diagram showing the use of an ultratrogel for the production of crude high mite excretion antigen
FIG. 4 is a view showing the result of gel filtration with AcA54.

FIG. 5 is a diagram showing the results of gel filtration of Dff-1 with Sepharose 6B.

FIG. 6 is a graph showing the results of ion exchange chromatography of Dff-1-2 using DEAE-Toyopearl.

FIG. 7 is a diagram showing the results of ion exchange chromatography of Dff-1-2 using DEAE-Toyopearl.

FIG. 8 is a view showing the result of gel filtration of low molecular weight mite excretion antigen using Sephadex G25.

FIG. 9 shows that low molecular weight crude mite excretion antigen was
FIG. 4 is a view showing the result of gel filtration with AcA54.

FIG. 10 is a graph showing the results of gel filtration of LM-c using Sephadex G25.

FIG. 11 is a diagram showing the results of gel filtration of LM-2B using Sephadex G25.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 1/16 G01N 33/50 T G01N 33/50 33/53 Q 33/53 A61K 37/02 ABF // C12N 5/06 C12N 5/00 E (72) Inventor Takeshi Wada 908 No. 334, Ono-cho, Saiki-gun, Hiroshima Prefecture (56) References Naturewissenschaften, 66 (9), p. 475-476 (1979) (58) Fields investigated (Int.Cl. ⁶ , DB name) C07K 14/435 A61K 38/00 A61K 39/35 A61K 49/00 C07K 1/14 C07K 1/16 G01N 33/50 G01N 33/53 C12N 5/06 BIOSIS (DIALOG) CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. A purified mite allergen having the following physicochemical and biological properties. It is included in the excrement extract during mite culture. It is a glycoprotein containing about 40% or more carbohydrates. Molecular weight: 1,500 to 5,000 (Sephadex G25 gel filtration method) It has allergen activity. 2. The excrement in the mite culture is saturated saline and / or
2. The method for producing a purified mite allergen according to claim 1, wherein the extract is subjected to an extraction treatment with a buffer having a moderate ionic strength, and the obtained extract is fractionated using a technique such as gel filtration. 3. An agent for treating mite allergic diseases, comprising the purified mite allergen according to claim 1 as an active ingredient. 4. A diagnostic agent for mite allergic disease, comprising the purified mite allergen according to claim 1 as an active ingredient.