JP3124338B2 - 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. As for mite allergic diseases, two species of house dust mite, Dermatophagoides pteronyssinus and Dermatophagoides farinae, have been reported to be important as mite allergens in indoor dust [J. Allergy: 42, 14-28, (196)
8)]. Conventionally, as a major mite allergen, a molecular weight of 24 to 28 contained in mite excrement and / or mite body
kD glycoprotein (pI 4.6-7.2) and / or molecular weight 14.5
-20 kD protein (pI 5-8.3) has been reported [J. Immuno
l.: 125,587-592, (1980) / J. Allergy Clin. Immuno
l.: 76,753-761, (1985) / Immunology: 46,679-687,
(1982) / Int. Arch. Allergy Appl. Immunol.: 81,2
14-223, (1986) / J. Allergy Clin. Immunol .: 75,686
-692, (1985) etc.].

[0003] 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 mite asthma patient serum.
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, a purified mite allergen capable of detecting mite allergic disease with high sensitivity has not been obtained.

[0004] 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. Therefore, quick and accurate diagnosis of mite allergic disease is important for appropriate treatment of mite allergic disease, and establishment of a diagnostic system is expected.

The object of the present invention is exactly at this point, and an object of the present invention is to provide a novel purified mite allergen which is extremely useful as a diagnostic agent for mite allergic disease. That is, a first object of the present invention is to extract from excreta in mite culture,
An object of the present invention is to provide a novel purified mite allergen having allergen activity. 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 diagnostic agent for mite allergic disease.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on allergens in extracts of Dermatophagoides farinae excreta for the purpose of developing diagnostic agents for mite allergic diseases. As a result, it was found that glycoprotein (LM-2) having a molecular weight of about 4,000 and a protein (LM-2p) having a molecular weight of about 2,400 obtained by removing the sugar portion of the glycoprotein have strong allergen activity, and further studies were conducted. To complete the present invention.

[0007] Namely, the present invention provides (1) the sugar moiety of a glycoprotein having allergenic activity of the molecular weight that is part of the excrement extract in mite culture of about 4,000 (Sephadex G50 gel filtration method) Is obtained by removing
A protein that has allergen activity with a molecular weight of about 2,400.
Purification mite allergens, (2) fractionating the excrement in the mite culture extracts treated with buffer saturated saline and / or moderate ionic strength, the resulting extract using a technique such as gel filtration min , then the method of producing the (1) purified mite allergen, wherein further comprising the step of removing the sugar moiety of a glycoprotein having obtained allergenic activity, (3) the (1) Symbol placement of purified mite The present invention relates to a mite allergic disease diagnostic agent comprising an 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; Ala alanine; Val valine;
Ile Isoleucine; Leu Leucine; Tyr
Tyrosine; Phe Phenylalanine; L
ys lysine; His histidine; Arg arginine; Pro proline; Cys cysteine Abbreviations for sugars are as follows. Pen pentose; dHex deoxyhexose; Hex hexose; HexN hexosamine

In the present invention, the purified mite allergen may be either a single mite allergen or a plurality of mite allergens (ie, a mixture of purified mite allergens).

Hereinafter, the purified mite allergen of the present invention will be described in more detail. (1) Color and properties: white (lyophilized product) (2) Water solubility: easily soluble (3) Molecular weight: about 4,000 glycoprotein (LM-2) or about 2,400 protein (LM-2p) [Intact From the molecular weight (approximately 4,000, Sephadex G50 gel filtration method) of a glycoprotein, the sugar content of the glycoprotein is about 40% (about 1,600)
Minus the value].

(4) Composition: 1) Molecular weight of the present invention
Purified mite allergen consisting of 4,000 glycoproteins (LM-
The amino acid composition of 2) and purified mite allergen (LM-2p) consisting of a protein having a molecular weight of about 2,400 can be obtained by the following operation. That is, 200 μl of a 0.2% sample solution was added to 1
It is mixed with 200 μl of 2M hydrochloric acid and hydrolyzed at 110 ° C. for 24 hours in a sealed N ₂ -substituted test tube. After dehydration, the operation 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 with an amino acid analyzer (Hitachi, Ltd.). Quantify. This gives the following result:

Amino acids of LM-2 (total 6.6 μmol / mg) Asp 0.9; Thr 0.4; Ser 0.6; Glu 1.2;
ly 1.6; Ala 0.5; Val 0.2; Cys 0.0; Ile
0.1; Leu 0.1; Tyr 0.0; Phe 0.1; Lys
0.2; His 0.1; Arg 0.1; Pro 0.5 Amino acid of LM-2p (total 9.6 μmol / mg) Asp 1.1; Thr 0.5; Ser 0.9; Glu 1.5; G
ly 2.3; Ala 0.7; Val 0.3; Ile 0.2; Leu
0.3; Phe 0.1; Lys 0.4; His 0.2; Arg
0.4; Pro 0.7

2) The sugar content of the purified mite allergen (LM-2) of the present invention comprising a glycoprotein having a molecular weight of about 4,000 is Gl
When the total neutral sugar was determined by the phenol-sulfuric acid method using ucose as a standard, it was about 40%. Each neutral sugar and amino sugar were acetylated by hydrolyzing the sample in 4M TFA (trifluoroacetic acid) at 100 ° C. for 4 hours, reducing with NaBH ₄ , and heating with acetic anhydride at 100 ° C. for 4 hours. Later, when quantified by the GLC method, the following results are obtained. LM-2 sugar (total 2.9 μmol / mg) Pen 1.4; dHex 0.1; Hex1.1; HexN 0.3 On the other hand, the sugar component of purified mite allergen (LM-2p) consisting of a protein having a molecular weight of about 2,400 is not detected.

3) The test of the purified mite allergen of the present invention by digestion with pronase can be performed by the following operation. That is, 100 μl of 0.5 M Tris-HCl buffer (pH 8.5) was added to 100 μl of 1% sample solution, 10 μl of 1 M calcium chloride, 10 μl of 1% pronase E solution,
Then, 780 μl of water and 780 μl were added, and the reaction was carried out at 37 ° C. for 48 hours. The reaction solution was boiled at 100 ° C. for 10 minutes to stop the reaction. This reaction solution is used for a histamine release test.

4) The test of the purified mite allergen of the present invention by periodate oxidation can be performed by the following operation. Dissolve 1 mg of sample in 0.5 ml of water and add 20 mM
0.5 ml of a sodium periodate solution is added, and the mixture is reacted at room temperature in a dark place for 25 hours. During the reaction period, consumption of periodic acid, generation of iodic acid, and generation of formic acid are monitored by an isotachophoresis apparatus to prevent peroxidation. afterwards,
25 μl of 1% ethylene glycol is added to convert the excess periodate to iodic acid, then the aldehyde formed in this reaction is reduced with sodium borohydride and used for the histamine release test.

(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. By an ordinary method, no anaphylactic reaction is observed in an experiment using a guinea pig.

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 antigen The purification of low-molecular-weight crude mite excretion antigen is carried out by (i) serum-specific IgE, IgG, rabbit anti-mite excretion antiserum, and rabbit anti-mite excretion of mite asthma patients. 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, the purification of the low molecular weight crude mite excretion antigen is performed by gel filtration of the low molecular weight crude mite excretion antigen using Sephadex G50 (Pharmacia LKB Biotechnology AB). The elution pattern at that time was monitored by the ability of mite asthma patients to release leukocyte histamine, ELISA-specific serum serum IgE and rabbit anti-mite excretion serum,
The protein content and the absorbance at 280 nm are divided into fractions using a guide. The active ingredient which is the purified mite allergen of the present invention can be obtained by appropriately combining the fraction 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 is, as described above, a glycoprotein having a molecular weight of about 4,000 (LM-2) and a protein having a molecular weight of about 2,400 (LM-2p) obtained by removing the sugar portion of the glycoprotein. However, the step of removing the sugar moiety can be performed by a commonly known method, and is not particularly limited. For example, various methods such as a method in which a sugar chain is cleaved by Smith decomposition and then oxidized with periodate, and a method in which a sugar chain is released using an enzyme such as glycopeptidase, N -glycanase, endo- N -acetylgalactosaminidase, etc., are mentioned. Any method may be used.
In the case of periodate oxidation, for example, the method described above is used.

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.

[0023]

[Table 1]

[0024]

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 crude mite excretion antigen: Rat M, hamster feed M (Oriental Yeast) at a temperature of 26
Dermatophagoides farinae are reared in an environment of ± 2 ° C. and humidity of 75% RH so that the mite density becomes 20,000 to 30,000 / g, and 1 liter of a saturated saline solution is added to 100 g of the mite culture medium, followed by sufficient stirring. did. After standing at room temperature for 30 minutes,
Centrifugation was performed at 00 rpm for 30 minutes. At this time, the mite bodies float on the surface of the supernatant, and are removed by filtration. 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 (2).
Times). The obtained extract is dialyzed against tap water overnight, and subjected to ultrafiltration with a molecular weight cut off of 10,000 (UF-20CS-10PS, Tosoh membrane)
Fractionated to a molecular weight of 10,000 or more. 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 tick culture medium, high molecular crude mite excretion antigen (HM) 12
7.0 g, low molecular weight mite excretion antigen (LM) 69.7 g
I got

Example 2 Fractionation of Allergen Active Fraction from Low-Molecule Crude Mite Excretion Antigen (LM) Low-Molecule Crude Mite Excretion Antigen (LM) 5 Obtained in Example 1
After dissolving 00 mg in 30 ml of 0.9% NaCl eluate, an Ultrogel AcA54 column (IBF, size 4.4 × 100 c
m), and using this eluate, gel filtration chromatography was performed at a flow rate of 120 ml / hour to fractionate components having allergen activity. For each fraction,
Antigenicity in response to antisera (not shown)
Further, the allergen activity was assayed by a histamine release test on leukocytes from mite allergic patients. As shown in the histogram of FIG.
Divided into two. Therefore, fractions No. 27-31 and No. 37
-39 was collected as fractions containing the allergen active component, and these 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 yield after each lyophilization was LM-1 fraction 23.4.
mg and the LM-2 fraction were 70.8 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 Preparation of Purified LM-2 Active Ingredient The LM-2 fraction was separated from Sephadex G50 (Pharmacia LKB).
Gel filtration was performed using Biotechnology AB) and fractionation was performed. At the same time ovalbumin, cytochrome C, and using the insulin β-chain and vitamin B _12, to calibrate, the molecular weight was estimated of active ingredient in the LM-2 fraction.
The result is shown in FIG. The conditions for gel filtration are as follows: sample amount 50 mg (concentration 50 mg / 3 ml 0.9% NaCl aqueous solution), eluent 0.9% NaCl, flow rate 30 ml / hour, column size 1.5 × 100 cm. Since the allergen active component contained in the LM-2 fraction has a low molecular weight, its reactivity with antiserum is weak and ELISA measurement is difficult.
All fractions were monitored for leukocyte histamine release activity.

In the figure, the histogram shows leukocyte histamine release activity, OVA shows the elution position of ovalbumin having a molecular weight of 45,000, Cyt. C shows the elution position of cytochrome C having a molecular weight of 13,500, Insulin β shows the insulin β chain, and B ₁₂ indicates the elution position of vitamin B ₁₂ . From the plot of the volume of each component up to the peak elution position and the semi-logarithm of each molecular weight, it was estimated that the molecular weight of the active component in the LM-2 fraction exhibiting leukocyte histamine releasing activity was about 4,000. Here, the fractions indicated by the arrows in the figure (fractions No. 24-27) were pooled, used as LM-2 active fractions, and lyophilized after dialysis. The yield was 15 mg. This fraction showed a strong intradermal reaction activity against mite allergy patients. This fraction was used as the purified LM-2 active ingredient.

Example 4 Properties of Purified LM-2 Active Ingredient The purified LM-2 active ingredient obtained in Example 3 was subjected to amino acid analysis and sugar analysis by the GLC method.
6.6 μmol / mg and sugar 2.9 μmol / mg were detected. The results of the composition analysis of amino acids and sugars were as follows. Amino acids: Asp 0.9; Thr 0.4; Ser 0.6; Glu
1.2; Gly 1.6; Ala 0.5; Val 0.2; Cys
0.0; Ile 0.1; Leu 0.1; Tyr 0.0; Phe 0.
1; Lys 0.2; His 0.1; Arg 0.1; Pro 0.5 sugar: Pen 1.4; dHex 0.1; Hex 1.1; HexN
0.3 When this purified LM-2 active ingredient was quantified by the phenol-sulfuric acid method, about 40% was neutral sugar.

Example 5 Preparation of Purified LM-2p Active Ingredient (a) Periodic Acid Oxidation of Purified LM-2 Active Ingredient Purified LM- containing about 40% of neutral sugar obtained in Example 3
Pronase digestion and periodate oxidation of the two active ingredients were performed.
The result is shown in FIG. As can be seen from this figure, the purified LM-2 active ingredient completely lost allergen activity by pronase digestion, but about 70% of the activity was preserved in the periodate oxidation treatment. Therefore, this purified L
Periodic acid oxidation of the M-2 active ingredient oxidized sugar moieties not directly related to activity. By this treatment, the allergen is reduced in molecular weight, and the components that pass through the dialysis membrane (hereinafter referred to as LM-2p
Fractions) and components that did not pass through. From 10 mg of the purified LM-2 active ingredient, 3 mg of the LM-2p fraction and 7 mg of the fraction that did not pass were obtained. All fractions showed histamine-releasing activity of mite-allergic patients and strong intradermal reaction activity on the patient's skin.

(B) Ion exchange chromatography of LM-2p fraction 3 mg of the LM-2p fraction obtained in (a) was subjected to QAE-Sephadex A25 (Pharmacia LKB Biotechnology AB,
Ion exchange chromatography was performed at a size of 1.5 × 30 cm) to perform fractionation. 20 minutes after the sample was injected using water as the eluent, elution was performed at a flow rate of 0.5 ml / min by a linear gradient of hydrochloric acid of 3 M / 20 hours. FIG. 4 shows the results. Here, the histogram shows the histamine release activity of leukocyte, は shows the ultraviolet absorption at 215 nm, and the solid line shows the concentration of hydrochloric acid. In the figure, among the histamine releasing activities indicated by the histograms, fraction No. 23-2
The fractions eluted in 4 were collected, dried under reduced pressure, dissolved in water, and freeze-dried to obtain 150 mg of the active fraction. In this fraction,
Leukocyte histamine release activity of mite allergic patients and strong intradermal reaction activity on the patient's skin were present.

(C) Reverse phase chromatography of LM-2p fraction 10 mg of LM-2p fraction obtained in (b) (concentration: 10 m
g / 0.1 ml) with TSKgel ODS-120T (Tosoh, size
(0.46 × 25 cm) by reverse phase HPLC. 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. The result is shown in FIG. 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 fraction Nos. 11-13. This operation was repeated 14 times, and the corresponding fractions were separated into LM-2p
Collected as fractions and lyophilized to give 1.2 mg. An intradermal reaction test was also performed on this fraction at the same time for a mite allergy patient, and a strong allergen activity was observed.

(D) Dual mode chromatography (DMC) of LM-2p fraction 1.1 mg of the LM-2p fraction obtained by (c) was
DMC was performed using a pack GS-320 (Asahi Kasei, size 0.76 × 50 cm) column to fractionate. Elution was carried out isocratically at a flow rate of 0.5 ml / min using 50 mM ammonium acetate as the eluent. FIG. 6 shows the result. Here, the histogram shows the leukocyte histamine releasing activity, and the solid line shows the ultraviolet absorption at 215 nm. As shown in the figure, histamine releasing activity was observed in fractions Nos. 10, 12, and 17, but in many cases, fraction No. 10 in which activity was detected in common was collected as an LM-2p fraction and freeze-dried. However, it could not be weighed with sufficient accuracy with a small 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-2p fraction The LM-2p fraction obtained in (d) (fraction No. 1)
0), TSKgel ODS-120T (Tosoh, size 0.46 × 25c)
m) Reversed phase HPLC was performed using a column and purified.
Using 0.1% trifluoroacetic acid (TFA) as an eluent,
Five minutes after injection of the sample, elution was carried out at a flow rate of 1 ml / min with a linear gradient of methanol of 1% / min. FIG. 7 shows the result. Here, the solid line is 215 nm.
And the broken line shows the concentration of methanol. In the figure, histamine releasing activity was detected in the fraction containing the peak indicated by the arrow. In addition, an intradermal reaction test for patients with mite allergy was also conducted, and strong allergen activity was observed. This fraction was collected and used as a purified LM-2p active ingredient.

Example 6 Properties of Purified LM-2p Active Ingredient When the purified LM-2p active ingredient obtained in Example 5 (e) was subjected to amino acid analysis and phenol-sulfuric acid analysis, 27.5 μg of amino acid was obtained. Detected, but no sugar was detected. The results of amino acid composition analysis were as follows. Asp 1.1; Thr 0.5; Ser 0.9; Glu 1.5; G
ly 2.3; Ala 0.7; Val 0.3; Ile 0.2; Leu
0.3; Phe 0.1; Lys 0.4; His 0.2; Arg
0.4; Pro 0.7

The molecular weight of this purified LM-2p active ingredient is
The molecular weight of the glycoprotein estimated in Example 3, which is about 4,000
By subtracting about 40% (about 1,600) of sugar content from
It was estimated to be about 2,400. The color and properties of such a purified mite allergen of the present invention, a protein having a molecular weight of about 2,400, were white (lyophilized) and easily soluble in water. In the anaphylactic reaction test using guinea pigs, no anaphylactic reaction is induced. These properties and properties were the same for the purified LM-2 active ingredient having a molecular weight of about 4,000, which is one of the purified mite allergens of the present invention.

Example 7 Preparation of a titration reagent for mite allergy diagnosis Using Hanks buffer as a solvent, the purified LM-2 active ingredient and the purified LM-2p active ingredient obtained in Examples 3 and 5 were each 1 mg / ml. To make a stock solution of the reagent for histamine release titration.

[0037]

The purified mite allergen of the present invention is a useful antigen for diagnostic agents from the viewpoint of efficacy and safety. The establishment of a rapid and accurate diagnostic system for mite allergic diseases using the purified mite allergen of the present invention 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 from mite allergic patients. In the figure, BD corresponds to the void volume and indicates 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 280n
m shows ultraviolet absorption.

Ovalbumin LM-2 fraction in Figure 2 Example 3, cytochrome C, with insulin β chain and vitamin B ₁₂ was subjected to gel filtration chromatography on Sephadex G50, in LM-2 fraction It is the figure which estimated the molecular weight of the active ingredient. In the figure, the histogram shows leukocyte histamine release activity, OVA shows the elution position of ovalbumin with a molecular weight of 45,000, and Cyt. C shows the molecular weight of 1
3,500 indicates the elution position of cytochrome C, Insulin β indicates the insulin β chain, and B ₁₂ indicates the elution position of vitamin B ₁₂ .

FIG. 3 is a graph showing the results of histamine releasing activity of 10 mg of the purified LM-2 active ingredient obtained in Example 3 after pronase digestion and periodate oxidation, and histamine releasing activity of each product. In the figure, .largecircle. Indicates intact, .DELTA. Indicates periodate-oxidized, and .quadrature. Indicates pronase digestion.

FIG. 4 shows the LM obtained in (a) in Example 5.
The results obtained by performing ion exchange chromatography on 3 mg of the 2p fraction using QAE-Sephadex A25 and fractionating it are shown. In the figure, the histogram shows the histamine release activity of leukocytes, は shows the ultraviolet absorption at 215 nm, and the solid line shows the concentration of hydrochloric acid.

FIG. 5 shows the LM obtained in Example 5 in (b).
The results of fractionation of 10 mg of the -2p fraction by reverse phase HPLC using TSKgelODS-120T are shown. In the figure, the histogram indicates the leukocyte histamine release activity, the solid line indicates the ultraviolet absorption at 280 nm, and the dashed line indicates the methanol concentration.

FIG. 6 shows the LM obtained in (c) in Example 5.
The results of DMC using 1.1 mg of -2p fraction using an Asahipack GS-320 column are shown. In the figure, the histogram shows leukocyte histamine release activity, and the solid line shows ultraviolet absorption at 215 nm.

FIG. 7 shows the LM obtained in (d) in Example 5.
-P fraction was subjected to reverse phase HPLC using TSKgel ODS-120T.
The result of performing is shown. In the figure, the solid line indicates ultraviolet absorption at 215 nm, and the broken line indicates the concentration of methanol.

──────────────────────────────────────────────────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 (3)

(57) [Claims] 1. A obtained by removing the sugar moiety of a glycoprotein having allergenic activity minute <br/> molecular weight that is part of the excrement extract in mite culture of about 4,000 (Sephadex G50 gel filtration method)
Allergen with a molecular weight of about 2,400
Purification mite allergens having sex. 2. The excrement in the mite culture is extracted with a saturated saline solution and / or a buffer having a moderate ionic strength.
The resulting extract was fractionated using a technique such as gel filtration, then purified mite allergen according to claim 1, further comprising a step of removing the sugar moiety of a glycoprotein having obtained allergenic activity Manufacturing method. 3. A mite allergic diseases diagnostic agent as an active ingredient purified mite allergen of claim 1 Symbol placement.