JP3323996B2 - Quantitative method for specifically quantifying immunoglobulins that react to allergens - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for quantifying immunoglobulin E which specifically reacts with mite allergen.

[0002]

2. Description of the Related Art Allergic diseases such as bronchial asthma, pediatric asthma, and atopic dermatitis have been found to be mainly caused by allergies to mites inhabiting indoor dust. Major mite allergen protein has been identified (Platts-Mills)
Et al., The Journal of Allergy and Clinical Immunology (J. Allergy Clin. Immunol.),
80, 755, 1987). This allergic disease is sensitized to mite allergens, and allergen-specific IgE antibodies (reagin antibodies) are produced in serum and tissues by sensitization to mite allergens. Cause an antigen-antibody reaction, and are thought to be caused by various symptoms occurring at that time.

[0003] As a method for treating this allergic disease, there is desensitization therapy. This method is a method of repeatedly administering a mite major antigen or a mite or a house dust extract containing this antigen little by little, and gradually increasing the dose according to symptoms. It is believed that this therapy suppresses IgE antibody production and suppresses histamine release from mast cells, resulting in a therapeutic effect. Therefore, although desensitization therapy with this major allergen is a very effective treatment, patients with Ig before, during and after treatment
It is necessary to quickly, simply and accurately quantify the amount of E, particularly IgE for a specific antigen. The currently used IgE measurement method includes the RAST method, which requires a facility capable of using a radioisotope by a method using a radioisotope, and has a problem in terms of simplicity and safety.

On the other hand, an IgE antibody measurement method using an ELISA system is excellent in terms of simplicity and safety. In this method, the sensitivity varies greatly depending on the amount of effective antigen coated on the ELISA plate. In addition, when the plate and the antigen bind at or near the IgE binding site, the antigen molecule does not participate in the reaction with IgE, or the reactivity decreases, resulting in a decrease in measurement sensitivity. In addition, when the antigen molecule is small or the hydrophobicity is low, there is a problem that the antigen is hardly adsorbed to the plate or easily peeled off.

[0005]

SUMMARY OF THE INVENTION The present inventors have found that these problems can be solved by coating mite allergen on an ELISA plate in the form of a fusion protein bound to an appropriate protein. Da It is therefore an object of the present invention, a mite allergen protein is prepared as a fusion protein with an appropriate protein using genetic engineering, using the same
To provide a immunoglobulin E (IgE) assay for two allergens.

[0006]

The present invention is based on the ELISA method.
Anti-mite allergen immunoglobulin E (Ig
In the quantification method of E), Ru Oh in fusion state and other proteins
Copy mite allergen or part of it to ELISA plate.
The mite allergen or a part thereof with serum and
A method for quantifying anti- mite allergen immunoglobulin E, which comprises reacting a labeled anti-immunoglobulin E antibody . The protein used in the present invention is preferably β-
It is a fusion protein with galactosidase or a part thereof. The allergen used is Dermatophago
ides farinae) major allergens, it is not preferable is DERF II or a portion thereof.

The major allergen of Dermatophagoides pteronyssinus ( Derf)
A protein or peptide having the biochemical and immunological properties of II) is produced as a fusion protein with an appropriate protein, and the anti- Derf II immunoglobulin can be measured using the protein or peptide. The major mite allergen, Derf II, fused to other proteins, is a gene disclosed by Yuki et al. (Allergy (JPN. J. Allergol.), 39, 557,
1990). Also obtained
Derf II gene and Protein Fusion & Purification System (Protein Fusion & Puri
fication System, New England Biolabs) can be used to prepare the allergen Derf II protein.
The Derf II protein prepared by these methods has a secondary advantage that the production amount is large and the purification is easy.

[0008]

The method according to the present invention, the anti-mite allergen immunoblot using an ELISA method for mite allele <br/> Gen was traditional hard
Globulin E can be measured easily and quickly,
Its measurement sensitivity is also high.

[0009]

The present invention will be described in more detail with reference to the following examples. Example 1 Preparation of mite allergen protein Derf II and fusion Derf II protein Mite ( Dermatophagoides farinae ) was subjected to temperature of 25 ° C and humidity of 75.
% Of the animal body in a sample for laboratory animal breeding for about 30 days, and the obtained insects (5 g) were put in a phosphate buffer (10 mM, pH 7.
0) Extracted with 50 ml. Solid ammonium sulfate was added to the extract to a final concentration of 55% saturation, and the mixture was stirred at 10 ° C. for 1 hour and centrifuged at 10,000 G for 30 minutes to obtain about 35 ml of the supernatant. Furthermore, ion exchange chromatography using DEAE-Sephacel (Pharmacia) and Sephacryl S-200 (Pharmacia)
Purified by gel filtration using. The obtained sample is SDS
Gel electrophoresis in the presence of (sodium lauryl sulfate) showed a single band.

On the other hand, a Derf II fusion protein was prepared using a plasmid containing the Derf II gene already disclosed by Yuki et al. (Yuki et al., Allergy (JPN. J. Allerg.
ol.), 39, 557, 1990). E. coli harboring plasmid pFL1 was transformed into L broth (1 μl containing 50 μg / ml ampicillin).
% Bactotripton, 0.5% yeast extract,
5 ml of 0.5% sodium chloride (pH 7.4) and inoculated at 30 ° C. overnight with shaking. It was inoculated in the same medium at 1% and cultured with shaking at 30 ° C. When the absorbance at 660 nm reached 0.6, add the same amount of the same medium preheated to 54 ° C, and add 42
Shaking culture was further performed at 2 ° C. for 2 hours. After culturing, the cells were collected and subjected to Derf II according to the method of Marston and Carroll et al. (DNA Cloning, IRL Press, Volume 3, Chapters 4 and 5, 1985).
The fusion protein was extracted. When the cells at this time were observed with a phase-contrast microscope (× 1000), the fusion protein was present as an inclusion body in the cells.

This fusion protein was purified using a ProtoSorb lacZImm. Column (Promega) and confirmed to be a single band by gel electrophoresis in the presence of SDS. Example 2 Sensitivity comparison by antigen protein used in ELISA method The Derf II fusion protein prepared in Example 1 and the antigen protein purified from insects were used in 10 μg / ml and 1 μg / ml solutions.
Add 150 μl to each well of the ELISA 96-well plate,
The binding was left for about 18 hours at 4 ° C. Phosphate buffer containing 1% bovine serum albumin and 0.05% Tween 20 (10m
(M, pH 7.4) three times to remove unadsorbed antigen protein. The mite allergic patient serum to be tested was appropriately diluted, and 100 μl thereof was added to each well, followed by reaction at room temperature for 1 hour.
Thereafter, the wells were washed three times with the above buffer solution, and the antibodies in the serum that did not react with the remaining antigen were removed from the wells. Thereafter, 1 μg of a biotin-labeled anti-human IgE antibody was added and reacted at room temperature for 1 hour. After washing three times with the above buffer, 50 μl of avidin-labeled peroxidase was added. It was left at room temperature for 1 hour and washed three times with the above buffer solution. There, a coloring substrate solution (0.15%, 2,2′-azino-bis (3-ethylbenzazoline-6-sulfonic acid)) and 0.0025% hydrogen peroxide solution
50 μl was added, the color was developed at room temperature for 30 minutes, and the reaction was stopped with 6% oxalic acid. The absorbance at 405 nm was measured with an ELISA plate reader. DerfII antigen removed as blank
Using a fusion protein portion (β-galactosidase only)
The value obtained by subtracting the blank value from the absorbance of the fusion Derf II protein was shown. The amounts of proteins used were all the same.
Table 1 shows the results.

[0012]

[Table 1] IgE content in serum (absorbance) Example 3 Antibody in Serum of Atopic Asthmatic Patients Positive for Mite Allergen
Quantification of Derf II IgE antibody Heparin blood was collected from an atopic asthmatic patient showing a positive reaction to mite allergen, and the sample was centrifuged at 3000 rpm for 5 minutes to obtain a supernatant. The fusion Derf II protein was coated on an ELISA plate in exactly the same manner as described in Example 2.
The antigen used was the same as in Example 2, and the protein concentration was 10 μm.
Performed at g / ml. Serum was undiluted and phosphate buffer (10 mM,
(pH 7.5, common salt 0.8%, KCl 0.02%) and used 10-fold. As a control, blood was similarly collected from a normal person and compared.
Table 2 shows the results. The values shown here are the values obtained by subtracting the blank value using β-galactosidase as a blank for each sample.

[0013]

[Table 2] Anti- Derf IIIgE amount (absorbance) in serum of atopic asthmatic patients

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yasushi Okumura 2-13-1 Omorikita, Ota-ku, Tokyo Asahi Breweries Co., Ltd. Applied Technology Research Laboratories (72) Inventor Tohru Ando 3-14 Minami-Yawata, Ichikawa-shi, Chiba No. 3 (72) Inventor Reiko Honma 3-14-3 Minami-Hachiman, Ichikawa-shi, Chiba (56) References WO 89/9260 (WO, A1) Jpn. J. Allergol 39 (6) p. 557-591 (1990)

Claims (3)

(57) [Claims] 1. Antiserum mite allergen in serum by ELISA method
In a method for quantifying immunoglobulin E (IgE), mite allergen or a part thereof fused to another protein is coated on an ELISA plate, and the mite allergen or
Contains serum and labeled anti-immunoglobulin E antibody
A method for quantifying anti-mite allergen immunoglobulin E. 2. A mite allergen Dermatophagoides (Dermat
The method according to claim 1, which is a major allergen of D. ophagoides farinae ), Derf II or a part thereof. 3. The method according to claim 1, wherein the other protein is β-galactosidase or a part thereof.