JP3060159B2 - Monoclonal antibody against mite antigen and its use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody against a tick antigen, in particular, Dermatop
hagoides farinae ), a major allergen
The present invention relates to a monoclonal antibody specific to, a cell line for producing the antibody, an immunological detection method and a quantitative method using the monoclonal antibody.

[0002]

2. Description of the Related Art House dust (room dust) is bronchial asthma,
It is an important allergen such as allergic rhinitis, and it is said that the allergen is mainly derived from mites (Sakamoto. Chemistry and Biology Vol.26, No.2, 1988). It is said that more than a percentage are positive for ticks (Hayakawa, Shinda, Japan Clinical Laboratory, Vol. 45, No. 8, 1987).
Dermatophagoides farinae ( Dermatophagoides farinae ) and Dermatophagoides pteronyssinus ( De )
rmatophagoides pteronysinuss ) (T. Miyamoto
et al., J. Allergy 42, 14, 1968).

Dermatophagoides farinae include Der f I (molecular weight 24,000) and Der f II (molecular weight 15,0) having allergen activity.
00-16,000) (H. Yasueda,
Int. Arch. Allergy ppl. Immunol., 81, 214, 1986), as well as multiple allergens of various molecular weights. Der f I is mainly contained in mite feces, and Der f II is contained in mite bodies (carcasses and debris). In general, it is most important in the treatment of allergies to identify the allergen, and in clinical practice, patient guidance such as avoidance of allergens is effective (Sakamoto, Chemistry and Biology, Vol. 26, No. 2, 198).
In 8), it is necessary to identify and measure the amount of mite allergens present in the patient's living environment. For this purpose, various methods for detecting mite allergens in house dust have been proposed. However, a color reaction between a protein derived from mite excrement in dust extracted with alcohol and an aromatic diazo compound (Japanese Patent Application Laid-Open No.
71459, JP-A-61-59261), the detection method is too complicated. In addition, identification and quantification of allergens cannot be performed only by confirming the presence of mite allergens. Color reaction between small animal body fluids and chemicals (JP-A-62-29682)
8, JP-A-62-296829), although the operation is simple, small animals other than mites are also detected, so that mite allergen cannot be identified and quantified. Further, a detection method using an antiserum obtained by immunizing an animal with a mite body extract (Japanese Patent Application Laid-Open No.
In 3-191961), the detection and quantification of living organisms of mites in dust can be performed, but the detection and quantification of dead bodies, debris and feces is unknown, and the identification and quantification of allergens is not possible.

Recently, a monoclonal antibody against the major mite allergen Der f I (PW Heymann et al., J. A.
llergy Clin. Immunol., 83 (6), 1055-1067 (1989);
PWHeymann and MD Chapman, Clinical Reviews i
n Allergy, 8, 51-68 (1990); AE Thomas and MD
Chapmann, J. Allergy Clin. Immunol., 80 (6), 755-77
5 (1987); Takao Yamai et al, Prog. Med., 12 (8), 20
24-2029 (1992)) and a monoclonal antibody against Der f II (Japanese Patent Application Laid-Open No. 5-207892), making it possible to detect and quantify Der f I using an anti-Der fI monoclonal antibody (H. Yasueda, et al., Clin. Exp. Allergy
24 (11), 1030-1035 (1994)).

[0005] In the treatment of mite allergy, desensitization therapy using mite allergen extract is generally performed. For hyposensitization therapy, determine the administered allergen,
Purification is desired (Eda Nippon Clinic, vol. 44, extra edition 1986). In this regard, the specificity of antibodies is not clear in conventional polyclonal antibody systems generated from mammals immunized with worms or feces themselves.

[0006]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the allergen identification and quantification system by using a monoclonal antibody that is specific to Der f I and recognizes a specific site. It is intended to identify and quantify Der f I in dust with high sensitivity and ease.

[0007]

Means for Solving the Problems The present inventors have proposed Der f I
We prepared a monoclonal antibody specific for, and examined the immunoassay method using this monoclonal antibody. As a result, they found that the monoclonal antibody according to the present invention was extremely useful as a reagent for measuring Der f I, and reached the present invention. That is, the present invention uses a monoclonal antibody capable of specifically binding to Der f I,
It is a method for identifying, quantifying, and measuring erf I.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
It can be produced by a known cell fusion method. More specifically,
The monoclonal antibody of the present invention comprises the following steps (1) to (4),
That is, (1) antibody-producing cell preparation step (2) fusion, screening, and cloning steps (3) hybridoma culture step (4) purification step performed as necessary. Hereinafter, each step will be described in detail.

(1) Step of Preparing Antibody-Producing Cells Anti-Der fI antibody-producing cells can be obtained from the spleen of a Balb / c mouse or A / J mouse by immunizing the antigen with a partial peptide of Der fI as an antigen. Repeating the intraperitoneal administration of an equal amount of Freund's complete adjuvant or incomplete adjuvant so that the Der f I partial peptide is 10 to 100 μg per mouse is repeated at intervals of 2 to 4 weeks. After confirming that the antibody titer in the blood is sufficiently high, the same amount is administered to the tail vein or intraperitoneal cavity without adjuvant for final immunization. Two to five days after the final immunization, antibody-producing cells are collected from the spleen of the mouse.

(2) Fusion, screening and cloning steps Fusion is carried out by mixing the above-mentioned mouse antibody-producing cells and known mouse myeloma cells (myeloma cells) by a known method in the presence of a fusion promoter. it can. Generally, mouse myeloma cells are preferably those that cannot grow on a hybridoma selection medium and that do not themselves produce antibodies. Such mouse myeloma cells include mouse myeloma cells P3-NS1-1-Ag4-
1 (hereinafter abbreviated as NS-1), Sp-2 / O-Ag1
4 (hereinafter abbreviated as SP-2) or the like.

The ratio between the two cells is usually 1 to 20 myeloma cells and 1 to 20 antibody-producing cells. As the cell fusion promoter, for example, polyethylene glycol is used, and preferably those having a molecular weight of 1,000 to 7,500 are often used. For culturing the hybridoma, for example, the fusion promoter is washed off, and the hybridoma suspended in the hybridoma selection medium is cultured.
100-200 μl each can be seeded on a 96-well plate and performed at about 37 ° C. in 5% carbon dioxide gas-air.

The screening of the desired hybridoma is performed by measuring the antibody titer in the culture solution. That is, the supernatant of the medium in which the hybridoma was sufficiently grown was added to each well of the assay plate that had been bound with Der f I and blocked with bovine serum albumin, and incubated sufficiently to allow an antigen-antibody reaction in the plate. rear,
Remove and wash the supernatant. Further, a biotinylated anti-mouse IgG antibody is allowed to act on this, and after washing, avidinated alkaline phosphatase is allowed to act. After washing, p-nitrophenyl phosphate serving as a substrate is added to develop color. A single hybridoma of interest can be prepared by cloning a hybridoma positive for antibody production by the limiting dilution method.

(3) Hybridoma culturing step If the cloned hybridoma obtained in the previous step is cultured in vitro or in vivo, the desired monoclonal antibody can be produced. In vitro culture can be performed, for example, by starting with culturing several hybridomas in a 96-well plate and gradually increasing the scale. For in vivo culture, for example, hybridomas can be injected intraperitoneally into mice treated with pristane (2,6,10,14-tetramethylpentadecane: Aldrich) to facilitate the growth of fused cells. This can be done by inoculation. After 7 to 15 days, ascites containing monoclonal antibodies accumulates.

(4) Purification Step The purification step, if necessary, is carried out by subjecting the monoclonal antibody obtained in the preceding step to usual physicochemical techniques, for example, salting out, centrifugation, dialysis, ion exchange chromatography and the like. However, when the antibody subclass of the monoclonal antibody is IgG1, the adsorption rate by the protein A column and the recovery rate by elution are poor as in the other subclasses. Elution yields higher recovery, and
It is simple. In the present invention, De
A monoclonal antibody recognizing a specific site of rfI was obtained.

[0015]

The monoclonal antibody that can bind to Der f I site-specifically obtained as described above is an antibody whose binding site is clear, so that, for example, the three-dimensional structure changes after rewinding, It can also bind to degraded proteins from Der f I. Therefore, the detection method using the monoclonal antibody has extremely high detection sensitivity. In addition, known immunoassays, such as the simpler Western
Detection of Der f I by the blotting method etc. became possible.

For example, a sample for which the presence or absence of Der f I is to be known is charged to an acrylamide gel which is usually used, and electrophoresis is carried out in a buffer in the presence of SDS. After the electrophoresis, the protein was removed from the gel by PVDF membrane (Millipore
And the transferred membrane is blocked with albumin. Thereafter, the above antibody is added to an appropriate concentration and allowed to react for a certain period of time. Excess antibody is removed by washing thoroughly, and an anti-mouse IgG antibody labeled with an enzyme such as peroxidase is added thereto, and the mixture is further reacted for a certain period of time. Thereafter, washing is performed again to remove excess labeled antibody. After the series of treatments, the PVDF membrane is transferred to a thin vat, and hydrogen peroxide as a substrate of the enzyme and a coloring agent, for example, 4-chloro-1-naphthol are added to form a color. Thus, if a Der f I standard product is simultaneously electrophoresed as a control, a band is detected at the same electrophoretic distance, so that it can be confirmed whether or not the allergen is present in the examined sample.

[0017]

According to the present invention, a monoclonal antibody capable of site-specifically binding to Der f I and a hybridoma producing the antibody (Biotechnology Research Institute of Biotechnology,
Accession number FERM P-15028) was obtained. Furthermore, Der f I can be detected with high sensitivity even at a concentration as low as 10 ng / ml by the method of the present invention. This method is a specific method for measuring Der f I and Der f I-derived proteins. It is much simpler than conventional methods using chemical reactions, and uses polyclonal antibodies obtained by immunizing Dermatophagoides farinae. Der f I with extremely high sensitivity and specificity compared to the conventional method
Can be detected and measured. Also, by using the monoclonal antibody obtained by the present invention,
Der f I can be easily purified from samples to high purity. In addition, the monoclonal antibody of the present invention can be applied to the analysis of the binding site of IgE antibodies in the serum of mite allergic patients, and is also very useful for the desensitization treatment of mite allergy.

[0018]

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to this. Example 1 Preparation of Anti-Der f I Monoclonal Antibody Preparation of anti-Der f I monoclonal antibody was performed by the following method. B) Immunity In the amino acid sequence of the Der f I molecule, 92
-A peptide corresponding to position 110 was synthesized using a synthesizer (MPS350, Adva
using the nced ChemTech Co., Ltd. (JP Tam)
Proc. Natl. Acad. Sci. USA, 85, 5409-5413, 198
In step 8), a peptide having eight such peptides was synthesized.
After purification, a peptide sequencer (Applied Biochemi
The amino acid sequence was confirmed using cal company, type 473A).

An equal volume of Freund's complete adjuvant (FCA Difco
And emulsified with Balb / c mouse (♂), and the mixed emulsion is administered to the abdominal cavity of 100 μg per animal. About 2
At intervals of about 4 weeks, the same amount of the Der f I partial peptide mixed and emulsified in Freund's incomplete adjuvant (FIA Difco) was boosted intraperitoneally five times. The final immunization was performed on mice whose blood antibody titer was confirmed to be positive 3 days before cell fusion in advance.
g containing PBS was administered intraperitoneally.

B) Preparation of cells Three days after the final immunization, the spleen of the mouse was excised and spleen cells were
% RPMI medium containing fetal bovine serum (FLOW Lab. Co., L
TD. Hereinafter, abbreviated as RPMI), filtered through a 200 m / s stainless steel mesh, and washed three times with RPMI containing no fetal bovine serum (hereinafter abbreviated as -RPMI). NS-1 was used as a mouse myeloma cell (myeloma) as a fusion partner. This was cultured in RPMI to which 30 μg / ml of 8-azaguanine had been added in advance up to about one week before cell fusion, and then those in the logarithmic growth phase cultured in RPMI were washed three times with -RPMI.

C) Cell fusion and selection of antibody-producing hybridomas 1 to 2 × 10 ⁸ cells of spleen cells and myeloma were mixed at a ratio of about 5: 1, and the mixture was centrifuged to obtain a pellet. This contains 50% polyethylene glycol (PEG4000 Kanto Chemical)-RPM
Add 1 ml of I and stir for 1 minute.
Add 8 ml of RPMI over 8 minutes, stir, and add 10 ml of RPMI.
The pellet was suspended at a density of 5 × 10 ⁶ / ml and seeded in a 96-well plate (Sumitomo Bakelite) in 100 μl portions. 0.1m hypoxanthine from the next day
RP containing M, aminopterin 0.4μM, thymidine 16μM
100 μl of MI (hereinafter abbreviated as HAT medium) to each well
After that, the HAT medium was replaced by 100 μl at a time for about 1 to 2 weeks until hybridoma colonies appeared. When hybridoma colonies appear in the wells, anti-Der
After fI antibody detection, cells in positive wells were subjected to limiting dilution, and after a culture period in RPMI containing 0.1 mM hypoxanthine and 16 μM thymidine, anti-Der After confirming the detection of the fI antibody, a second limiting dilution was performed and cloning was performed. Thus, a hybridoma producing an anti-Der f I monoclonal antibody was obtained.

D) Production of Monoclonal Antibody The cloned monoclonal antibody-producing hybridoma was grown in RPMI and then injected into the peritoneal cavity of Balb / c mice to which 0.5 ml of pristane had been administered intraperitoneally two weeks before. ^The transplant was performed at ⁶ to 10 ⁷ cells. Approximately two weeks later, the collected ascites fluid was collected, and the monoclonal antibody was purified therefrom.

E) Purification of monoclonal antibody The ascites fluid was centrifuged at 10,000 rpm for 20 minutes to remove the precipitate, and 0.3 μm
The protein concentration of the filtrate filtered through a sterilizing filter (Milesque 0.3 μm, Millipore) was measured by the Lowry method. Filtrate for 100-150 mg protein was treated with a commercially available protein G column kit (Mab Trap G Pharmacia), the adsorbed fraction was dialyzed into PBS, subjected to SDS-PAGE electrophoresis, and a single band was obtained by CBB staining. After confirmation, the antibody was used as a purified monoclonal antibody.

One of the obtained monoclonal antibody-producing hybridoma cell lines was deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology. The accession numbers are as follows. Indication for identification given by the depositor Accession number 12F1 FERM P-15028 f) Preparation of enzyme-labeled antibody The antibody 12F1 purified by the above e) was purchased from a commercially available peroxidase labeling kit (Immuno-Link ™ HRP, Cambridge Re
search Biochemicals). The method is
This was performed according to the instruction manual attached to the kit.

Example 2 Quantification of Der f I Der f I and Der f II were prepared at each concentration in the range of 0 to 2000 ng / ml, and 50 μl of the solution was added to a 96-well microtiter plate. And left overnight. After washing, blocking was performed with a 3% BSA solution. To each well of the blocked plate, 50 μl of the PBS solution (100 μg / ml) of the purified antibody 12F1 obtained in e) of Example 1 above was added.
The reaction was performed at 37 ° C. for 3 hours. After the reaction, the plate was thoroughly washed to remove the antibody 12F1 remaining without binding. Next, a goat-derived peroxidase-labeled anti-mouse IgG antibody was reacted. Excess enzyme-labeled anti-mouse antibody was removed by washing. Then, add hydrogen peroxide as a substrate and orthophenylenediamine as a color former to the wells, and add them at room temperature for 10 hours.
After reacting for 2 minutes, the reaction was stopped with 2N sulfuric acid, and the absorbance at 490 nm was measured. Anti-Der f I antibody 12F1
II did not react at all and showed the same absorbance as the background, whereas Der f I developed color at a concentration of 20 ng / ml or more. The quantification range was 50-1000 ng / ml.

Example 3 Detection of Der f I A freeze-dried preparation of Dermatophagoides farinae body extract was dissolved in distilled water to a concentration of 1 mg / ml, and 10 μl thereof was double-concentrated with SDS sample buffer (1.52 g Tris, 20 ml glycerin). , 2
g SDS, 2 ml of 2-mercaptoethanol and 1 mg of bromophenol blue are dissolved in 40 ml of water, and pH is adjusted with 1N hydrochloric acid.
10 μl) and heated in a boiling water bath for 3 minutes. After cooling, 10 μl of a sample well of a 16% concentration acrylamide gel (1 mm thick, manufactured by TEF Corporation) was charged, and electrophoresed in a buffer for SDS-PAGE under a constant current of 18 mA for 100 minutes. In the other wells, Der f I and II purified from insects were electrophoresed in the same manner as a standard. After electrophoresis, the separated proteins on the gel
It was electrically transferred to the membrane. After the transferred PVDF membrane was washed and SDS was removed, it was blocked with 3% BSA, and the peroxidase-labeled anti-Derf described in Example 1 f) above).
I antibody (12F1) was added and reacted at room temperature for 3 hours.
After the reaction, the antibody remaining unreacted was removed by washing. Then, the substrate, hydrogen peroxide, and the color former, 4
-Chlornaphthol was added to detect Der f I.
Standard Der f I and Der f in lyophilized mite preparation
A band was observed only at position I, but the standard Der f I
No bands were observed at positions other than Der f I, as well as the position of Derf II in the freeze-dried preparations of I and mite.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91) (72) Inventor Toshifumi Yuki Ota, Tokyo 2-13-1 Omorikita-ku, Asahi Breweries, Ltd. Central Research Laboratory (72) Inventor Yasushi Okumura 2-13-1 Omorikita, Ota-ku, Tokyo Asahi Breweries, Ltd. Central Research Laboratory (56) References JPN. J. Allergol. , Vol. 43, No. 5, (1994), 634-644 Clin. Exp. Allergy, Vol. 24, No. 11 (1994), pp. 1030-1035 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C07K 1/00-16/46 C12N 5/00-5/28 C12P 21/00-21/08 G01N 33/53 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (5)

(57) [Claims] (1) Dermatophagoides far
inae ) derived from the N-terminus of the antigen Der f I
IgG obtained by immunizing mammals with a partial peptide consisting of the 10th amino acid residue and selectively recognizing Der f I
Anti-Der f I monoclonal antibody belonging to 2. A hybridoma cell line which produces the monoclonal antibody according to claim 1. 3. A labeled antibody obtained by labeling the monoclonal antibody according to claim 1 with an enzyme, a fluorescent dye, a chemiluminescent substance, or a radioisotope or a combination thereof. 4. A method for detecting or quantifying Der f I from a sample in which a mite antigen or a tick is considered to be present using the monoclonal antibody according to claim 1. 5. 5F1 (Accession No. FERM P-15028)
The hybridoma cell line according to claim 2, wherein