JPH05222098A - Reagent for classification and identification of candida - Google Patents

Abstract

PURPOSE:To provide the subject reagent capable of quickly, accurately and easily identifying candida, bearing a marker, etc., or group to bind it on an antibody secreted by fused cells produced from anti-candida antibody-producing cells and cells capable of longterm in vitro subculture. CONSTITUTION:A slant agar medium is inoculated with Candida-albicans ATCC 752 strain to make a three-day culture in an incubator at 37 deg.C followed by centrifugation to collect microbial cells, which are then treated with 1.0% glutaraldehyde, washed, and suspended in a buffer solution. Thence, the resulting suspension as immunogen is injected into the peritoneal cavity of a BALB/c mouse to immunize it followed by boosting and taking the splenocytes, which are then fused with mouse myeloma cells, and the resulting fused cells are cultured in a HAT medium to proliferate hybridoma, and antibody-producing clone is selected, and cultured after cloning to obtain IgG or IgM antibody, into which a radioactive substance, fluorescent substance, enzyme, or a marker for electron microscopic observation (or structure to secondarily binding it), thus obtaining the objective reagent for quickly, accurately identifying candida.

Description

Detailed Description of the Invention

The present invention relates to a derivative of an antibody secreted by a fused cell (hereinafter referred to as hybridoma) between an antibody-producing cell against Candida and a cell capable of long-term subculture in vitro, and Candida containing the same. The present invention relates to reagents for classification and identification of bacteria.

The term "antibody derivative" as used herein refers to a group containing a radioactive substance, a fluorescent dye, an enzyme, a marker for electron microscope observation, or a structure for secondarily binding them to the antibody. It is a chemically coupled product.

Infections due to bacteria have been remarkably reduced in recent years due to the development of preventive medicine and the spread of antibiotics, but diseases due to fungi are on the rise worldwide. Many of the fungi that cause these infectious diseases are bacteria that are resident in normal environments, and are present in the oral cavity, digestive tract, pharynx, skin, vagina, etc. of healthy people. As mycosis, endocarditis, pneumonia, urinary tract disease, meningitis, bone and joint diseases, skin diseases and the like have been reported.

[0004] It is also known that mycosis is accompanied by chronic wasting diseases such as tuberculosis and cancer, and significantly worsens the symptoms. On the other hand, there are few treatments such as antibiotics that are effective against mycosis, and the side effects are strong. Therefore, the rapid detection of fungi and their accurate identification have clinical significance.
There is a great need for effective treatments for mycosis. In addition, the brewing process requires rapid and accurate discrimination between harmful fungi and brewer's yeast.

The derivatives of the present invention provide a very effective means for the classification, identification and treatment of candidiasis of the genus Candida.

Conventionally, Candida has been classified and identified based on its morphological and biochemical properties, but these are lacking in promptness and require skill. In addition, a serological identification method using a factor serum obtained by immunizing an animal such as a rabbit and absorbing it with cells of a species different from that used for immunization is also performed. However, this factor serum is a serum obtained by immunizing an animal, and is a mixture of antibodies with various specificities. Therefore, it is inevitable that the peculiarity and potency of each lot vary to some extent.
In addition, there is a possibility that an unexpected side reaction may occur due to foreign substances, resulting in incorrect identification. In addition, because the fungi belonging to the same genus are antigenically similar to each other, the titers of the resulting factor sera are generally low. Therefore, there is a limit to the reliability of the method for classifying and identifying Candida using such factor sera, and the possibility of erroneous identification cannot be denied.

[0007] The present inventors have established a reliable fungal classification,
As a result of repeated research on the identification method, it was found that the antibody secreted by the hybridoma between the antibody-producing cells against Candida and the cells capable of long-term subculture in vitro can be used for serological classification and identification of Candida. The present invention has been completed by further studying the headings and further the classification and identification methods using them.

[0008]

[Means and Actions] Since the antibody for producing the derivative of the present invention (hereinafter referred to as antibody A in the "Means and actions and effects" section) has a highly specific reactivity with an antigen, Compared with the conventional method, the present invention enables rapid and highly reliable identification without complicated procedures. The class of antibody A is IgG or IgM.

The sample that can be tested by using the present invention may be any Candida fungus body, regardless of its origin. For example, clinical materials obtained from a patient suspected of having candidiasis clinically, such as canopy, urine, vaginal secretion, or tissue, can be used by separating and culturing Candida bacterial cells, and brewing processes such as beer brewing. In order to identify harmful fungi contained in yeast, the brewing yeast can be used as a sample.

For identification, it is convenient to contact a specimen containing Candida cells with a test solution containing a derivative of antibody A and use the antigen-antibody reaction (aggregation reaction etc.) that occurs at that time. Identification can also be performed by immunofluorescence microscopy, immunoelectron microscopy, radioactive binding, enzyme immunoassay, complement fixation reaction, or the like. When the direct method of immunofluorescence microscopy is used, the antibody A is fluorescently labeled with fluorescein, rhodamine or the like, and when immunoelectron microscopy is used, the marker is labeled with ferritin or the like, radioactivity It is convenient to use radioisotope-labeled with ¹²⁵ I, ¹³¹ I, etc. by the binding method, and enzyme-labeled with peroxidase, alkaline phosphatase, β-galactosidase, etc. by the enzyme immunoassay. Of course, the indirect method can be performed by using a secondary antibody or a binding substance instead thereof (for example, an indirect method can be performed by using a biotin-labeled anti-Candida antibody and using avidin instead of the secondary antibody). You can also The above are specific examples of the derivative in the present invention. Instead of the antibody A itself, it is also possible to use a portion of the antibody A obtained by limiting decomposition of the antibody A by chemical and / or enzymatic treatment, for example, F (ab ′) ₂ . However, this is not directly related to the present invention.

Any of the Candida bacteria to which the present invention can be applied may be exemplified by the following.

Candida albicans Serotype A, Candida albicans Serotype B, Candida tropicalis , Candida guilliermondii , Candida krusei , Candida parapsilosis , Candida pseudotropicalis .

The antibody which can be used in the method of the present invention may be any antibody as long as it is an antibody secreted by a hybridoma between an acid cell of an antibody against Candida and a cell capable of long-term subculture in vitro. The antibody secreted by the hybridoma as shown in 1 can be exemplified.

The antibody used in the present invention is produced, for example, as follows.

The antibody A which can be used in the present invention may be any antibody A secreted by a hybridoma of a cell producing an antibody against Candida and a cell capable of long-term subculture in vitro, and shown in Table 1 below. The antibody A secreted by such a hybridoma can be exemplified.

The antibody A used in the present invention is produced, for example, as follows.

A. Preparation of antibody-producing cells In order to obtain the hybridoma and antibody A used in the present invention, antibody-producing cells against Candida and cells capable of long-term subculture in vitro are required. By fusing the two, a hybridoma that produces antibody A against Candida and that can be subcultured for a long time in vitro can be obtained.

The antibody-producing cells against Candida may be obtained from any animal species including humans, and it is not essential to carry out immunization in advance, but by doing so, the efficiency of collection of the desired hybridoma can be improved. Can be significantly increased.

When human cells are used, those having a history of Candida infection or those having a high antibody titer against Candida in serum can be selected. When it is attempted to obtain it from an artificially immunized living body, the immunogen may be a viable cell or a cell whose growth is lost by glutaraldehyde treatment, mitomycin treatment, heat treatment or the like. You may use what isolate | separated and refined the surface antigen by suitable methods, such as an enzyme treatment. The bacterial species can be selected from the following bacterial species. The form thereof may be any of mycelium, yeast, chlamydospore, and the like.

Candida albicans Serotype A Candida albicans Serotype B Candida tropicalis Candida guilliermondii Candida krusei Candida parapsilosis Candida pseudotropicalis In the immunization, an adjuvant such as Freund's complete or incomplete adjuvant can be mixed and used as an immunogen.
The immunogen administration method for immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, etc., but subcutaneous injection or intraperitoneal injection is preferred. Immunization may be performed once or repeatedly at appropriate intervals, preferably 1 to 5 weeks. If the antibody titer against Candida in the serum of the immunized animal is measured and antibody-producing cells are obtained from the animal having a sufficiently high antibody titer, the efficiency of the subsequent operation can be increased. For fusion, it is preferable to use animal-derived antibody-producing cells 3 to 5 days after the final immunization. The antibody-producing cells are plasma cells and their precursor cells, lymphocytes, which may be obtained from any part of the individual, but generally can be obtained from the spleen, lymph nodes, peripheral blood or combinations thereof. ..

B. Cell fusion In vitro, any cell that can be subcultured for a long time may be any cell that fuses with an antibody-producing cell to produce a hybridoma suitable for the purpose, but a high probability is leukemia cells such as myeloma. .. The species of origin may be any of human, rat, mouse and the like. As described below, it is preferable to use a hypoxanthine guanine phosphoribosyl transferase-deficient cell line or a thymidine kinase-deficient cell line in order to remove parent cells mixed after fusion.

For example, human-derived GM-1500 6TG-A
1-2, RPMI8226, mouse-derived P3-X63-Ag8, P3-
NSI / 1-Ag4-1, Sp2 / 0-Ag14, X63-Ag 8.653 and the like can be used.

It is not essential that the species from which the above-mentioned antibody-producing cells are derived and the species from which cells can be subcultured for a long time are the same, but the efficiency of fusion, the stability of the properties of the cells after fusion, In general, it is often advantageous to use the same one in terms of easiness in culturing in the body. In particular, P3-X63-A derived from mouse as a cell capable of long-term subculture
g8, P3-NSI / 1-Ag4-1, Sp2 / 0-Ag14 or X63
-When using Ag8.653, BAL, a syngeneic mouse
It is advantageous to use B / c or a hybrid mouse thereof.

Upon fusion, a fusion promoter such as Sendai virus or polyethylene glycol is preferably used, and particularly polyethylene glycol 1000, 1540, 2000, 4000 or 6000 is preferably used. The fusion is carried out in a solution containing about 30-55%. Dimethyl sulfoxide may be further added as an auxiliary agent.

C. In addition to the hybridoma, antibody-producing cells that are parent cells and cells that can be subcultured for a long time in vitro remain in the mixture after establishment and fusion of the hybridomas. The former usually does not endure in vitro culture for a long period of time, so there is no problem, but the latter may be proliferated together with the target hybridoma, and therefore it is desirable to exclude this. Therefore, as the latter, after using hypoxanthine guanine phosphoribosyl transferase or thymidine kinase deficient cell line and fusing,
Culture in medium containing hypoxanthine, aminopterin and thymidine. As a result, only the hybridoma can be selectively grown. When a hypoxanthine guanine phosphoribosyl transferase or thymidine kinase deficient cell line is not used as a parent cell, by treating the cell with emetine and actinomycin D prior to the fusion to make the cell lose its proliferative property,
The hybridoma may be selected from a mixture with parental cells.

The hybridoma group thus obtained is
In general, it often contains two or more clones, and is not a population of cells having completely the same property. When it is desired to separate individual clones, it is necessary to carry out cloning. Cloning is, of course, for producing antibodies with a single specificity, but it also means preventing changes in population that often occur during long-term culture in a system in which multiple clones are mixed. It is also effective and desirable to do. As a cloning method, a limiting dilution method, a soft agar method, a fibrin gel method or the like can be used. It is also possible to separate cells during cloning using a fluorescence activated cell sorter. In addition, against the appearance of mutant strains that occur during long-term culture, it is possible to preserve cells having the characteristics of the original cells by performing cloning occasionally.

As an example of the hybridoma producing the antibody A against Candida produced by the above-mentioned production method, as shown in the Examples below, CD-1, CD-2,
Examples include CD-3, CD-4, CD-5, CD-6, CD-7, CD-8 and CD-9.

As a method for maintaining the hybridoma, in addition to subculture in vitro and in vivo, it can be cryopreserved according to a conventional method.

D. Production of Antibody A In producing antibody A, a hybridoma producing an antibody against Candida is cultured in vitro or in vivo.

In the case of in vitro culture, a nutrient medium suitable for the hybridoma, eg 10% (V / V)
RPMI 1640 medium containing fetal bovine serum, 5 × 10 ⁻⁵ M β-mercaptoethanol, 1 mM sodium pyruvate and antibiotics can be used. RPMI
Dulbec containing 4.5 g / L glucose instead of 1640 medium
co's modified Eagle's MEM (hereinafter Dulbecco '
s MEM) medium may be used. The appropriate initial concentration for growing cells varies depending on each hybridoma, but is generally about 10 ⁵ cells / ml, and it is desirable that the cell concentration in the culture does not exceed 2 × 10 ⁶ cells / ml.

It is also possible to produce the antibody A secreted by the hybridoma by transplanting this hybridoma into a living body, allowing it to grow in a solid or ascites form, and collecting a body fluid, preferably serum or ascites, from the living body. .. The crude antibody solution obtained by this method has the drawback that it contains various substances derived from the living body as hosts as impurities, while it has a significantly higher concentration of the target antibody A than the antibody solution obtained by in vitro culture. It is excellent in that it includes it.
When hybridomas are transplanted into the abdominal cavity and proliferated, pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered before transplantation, preferably 3 to 9 weeks before the production of crude Although the yield of antibody solution can be increased, this treatment is not essential. The living body used as a host is preferably an animal of the same strain and the same strain as the parent cell of the hybridoma to be transplanted. In this case, the hybridoma usually grows in the living body without any special treatment. However, when the histocompatibility identities of the hybridoma and the host do not match, generally, the host body is administered with anti-lymphocyte antibody and X-ray irradiation. It is necessary to take measures such as these in advance. After transplantation, it usually takes about 1 week for the cells to grow.
It takes 3 weeks.

Can manufactured by the above manufacturing method
Examples of antibody A against Dida albicans, as shown in the Examples below, antibody A which reacts with Candida albicans, Candida genus Candida tropicalis, Candida gui
lliermondii , Candida krusei , Candida parapsilo
Antibody A that also reacts with sis and Candida pseudotropicalis , and antibody A that also reacts with other fungi. The specificity and immunoglobulin class are shown in Table 1 below.
As shown in.

By the conventional method, rabbits were treated with Candida a
Antiserum obtained by immunization with lbicans was treated with Candida tropica
Antibody A obtained by absorption with lis is Candida albicans
Not only other Candida species ( Candida parapsilosi
s ) also reacted.

The antibody A may be used as a crude antibody solution as it is, but according to a conventional method for immunoglobulin purification such as ammonium sulfate fractionation or ion exchange chromatography, or by affinity chromatography with Protein A or an antigen. It can be purified before use.

Further, the obtained antibody A is C as described above.
It is effective for the classification and identification of andida albicans and the treatment and prevention of candidiasis, and can be used in a wide range when purifying an antigen substance by affinity chromatography and the like.

If desired, the above antibodies may be mixed and used.

[0037]

Examples Example 1 (1) Preparation of immunogen : Candida albicans ATCC 7
52 strains were inoculated on slope agar containing Sabouraud's medium and cultured in an incubator at 37 ° C for 3 days. After culturing, add phosphate buffered saline (hereinafter PBS, abbreviated as pH 7.2), suspend the bacteria by pipetting, and centrifuge (1000
× g, 4 ° C, 10 minutes) to obtain a precipitate (bacteria). After repeating this washing operation three times, 1.0% glutaraldehyde was added to the cells and treated at 0 ° C for 30 minutes. Next, the cells were washed 5 times with PBS, adjusted to a concentration of 2 × 10 ⁷ cells / ml with PBS, and used as an immunogen suspension in the following experiments.

(2) Preparation of antibody-producing cells : Female BALB / c mice aged 8 weeks (Charles River Japan) were immunized intraperitoneally with 0.2 ml of the above immunogen suspension. Furthermore, immunization was repeated at 10-day intervals, and 100 days after the start of immunization, a booster was performed by intravenously administering 0.2 ml of the above immunogen suspension, and after 3 days, the mice were exsanguinated and killed on a clean bench ( The spleen was aseptically removed in Hitachi. Next, the spleen was put into a petri dish containing RPMI1640 medium, loosened into small pieces with tweezers, gently pipetting, and then the above-mentioned spleen suspension was filtered with a stainless wire mesh to give a spleen cell suspension. Obtained. This suspension contains 0.747% ammonium chloride in the cell pellet obtained by centrifugation (500 × g, 10 minutes)
1.7 mM Tris / HCl buffer (pH 7.65) was added and suspended to destroy and remove red blood cells. Then, the cell pellet obtained by centrifugation (500 × g, 3 minutes) of this spleen cell suspension was washed 3 times with RPMI1640 medium to obtain RP.
The concentration was adjusted to 10 ⁸ cells / ml with MI1640 medium.

(3) Cell fusion and preparation of hybridoma
Preparation : Mouse myeloma cells Sp2 / 0-Ag14 1 × 10 ⁷ cells previously cultured in vitro and the above splenocyte suspension (1 ×
Was mixed with 10 ⁸ cells and centrifuged (500 × g, 5 minutes), and the supernatant was removed to obtain a cell pellet. After loosening the pellet by gently tapping the bottom of the container, 37
50% (V / V) polyethylene glycol kept at ℃
1 ml of RPMI1640 medium containing 4000 was added and left for 1 minute. Next, the polyethylene glycol solution and the cell pellet were mixed by placing them in a 37 ° C thermostat and gently rotating the container for 1 minute. Next, RPM kept at 37 ℃
After adding a total of 10 ml of I1640 medium at a rate of 1 ml / 30 seconds,
Centrifugation (500 xg, 5 minutes) was performed. After removing the supernatant, the cell pellet was suspended in RPMI1640 medium and centrifuged (500 × g, 5 minutes) to obtain a cell pellet. After repeating this washing operation again, the cell pellet was incubated at 37 ° C. in HAT medium, that is, 20% fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, 4.5 g / L glucose, 5 × 10 ⁻⁵ M β. -Mercaptoethanol, 1x
10 ^-4 M hypoxanthine, 4 × 10 ^-7 M aminopterin,
20 ml of RPMI1640 medium containing 1.6 × 10 ^-5 M thymidine and 50 mg / L kanamycin sulfate was added and well suspended. This cell suspension was added to each well of a 96-well tissue culture plate (Nunc 167008, Nunc, Denmark).
100 μl of each was dispensed, and the culture was started at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. 24 hours after the start of culture, 100 μl of HAT medium was added. Then 2 ~
Remove 100 μl of medium from each well at 3 day intervals and add fresh H
Culturing is performed by adding 100 μl of AT medium,
Hybridomas having the ability to grow in AT medium were selected.

Two weeks after the start of culture, the growth of hybridomas was observed, and the presence or absence of produced antibody in the culture supernatant in each well was examined by the method described in (4) below.

(4) Establishment of antibody-producing hybridoma :
The presence or absence of antibody production in the culture supernatant obtained above was examined by enzyme immunoassay. That is, each well of a 96-well tissue culture plate (Nunc 167008, Nunc, Denmark) was treated with anti- Candida albicans antibody (2.5 at 100 ° C).
Serum obtained by intravenously immunizing rabbits with 2 × 10 ⁷ Candida albicans heat-treated for 5 times was fractionated by ammonium sulfate salting-out (IgG fraction) with 30 μg of 0.1 M sodium bicarbonate / 50 μl of the solution adjusted to the protein concentration of ml
Each was dispensed and left at 4 ° C for 24 hours. Next, after thoroughly washing each well with distilled water, Candida albicans ATC
30 μl of C 752 bacterial cell fluid (2 × 10 ⁷ cells / ml) was dispensed and reacted at room temperature. Further, the plate wells were dried by treatment at 70 ° C for 3 hours. This plate is -20 until use
Stored at ° C. Then in each well of this plate, add 0.5
50 μl of PBS containing% glutaraldehyde was dispensed, allowed to stand at room temperature for 30 minutes, and each well was washed 3 times with PBS containing 0.05% Tween 20 (registered trademark). Add 100 μl of the test sample (culture supernatant of each well) to each well after washing, and add 37
The reaction was carried out at ℃ for 1 hour. After washing three times with PBS containing 0.05% Tween 20 (registered trademark), a horseradish-derived peroxidase-conjugated goat anti-mouse immunoglobulin antibody (Cappel, USA) was diluted 1000 times with horse serum.
50 μl was dispensed into each well and reacted at 37 ° C. for 1 hour. After completion of the reaction, each well was washed 5 times with PBS containing 0.05% Tween 20 (registered trademark), and 0.1 mg containing 0.1 mg / ml o-phenylenediamine and 0.04% (V / V) 31% hydrogen peroxide solution. 100 μl of M citrate buffer (pH 4.5)
It was added to each well and reacted at room temperature for 30 minutes. In each well
The enzymatic reaction was stopped by adding 50 μl of 12.5% sulfuric acid, and the identification was performed by measuring the absorbance at 492 nm.
As a result, antibody production was observed in 22 of the 192 wells.

Next, the hybridomas in the wells in which antibody production was observed were cloned. That is, spleens were extracted from untreated 8-week-old female BALB / c mice as feeder cells, and spleen cells were obtained by the same method as in (2) above, and the spleen cells were expressed in HAT medium at 5 × 10 ⁶ cells / The concentration was adjusted to ml. Then, the above hybridoma was added to this spleen cell suspension at a rate of 2 cells / ml, and after stirring well, a 96-well tissue culture plate (Nunc 167008, N
100 μl was dispensed to each well (unc, Denmark). After 24 hours, 100 μl of HAT medium was dispensed into each well and cultured at 37 ° C. in an incubator containing 5% carbon dioxide gas.

After 2 weeks of cloning, the growth of hybridomas was observed, and the presence or absence of antibodies in the culture supernatant in each well was examined by the method described above. As a result, 2 to 80 antibody-producing clones were obtained for each well cloning. From these clones, a clone having high antibody secretory ability, excellent proliferative property, and stable cell was selected, and cloned again in the same manner as above to produce antibody-producing hybridomas CD-1, CD-2 and Established CD-3.

(5) Production of antibody : ( Production by in vitro culture ); Hybridoma CD-
1, CD-2 or CD-3, 20% fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, 4.5 g / L glucose, 5
1 × 10 ⁵ was added to RPMI1640 medium containing × 10 ⁻⁵ M β-mercaptoethanol and 50 mg / L kanamycin sulfate.
Cells / ml and suspend 25 ml of this cell suspension at 75
A cm ² tissue culture flask (Corning, USA) was dispensed and cultured at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. The culture supernatant was collected on the 4th day when the growth reached almost steady. The number of cells at this time is about 2 × 10 ⁶ cells / ml
And the antibody contents of the supernatant are 3.0 μg / ml and 2.3 μg, respectively.
/ Ml and 2.8 μg / ml.

( Production by in vivo culture ): 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to the BALB / c mouse intraperitoneally, 10 to 30 days after the in vitro administration. Proliferated hybridoma CD-1,
5 × 10 ^{6 of} CD-2 or CD-3 was inoculated. Ascites was collected 2 to 3 weeks after inoculation and centrifuged (1000 × g, 4 ° C, 1
Ascites supernatant was obtained by (5 minutes). About 30 ml of ascites supernatant was obtained from 10 mice for each hybridoma, and their antibody contents were 1.5 mg / ml, 2.3 mg / ml and 1.8 mg / ml, respectively.

(6) Specificity and properties of antibody : ( Study of specificity ): In addition to Candida albicans ATCC752, Candida albicans IF as an allogeneic antigen cell
O0588, Candida albicans IFO1385, Candida alb
icans IFO1389, Candida albicans IFO1594 and Candida albicans IFO1269 as Candida tropicalis ATCC750, Candida
guilliermondii IFO0679, Candida krusei IFO
1395, Candida parapsilosis IFO1396 and Candi
The da pseudotropicalis IFO0432 strain was cultured in the same manner as in (1) above, treated with formalin, and adjusted to 1 × 10 ⁸ cells / ml with PBS containing 0.05% Tween 20 (registered trademark). 0.3 ml of this antigen cell suspension was dispensed into a siliconized test tube with a diameter of 1.2 cm and centrifuged (1000 xg,
After removing the supernatant for 5 minutes), 0.5 ml of the supernatant of the hybridoma CD-1, CD-2 or CD-3 in vitro culture obtained in (5) above was added and reacted at 37 ° C for 1 hour. .. After completion of the reaction, add 3% PBS containing 0.05% Tween 20 (registered trademark).
Washed twice, then horseradish-derived peroxidase-conjugated anti-mouse immunoglobulin antibody (Kappel, USA)
0.5 ml of a 1,000-fold diluted horse serum solution was added, and the mixture was incubated at 37 ° C.
The reaction was carried out for 1 hour. After completion of the reaction, the plate was washed 5 times with PBS containing 0.05% Tween 20 (registered trademark), and then 1 mg / ml.
O-phenylenediamine and 0.04% (V / V) 31%
Add 1 ml of 0.1M citrate buffer containing hydrogen peroxide,
The reaction was carried out at room temperature for 30 minutes. And 1 as a reaction terminator
0.5 ml of 2.5% sulfuric acid was added, and the identification was performed by measuring the absorbance at 492 nm. The results are shown in Table 1 below.

( Investigation of properties ); 50 μl of a solution of anti-mouse IgG antibody, anti-mouse IgA antibody and anti-mouse IgM antibody (Miles, USA) diluted 100-fold with 0.1 M sodium bicarbonate was added to 96-well flat bottom tissue. Dispense into a culture plate (Nunc 167008, Nunc, Denmark), and
It was left at 24 ° C for 24 hours. After thoroughly washing each well with PBS containing 0.05% Tween 20 (registered trademark), 100 μl of the culture supernatant of the hybridoma CD-1, CD-2 or CD-3 obtained in the above (5) was added, and 37 The reaction was carried out at ℃ for 1 hour. After completion of the reaction, add 3% PBS containing 0.05% Tween 20 (registered trademark).
After washing twice, 50 μl of a solution of horseradish-derived peroxidase-conjugated anti-mouse immunoglobulin antibody (Kappel, USA) diluted 1000-fold with horse serum was dispensed into each well and reacted at 37 ° C. for 1 hour. After completion of the reaction, each well was washed 5 times with PBS containing 0.05% Tween 20 (registered trademark), and 1 mg / ml o-phenylenediamine and 0.04% (V
/ V) 0.1M citrate buffer (pH 4.5) containing 31% hydrogen peroxide solution (100 μl) was added to each well and reacted at room temperature for 30 minutes. The enzymatic reaction was stopped by adding 12.5% sulfuric acid to each well, and the identification was performed by measuring the absorbance at 492 nm.

The results are shown in Table 1 below.

Example 2 (1) Preparation of immunogen : Candida albicans IFO1594
The strain was inoculated on slope agar containing Sabouraud's medium and cultured in an incubator at 37 ° C for 3 days. After culturing, PBS (pH
7.2) was added, the bacteria were suspended by pipetting, and centrifugation (1000 × g, 4 ° C., 10 minutes) was performed to obtain a precipitate (bacteria). After repeating this washing operation 3 times, P
The concentration was adjusted to 5 × 10 ⁵ cells / ml with BS and used as the immunogen suspension in the following experiments.

(2) Preparation of antibody-producing cells : Female CDF mice aged 8 weeks (CLEA Japan, Inc.) were subjected to the above immunogen suspension.
Immunization was performed by intravenously administering 0.2 ml. Further, immunization was repeated at 14-day intervals, and 70 days after the start of immunization, a booster was performed by intravenously administering 0.2 ml of the above immunogen suspension, and after 3 days, the mice were exsanguinated to death and the clean bench ( The spleen was aseptically removed in Hitachi. Then, the spleen was placed in a Petri dish containing Dulbecco's MEM medium, and 1 × 10 6 was prepared in the same manner as in Example 1 (2).
A splenocyte suspension of ⁸ cells / ml was obtained.

(3) Cell fusion and preparation of hybridoma
Ltd.: a pre mouse myeloma cells P3-X63-Ag8 1 × 10 7 cells cultured in vitro, the spleen cell suspension (1 ×
Was mixed with 10 ⁸ cells and centrifuged (500 × g, 5 minutes), and the supernatant was removed to obtain a cell pellet. After loosening the pellet by gently tapping the bottom of the container, 37
It was kept warm at ℃. 1 ml of Dulbecco's MEM medium containing 45% polyethylene glycol 4000 kept at 37 ℃
Was gradually added over about 1 minute. After keeping it at 37 ℃ for 7 minutes, keep it warm at 37 ℃ while rotating the container slowly.
Dulbecco's MEM medium (15 ml) was added to the vessel wall over a period of about 5 minutes. About 25 ml of Dulbecco's
After adding the MEM medium, centrifugation (500 xg, 5 minutes) was performed to remove the supernatant.

Dulbecco's MEM medium containing 10% fetal bovine serum kept at 37 ° C. was added to the cell pellet, and 1 × 10 ⁶ was added.
24 ml after gently adjusting with a pipette
Dispense 1 × 10 ⁶ cells into each well of a well tissue culture plate (Nunclon, Nunc, Denmark) at 5% at 37 ℃.
The cultivation was started in a carbon dioxide incubator containing carbon dioxide.
24 hours after the start of the culture, 1 ml of HAT medium was added.
Then remove 1 ml of medium in each well every 2-3 days,
Culture by adding 1 ml of new HAT medium,
Hybridomas capable of growth in HAT medium were selected.

Two weeks after the start of the culture, the growth of the hybridomas was observed, and the produced antibody in the culture supernatant in each well was examined by the method described in Example 1 (4). As a result, antibody production was observed in 10 of the 48 wells.

(4) Establishment of antibody-producing hybridoma : Next, the hybridoma in the well in which antibody production was observed was cloned by the soft agar method. That is,
30% and 10% 2.5% agar (Difco, Germany) kept at 45 ℃
3 ml of double concentration Dulbecco's MEM medium was mixed and mixed with this.
117 ml of Dulbecco's MEM medium kept at 45 ° C was added. Untreated 8-week-old female CDF ₁ mouse splenocytes were added to this agar solution as feeder cells at 5 × 10 ⁵ cells / ml.
And then add it to a 10 cm diameter Petri dish (Falcon
300 ml, Becton-Dickinson, USA) was dispensed in 10 ml aliquots and left at room temperature for 15 minutes for gelation. Then, Dulbecco's MEM containing about 2 ml of the hybridoma suspension in the wells positive for antibody production and 0.5% agar in the same amount.
The medium was mixed and dispensed in 2 ml portions so that the cells were evenly distributed on the gelled layer. The culture was performed at 37 ° C in a carbon dioxide incubator containing 5% carbon dioxide. After 10 days from the start of the culture, collect the colonies of cells generated on soft agar with a Pasteur pipette, transfer them to a 96-well flat bottom plate for tissue culture, and add 0.2 ml of Dulbecco's MEM medium.
Culturing was carried out at 5 ° C in a carbon dioxide incubator containing 5% carbon dioxide. Then, while observing the growth of the hybridoma, the presence or absence of the antibody in the culture supernatant in each well was determined in Example 1.
It was inspected by the method described in (4).

From the hybridomas positive for antibody production, clones having high antibody secretory ability, excellent proliferative property, and stable were selected, and cloned again in the same manner as described above to obtain antibody-producing hybridomas CD-4 and Established CD-5.

(5) Production of antibody : ( Production by in vitro culture ): Hybridomas CD-4 and CD-5 were cultured by the method described in Example 1 (5) to obtain a culture supernatant.

( Production by In Vivo Culture ); Example 1
Hybridomas CD-4 and CD-5 were intraperitoneally transplanted into a CDF ₁ mouse by the method described in (5), and 2 to 3
Ascites was collected at the week to obtain an ascites supernatant. About 30 ml of supernatant was obtained from 10 mice.

(6) Specificity and properties of antibody : Hybridomas CD-4 and C were prepared by the method described in Example 1 (6).
The specificity of the antibody and the class of immunoglobulin contained in the culture supernatant of D-5 were examined. The results are shown in Table 1 below.

Example 3 (1) Preparation of immunogen : Candida albicans IFO0588
The strain was inoculated on slope agar containing Sabouraud's medium and cultured in an incubator at 37 ° C for 3 days. After completion of the culture, the bacteria were collected with a platinum loop, suspended in PBS (pH 7.2), and centrifuged (1000 × g, 4 ° C., 10 minutes) to obtain a precipitate (bacteria). After repeating this washing operation three times, the bacterial cells at 100 ° C
It was heat-treated for 2.5 hours.

The cells were washed 3 times with PBS and then washed with PBS.
The concentration was adjusted to 2 × 10 ⁷ cells / ml and used as an immunogen suspension in the following experiments.

(2) Preparation of antibody-producing cells : Female BALB / c mice (Charles River Japan) of 8 weeks of age were immunized by intraperitoneally administering 0.2 ml of the above immunogen suspension. Immunization was repeated at 2-week intervals, and a booster was performed by intravenously administering 0.2 ml of the above immunogen suspension 10 weeks after the start of immunization, and after 3 days, the mice were exsanguinated and killed by a clean bench (Hitachi, Ltd.). ), The spleen was aseptically removed. Next, the spleen was placed in a petri dish containing RPMI1640 medium, and 5 × 10 5 cells were prepared in the same manner as in Example 1 (2).
A splenocyte suspension having a concentration of ⁷ cells / ml was obtained.

(3) Cell fusion and preparation of hybridoma
Ltd.: pre vitro by the mouse myeloma cell P3-NSI / 1-Ag4-1 0.5 × 10 7 cells were cultured in the spleen cell suspension (5 × 10 ⁷ cells) were mixed, centrifuged (500 × g, 5 minutes) and the supernatant was removed to obtain a cell pellet. After gently loosening the bottom of the container to loosen the pellet, 42.5% polyethylene glycol 15 kept at 37 ℃ was used.
RPMI 1640 with 40 and 15% dimethyl sulfoxide
0.5 ml of medium was added and reacted for 1 minute. At this time, the polyethylene glycol solution and the cell pellet were mixed by slowly rotating the container with fingers and gently rotating it. After 1 minute, while rotating the container in the same manner, add 10 ml of RPMI1640 medium kept at 37 ° C at a speed of 1 ml / 30 seconds, and then centrifuge (500 xg, 5 minutes).
Was done. After removing the supernatant, replace the cell pellet with RPM.
The cells were suspended in I1640 medium and centrifuged (500 × g, 5 minutes) to obtain a cell pellet. After repeating this washing operation again, the culture medium kept at 37 ° C in the cell pellet,
That is, 10 ml of RPMI1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 1 mM pyruvic acid, 4.5 g / L glucose, 5 × 10 ^-5 M β-mercaptoethanol and 50 mg / L kanamycin sulfate was added and well suspended. Let This cell suspension was added to a 96-well tissue culture plate (Nunc
167008, Nunc, Denmark) 200 μl per well
Each was aliquoted, and cultivation was started at 37 ° C in a carbon dioxide incubator containing 5% carbon dioxide. Twenty-four hours after the start of the culture, the supernatant was halved, and 1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ was added to the HAT medium kept at 37 ° C., that is, the above culture medium. 100 with M thymidine added
μl was added. After that, culture medium in each well at 2-3 day intervals 1
After culturing by removing 100 μl of HAT medium from 100 μl, hybridomas having a growth ability in HAT medium were selected.

Two weeks after the start of culture, the growth of hybridomas was observed, and the produced antibody in the culture supernatant in each well was examined by the method described in Example 1 (4). However, Candida albicans I can be used as the cells to bind to the plate.
The FO0588 strain was used. As a result, antibody production was observed in 3 of 48 wells.

(4) Establishment of antibody-producing hybridoma :
Then, the hybridomas in the wells in which antibody production was observed were cloned by the method described in Example 1 (4) to establish antibody-producing hybridomas CD-6 and CD-7.

When the hybridoma is replaced or subcultured, the HAT medium is replaced with the HT medium (1 × 10 6 as the culture medium).
^-4 M hypoxanthine and 1.6 × 10 ^-5 M thymidine) were used to gradually change the medium to HT medium, and after further culturing in HT medium for 2 weeks or more, H
By replacing the T medium with a culture medium containing no hypoxanthine or thymidine, the culture was gradually adapted from the culture in the selective medium to the culture in the usual culture medium.

(5) Production of antibody : ( Production by in vitro culture ): Hybridomas CD-6 and CD-7 were cultured by the method described in Example 1 (5) to obtain a culture supernatant. However, the cell concentration at the start of the culture was 2 × 10 ⁵ cells / ml, and the antibody content of the culture supernatant after 4 days was 2.5 μg / ml and 3.0 μg / ml, respectively.

( Production by In Vivo Culture ); Example 1
According to the method described in (5), 1 × 10 ⁷ hybridoma CD-6 or CD-7 was inoculated into the abdominal cavity of BALB / c mouse, and ascites was collected at 2 to 3 weeks after the inoculation, and the ascites supernatant was collected. Got About 30 ml of supernatant was obtained from each of 10 mice.

(6) Specificity and properties of antibody : Hybridomas CD-6 and C were prepared by the method described in Example 1 (6).
The specificity of the antibody and the immunoglobulin class contained in the culture supernatant of D-7 were examined. The results are shown in Table 1 below.

Example 4 (1) Preparation of immunogen : Candida albicans ATCC 7
The 52 strains were inoculated on plate agar containing corn meal (Nissui Pharmaceutical Co., Ltd.) and cultured in an incubator at 25 ° C for 10 days. After the culture was completed, the mycelium and the portion rich in chlamydospores were collected with a spoon and suspended in PBS (pH 7.2) for homogenization. Then, centrifugation (1000 × g, 4 ° C., 10 minutes) was performed to obtain a precipitate (bacteria). After repeating this washing operation three times, the volume was adjusted to 1% with PBS and used as the immunogen suspension in the following experiments.

(2) Preparation of antibody-producing cells : Female BALB / c mice (Charles River Japan) of 8 weeks of age were immunized by intraperitoneally administering 0.2 ml of the above immunogen suspension. Immunization was repeated at 14-day intervals, and a booster was performed 70 days after the start of immunization by intraperitoneally administering 0.2 ml of the above immunogen suspension. After 3 days, the mice were exsanguinated and killed by a clean bench (Hitachi Ltd. ), The spleen and mesenteric lymph nodes were aseptically removed. Hereinafter, Example 1 (2)
A splenocyte suspension of 10 ⁸ cells / ml was obtained according to the method described in 1.

(3) Cell fusion and preparation of hybridoma
Ltd.: pre mouse myeloma cells cultured in vitro X63-Ag8.6 5 3 1 × 10 7 cells and the spleen cell suspension (1 ×
(10 ⁸ cells) were mixed, and cell fusion was performed by the method described in Example 2 (3) to prepare hybridomas. As a result, antibody production was observed in 10 of 48 wells.

(4) Establishment of antibody-producing hybridoma :
Then, the hybridomas in the wells in which antibody production was observed were cloned by the fibrin gel method. That is, 2.5 mg / ml fibrinogen (Miles,
USA), 8 mg / ml sodium chloride, 0.5 mg / ml potassium chloride and sodium citrate 1 ml solution
Petri dish (Falcon 3002, Beckton- Dickinson,
(America) and spread evenly on the bottom, then 10m
U / ml thrombin (Miles, USA) and 20
4 ml of Dulbecco's MEM medium containing 15% fetal bovine serum was added, and the mixture was allowed to stand at 37 ° C. for 1 hour for gelation. Next, 100 μl of 1 × 10 ⁴ hybridomas / ml was added to the gel to spread the cells uniformly, and the cells were cultured at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. From the 10th day after the start of culture, the cell colonies formed on the gel were collected with a Pasteur pipette, transferred to a 96-well flat bottom plate for tissue culture, and 0.2 ml of Dulbecco's MEM medium was further added, followed by incubation at 37 ° C.
The cells were cultured in a carbon dioxide incubator containing 5% carbon dioxide. Then, the growth of the hybridoma was observed, and the presence or absence of the antibody in the culture supernatant in each well was examined by the method described in Example 1 (4). From among the hybridomas producing positive antibody, clones having high antibody secretory ability, excellent proliferative ability, and stable, were selected and cloned again in the same manner as described above to obtain antibody-producing hybridomas CD-8 and C.
Established D-9.

(5) Production of antibody : The above hybridomas CD-8 and CD were prepared by the method described in Example 1 (5).
-9 was cultured in vitro and in vivo to obtain a culture supernatant and an ascites supernatant.

(6) Specificity and properties of antibody : The specificity and immunoglobulin class of the antibody contained in the culture supernatant of hybridomas CD-8 and CD-9 were examined by the method described in Example 1 (6). It was The results are shown in Table-1.

[0075]

[Table 1]

Example 5 The antibodies obtained in Examples 1 to 4 were administered to 10 ICR mice per group at 2 g / kg orally, 400 mg / kg intraperitoneally or 200 mg / kg.
After intravenous administration and observation of life and death for 14 days, no death due to these antibodies was observed.

Further, the shapes, sizes and properties of the hybridomas CD-1 to CD-9 obtained in Examples 1 to 4 are shown in Table-2.
Shown in.

[0078]

[Table 2]

Example 6 Identification of Candida by Antibody (i) Specimens : Sputum, urine or vaginal secretions were collected from 5 patients suspected of having Candida infection and smeared on a petri dish containing Sabouraud-glucose agar medium. Culturing was carried out for 5 days in an incubator at 37 ° C. The colony that appeared on the agar plate was used as a test sample.

(Ii) Identification method : The antibody secreted by the hybridoma CD-1 (ascites supernatant) was used for identification by the slide agglutination method and the fluorescent antibody method.

That is, in the slide agglutination method, one drop of the above antibody solution was dropped on a slide glass, a small amount of bacteria scraped off with a platinum loop from the test colony was stirred, and the presence or absence of agglutination was observed visually or under a microscope. ..

In the fluorescent antibody method, a small amount of bacteria scraped off with a platinum loop from the test colony was smeared on a slide glass, and 1.0
After fixing with 4% formalin overnight at 4 ℃, PBS (pH 7.2)
After that, 1 drop of a 100-fold diluted solution of the above antibody solution was added dropwise, and the mixture was reacted at 37 ° C for 1 hour. PBS (pH 7.2)
After thoroughly washing with, to remove unreacted antibody, one drop of a 100-fold diluted solution of fluorescein-conjugated anti-mouse immunoglobulin (Cappel, USA) was added dropwise and reacted at 37 ° C. for 1 hour. Unreacted fluorescein-conjugated anti-mouse immunoglobulin was removed by washing with PBS (pH 7.2), and the presence or absence of fluorescence of the test bacteria on the slide was examined using a fluorescence microscope (Olympus Vanox, Olympus, Japan). I observed it.

At the same time, morphological and biochemical identification was performed. That is, morphologically, the presence or absence of chlamydospore formation by the slide culture method using corn meal agar medium, and biochemically, the presence or absence of fermentation ability of glucose, saccharose, maltose and lactose was searched by a conventional method. ..

(Iii) Results : As shown in Table 3 below,
The Candida identification results using this antibody were completely consistent with the morphological and biochemical identification results.

[0085]

[Table 3]

Example 7 (1) Hybridomas CD-1, CD-4, CD-6 and
Peroxidase labeling of CD-8 secretory antibody : 4 mg of horseradish peroxidase (Sigma Type VI, Sigma, USA) was dissolved in 1 ml of distilled water and prepared immediately before use.
60 ml of 0.1M Na IO ₄ solution was added and mixed at room temperature for 20 minutes. The reaction solution is 1 mM sodium acetate buffer (pH 4.4)
After dialysis against 1 night, 20 ml of 0.2M sodium carbonate solution
Was added. Immediately, 1 ml of 0.01M carbonate buffer (pH 9.5)
Hybridoma CD-1, CD-4, CD-6 or CD-8 secreting antibody (antibody ascites supernatant purified, protein content 10 mg) dissolved in the above was added and reacted for 2 hours with stirring at room temperature. After the reaction, freshly prepared Na BH ₄
Aqueous solution (4 mg dissolved in 1 ml distilled water) 0.1 ml
Was added, the mixture was allowed to stand at 4 ° C. for 2 hours, and dialyzed against PBS overnight. The mixture thus obtained is used as Sephadex G-100.
It was applied to a column of (registered trademark) (Pharmacia, Sweden), eluted with PBS, and the first fraction at which the absorbance at 280 nm and the absorbance at 403 nm coincided was collected. To 1 ml of this fraction (enzyme / antibody conjugate), 10 mg of rabbit serum albumin was added and dissolved, and stored at -70 ° C until use.

(2) Hybridoma CD-1, CD-4,
Alkaline phosphaters of CD-6 and CD-8 secretory antibodies
ZE Labeling : Alkaline phosphatase Type VII (Sigm) that has been dialyzed against PBS in advance to remove ammonium sulfate
a, U. S. A. ) 5 mg and hybridoma CD-1, CD-
4, 17 mg of CD-6 or CD-8 secreting antibody (antibody obtained by purifying ascites supernatant) was dissolved in PBS to a total volume of 1 ml. to this
10 μl of 20% glutaraldehyde solution was added, and the mixture was reacted at room temperature for 2 hours with stirring. After completion of the reaction, Sephadex equilibrated with Tris-HCl buffer (pH 7.6)
G-200 (registered trademark) (Pharmacia, Sweden)
It was applied to a column and eluted with the same buffer. Void Volume
To the IgG outflow position were collected, bovine serum albumin was added to this fraction at 5% (W / V), and sterilized through a Millipore filter (0.22μ, Millipore). Stored at 4 ° C protected from light until use.

(3) Hybridoma CD-1, CD-4,
Β-galactosidase standard for CD-6 and CD-8 secretory antibodies
Knowledge : By the same operation as (2) above, hybridoma C
The D-1, CD-4, CD-6 and CD-8 secretory antibodies were labeled with β-galactosidase to obtain β-galactosidase labeled antibodies. In this case, β-galactosidase is β-galactosidase.
Galactosidase Sigma grade IV (Sigma, USA)
Was used.

(4) Hybridoma CD-1, CD-4,
¹²⁵ I-labeling of CD-6 and CD-8 secreting antibodies : 100 mCi /
ml Na ¹²⁵ I (carrier free, Amersham, USA)
Hybridoma CD-1, CD-4, CD-6 in 10 μl of solution
Alternatively, a CD-8 secretory antibody solution (an antibody obtained by purifying ascites supernatant,
Protein content 1.0 mg / ml) 50 μl, 0.30 mg / ml
0.5M phosphate buffer (pH 7.2) containing chloramine T
30 μl was added and mixed well, and 15 seconds later, 100 μl of PBS saturated with L-tyrosine was added and immediately mixed.
The obtained reaction solution was applied to a column packed with Amberlite IRA 400 and eluted with PBS containing 1% bovine serum albumin. The eluted fraction was collected and stored at 4 ° C until use. The specific radioactivity of the obtained labeled product is 1.0 μCi /
It was mg antibody protein.

(5) Hybridoma CD-1, CD-4,
Fluorescein labeling of CD-6 and CD-8 secreting antibodies :
1 ml of 10 mg / ml hybridoma CD-1, CD-4, CD-6 or CD-8 secretory antibody (antibody obtained by purifying ascites supernatant)
To this was added 0.1 ml of 0.5 M carbonate buffer (pH 9.3), and then 0.1 mg of fluorescein isothiocyanate powder was added. The mixture was reacted at 4 ° C for 6 hours while stirring so as not to generate bubbles. Immediately after completion of the reaction, the unreacted low molecular weight substance was removed by applying a Sephadex G-25 (registered trademark) (Pharmacia, Sweden) column to obtain the desired fluorescein isothiocyanate-labeled antibody in the high molecular fraction. It was Stored at 4 ° C protected from light until use.

(6) Hybridoma CD-1, CD-4,
Tetramethylrhodamine, a CD-6 and CD-8 secreting antibody
Label : 10 mg / ml hybridoma CD-1, CD-4, CD
-6 or CD-8 secretory antibody (antibody ascites supernatant purified) solution 1ml, 0.5M carbonate buffer (pH 9.3) 0.1ml was added,
Further, 0.2 mg of tetramethylrhodamine isothiocyanate powder was added. While stirring to avoid foaming 4
The reaction was carried out at 0 ° C for 20 hours. Sephadex immediately after the reaction
An unreacted low-molecular substance was removed by applying to a column of G-25 (registered trademark) (Pharmacia, Sweden) to obtain a target tetramethylrhodamine-labeled antibody in a high-molecular fraction. Stored at 4 ° C protected from light until use.

(7) Hybridoma CD-1, CD-4,
Biotin labeling of CD-6 and CD-8 secretory antibodies : 244 mg
(1 mM) d-biotin (Wako Pure Chemical Industries) and 173 mg (1.5 m
M) N-hydroxysuccinimide (Eastman Kod
ak, USA) with dimethyl sulfoxide (Wako Pure Chemical Industries)
Dissolve it in a mixed solution of 8 ml and 5 ml of 1,2-dimethoxyethane (Hanai Chemical), and add 206 mg (1 mM) of N, N'-dicyclohexylcarbodiimide (Kanto Chemical) to 0.5 ml of 1,
A solution dissolved in 2-dimethoxyethane was added, and the mixture was reacted overnight at 4 ° C. The formed precipitate was removed by filtration to obtain a filtrate. The solvent in the filtrate was removed by concentration under reduced pressure, and the remaining oily substance was dissolved in 10 ml of dichloromethane (Wako Pure Chemical Industries, Ltd.)
Cooled to. 0.1M NaHCO ₃ solution cooled to 4 ℃
10 ml was added to this and mixed well by shaking. The formed dichloromethane layer was removed, and 10 ml of 0.1M NaHCO ₃ was added,
Then, 10 ml of distilled water cooled to 4 ° C. was added, the same operation was repeated, and then anhydrous sodium sulfate powder (Koizumi Kagaku) was added to the produced dichloromethane layer for dehydration treatment. After the powder was filtered off, n-hexane was added to the filtrate until it became cloudy. This solution was cooled to -20 ° C, the precipitated crystals were placed in a desiccator, the solvent was removed and dried, and biotin-
N-hydroxysuccinimide ester was obtained.

This biotin-N-hydroxysuccinimide was dissolved in dimethyl sulfoxide to adjust the concentration to 1 mg / ml, and 60 μl of this solution and hybridoma CD-1,
1 ml of a CD-4, CD-6 or CD-8 secreting antibody solution (antibody ascites supernatant purified, protein content 1 mg / ml) was mixed and reacted at room temperature for 4 hours. After the reaction, PB
It was dialyzed against S for 3 days at 4 ° C. The external dialysate was changed 3 times. The solution in the dialysis tube after dialysis was stored as a biotin-labeled antibody at 4 ° C until use.

Example 8 (1) Test Bacteria : Candida albicans ATCC 752 strain,
Candida tropicalis ATCC 750 strain, Candida guill
The iermondii IFO0679 strain was used. Slope agar containing Sabouraud's medium was inoculated into each well and cultured for 3 days in an incubator at 37 ° C. After completion of the culture, the bacteria were collected by a platinum loop and used as test bacteria.

(2) Identification method by fluorescent antibody method : Example 7
Fluorescein-bonded C produced by the method described in (5)
D-1, CD-4, CD-6 and CD-8 secreting antibodies were used.
A small amount of each test bacterium was smeared on a slide glass, fixed with 1.0% formalin at 4 ° C. overnight, and washed with PBS (pH 7.2). One drop of a 10-fold diluted solution of the above fluorescein-conjugated antibody solution was added dropwise and reacted at 37 ° C for 1 hour. It was thoroughly washed with PBS to remove unreacted antibody. The presence or absence of fluorescence of the test bacteria on the slide fluorescent microscope (OlympusVanox, Olympus, Japan) was observed using, Candida alb
Fluorescence was observed in the icans ATCC strain 752 strain.

The rhodamine-labeled hybridomas CD-1, CD-4, CD-6 and C prepared in Example 7 (6) were also prepared.
When a similar operation was performed using a D-8 secretory antibody, C
Fluorescence was observed in the andida albicans ATCC 752 strain.

Biotin-labeled hybridoma CD-1, CD
In the case of -4, CD-6 and CD-8 secretory antibodies, the biotin-conjugated antibody prepared in Example 7 (7) was bound to the test bacteria by the same procedure as described above, and after thoroughly washing with PBS, ,
Fluorescein-conjugated avidin (Funakoshi Pharmaceutical) 5 μg
One drop of the / ml solution was dropped on the slide and reacted at 37 ° C. for 1 hour. After the completion of the reaction, the cells were thoroughly washed with PBS, and the presence or absence of fluorescence of the test bacteria on the slide was observed using a fluorescence microscope. As a result, fluorescence was observed in the Candida albicans ATCC 752 strain bacterial cells.

(3) Identification by enzyme immunoassay : 0.3 ml of the bacterial suspension was dispensed into a siliconized test tube having a diameter of 1.2 cm, and the supernatant was removed by centrifugation (1000 × g, 5 minutes). Then, the peroxidase, alkaline phosphatase or β-galactosidase labeled hybridoma CD-1, CD-4, C prepared in Example 7 (1), (2) or (3) was used.
0.5 ml of a solution prepared by diluting the D-6 or CD-8 secretory antibody 100 times with horse serum was added, and the mixture was reacted at 37 ° C for 1 hour. After completion of the reaction, the plate was washed 5 times with PBS containing 0.05% Tween 20 (registered trademark), and then 1 ml of the substrate solution was added.

That is, in the case of peroxidase-conjugated antibody, 1 mg / ml of o-phenylenediamine and 0.04% (V
/ V) 0.1M citrate buffer (pH 4.5) containing 31% hydrogen peroxide solution (1 ml) was added and reacted at room temperature for 30 minutes,
0.5 ml of 12.5% sulfuric acid was added as a reaction terminator, and the absorption at 492 nm was measured.

In the case of an alkaline phosphatase-conjugated antibody,
1 mg / ml p-nitrophenyl phosphate (Sigma, USA), 9.7% (V / V) diethanolamine and
Substrate solution containing 0.01% magnesium chloride hexahydrate (pH
1 ml of 9.8) was added and reacted at room temperature for 30 minutes, 0.5 ml of 5M sodium hydroxide was added as a reaction terminator, and the absorption at 405 nm was measured.

In the case of β-galactosidase binding antibody, 1
Add 1 ml of borate buffer (pH 8.5) containing mg / ml 4-methylumbelliferyl-β-galactoside, and add 10 ml at 30 ℃.
After reacting for 1 minute, 0.5 ml of 0.1 M glycine / sodium hydroxide buffer (pH 10.3) was added as a reaction terminator, and 4-methylum released by Hitachi Fluorescence Spectrophotometer (excitation wavelength 360 nm, fluorescence wavelength 450 nm) The amount of beryferon was measured.

As a result, these enzyme-linked labeled antibodies were labeled with C
A reaction was observed with the andida albicans ATCC 752 strain.

(4) Identification by radioimmunoassay : using ¹²⁵ I-labeled hybridomas CD-1, CD-4, CD-6 and CD-8 secreting antibodies prepared by the method described in Example 7 (4). I was there. Suspend the test bacteria in PBS containing 0.05% Tween 20 (registered trademark), dispense 1 ml of them into a siliconized test tube with a diameter of 1.2 cm, and centrifuge (1000 × g, 10 minutes) The body (sediment) was obtained. After washing this precipitate twice with PBS containing 0.05% Tween 20 (registered trademark),
50 μl of ¹²⁵ I-labeled antibody (specific activity, 1 μCi / mg antibody) was added and reacted at 37 ° C. for 1 hour. After completion of the reaction, the plate was washed 5 times with PBS containing 0.05% Tween 20 (registered trademark), and then γ-
Counter (Beckman Gamma 8500, Beckman,
The radioactivity bound to the test bacteria was measured in USA).

As a result, as shown in Table 4, Candida a
Radioactivity was observed in the Lbicans ATCC 752 strain.

[0105]

[Table 4]

Example 9 Urine and Candida from three patients with suspected urinary candidiasis
Each of the albicans ATCC 752 strains was inoculated into Sabouraud-glucose agar medium and cultured in an incubator at 37 ° C for 5 days. The yeast-like colonies that appeared on the agar plate were scraped off with a platinum loop, and PBS 3 containing 0.05% Tween 20 (registered trademark) was used.
The suspension was suspended in ml and homogenized with a homogenizer, and the obtained bacterial cell suspension was used as a test sample.

(1) Identification by enzyme immunoassay : 0.3 ml of the bacterial suspension was dispensed into a siliconized test tube with a diameter of 1.2 cm, and the supernatant was removed by centrifugation (1000 × g, 5 minutes). Then, 0.5 ml of a solution prepared by diluting the peroxidase-, alkaline phosphatase- or β-galactosidase-conjugated antibody prepared in Example 7 (1), (2) or (3) 100 times with horse serum was added, and the mixture was incubated at 37 ° C for 1 hour. It was made to react. After completion of the reaction, the plate was washed 5 times with PBS containing 0.05% Tween 20 (registered trademark), and then 1 ml of the substrate solution was added.

That is, in the case of a peroxidase-conjugated antibody, 1 mg / ml of o-phenylenediamine and 0.04% (V
/ V) 0.1M citrate buffer (pH 4.5) containing 31% hydrogen peroxide solution (1 ml) was added and reacted at room temperature for 30 minutes,
0.5 ml of 12.5% sulfuric acid was added as a reaction terminator, and the absorption at 492 nm was measured.

In the case of an alkaline phosphatase-conjugated antibody,
1 mg / ml p-nitrophenyl phosphate (Sigma, USA), 9.7% (V / V) diethanolamine and
Substrate solution containing 0.01% magnesium chloride hexahydrate (pH
1 ml of 9.8) was added and reacted at room temperature for 30 minutes, 0.5 ml of 5M sodium hydroxide was added as a reaction terminator, and the absorption at 405 nm was measured.

In the case of β-galactosidase binding antibody, 1
Add 1 ml of borate buffer (pH 8.5) containing mg / ml 4-methylumbelliferyl-β-galactoside, and add 10 ml at 30 ℃.
After reacting for minutes, 0.5 ml of 0.1 M glycine / sodium hydroxide buffer (pH 10.3) was added as a reaction terminator, and a Hitachi Fluorescence Spectrophotometer (excitation wavelength 360 nm, fluorescence wavelength 45
The amount of 4-methylumbelliferone released was measured at 0 nm).

The results are summarized in Table-5.

(2) Identification by Fluorescent Antibody Method : The bacterial suspension was smeared on a slide glass, fixed with 1.0% formalin overnight, and washed with PBS. Next, Example 7 (5),
A drop of a 100-fold diluted fluorescein, rhodamine, or biotin-conjugated antibody prepared in (6) or (7) with horse serum was added dropwise to the sample, and the mixture was allowed to react at 37 ° C for 1 hour.
It was thoroughly washed with BS to remove unreacted antibody.

In the case of the fluorescein- or rhodamine-conjugated antibody, the presence or absence of fluorescence of the test bacteria on the slide was observed using a fluorescence microscope (Olympus Vanox, Olympus, Japan).

For biotin-conjugated antibody, 5 μg / ml fluorescein-conjugated avidin D (Funakoshi Yakuhin, Japan)
1 drop was added, and the mixture was reacted at room temperature for 1 hour and thoroughly washed with PBS. The presence or absence of fluorescence of the test bacteria on the slide was observed using a fluorescence microscope.

The results are summarized in Table-5.

[0116]

[Table 5]

When morphological and biochemical identification tests were carried out at the same time, urine-derived colonies of patients F and G were identified as Candida albicans , which was completely in agreement with the results using the above-mentioned labeled antibody. The urine-derived colony of Patient H was identified as Saccharomyces cerevisiae .

Example 10 Urine of 20 ml and Can in two patients with suspected urinary candidiasis
1 ml of a solution prepared by suspending a small amount of dida albicans ATCC 752 strain in PBS containing 0.05% Tween 20 (registered trademark)
Was dispensed into a siliconized test tube with a diameter of 1.2 cm,
The sediment was obtained by centrifugation (1500 xg, 20 minutes).

This sediment was added to 0.05% Tween 20 (registered trademark).
Obtained in Example 7 (4) after washing twice with PBS containing
¹²⁵ I-labeled hybridoma CD-1 derived antibody (specific activity 1 μ
Ci / mg antibody) 50 μl or ¹²⁵ I-labeled mouse immunoglobulin (specific activity 1.5 μCi / mg protein) was added at 37 ° C.
The reaction was carried out for 1 hour. After completion of the reaction, the plate was washed 5 times with PBS containing 0.05% Tween 20 (registered trademark), and then γ-counter (Beckman Gamma 8500, Beckman, USA).
The radioactivity was measured at. At the same time
It was inoculated on dextrose agar medium and cultured in an incubator at 37 ° C for 5 days to perform morphological and biochemical identification of the obtained colonies. The results are shown in Table-6.

[0120]

[Table 6]

As a result, as shown in Table 6, all of these specimens bound to ¹²⁵ I-labeled antibody, and Candida albic
It turned out to contain ans . On the other hand, as a result of morphological and biochemical identification performed at the same time, Candi
It was found to contain da albicans .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location G01N 33/533 8310-2J 33/534 8310-2J 33/535 8310-2J 33/569 F 9015- 2J // A61K 39/40 8413-4C C12N 5/20 15/06 C12P 21/08 8214-4B C12Q 1/04 6807-4B (C12P 21/08 C12R 1:91) (72) Inventor Takao Ando Tokyo 3-10-13 Nerima, Nerima No. 24 Kurehaso, Nerima, Nerima-ku No. 24 (72) Isamu Motokawa 5-3-4 Hinodai, Hinodai, Hino-shi, Tokyo No. 404, Omori Heights 404 (72) Katsuo Sakurai 100 people, Shinjuku-ku, Tokyo 3-26-1 Machi Kureha Chemical Co., Ltd. Okubosha No.403 (72) Inventor Chikio Yoshipu 2-19-46 Higashi, Kunitachi, Tokyo

Claims (18)

[Claims] 1. An IgG or IgM antibody secreted by a hybridoma between an anti-Candida antibody-producing cell and a cell capable of long-term subculture in vitro is treated with a radioactive substance, a fluorescent dye, an enzyme, and an electron microscope. A derivative of an IgG or IgM antibody to which a marker or a group containing a structure for secondary binding thereof is chemically bonded. 2. The derivative according to claim 1, wherein the radioactive substance is ¹²⁵ I. 3. The derivative according to claim 1, wherein the fluorescent dye is fluorescein or rhodamine. 4. The derivative according to claim 1, wherein the enzyme is peroxidase, alkaline phosphatase or β-galactosidase. 5. The derivative according to claim 1, wherein the structure for secondarily binding a radioactive substance, a fluorescent dye, an enzyme or a marker for electron microscope observation is biotin. 6. The derivative according to claim 1, wherein the Candida bacterium is Candida albicans . 7. The derivative according to claim 1, wherein the antibody is an antibody against Candida albicans . 8. The antibody is hybridoma CD-1, CD-
2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8
Alternatively, the derivative according to claim 7, which is an antibody secreted by CD-9. 9. An IgG or IgM antibody secreted by a hybridoma between an anti-Candida antibody-producing cell and a cell capable of long-term subculturing in vitro is added to a radioactive substance, a fluorescent dye, an enzyme, or an electron microscopic observation. Reagent for classification and identification of Candida containing a derivative for IgG or IgM antibody chemically bound to a marker for or a group containing a structure for secondary binding thereof. 10. The reagent according to claim 9, which contains a carrier or a diluent. 11. The reagent according to claim 9, wherein the radioactive substance is ¹²⁵ I. 12. The reagent according to claim 9, wherein the fluorescent dye is fluorescein or rhodamine. 13. The reagent according to claim 9, wherein the enzyme is peroxidase, alkaline phosphatase or β-galactosidase. 14. The structure for secondarily binding a radioactive substance, a fluorescent dye, an enzyme or a marker for electron microscope observation is biotin.
The reagent according to. 15. The reagent according to claim 9, wherein the Candida bacterium is Candida albicans . 16. The reagent according to any one of claims 9 to 15, wherein the antibody is an antibody against Candida albicans . 17. The antibody is hybridoma CD-1, CD
-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8
The antibody according to claim 16, which is also an antibody secreted by CD-9. 18. The reagent according to any one of claims 9 to 17, which is used for identifying a human or animal Candida infection.