JP3124337B2 - Refined mite allergen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a purified mite allergen having allergenic 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 that administers an allergen that is a cause of allergy to desensitize has been regarded as the most important fundamental therapy, particularly when 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.

As for mite allergic disease, Dermatop as a 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 (p
I 5 to 8.3) have been reported [J. Immunol .: 125,587-59.
2, (1980) / J. Allergy Clin. Immunol .: 76,753-761,
(1985) / Immunology: 46,679-687, (1982) / Int. Ar
ch. Allergy 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 a mite body extract have been conducted mainly by interviews, and in rare cases, the 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 excretions in mite culture. A second object of the present invention is to
An 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]

Means for Solving the Problems The present inventors have intensively studied allergens in extracts of Dermatophagoides farinae excreta for the purpose of developing therapeutic agents and diagnostic agents for mite allergic diseases. As a result, they have found that glycoproteins having a molecular weight of about 13,000 have strong allergen activity, and have further studied to complete the present invention.

That is, the present invention is a purified mite allergen having the following physicochemical and biological properties. Included in mite culture excrement extract, fractional molecular weight 10,
000 is a component that passes through the ultrafiltration membrane. It is a glycoprotein containing about 30% carbohydrate. Molecular weight (Sephadex G50 gel filtration method): about 13,0
00 Has allergen activity. In addition, the present invention provides an extraction treatment of excreta during mite culture with a saturated saline solution and / or a buffer having a moderate ionic strength,
The method for producing a purified mite allergen as described above, wherein 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 purified mite allergen as an active ingredient.

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. Abbreviations for amino acid residues are as follows. Asp Aspartic acid; Thr Threonine; Se
r Serine; Glu Glutamic acid; Gly Glycine; Al
a Alanine; Val valine; Ile Isoleucine; Le
u leucine; Phe phenylalanine; Lys lysine; His
Histidine Arg Arginine; Pro Proline;

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, the purified mite allergen of the present invention will be described in more detail. That is, the properties of the purified mite allergen of the present invention (for example, the purified LM-1 active ingredient obtained in Examples described later) are as follows. (1) Color and properties: white (lyophilized product) (2) Water solubility: easily soluble (3) Molecular weight: about 13,000 by Sephadex G50 gel filtration method. (4) Composition: According to analysis from the composition, the sugar content is about
Accounts for 30%.

The amino acid composition of the purified mite allergen of the present invention can be obtained by the following operation. In other words, the 0.2% sample solution 200 [mu] l was mixed with 12N hydrochloric acid 200 [mu] l, in a sealed test tube with N ₂ substitution, 24 hours hydrolyzed at 110 ° C.. After dehydration, a procedure of adding a small amount of water and re-drying is repeated three times to remove hydrochloric acid. This is dissolved in 1 ml of a diluting buffer for an amino acid analyzer, and the solution is analyzed by an amino acid analyzer (Hitachi, Ltd.). Quantify. This gives the following result: Amino acids (total 7.6 μmol / mg) Asp 0.9; Thr 0.4; Ser 0.7; Glu 1.0; G
ly 1.5; Ala 0.6; Val 0.3; Ile 0.3; Leu 0.4; Ph
e 0.2; Lys 0.3; His 0.1; Arg 0.3; Pro 0.6

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

(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.

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 antigen, for example, any of 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 is 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.
Examples include lactate buffer, acetate buffer, citrate buffer, Tris-HCl buffer, 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, this crude mite excretion extract is passed through, for example, an ultrafiltration membrane (UF-20CS-10PS) having a molecular weight of 10,000 cuts, and a high molecular crude mite excretion antigen that does not pass through this membrane and a low molecular crude that passes therethrough. Fractionation into mite excretion antigens.

B) Purification of low molecular weight crude mite excretion antigens The purification of low molecular weight crude mite excretion antigens is carried out by (i) mite asthma patient serum-specific IgE, IgG, rabbit anti-mite excretion antiserum, rabbit anti-mite excretion Antibody, measurement of antigen activity of each fraction by measuring the reaction with mouse monoclonal antibody specific for excretion antigen by enzyme immunoassay (ELISA) (Immunochemistry: 8, 871, (1971)), ( ii) Measurement of antigen activity of each fraction by radioimmunoassay using rabbit anti-mite excretion antiserum, (iii) Measurement of allergen activity of each fraction by intradermal reaction activity, (iv) Histamine release from mite allergy patient A known purification method, such as gel filtration chromatography, ultrafiltration, or ion exchange chromatography, under monitoring such as measurement of the allergen activity of each fraction by activity. Affinity chromatography, hydrophobic chromatography, focusing electrophoresis, can be purified gel electrophoresis and the like, alone or in combination.

For example, specifically, a low-molecular-weight crude mite excretion antigen is separated from Sephadex G50 (Pharmacia LKB Biotechnol).
ogy AB). The elution pattern at that time is monitored by the leukocyte histamine releasing ability of the mite asthma patient, the reactivity to the serum-specific IgE of the patient by ELISA and the serum of rabbit anti-mite excreta, and the like. The active ingredient which is the purified mite allergen of the present invention can be obtained by appropriately combining the fractions showing strong intradermal reaction activity with purification means such as ion exchange chromatography, reverse phase chromatography, dual mode chromatography and reverse phase HPLC. Can be purified.

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 means 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, an oral, intradermal, subcutaneous, intramuscular, intraperitoneal or the like administration method. Furthermore, it can also 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.

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. Measure the amount of histamine released from basophils (a type of leukocyte) [Allergy: 33,692, (198
4) / Allergy: 33,733, (1984)]. In this histamine release titration, 50% of the maximum release amount (inflection point of titration curve)
The amount of histamine released from the water 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.

Accordingly, 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.

[Table 1]

[0023]

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 low molecular weight mite excretion antigen: In a feed M for rats, mice and hamsters (Oriental Yeast Co., Ltd.), at a temperature of 26 ± 2 ° C. and a humidity of 75% RH, the mite density was 20 to 30,000. Breeding Dermatophagoides farinae so that the number of animals per gram of the medium is 100 g / m.
One liter of a saturated saline solution was added, and the mixture was stirred well. 3 at room temperature
After leaving still for 0 minutes, centrifugation was performed at 3000 rpm for 30 minutes. At this time, the mite bodies float on the surface of the supernatant,
This was filtered off. Saturated saline was added again to the precipitate, and the same operation was repeated. Further, 1 liter of 10 mM phosphate buffer was added to the precipitate, and the same operation as in the case of saturated saline was repeated (twice). The resulting extract was dialyzed against tap water overnight, and subjected to ultrafiltration with a molecular weight cut off of 10,000 (membrane: UF-2
(0CS-10PS, Tosoh)). Each fraction was concentrated and freeze-dried to obtain a high molecular weight crude mite excretion antigen and a low molecular weight crude mite excretion antigen, respectively. From 3.7 kg of the mite culture medium, 127.0 g of a high molecular weight crude mite excretion antigen (HM) and 69.7 g of a low molecular weight crude mite excretion antigen (LM) were obtained.

Example 2 Fractionation of Allergen Active Fraction from Low Molecular Crude Mite Excretion Antigen (LM) Low Molecular Crude Mite Excretion Antigen (LM) 5 Obtained in Example 1
00mg (concentration 500mg / 30ml 0.9% NaC
l) with an Ultrogel AcA 54 column (IBF, size 4.4 × 100)
cm), and fractionated by gel filtration using 0.9% NaCl as an eluate at a flow rate of 120 ml / hour. Each fraction was tested for antigenicity in response to antiserum (not shown) and for allergen activity in a histamine release test on leukocytes from mite allergic patients. As shown in the histogram of FIG. The release activity was largely divided into two. So the fraction
No. 27-31 and No. 37-39 were collected as fractions containing the allergen active component, and were designated as LM-1 fraction and LM-2 fraction, respectively. In the figures, BD corresponds to the void volume, which is the elution position of blue dextran, BSA is the elution position of bovine serum albumin, and TV is the elution position of K ₂ CrO _{4 in} the total volume. The dashed line indicates ultraviolet absorption at 280 nm. The lyophilized yield of each fraction was 23.4 mg for the LM-1 fraction and 70.8 for the LM-2 fraction.
mg. In addition, when an intradermal reaction test for mite-allergic patients was simultaneously performed, strong allergen activity was observed in both fractions.

Example 3 Purification of Active Ingredient in LM-1 Fraction (a) Gel Filtration Chromatography of LM-1 Fraction on Sephadex G50 Sephadex G50 (Pharmaci
a Gel filtration chromatography was performed using LKB Biotechnology AB) to fractionate the LM-1 fraction. At the same time, calibration was performed using ovalbumin and cytochrome C, and the molecular weight of the active ingredient in the LM-1 fraction was estimated. The result is shown in FIG. The gel filtration conditions were as follows: sample amount 10 mg (concentration 10 mg / 3 ml), eluent 0.
9% NaCl, flow rate 30 ml / hour, column size 1.5
× 100 cm. Each fraction was monitored for leukocyte histamine release activity along with the antiserum response. In the figure, the histogram shows the histamine release activity of leukocyte, OV
A indicates the elution position of ovalbumin having a molecular weight of 45,000, and Cyt. C indicates the elution position of cytochrome C having a molecular weight of 13,500. From the plot of the volume up to the elution position of the peak of each component and the semilogarithm of each molecular weight, the molecular weight of the component showing leukocyte histamine release activity in the LM-1 fraction is:
It was estimated to be about 13,000. Here, the fractions indicated by the arrows in the figure (fractions No. 13-16) were pooled,
It was used as an M-1 active fraction and lyophilized. The yield is 3
mg.

(B) Ion exchange chromatography of LM-1 active fraction 10 mg of LM-1 active fraction obtained in (a) (concentration 10 mg)
/0.1 ml) was fractionated by ion exchange chromatography using an HPLC column DEAE GM (Kurita Kogyo, size 0.78 × 10 cm). 20 mM Tris-HCl buffer (p
H8.0), 10 minutes after the sample was injected, Na
With a linear gradient of 500 mM / 40 min of Cl,
Elution was performed at a flow rate of 1 ml / min. The result is shown in FIG. Here, the histogram shows the leukocyte histamine release activity, the solid line shows the ultraviolet absorption at 215 nm, and the broken line shows the N
Shows the concentration of aCl. In the figure, as can be seen from the histogram, the histamine releasing activity was determined by fraction Nos. 19-20.
Eluted only at a position corresponding to about 250 mM NaCl. This acidic fraction was collected as an LM-1 active fraction, dialyzed and lyophilized to obtain 2 mg of an active fraction.

(C) Reversed phase chromatography of LM-1 active fraction 10 mg of LM-1 active fraction obtained in (b) (concentration: 10 mg)
/0.1 ml) with TSKgel ODS-120T (Tosoh, size 0.
Further, reversed phase HPLC was carried out at 46 × 25 cm) to fractionate. Using 0.1% trifluoroacetic acid (TFA) as an eluent, elution was performed at a flow rate of 1 ml / min with a linear gradient of 1% / min of methanol 10 minutes after the sample was injected. FIG. 4 shows the results. Here, the histogram shows white blood cell histamine release activity, the solid line shows ultraviolet absorption at 280 nm, and the broken line shows the concentration of methanol. In the figure, as can be seen from the histogram, the histamine releasing activity eluted in fractions No. 15-16. This fraction was collected as an LM-1 active fraction, freeze-dried after dialysis, but could not be weighed with sufficient precision due to the trace amount. However, when an intradermal reaction test was also performed on mite allergic patients at the same time, strong allergen activity was observed.

(D) Dual mode chromatography (DMC) of the LM-1 active fraction The LM-1 active fraction obtained by (c) was subjected to Asahipack GS-320.
(Asahi Kasei, size 0.76 × 50 cm) and further purified by DMC. Using 50 mM ammonium acetate as the eluent,
Elution was carried out isocratically at a flow rate of 0.5 ml / min. The result is shown in FIG. Here, the solid line is 215 nm
Shows the ultraviolet absorption of In the figure, a sharp peak was given at 12.1 minutes, and this peak was collected as an LM-1 active fraction, freeze-dried after dialysis, but could not be weighed with sufficient precision due to the trace amount. However, when an intradermal reaction test was also performed on mite allergic patients at the same time, strong allergen activity was observed.

(E) Purification of LM-1 active ingredient In order to check the homogeneity of the LM-1 active fraction obtained in (d),
Reversed phase HPLC was performed on TSKgel ODS-120T (Tosoh, size 0.46 × 25 cm). Using 0.1% trifluoroacetic acid (TFA) as an eluent, 10 minutes after the sample was injected, a linear gradient of 1% / min of methanol was used.
Elution was performed at a flow rate of 1 ml / min. FIG. 6 shows the result. Here, the solid line indicates ultraviolet absorption at 215 nm. In the figure, in the fraction containing the peak indicated by the arrow, leukocyte histamine releasing activity and intradermal reaction activity for mite allergic patients were observed. This fraction was separated and purified LM-1
The active ingredient.

Example 4 Properties of Purified LM-1 Active Ingredient The purified LM-1 active ingredient obtained in (e) of Example 3 was subjected to amino acid analysis and sugar analysis by the phenol-sulfuric acid method. 3.0 μg in terms of glucose
Of neutral sugars (ie, about 30% in sugar content) was detected.
The results of amino acid composition analysis were as follows. Asp 0.9; Thr 0.4; Ser 0.7; Glu 1.0; G
ly 1.5; Ala 0.6; Val 0.3; Ile 0.3; Leu 0.4; Ph
e 0.2; Lys 0.3; His 0.1; Arg 0.3; Pro 0.6 The color and properties of this purified LM-1 active ingredient were white (lyophilized product) and easily soluble in water.
In an anaphylactic reaction test using guinea pigs, no anaphylactic reaction is induced.

Example 5 Preparation of Antigen Preparation for the Treatment of Desensitization A 0.9% saline solution containing 0.5% phenol was used as a solvent.
1 mg / m of the purified LM-1 active ingredient obtained in Example 3
1 to make a stock solution of the antigen for desensitization treatment.

Example 6 Preparation of titration reagent for mite allergy diagnosis Purified L obtained in Example 3 using Hanks buffer as a solvent
The M-1 active ingredient is dissolved to a concentration of 1 mg / ml, and used as a stock solution of a histamine free titration reagent.

[0033]

The purified mite allergen of the present invention is a useful antigen for the treatment of desensitization from the viewpoint of efficacy and safety.
It is useful as a therapeutic or preventive agent for desensitization to mite allergic disease. In addition, the therapeutic agent for desensitization has no anaphylaxis-inducing effect and can be safely used on the human body, and is important for prompt and accurate diagnosis of mite allergic disease and for appropriate treatment of mite allergic disease Therefore, establishment of a diagnostic system is expected.

[Brief description of the drawings]

FIG. 1 shows the results of assaying allergen activity of each fraction obtained by gel filtration of LM in Example 2 in a histamine release test on leukocytes of mite allergic patients. Here, BD in the figure corresponds to the void volume, the elution position of blue dextran, BSA indicates the elution position of bovine serum albumin, and TV indicates the elution position of K ₂ CrO _{4 in} the total volume. The broken line is 280
Shows ultraviolet absorption in nm.

FIG. 2 shows that the LM-1 fraction was combined with ovalbumin and cytochrome C in Sephadex G in Example 3.
Perform gel filtration chromatography on LM-1
It is the figure which estimated the molecular weight of the active ingredient in a fraction. Here, the histogram in the figure shows the leukocyte histamine release activity,
OVA indicates the elution position of ovalbumin having a molecular weight of 45,000, and Cyt. C indicates the elution position of cytochrome C having a molecular weight of 13,500.

FIG. 3 shows the LM obtained in (a) in Example 3.
1 is a diagram showing the results of fractionation of 10 mg of the 1-active fraction by ion exchange chromatography using an HPLC column DEAE GM.

FIG. 4 shows the LM obtained in (b) in Example 3.
10 mg of the active fraction was reverse-phased with TSKgel ODS-120T.
It is a figure which shows the result of having performed LC and fractionation.

FIG. 5 shows LM-1 obtained in Example 3 in (c).
It is a figure which shows the result of having performed DMC of the active fraction with Asahipack GS-320.

FIG. 6 shows LM-1 obtained in (d) in Example 3.
FIG. 4 shows the results of reversed-phase HPLC performed on TSKgel ODS-120T to check the homogeneity of the active fraction.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Takeshi Wada 908 No. 334, Ono-cho, Saeki-gun, Hiroshima Prefecture (56) References JP-A-3-81300 (JP, A) (58) Fields investigated (Int. . ^7, DB name) C07K 14/435 BIOSIS (DIALOG) MEDLINE (STN)

Claims (4)

(57) [Claims] 1. A purified mite allergen having the following physicochemical and biological properties. Included in mite culture excrement extract, fractional molecular weight 10,
000 is a component that passes through the ultrafiltration membrane. It is a glycoprotein containing about 30% carbohydrate. Molecular weight (Sephadex G50 gel filtration method): about 13,0
00 Has allergen activity. 2. The excrement in the mite culture is extracted with a saturated saline solution and / or a buffer having a moderate ionic strength.
The method for producing a purified mite allergen according to claim 1, wherein 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.