JP3072919B2 - Monoclonal antibody and diagnostic method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention is useful for the diagnosis of various diseases which are thought to cause abnormalities in the superoxide production mechanism, especially for the diagnosis of congenital chronic granulomatosis, and the biochemistry and histological analysis of said diseases. New substances useful for research.
More specifically, human polynuclear cell cytoplasmic factor-47KD protein (hereinafter, referred to as
The present invention relates to a monoclonal antibody against NCF-47KD) and -67KD protein (hereinafter, NCF-67KD), a hybridoma secreting the antibody, and a method for diagnosing human autosomal recessive chronic granulomatosis using the antibody.

[0002]

2. Description of the Related Art Chronic Granulomatous Diet
sease (hereinafter, CGD) is the most frequent hereditary phagocytic dysfunction in which superoxide-producing ability is lacking in polynuclear cells, monocytes, and the like. In this disease, the bactericidal activity of polynuclear cells and macrophages is lost, and patients suffer from repeated severe bacterial infections. In particular, the bactericidal activity of catalase-positive bacteria and fungi that do not produce H ₂ O ₂ is significantly reduced. Advances in antibiotics and prophylactic administration of Sulfamethoxazole-trimethoprim (ST) combination drugs have made it possible to control bacterial infections well, but antibiotic-resistant bacterial infections and fungal infections have Patients often die in late puberty. Therefore, the importance of early diagnosis of CGD and prophylactic administration of ST combinations and antifungal agents is increasing. Furthermore, in recent years, interferon-γ therapy has been considered to be effective in certain types of CGD, and its classification is becoming more important from the viewpoint of preventive treatment.

[0003] The superoxide-producing enzyme complex composed of three cell membrane factors and at least three cytoplasmic factors is greatly involved in the superoxide production mechanism of human polynuclear cells. In order to produce superoxide and to properly exert the phagocytic function (bactericidal activity), it is considered essential that this enzyme complex be activated by the action of each of the above-mentioned factors. It is presumed that loss or mutation leads to diseases such as CGD.

The genotypes of CGD include sex-linked recessive genotypes (cytochrome b558 membrane protein heavy chain deficiency) and autosomal recessive genotypes (cytochrome b558 membrane protein light chain deficiency, NCF-47
KD and NCF-67KD deficiency) are known,
Conventionally, diagnosis has been made by a human polynuclear cell NBT (nitroblue tetrazolium) reducing ability test, a cytochrome C reducing ability test, and the like.

[0005] Regarding the pathologic classification, sex-linked recessive genotype diagnosis is performed by flow cytometry using an anti-cytochrome b membrane protein heavy chain monoclonal antibody, while autosomal recessive diagnosis is performed by NCF-47KD and NCF-47KD. B1 polyclonal antibody that simultaneously recognizes NCF-67KD (Volpp, BD, Sc
ience, 242: 1295, 1988) .It is necessary to separate the polynuclear cells from peripheral blood, ultracentrifuge the cytoplasm, further perform electrophoresis, and perform Western blotting with the B1 antibody. , A very complicated procedure was required. Further, the diagnostic method using the B1 antibody cannot accurately detect defects of each of NCF-47KD and NCF-67KD, and is insufficient as a diagnostic method for classification of disease types. There are still many unclear points about the relationship between the various factors mentioned above and the pathology of the disease, and if we can grasp their trends, it is considered to be very important in the fields of basic medicine and clinical medicine. Can be

[0006] Monoclonal antibodies are specific for a single antigenic determinant and can be stably supplied by a hybridoma producing the same. Therefore, analysis of the function and structure of the antigen protein, or immunoassay. (EIA, RIA) and the like have recently come into wide use.

[0007]

The present invention can be used for the diagnosis of autosomal recessive chronic granulomatosis, and is also used for various other diseases in which an abnormality in the superoxide production mechanism is considered to be the etiology.
The development of a highly specific monoclonal antibody that can be easily measured and a simple measurement method has been eagerly desired. However, no report has been made on the monoclonal antibodies reactive with NCF-47KD and NCF-67KD. This is because NCF-47KD and NCF-67 are used to produce monoclonal antibodies that react with the desired NCF-47KD and NCF-67KD.
This was thought to be due to the difficulty in preparing the KD antigen.

[0008]

SUMMARY OF THE INVENTION The present inventors have determined that NCF-47KD and NCF-67 are defective in autosomal recessive chronic granulomatosis.
As a result of intensive studies on monoclonal antibodies recognizing KD, the present inventors have reached the present invention which can provide a desired monoclonal antibody. The monoclonal antibody of the present invention that specifically recognizes NCF-47KD and NCF-67KD belongs to the IgG class,
Specific examples thereof include those named C-47 / -1,2,3 and C-67 / 1,2,3. The monoclonal antibody obtained according to the present invention, by using the FITC label, allows a simpler diagnosis of CGD by a flow cytometer method, and also enables tissue staining by biotinylation.

The hybridoma producing the monoclonal antibody of the present invention is obtained by the method of Keller and Milstein (Kohler an
d Milstein, Nature: 256 p. 495-497 (1975)). That is, after immunizing a mouse with an antigen prepared by a suitable method, NCF-47KD and NCF-67KD, spleen cells of this mouse are fused with mouse myeloma cells, and a target antibody is produced from the obtained hybridoma. The secreting hybridomas can be selected and isolated.

Hereinafter, a method for preparing a monoclonal antibody and a method for using the same in the present invention will be outlined.

(1) Isolation and purification of antigen; NCF-47KD and NCF-67KD used as antigens can be prepared from human polynuclear cells as a raw material by a method combining biochemical techniques. The present inventors used genetic engineering techniques to express E. coli,
The CF-47KD and NCF-67KD cDNAs were incorporated into E. coli using a suitable vector, and IPTG (isopropyl-thiog
After treatment with alactoside (isopropyl thiogalactoside), each expressed protein was subjected to SDS electrophoresis, and elution was performed after electrophoresis from each protein band to overcome the difficulty of antigen preparation. The recombinant NCF-47KD and NCF-
67KD was used as the immunizing antigen.

(2) Immunization of mice; BALB / c mice of 4 to 8 weeks of age can be preferably used as immunized animals, but mice of other strains can also be used. During immunization, the immunization schedule and antigen concentration should be chosen so that a sufficient amount of antigen-stimulated lymphocytes is formed. For example, the above recombinant NCF-47KD / -67KD together with a suitable adjuvant is injected intraperitoneally into mice.
50 μg / animal is administered. Thereafter, every two weeks, the same antigen as that used for the first immunization is administered 2 to 5 times in the same amount. On the 4th to 7th days after the third immunization, blood is collected from the fundus vein to examine antibodies in the blood that react with NCF-47KD / -67KD. Antibody measurement can be suitably performed by Western blotting.

In order to provide for cell fusion, 4 to 8
50μ of NCF-47KD / -67KD protein was added to mice immunized the day before.
g / animal is intraperitoneally administered, and after this booster, the spleen is aseptically removed and spleen cells are prepared therefrom.

(3) Cell fusion; The spleen single cell suspension prepared as described above is subjected to cell fusion with mouse myeloma cells derived from a suitable cell line using a suitable fusion promoter. The preferred ratio of spleen cells to myeloma cells ranges from about 20: 1 to about 2: 1.

There are no particular restrictions on the mouse myeloma cells used for cell fusion, but in the present invention, 8-azaguanine-resistant mouse myeloma cells P3-U1 (Yelton, D .;
E. et al. Current Topics in Microbiology and Immuno
logy, 81 p.1 (1978)).

As a preferred fusion promoter, for example, polyethylene glycol having an average molecular weight of 1,000 to 4,000 can be suitably used. Other fusion promoters known in the art can also be used.

(4) Selection of fused cells; a mixture of unfused spleen cells, unfused mouse myeloma cells and fused hybridomas in a separate container, and selection medium not supporting unfused mouse myeloma cells , For example, HAT
Dilute with medium and incubate for a time sufficient to kill unfused cells. In this selective medium, unfused mouse myeloma cells die, and unfused spleen cells die after a certain period of time. On the other hand, the fused cells can survive in the selective medium because they have the properties of the spleen cells and the tumor properties of the parental cells of myeloma.

(5) Confirmation of antibody produced by hybridoma
(Screening); After the hybridoma is detected by the above operation, the culture supernatant is collected and screened for antibodies against NCF-47KD and NCF-67KD. This screening can be suitably performed, for example, by Western blotting.

(6) Cloning of hybridoma producing the desired antibody and production of the antibody; After cloning the hybridoma producing the desired antibody by an appropriate method (for example, limiting dilution method), It can be produced in two different ways. One is a method in which the hybridoma is cultured for a certain period of time in an appropriate medium, and a monoclonal antibody produced by the hybridoma can be obtained from the culture supernatant. A second method is to inoculate the hybridoma into the peritoneal cavity of a mouse that has an isogenic or semi-isogenic gene. A desired monoclonal antibody produced by the hybridoma can be obtained from the blood and ascites of the inoculated mouse after a certain period of time.

(7) Purification of Monoclonal Antibody The objective monoclonal antibody is purified from the culture supernatant of the hybridoma or from the blood and ascites of the mouse by a widely used biochemical purification method. For example, a combination of ammonium sulfate salting out, ion exchange chromatography, affinity chromatography, or the like can be considered.

(8) Method for diagnosing human autosomal recessive chronic granulomatosis using monoclonal antibodies; various methods based on immunoassays can be considered, including flow cytometry, Western blotting, and EIA.
The law and the like are suitably applied.

A typical example of the hybridoma producing the monoclonal antibody of the present invention is the microorganism No. 11953 (FERM P-11953) of the Institute of Microbial Industry and Technology (MIC).
And accession number 11954 (FERM P-11954).

Hereinafter, the present invention will be described with reference to examples for better understanding of the present invention, but the present invention is not limited to these examples.

Example 1 (1) Preparation of antigen for immunization NCF-47KD and NCF-67KD were expressed in Escherichia coli using a gene recombination technique. CDNAs corresponding to NCF-47KD and NCF-67KD were isolated from differentiated HL-60 cells by Blue
It was cloned from the cDNA library incorporated into ScriptVector. Next, E. coli (X
L-1) was transformed, and after stimulation with IPTG, desired proteins, NCF-47KD and NCF-67KD were expressed. After solubilizing each Escherichia coli with SDS, it was subjected to acrylamide gel electrophoresis, and bands corresponding to NCF-47KD and NCF-67KD were electrophoresed and eluted to prepare an antigen.

(2) Preparation of spleen cells from immunized mice Eight-week-old BALB / c male mice were treated with 50 μl of Freund's incomplete adjuvant (manufactured by DIFCO), the antigen protein prepared by the above method, recombinant NCF-47KD and NCF -67KD each 50μg /
The animals were immunized by intraperitoneal administration. Thereafter, the same antigen was administered to the peritoneal cavity every one to two weeks, and immunization was performed twice or more. After the third immunization, blood was collected from the fundus vein 4 to 5 days after the immunization, and anti-NCF-47KD / -67KD antibodies in the serum were confirmed by the following Western blotting method.

Human multinuclear cell cytoplasm was subjected to 10% SDS electrophoresis and transferred to a nitrocellulose membrane. First green staining was performed to prepare a bundle containing the protein band of interest. The culture supernatant of the hybridoma was added as a first antibody to a 96-well ELISA plate, and the above-mentioned bunched nitrocellulose membrane was placed therein.
It was left for 4 hours, possibly overnight. After washing three times with a Tris / NaCl buffer (pH 7.0), a rabbit anti-mouse IgG / peroxidase-labeled antibody (Immuno Re
search Laboratory) was sufficiently dispensed into each well and allowed to react at room temperature for 2 hours. After washing three times with a Tris / NaCl buffer, a peroxidase substrate solution (4-Methoxyl-l-na
Phthol naphtal was dissolved in ethanol, Tris buffer was added, and a solution was added immediately before use with hydrogen peroxide) to add color, and an appropriate band was confirmed.

Spleen cells were prepared from mice in which antibodies against NCF-47KD and NCF-67KD were confirmed, and used for cell fusion.

(3) Preparation of mouse myeloma cells 8-azaguanine-resistant mouse myeloma cells P3-U1 were cultured in a normal medium (glutamine 1.5 mM in RPMI-1640, 2-mercaptoethanol 5 × 10 ⁻⁵ M, gentamicin 10 μg / ml). And a medium supplemented with 10% fetal calf serum) at 37 ° C. in a CO ₂ incubator to obtain 2 × 10 ⁷ or more cells after 4 days.

(4) Cell fusion and culturing of hybridoma The spleen cells were cultured with mouse myeloma cells P3-U1 at a cell number of 1: 5.
, The supernatant was removed by centrifugation (1,200 rpm / 5 minutes), and the precipitated cell mass was sufficiently loosened.
1 ml of the mixed solution (polyethylene glycol-
4,000 (2g), MEM (Modified Essential Medium: 2ml),
Dimethylsulfoxide). After incubation at 37 ° C. for 5 minutes, MEM was slowly added so that the total volume of the solution was 50 ml. After centrifugation (900 rpm / 5 min), the supernatant was removed and the cells were loosened gently. Add normal medium (R
100 ml of PMI-1640 medium supplemented with 10% fetal calf serum) was added thereto, and the cells were gently suspended using a female pipette.

The suspension was dispensed into a 24-well culture plate (1 m).
(l / well) The cells were cultured in an incubator containing 5% carbon dioxide at 37 ° C. for 24 hours. Next, 1 ml / well of HAT medium (hypoxanthine (1 × 10 ⁻⁴ M), thymidine (1.5 × 10 ⁻³ M) and aminopterin (4 × 10 ⁻⁷ M) in a normal medium) was added, and an additional 24 ml / well was added. Cultured for hours. Thereafter, every 24 hours for 2 days, 1 ml of the culture supernatant was added to the same amount of HT medium (aminopterin was removed from HAT medium).
And cultured for 10 to 14 days in the same manner as described above.

Colony-grown fused cells (about 300 cells)
1 ml of the culture supernatant was replaced with the same amount of the HT medium in each well in which was observed, and after culturing for 1 day, the medium was gradually replaced with a normal medium.

After culturing for 3 to 4 days in a normal medium, a part of the culture supernatant was collected and subjected to screening by the Western blot method described in Example 1 (2) to select the desired hybridoma.

Cloning was repeated twice in the wells in which the antibody titer was recognized by the limiting dilution method, and the clone in which the antibody titer was stably recognized was replaced with the anti-NCF-47KD / -67KD monoclonal antibody-producing hybridoma strain (P5- 13-6, P4-90-3).

(5) Purification of monoclonal antibody Pristane treatment (2,6,10,14-tetramethylpentadecane
0.5 ml / animal was intraperitoneally administered and bred for 1 to 2 weeks) and 4 × 10 ⁸ cells /
The animals were inoculated intraperitoneally. After 10 to 21 days, the ascitic fluid (4 to 10 ml / animal) was collected from the mouse with ascitic carcinoma of the hybridoma strain, and the solid matter was removed by centrifugation. The supernatant was salted out with 40% ammonium sulfate, dissolved in PBS (pH 7.2), dialyzed for 2 days, and used as a crude purified monoclonal antibody.

Example 2 Antigen Specificity of Monoclonal Antibody 1) Confirmation of Antigen Specificity by Western Blot Method NCF-47KD / -67KD Deficiency Patient Multinuclear Cell Cytoplasm and NCF-91K
SD deficient patient multinuclear cell cytoplasm and normal multinuclear cell cytoplasm
After S electrophoresis, the antigen specificity of the obtained monoclonal antibody was confirmed by Western blotting in the same manner as described in Example 1, (2). FIG. 1 shows the results. That is, the anti-NCF-47KD monoclonal antibody is NCF-47
KD and anti-NCF-67KD monoclonal antibody is NCF-67
It was found that it reacts specifically with KD.

2) Confirmation of antigen specificity by flow cytometer Normal human multinucleated cells and NCF-47KD / -67KD deficient patient multinuclear cells were sufficiently fixed with 1% paraformaldehyde / saline. After washing with PBS, the cells were reacted with an anti-NCF-47KD / -67KD monoclonal antibody for 1 hour. FITC after washing 3 times with PBS
After further reacting with a labeled anti-mouse IgG antibody for 1 hour,
FITC fluorescence was detected using EPICS-V (Coulter Corp.). FIG. 2 shows the results.

[0037]

Industrial Applicability According to the present invention, there is provided a monoclonal antibody effective for detecting NCF-47KD / -67KD deficient patients among autosomal recessive chronic granulomatosis. At the same time, the monoclonal antibody of the present invention is used for various diseases that are thought to cause abnormalities in the mechanism of superoxide production, such as various infectious diseases, collagen diseases, mesangial proliferative renal diseases, lung diseases, and histioproliferative diseases. It is considered to be useful for diagnosis of pathological conditions such as.

[0038]

[Brief description of the drawings]

FIG. 1 shows the results of a Western blot for confirming the specificity of the monoclonal antibody of the present invention, and FIG. 2 shows the flow cytometry clarifying the specificity of the monoclonal antibody of the present invention. . In FIG. 2, the number of cells is plotted on the vertical axis, and the fluorescence intensity is plotted on the horizontal axis.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI G01N 33/577 C12N 15/00 C // A61K 39/395 A C12N 5/10 5/00 B (C12P 21/08 C12R 1: 91) (56) References Science, Vol. 242, (1988), p. 1298-1301 Science, Vol. 242, (1988), p. 1295-1297 (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 14/00-16/46 C12N 15/00-15/90 C12P 21/00-21/08 G01N 33/53-33 / 98 A61K 39/00-39/44 C12N 5/00-5/28 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] The present invention does not show reactivity with human polynuclear cell cytoplasmic factor-67KD protein (hereinafter sometimes referred to as NCF-67KD), and does not exhibit reactivity with human polynuclear cell cytoplasmic factor-47KD protein (hereinafter referred to as NCF-47KD). Monoclonal antibody that specifically recognizes 2. Accession No. 11954 (FERM P-119) from the Institute of Microbial Industry and Technology (MIC)
The monoclonal antibody according to claim 1, which is prepared from the hybridoma deposited as (54). 3. It does not show reactivity with NCF-47KD,
A monoclonal antibody that specifically recognizes CF-67KD. 4. An accession number: 11953 (FERM P-119) to the Research Institute of Microbial Industry (MIC) of the National Institute of Advanced Industrial Science and Technology.
The monoclonal antibody according to claim 3, which is prepared from the hybridoma deposited as (53). 5. A flow cytometer method, a Western blot method and an EIA using polynuclear cells or polynuclear cell cytoplasm as a test sample and using the monoclonal antibody according to any one of claims 1 to 4. An immunological measurement method for a disease caused by an abnormality in a superoxide production mechanism selected by the method. 6. The immunoassay according to claim 5, wherein the disease caused by the abnormality of the superoxide production mechanism is human autosomal recessive chronic granulomatosis.