GB2138444A - Antibody to candida fungi - Google Patents

Abstract

Monoclonal antibody to a surface antigen of a fungus belonging to the genus Candida is produced by preparing a hybridoma between a cell which produces a said antibody and a cell which can be permanently maintained in vitro by subculture and recovering the said antibody secreted by the hybridoma. The antibody is useful for the identification and/or classification of a fungus of the genus Candida and for treating candidiasis.

Description

SPECIFICATION Antibody to candida fungi The present invention relates to the fieid of cell fusion techniques, in particular to an antibody produced by a fused cell between a cell capable of producing an antibody and a cell capable of proliferating permanently in vitro by subculture, the fused cell being hereinafter referred to as "hybridoma".

Recently, infectious diseases caused by bacteria have decreased due to the development of preventive medicine and the spread of antibiotics, while, on the contrary, diseases caused by fungi, i.e. mycoses, have tended to increase in the world. Many of such fungi causing mycoses are usually present in the normal environment, for example in the mouth, digestive tract, pharynx, skin or vagina of a healthy body. Among mycoses, endocarditis, pneumonia, uropathy, meningitis, osteopathy, arthritis, dermatosis and the like have been reported. It is also known that mycoses concur with chronic consumption diseases such as phthisis and cancer and worsen the symptoms thereof to a great extent.

At present, few remedies for the effective cure of mycoses are known. Furthermore, presently available remedies are accompanied by severe side effects. On the other hand, antifungal antibiotics such as amphotericin B and 5-fluorocytosine have hitherto used for curing mycoses.

Their effect on fungi is however insufficient and side effects occur on the use thereof.

A treatment of such diseases by an antibody has also been attempted by e.g. administering a human gamma-globulin preparation against an infection to a patient. Such preparation is, however, considered to have a low content of an antibody specific to the infection-causing fungus since it is not subjected to e.g. an immunization in the preparation process thereof, and therefore the activities are limited to some extent. For a treatment with an antibody having an enhanced titer due to immunization, a serotherapy is already known in an acute poisoning by for example snake. Although such therapy shows some remarkable effect, an immune reaction to the foreign antigen may be caused and an allegic response may occur since such serum is a foreign serum of e.g. horse and contains a number of contaminants othe than the specific antibody.Such serotherapy has not been applied to the treatments of mycoses.

The rapid detection and the accurate identification of those fungi, therefore, are important for a clinical diagnosis and an effective curing method for mycosis is required. On the other hand, it is necessary to recognize and separate a ferment yeast from harmful fungi in the fermentation and brewing process. Candida, which constitutes the subject matter of the present invention, is one typical example of such fungi.

Up to now, the classification and identification of fungi of the genus Candida are based on the morphological and biochemical properties. Such method necessitates long, troublesome experimental analyses and therefore it is unsuitable for the practical use. A serological method of identification is also known which uses serum factors obtained by immunization of an animal such as rabbit followed by absorption with a cell of a different species from that having been used on the immunization. The serum factor is a mixture of various antibodies having different specificities, and therefore the specificity or titer inevitably scatters between lots to a certain extent. Further, an unexpected side reaction due to contaminants may occasionally take place.

On the other hand, the titer of the obtained serum factor is generally relatively low since fungi belonging to the same genus resemble each other in antigenic properties. Moreover, such an immunization comprises troublesome procedures. Thus, these methods of classification and/or identification of Candida are limited to a certain extent from the view point of reliability and complexity.

The inventors of the present invention have made a great effort to research a method for preparing a monoclonal antibody against a fungus of the genus Candida and attained the invention wherein such antibody with a high specificity, a high titer and a low content of contaminants can be obtained from a hybridoma between a cell capable of producing an antibody against a fungus of the genus Candida (hereinafter sometimes referred to as "an anticandida antibody producing cell", or simply as "an antigen producing cell") and a cell capable of permanent in vitro subculture (hereinafter sometimes referred to as "subcultured cell"). The present inventors have also discovered that such antibody is useful for classification and/or identification of a fungus of the genus Candida.The antibody can also be useful for providing an effective curing method of an infection with such fungus.

It is already known that a hybridoma which may produce a specific antibody may be obtained by cell fusion from a cell capable of producing an antibody and a myeloma cell. See e.g. Köhler et al., Nature, 256, 495-497 (1975) and Eur. J. Immunol., 6, 511-519 (1976). A hybridoma capable of producing an antibody to fungus such as Candida, however, has not been known before the present invention.

An object of the present invention is therefore to provide a method for preparation of an antibody specific to the surface antigen of a fungus belonging to the genus Candida.

An another object of the invention is to provide a highly specific anti-candida antibody and a method of preparation thereof by cell fusion technique.

A still another object of the invention is to provide a hybridoma capable of producing such antibody.

A further object is to provide a method of classification and/or identification of the fungus of the genus Candida by such an antibody.

Also, an object of the present invention is to provide a remedy for candidiasis containing such antibody as an effective component.

The present invention also has an object to provide a derivative of the antibody effective in the use for the classification, identification and/or treatment of the diseases caused by fungi of the genus Candida.

Other objects of the present invention will be apparent from the following detailed descriptions on non-limitative, specific embodiments.

The method for preparing an anti-candida antibody of the present invention comprises preparing a hybridoma from an anti-candida antibody producing cell and a subcultured cell, particularly myeloma, and recovering the antibody secreted by the hybridoma.

The method of the invention will be described in more detail in the followings.

A. PREPARATION OF ANTIBODY PRODUCING CELL In the present invention, a cell capable of producing an antibody to a fungus belonging to the genus Candida and a cell which can permanently be maintained by in vitro subculture are essential for obtaining a hybridoma capable of producing the anti-candida antibody and proliferating permanently by in vitro subculture.

The anti-candida antibody producing cell may be obtained from any animal species including human. An immunization of the animal is not essential although such preliminary immunization may remarkably improve the collection efficiency of the hybridoma to be desired.

When the origin of such cell is human, anyone who has a past history of candidiasis or a high titer value of serum to the fungus of Candida may be chosen. Alternatively, such cell may be obtained from a living body immunized with an immunogen. The immunogen may be a viable cell, a cell which is treated with glutaraldehyde, mitomycin or heat and thereby can not proliferate, or the surface antigen separated from a cell by an appropriate treatment with e.g. an enxyme and purified.

Any species of the genus Candida may be used in the invention and any morphology may be acceptable, for example hypha, yeast, spore with thick membrane (chlamydospore), etc. An example of such species is: Candida albicans Serotype A, Candida albicans Serotype B, Candida tropicalis, Candida guilliermondii, Candida krusei, Candida parapsilosis or Candida pseudotropicalis.

The immunogen used on the immunization may be mixed with an adjuvant such as Freund's complete and incomplete adjuvant. The immunogen may be administered by any conventional means such as subcutaneous, intraperitoneal, intravenous, intradermal and intramuscular injections. A subcutaneous or intraperitoneal injection may be preferable. One immunization may be sufficiently possible although the immunization can be repeatedly carried out several times with an appropriate interval for example from one to five weeks. By measuring the antibody titer in serum of the immunized animal, the animal whose titer has been sufficiently high may be used to obtain the antibody producing cell, resulting in improvement of the efficiency of the subsequent procedures. The preferable cell is derived from the animal on the 3-5th day after the final immunization.The antibody producing cell is plasmocyte or lymphocyte which is a precursor cell thereof and may be derived from any site of the body, generally spleen, lymph node, peripheral blood or any combination thereof.

B. CELL FUSION A cell which can permanently be maintained by in vitro subculture may be any appropriate cell which can be fused with the antibody producing cell to give a hybridoma capable of producing a desired antibody. Preferable one of such cells is leukemia cell such as myeloma.

Such a cell may be derived from any species such as human, rat, mouse, etc. The cell which is deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or thymidine kinase (TK) is preferable since such parent cells can grow in a selective medium.

Preferred cell lines, which are available to the public, are GM-1500 6TG-AI-2 and Rim18226 derived from humans and P3-X63-Ag8, P3-NSl/1-Ag4-1, Sp2/0- Ag14 and X63-Ag8.653 derived from mice.

It is preferable to use an antibody producing cell and a subcultured cell, both of which are derived from the same species, although not essential, from the view points of fusion efficiency, stability of the properties of the fused cells and facility of in vivo culture thereof. Particularly, when the cell line P3-X63-Ag8, P3-NSI/1-Ag4-1, Sp2/0-Ag14 or X63-Ag8.653 is used as the subcultured cell, an inbred BALB/c mouse or its hybrid mouse may be preferably used.

For fusion, an accelerator such as Sendai virus (HVJ) and polyethylene glycol may be used, particularly polyethylene glycol 1000, 1540, 2000, 4000 or 6000 may preferably be used.

The cell fusion may be performed in a solution containing approximately 30-55 % of such polyethylene glycol. Further, dimethylsulfoxide may be present in the solution.

C. SELECTION OF HYBRIDOMA In the solution after fusion, there are present, in addition to the fused cell, the remaining parent antibody producing cell and the parent subcultured cell. The former can not survive during the subsequent in vitro culture while the latter may propagate together with the desired hybridoma. It is therefore preferable or even necessary to remove the subcultured cell from the solution containing the mixed cells. For this, HGPRT or TK-deficient cell is preferably used as the parent subcultured cell and the mixed cells containing the parent cells are cultured in a selection medium containing hypoxanthine, aminopterin and thymidine after the cell fusion, which enables selective growth of the desired hybridoma only.Alternatively, the parent subcultured cell, when this is not such a cell as deficient in HGPRT or TK, may be treated with emetine and actinomycin D prior to the cell fusion. The parent cell can not proliferate by such treatment and therefore the desired hybridoma can be easily selected from the mixed cells.

The thus obtained hybridomas generally contain two or more clones, thus those may not have the completely same properties. For separating them into the individual clones, cloning may be necessary and even desired when a monoclonal antibody may be required. The cloning is also effective in the view point of prevention of the change of population which may often occur in a long-term culture of a system with a number of clones being mixed. The cloning may be performed by limiting dilution culture, soft agar culture or fibrin gel culture. An apparatus for sorting out fluorescence activated cells can also be used to separate the cells on the cloning.

The cloning may also be used for separating possible variant cells occurring during the long culture and for retaining the cells having the same properties as the original hybridoma.

The thus obtained hybridoma may be maintained by in vitro or in vivo subculture or in the freeze-dried form by any conventional method.

D. PRODUCTION AND RECOVERY OF ANTIBODY For production of antibody, the hybridoma producing an anti-candida antibody is cultured in vitro or in vivo.

In in vitro culture, an appropriate nutrient broth for the hybridoma of the present invention may be chosen, for example, RPMI 1 640 medium containing 10 % (V/V) fetal bovine serum, 5 x 10-5 M ss-mercaptoethanol, 1 mM sodium pyruvate and antibiotics, or, Dulbecco's modified Eagle's MEM (hereinafter referred to as Dulbecco's MEM) containing 4.5 g/L glucose instead of RPMI 1 640 medium. An appropriate initial cell concentration for proliferation may generally be approximately 105 per ml while this may change depending on each hybridoma, and the cell concentration during the culture is preferably upto 2 X 106 per ml.

In in vivo culture, the hybridoma is transplanted to a living body and grown in the solid form or in ascites. A body fluid, preferably serum or ascites, is taken out of the living body for recovering the antibody secreted by the hybridoma. The obtained crude solution of the antibody may contain as impurities (contaminants) various substances originating from the host living body, nevertheless it is more remarkable than the antibody solution obtained in vitro because of a higher concentration of the desired antibody. When the hybridoma is transplanted intraperitoneally, pristane (2,6,10,1 4-tetramethylpentadecane) can be intraperitoneally administered prior to transplantation, preferably 3-9 weeks before the transplantation. This treatment can enhance the yield of the crude antibody solution but not essential.As a host, an animal of the same species and the same inbreeding as the animal from which the parent cell or cells is/are derived is preferable and in this case the hybridoma can grow in the animal even if not specially treated.

When the serotypes of the histo-compatability of antigens do not coincide with each other between the hybridoma and the host, the host is necessarily preliminarily treated with for example an administration of anti-lymphocyte antibody or an irradiation of X rays. The cells begin to grow from 1-3 weeks after the transplantation.

The obtained antibodies include antibodies reacting with Candida albicans, antibodies reacting with the other species of the genus Candida such as Candida tropicalis, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida pseudotropicalis as well as Candida albicans, and antibodies also reacting with the other fungi.

For comparison, the antibodies obtained by absorption of antiserum from rabbit immunized with Candida albicans by Candida tropicalis react with the other species of the genus Candida (Candida parapsilosis) as well as Candida albicans. Furthermore, the antibody obtained by absorbing the antiserum by Candida tropicalis, Candida guilliermondii, Candida krusei, Candida Parapsilosis or Candida pseudotropicalis has a very low titer and seldom reacts with Candida albicans.

The antibodies of the invention can be used as it is in the crude antibody solution, or they can be purified for use by any method conventionally employed for immunoglobulin, for example, ammonium sulfate fractionation or ion exchange chromatography, or by affinity chromatography with Protein A or an antigen. Such purified antibodies may be used independently or in a mixture thereof.

According to the method for preparation of the anti-candida antibody of the present invention, the defect attached to the conventional methods can substantially be overcome. The hybridoma of the invention can be subcultured and substantially permanently proliferated both in vitro and in vivo. Further, the hybridoma can produce an antibody to a specific antigenic determinant. The produced antibody, therefore, may have a monoclonal specificity, is an antibody to a fungus of the genus Candida and consists essentially of a single molecular species. The method of the present invention permits production of a necessary amount of the antibody depending on demand, avoidance of scatter between lots and further preparation of a solution containing the antibody with a high titer. In addition, the method does not require a troublesome absorption which has conventionally been inevitable.On the other hand, non-purified antigen such as a fungus per se of the genus Candida can be used without difficulty in the method and even in such a case the highly specific antibody can be obtained. Such a higher reactivity of the antibody with an antigen permits rapid identification of a fungus of the genus Candida with a high reliability without the conventional troublesome procedures. Moreover, the purity of the antibody is so high that allergic reactions due to contaminants inevitably contained in the conventional preparation seldom appear, permitting the use as a remedy for candidiasis.

The method of classification and/or identification of the present invention can be applied to a subject from any source. For example, fungal cells are separated from clinical materials such as sputum, urine, vaginal secretion or tissue obtained from a patient who may possibly suffer from candidiasis in the clinical view point and then cultured, or brewer's yeast can be used in order to distinguish harmful fungi from the yeast in brewage processes such as a brewery.

In the identification, a reagent containing the antibody of the present invention is contacted with a subject containing cells of Candida. It conveniently utilizes simply an antibody-antigen reaction (agglutination reaction etc.), or it may utilize immunofluorescence microscopy, immunoelectromicroscopy, radioactive binding assay, enzyme im munoassay, complement fixation test, etc. In a direct immunofluorescence microscopy, the antibody of the invention may conveniently be used. after labelling it with a fluorescent dye such as fluorescein and rhodamine.The other convenient form of the antibody of the invention may be one labelled with a marker such as ferritin for an immunoelectromicroscopy, one labelled with a radioisotope such as 1251 and 1311 for a radioactive binding assay or one labelled with an enzyme such as peroxidase, alkaline phosphatase and ssgalactosidase for an enzyme immunoassay. Of course, an indirect method can be performed with the aid of a secondary antibody or its binding product as a substitute, for example a biotin-labelled anti-candida antibody can be used together with avidin as a secondary antibody. Also, a part of the antibody obtained by restrictive cleavage with a chemical and/or enzyme treatment such as F(ab')2 can be used instead of the antibody per se.Such various derivatives and restriction products are useful for the method of classification and/or identification of the invention and therefore included within the scope of the present invention.

The antibody, its derivatives and restriction products may be mixed for use if necessary.

Furthermore, the reagent for classification and/or identification of the invention may further contain a carrier or a diluent conventionally used.

The present invention also relates to a curing agent (remedy) for candidiasis of urinary tract, lung, kidney, etc. infected with a fungus of the genus Candida such as Candida albicans, Candida tropicalis, Candida guilliermondii, Candida parapsilosis, Candida pseudotropicalis, Candida krusei, etc.

The curing effect of the pharmaceutical agent of the invention may be estimated and established by protection from the fatal infection with a fungus of the genus Candida, as seen in Example hereinbelow described. The mechanism of the protection may be considered to accelerate a complement fixation reaction and attack of macrophage and/or other immunocytes to the fungus of the genus Candida as the administered antibody binds with the surface antigen of the fungus. These reactions may also be confirmed even in vitro.

The antibodies of the present invention may be independently administered per se or in a mixture thereof. When the antibody is chemically combined with a germicide or an antifungal agent such as amphotericin B, 5-fluorocytosine, nystatin and griseofulvin, more advantages may be effected. A part of the antibody obtained by restrictive cleavage with a chemical or enzyme treatment, for example F(ab')2, can also be used for the curing agent of the invention instead of the antibody per se. In this case a possibility can be avoided that the tissue of a host may be affected by a damage due to non-specific complement fixation reaction etc. These derivatives (combination products with antifungal agent of germicide) and restriction products may be used either independently or in a mixture thereof as the curing agent of the present invention.

For evaluating the acute toxicity of the antibodies, its derivatives or restriction products, the curing agent of the invention was administered to three groups (each of 10 animals) of ICR mice; first in an amount of 2 g/kg perorally, second in an amount of 400 mg/kg intraperitoneally and third in an amount of 200 mg/kg intravenously, and no death was observed for 14 days. The curing agent of the invention may therefore be safe as a remedy for infectious diseases by fungi of the genus Candida.

The pharmaceutical agent of the invention for treatment of candidiasis of human and animal can be administered subcutaneously, intramuscularly or intravenously, preferably by subcutaneous or intramuscular injection. The curing agent can perorally be administered since a part of the administered agent can be absorbed through the intestinal tract while retaining the structure as an antibody, which has been confirmed by the present inventors. A preparation for injection can be prepared by for example dissolving or suspending 10 mg of the antibody or its derivative in distilled water to 10 ml, sterilizing by an conventional way, dividing into ampoules of 2 ml in each and freeze-drying. The obtained product will be dissolved in saline on use.An injective preparation may contain in addition to the antibody a carrier, a diluent, a buffer, a stabilizer, an isotonic agent, etc., these agent being known to those skilled in the art. The injectable preparation can be prepared in any form of subcutaneous, intramuscular or intravenous injection. An orally administrable agent may be prepared by any conventional method into an enteric drug. A dose rate of the curing agent of the invention may depend mainly on the symptoms, and generally 0.001 mg to 10 g a day per kg of body weight in animal such as mouse and 0.01-3,000 mg a day per kg of body weight in human.

The present invention will be illustrated by the following non-limitative examples. All the strains of microorganism listed in Table 1 are available to the public EXAMPLE 1 (1) PREPARATION OF IMMUNOGEN Candida albicans ATCC 752 was inoculated in a slant agar containing Sabouraud's medium and incubated at 37"C for three days. After adding phosphate buffered saline (PBS, pH 7.2) to the medium, the cells were suspended by pipetting and centrifuged at 1000 X g, 4"C, for 10 minutes to obtain a cell pellet. This washing procedure was repeated three times, 1.0 % glutaraldehyde was then added to the cell pellet and the cells were fixed at 0 C for 30 minutes.

Then, the cell was washed with PBS five times. The cell concentration was adjusted with PBS to 2 x 107 per ml to obtain a suspension of immunogen which was used in the following experiments.

(2) PREPARATION OF ANTIBODY PRODUCING CELL The immunogen suspension was intraperitoneally administered in an amount of 0.2 ml to female BALB/c mice of 8 weeks old (NIHON CHARLES LIVER, JAPAN) for immunization. The immunization was repeated every 10 days and boosted by an intravenous injection of 0.2 ml of the immunogen suspension on the 100th day from the beginning of immunization. Three days after the boosting, mice were dehematized to death and spleen was aseptically taken out in a clean bench (HITACHI LTD., JAPAN). The spleen was placed in a petri dish containing RPMI 1 640 medium, divided by forceps into fine pieces, gently pipetted and filtered by a stainless mesh to obtain a suspension of spleen cell.After centrifugation (500 X g, 10 minutes), 1.7 mM tris-HCI buffer (ph 7.65) containing 0.747 % ammonium chloride was added to suspend the cell pellet and to disrupt and remove erthyrocytes. The resultant suspension of spleen cell was again centrifuged at 500 X g for 3 minutes. After washing the resulting pellet three times with RPMI 1640 medium, the cell concentration was adjusted to 108 per ml by adding RPMI 1640 medium.

(3) CELL FUSION FOR PREPARATION OF HYBRIDOMA The spleen cell suspension prepared in (2) (1 X 108 cells) was mixed with 1 X 107 of mouse myeloma cell line Sp2/0-Ag14 preliminarily cultured in vitro. After centrifugation of the cell mixture at 500 x g for 5 minutes, the supernatant was discarded to obtain a cell pellet. After unfastening the cell pellet by lightly tapping the vessel's bottom, 1 ml RPMI 1 640 medium containing 50 % (V/V) polyethylene glycol 4000 (37'C) was added to the vessel and allowed to stand for one minute. The vessel was placed in a thermoplastic oven (37"C) and gently turned round for one minute to blend the polyethylene glycol solution with the cell pellet.RPMI 1 640 medium, maintained at 37"C, of 10 ml in total amount was added to the mixture at a rate of 1 ml in 30 seconds and centrifuged at 500 x g for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in RPMI 1640 medium and centrifuged at 500 X g for 5 minutes to obtain a cell pellet. This washing procedure was repeated once again. The resultant cell pellet was well suspended in 20 ml HAT medium (RPMI 1 640 medium containing 20 % fetal bovine serum, 2 mM glutamine, 1 mM pyruvic acid, 4.5 g/L glucose, 5 X 10-5 M ss- mercaptoethanol, 1 x 10-4 M hypoxanthine, 4 x 10-7 M aminopterin, 1.6 x 10-5 M thymidine and 50 mg/L kanamycin sulfate).The resultant cell suspension was placed into 96 well tissue culture plate (Nunc 167008, NUNC INC., DENMARK) with 100 iLl in each well and incubated in a CO2 incubator containing 5 % carbon dioxide at 37"C. After 24 hours from the initiation of the incubation, HAT medium was added in each 100 ,ul. The incubation was continued while removing 100 yl of the medium of each well and adding 100 ,ul of fresh HAT medium to each well every 2 or 3 days, and hybridomas capable of proliferating in the HAT medium were selected.

Since two weeks after the initiation of the incubation, the growth of the hybridoma was observed and the production of antibodies in the supernatant of each well was examined in the same manner as described in (4) below.

(4) SELECTION AND CLONING OF ANTIBODY PRODUCING HYBRIDOMA Enzyme immunoassay was used to determine the presence of antibodies secreted in the supernatant of the culture wells. Namely, 2 x 107 cells of Candida albicans pretreated at 1 00 C for 2.5 hours were intravenously injected five times to rabbit for immunization and the serum obtained from the rabbit was subjected to ammonium sulfate salting out to obtain an IgG fraction. The fraction (anti-Candida albicans antibody) was adjusted by 0.1 M sodium bicarbonate to 30 ,ug protein per ml. Fifty iL1 of the resultant solution was poured into each well of 96 well plate for tissue culture (Nunc 167008, Nunc Inc., Denmark) and allowed to stand at 4"C for 24 hours.Each well was thoroughly washed with distilled water and 30 ,ul of a cell suspension of Candida albicans ATCC 752 (2 X 107 per ml) was placed into each well and reacted at room temperature. The plate wells were dried at 70"C for 3 hours and stored at - 20'C until use. Fifty yl of PBS containing 0.5 % glutaraldehyde was poured into each well of the plate and allowed to stand at room temperature for 30 minutes. After washing each well three times with PBS containing 0.05 % Tween 20, 100 ,ul of the subject (the supernatant of each well) was added to each well and reacted at 37"C for one hour.After washing three times each well with PBS containing 0.05 % Tween 20, 50 ,ul of a solution of goat anti-mouse immuno globulin antibody bound with peroxidase derived from horseradish (Cappel Corp., USA), diluted 1000-fold with horse serum, was poured into each well and reacted at 37'C for one hour. After reaction, each well was washed five times with PBS containing 0.05 % Tween 20, and 100 iL1 of 0.1 M citrate buffer (pH 4.5) containing 1 mg/ml o-phenylenediamine and 0.04-% (V/V) of 31 % aqueous hydrogen oxide solution was added to each well and reacted at room temperature for 30 minutes.The enzymatic reaction was stopped by adding 50 ,ul of 1 2.5 % sulfuric acid to each well and the measurement of absorption at 492 nm showed antibody production in 22 wells among 192 wells.

Hybridomas in the wells assigned to produce antibodies were then cloned. Namely, spleen cells as feeder cells were obtained in the same manner as described in (2) above from spleen taken out of non-treated (control) female BALB/c mice of 8 weeks old and the cell concentration was adjusted to 5 X 106 per ml with HAT medium. The aforementioned hybridoma was added to the resultant spleen cell suspension in an amount of 2 cells per ml and thoroughly stirred.

Hundred ,ul of the cell mixture was inoculated in each well of 96 well tissue culture plate (Nunc 167008, Nunc Inc., Denmark). After 24 hours, 100 pl HAT medium was added to each well and incubated at 37"C in an incubator containing 5 % carbon dioxide.

After two weeks of cloning, the growth of the hybridoma was observed and the presence of antibody in the supernatant of each well was examined in the abovementioned manner. From 2 to 80 clones producing antibody were obtained by such cloning from each well. Among these clones, stable clones capable of producing a large amount of antibody and highly proliferating were chosen and re-cloned in the aforementioned manner to obtain the antibody producing hybridomas CD-1, CD-2 and CD-3.

(5) PRODUCTION OF ANTIBODY (IN VITRO CULTURE) The hybridoma CD-1, CD-2 or CD-3 was suspended in RPMI 1640 medium containing 20 % fetal bovine serum, 2 mM glutamine, 1 mM pyruvic acid, 4.5 g/L glucose, 5 X 10-5 M ss- mercaptoethanol and 50 mg/L kanamycin sulfate, in a cell concentration of 1 X 105 per ml. The resultant suspension (25 ml) was placed in 75 cm2 flask for tissue culture (Corning Glass Works, USA) and incubated at 37"C in a CO2 incubator containing 5 % CO2. On the fourth day, the supernatant was collected from the flask in which the growth was in the stationary state. The number of grown cells was approximately 2 x 106 ml and the antibody contents of the supernatant were 3.0 g/ml (CD-1), 2.3 yg/ml (CD-2) and 2.8 yg/ml (CD-3), respectively.

(IN VIVO CULTURE) Pristane (2,6,10,14-tetramethylpentadecane) in an amount of 0.5 ml was intraperitoneally injected to BALB/c mice and on the 10-30th day after the injection 5 X 106 cells of the hybridoma CD-1, CD-2 or CD-3, which had been proliferated in vitro, were inoculated intraperitoneally to the mice. After two or three weeks, ascites was collected and centrifuged at 1000 X g, 4"C, for 15 minutes to obtain ascitic supernatant. Approximately 30 ml of the ascitic supernatant were obtained from ten mice on each hybridoma and the antibody contents were 1.5 mg/ml (CD-1), 2.3 mg/ml (CD-2) and 1.8 mg/ml (CD-3), respectively.

(6) SPECIFICITY AND PROPERTIES OF ANTIBODY (SPECIFICITY): Cells used were Candida albicans ATCC 752, (as other strain of the same species) Candida albicans IFO 0588, 1385, 1389, 1 594 and 1269, (as other species belonging to the same genus) Candida tropicalis ATCC 750, Candida guilliermondii IFO 0679, Candida krusei I FO 1395, Candida parapsilosis IFO 1396 and Candida pseudotropicalis IFO 0432. Each strain was cultured by the method described in (1) above, treated with formalin and adjusted to 1 X 108 per ml by PBS containing 0.05 % Tween 20. The cell suspension was poured into silicon-treated test tubes of 1.2 cm in diameter in each 0.3 ml and centrifuged at 1000 X g for 5 minutes.After discarding the supernatant, 0.5 ml of the supernatant derived from the in vitro culture of hybridoma CD-1, CD-2 or CD-3 obtained in (5) above was added in each tube and reacted at 37"C for one hour. After reaction, each tube was washed three times with PBS containing 0.05 % Tween 20, and then 0.5 ml of a solution of horseradish-derived peroxidase-bound anti-mouse immunoglobin antibody (Cappel Corp., USA), diluted 1000-fold with horse serum, was added to each tube and reacted at 37"C for one hour. After washing five times with PBS containing 0.05 % Tween 20, 1 ml of 0.1 M citrate buffer containing 1 mg/ml o-phenylenediamine and 0.04 % (V/V) of 31 % aqueous hydrogen peroxide was added to each tube and maintained at room temperature for 30 minutes.The reaction was stopped by adding 0.5 ml of 12.5 % sulfuric acid and absorption at 492 nm was measured to identify the antibody produced. The results are shown in Table 1 below.

(PROPERTIES): Fifty ,ul 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 poured into each well of 96 well flat-bottomed tissue culture plate (Nunc 167008, Nunc, Denmark) and allowed to stand at 4"C for 24 hours. After thoroughly washing each well with PBS containing 0.05 % Tween 20(E), 100 yl of the supernatant from the culture of CD-1, CD-2 or CD-3 obtained in (5) above was added to each well and incubated at 37"C for one hour.After reaction, each well was washed three times with PBS containing 0.05 % Tween 208, and 50 yl of a solution of horseradish-derived peroxidase-bound anti-mouse immunoglobulin antibody (Cappel, USA), 1000-fold diluted with horse serum, was added to each well and incubated at 37"C for one hours. After washing each well five times with PBS containing 0.05 % Tween 2O, 100 yl of 0.1 M citrate buffer (pH 4.5) containing 1 mg/ml o-phenylenediamine and 0.04 % (V/V) of 31 % aqueous hydrogen peroxide was added to each well and incubated at room temperature for 30 minutes. The enzymatic reaction was stopped by adding 12.5 % sulfuric acid to each well and absorption at 492 nm was measured to identify the antibody produced. The results are shown in Table 1 below.

EXAMPLE 2 (1) PREPARATION OF IMMUNOGEN Candida albicans IFO 1 594 was inoculated in a slant agar containing Sabouraud's medium and incubated at 37"C for 3 days in an incubator. PBS (pH 7.2) was added, the cells were suspended by pipetting, centrifuged 1000 X g, 4"C, 10 minutes and a cell pellet was obtained.

After repeating three times this washing procedure , cell concentration was adjusted to 5 X 105 per ml by PBS and used as an immunogen suspension.

(2) PREPARATION OF ANTIBODY PRODUCING CELL The immunogen suspension was intravenously administered in each 0.2 ml to immunize female CDF, mice of 8 weeks old (NIPPON KREA, JAPAN). The mice were repeatedly immunized every two weeks. On the 70th day after beginning of immunization, the mice were boosed by intravenous injection of 0.2 ml of the suspension. On the third day from the booster, the mice were dehematized to death and spleen was taken out aseptically in a clean bench (HITACHI LTD. JAPAN). The spleen was then placed into a petri dish containing Dulbecco's MEM medium and a spleen cell suspension of 1 X 108 per ml was obtained in the same manner as in Example 1(2).

(3) CELL FUSION FOR PREPARATION OF HYBRIDOMA Mouse myeloma cell line P3-X63-Ag8 (1 x 107) preliminarily cultured in vitro was mixed with the spleen cell suspension (1 x 108), centrifuged (500 X g, 5 minutes) and the supernatant was discarded to obtain a cell pellet. After unfastening the pellet by gently tapping the vessel's bottom, the temperature was maintained at 37"C. Dulbecco's MEM medium, 1 ml, containing 45 % polyethylene glycol 4000 (37"C) was gradually added in approximately one minute. After maintaining at 37"C for 7 minutes, 1 5 ml of Dulbecco's MEM medium, 37"C, was added along the vessel's wall in approximately five minutes while the vessel was gently turned round.

Approximately 25 ml of Dulbecco's MEM medium was then added and centrifuged (500 X g, 5 minutes) and the supernatant was discarded.

Dulbecco's MEM medium containing 10 % fetal bovine serum, 37"C, was added to the cell pellet, adjusted to 1 X 106 per ml, gently mixed by a pipette, placed into each well of 24 well plate for tissue culture (Nunclon, Nunc, Denmark) in each 1 X 106 and incubated at 37"C in an incubator containing 5 % carbon dioxide. After 24 hours from the initiation of the incubation, 1 ml HAT medium was added to each well. During culture, 1 ml of the medium in each well was removed and 1 ml of fresh HAT medium was added to each well every 2 or 3 days. The hybridoma capable of proliferating in the HAT medium was selected.

Since two weeks after the initiation of the incubation, the growth of hybridoma was observed and the antibody produced in the supernatant of each well was investigated by the method described in Example 1 (4). Ten wells showed the antibody production among 48 wells.

(4) ESTABLISHMENT OF ANTIBODY PRODUCING HYBRIDOMA The antibody producing hybridomas of 10 wells were cloned by using a soft agar. Namely, 30 ml of 2.5 % agar (Difco, West Germany), 45"C, and 3 ml of Dulbecco's MEM medium of 10-fold concentration were mixed and 117 ml of Dulbecco's MEM medium, 45"C, was added.

To this agar solution, spleen cells of non-treated (control) female CDF1 mice of 8 weeks old were added in an amount of 5 X 105 per ml as feeder cells. Each 10 ml of the resultant agar solution was placed into each petri dish (Falcon 3003, Becton-Dickinson, USA) of 10 cm in diameter and allowed to stand at room temperature for 1 5 minutes to gel. Approximately 2 ml of the hybridoma suspension in each well showing positive in antibody production and the equivalent amount of Dulbecco's MEM medium containing 0.5 % agar were mixed and each 2 ml of the resultant mixture was placed on the gel obtained above so that the cells were uniformly distributed on the gel layer. Incubation was carried out at 37"C in a 5% CO2 incubator.After the tenth day from the initiation of incubation, colonies growing on the soft agar were collected by a Pasteur's pipette and transferred to 96 well tissue culture plate with flat bottom, 0.2 ml of Dulbecco's MEM medium was further added to each well and incubated at 37"C in a 5% CO2 incubator. The growth of hybridoma was observed and the presence of antibody in the supernatant of each well was simultaneously investigated by the method described in Example 1 (4).

Stable clones producing a large amount of the antibody and propagating well were chosen among the positive hybridomas, re-cloned in the same manner as described above and the antibody producing hybridomas CD-4 and CD-5 were obtained.

(5) PRODUCTION OF ANTIBODY (IN VITRO CULTURE) The hybridomas CD-4 and CD-5 were cultured by the method described in Example 1 (5) to obtain the supernatants.

(IN VIVO CULTURE) According to the method of Example 1 (5), the hybridomas CD-4 and CD-5 were intraperitoneally transplanted to CDFt mice, ascites was collected after 2 or 3 weeks and ascitic supernatant was obtained. Approximately 30 ml of such supernatant was obtained from ten mice.

(6) SPECIFICITY AND PROPERTIES OF ANTIBODY The specificities and the classes of immunoglobulin of the antibodies contained in the supernatants of the culture of the hybridomas CD-4 and CD-5 were investigated according to the method of Example 1 (6). The results are shown in Table 1 below.

EXAMPLE 3 (1) PREPARATION OF IMMUNOGEN Candida albicans IFO 0588 was inoculated in a slant agar containing Sabouraud's medium and incubated at 37"C for three days. After incubation, cells were collected by a platinum loop, suspended into PBS (pH 7.2) and centrifuged 1000 x g, 4"C, 10 minutes to obtained a cell pellet. After three times repeating this washing procedure, cells were heated at 100"C for 2.5 hours.

After washing the cells three times with PBS, the cell concentration was adjusted by PBS to 2 x 107 per ml and used as an immunogen suspension.

(2) PREPARATION OF ANTIBODY PRODUCING CELL The immunogen suspension was intraperitoneally injected in an amount of 0.2 ml to immunize female BALB/c mice (NIHON CHARLES LIVER, JAPAN) of 8 weeks old. The immunization was repeated every two weeks and 0.2 ml of the immunogen suspension was intravenously administered to boost in the tenth week from the first immunization. After three days, the mice was dehematized to death and spleens were aseptically taken out in a clean bench (HITACHI LTD., JAPAN). The spleen was placed in a petri dish containing RPMI 1 640 medium and a spleen cell suspension of 5 X 107 per ml was obtained according to the method of Example 1 (2).

(3) CELL FUSION FOR PREPARATION OF HYBRIDOMA Mouse myeloma cell line P3-NSI/1-Ag4-1 preliminarily cultured in vitro (0.5 X 107 cells) and the spleen cell suspension (5 X 107 cells) were mixed, centrifuged (500 X g, 5 minutes) and the supernatant was removed to obtain a cell pellet. After unfastening the cell pellet by gently tapping the vessel's bottom, 0.5 ml RPMI 1 640 medium containing 42.5 % potyethylene glycol 1540, 37"C, and 1 5 % dimethylsulfoxide was added and allowed to react for one minute. During the reaction, the vessel was gently turned round by a finger to thoroughly blend the polyethylene glycol solution with the cell pellet.After one minute of mixing, RPMI 1640 medium, 37 C, was added in a rate of 1 ml per 30 seconds in a total amount of 10 ml while similarly turning round the vessel, and the mixture was subjected to centrifugation 500 X g, 5 minutes. After removing the supernatant, the cell pellet was suspended in RPMI 1640 medium and centrifuged (500 X g, 5 minutes) to obtain a cell pellet again. After washing again in this manner, to the cell pellet was added 10 ml of RPMI 1640 medium containing 10 % fetal bovine serum, 2 mM glutamine, 1 mM pyruvic acid, 4.5 g/L glucose, 5 X 10-6 M ss- mercaptoethanol and 50 mg/L kanamycin sulfate as a culture medium, 37"C, and well suspended.Each 200,us of the cell suspension was placed into each well of 96 well plate for tissue culture (Nunc 167008, Nunc, Denmark) and incubated at 37'C in a CO2 incubator. After 24 hours, a half of the supernatant was discarded and 100 ,u11 HAT medium, 37"C, that was the aforementioned culture medium added with 1 x 10-4 M hypoxanthine, 4 X 10-7M aminopterin and 1.6 X 10-5M thymidine, was added. 100 iL1 of the medium in each well was removed every 2 or 3 days and instead thereof 100 ,tbl of fresh HAT medium was added to each well. The hybridomas capable of proliferating in HAT medium were chosen.

After two weeks culture, the growth of hybridomas was observed and simultaneously the antibody in the supernatant of each well was investigated in the manner of Example 1 (4). In this example, Candida albicans IFO 0588 was used as a cell to be bound to the plate. Three wells showed the antibody production among 48 wells.

(4) ESTABLISHMENT OF ANTIBODY PRODUCING HYBRIDOMA The hybridomas in three antibody producing wells were cloned by the method of Example 1 (4) and the antibody producing hybridomas CD-6 and CD-7 were obtained.

By using HT medium (that is the afore-mentioned culture medium added with 1 X 10-4 M hypoxanthine and 1.6 x 10-5 M thymidine) instead of HAT medium, the culture medium was exchanged gradually to HT medium. After culturing in HT medium for two weeks, the HT medium was exchanged by the culture medium without hypoxanthine, aminopterin nor thymidine and in this manner the hybridomas were rendered to be capable of proliferating in a normal culture medium instead of the selection medium.

(5) PRODUCTION OF ANTIBODY (IN VITRO CULTURE) The hybridomas CD-6 and CD-7 were cultured in the method of Example 1 (5) to obtain the supernatants. In this example, the initial cell concentration was 2 X 105 per ml and the antibody contents in the supernatants were 2.5 yg/ml (CD-6) and 3.0 yg/ml (CD-7), respectively.

(IN VIVO CULTURE) The hybridomas CD-6 and DC-7 were intraperitoneally inoculated to BALB/c mice by the method of Example 1 (5) in each amount of 1 X 107, ascites was taken out after 2 or 3 weeks from the inoculation and the ascitic supernatants were obtained. Approximately 30 ml of supernatant was obtained from ten mice.

(6) SPECIFICITY AND PROPERTIES OF ANTIBODY According to the method of Example 1 (6), the specificities and the classes of immunoglobulin of the antibodies in the supernatant of the culture of the hybridomas CD-6 and CD-7 were examined. The results are shown in Table 1 below.

EXAMPLE 4 (1) PREPARATION OF IMMUNOGEN Candida albicans ATCC 752 was inoculated in an agar plate containing corn mean (NISSUI SEIYAKU, JAPAN) and incubated at 25"C for 10 days. After incubation, a part rich in mycelia and chlamydospores was collected by a spoon, suspended in PBS (pH 7.2), homogenized and centrifuged 1000 X g, 4"C, 10 minutes to obtain a cell pellet. After repeating three times this procedure, PBS was used to adjust 1% (W/V) and the resultant suspension of immunogen was subjected to subsequent experimental procedures.

(2) PREPARATION OF ANTIBODY PRODUCING CELL Female BALB/c mice (NIHON CHARLES LIVER, JAPAN) of 8 weeks old were immunized by intraperitoneally injecting 0.2 ml of the immunogen suspension. The mice were further immunized every 14 days. On the 70th day after the beginning of the immunization, 0.2 ml of the immunogen suspension was intraperitoneally boosted. After three days, the mice were dehematized to death and the spleen and mesenteric lymph node were aseptically taken out of the mice in a clean bench (HITACHI LTD., JAPAN). The spleen cell suspension of 108 per ml was obtained according to the method of Example 1 (2).

(3) CELL FUSION FOR PREPARATION OF HYBRIDOMA Mouse myeloma cell line X63-Ag8.653 (1 > c x 107) preliminarily cultured in vitro and the spleen cell suspension (1 X 108) were mixed and cell fusion was carried out as Example 2 (3) to prepare hybridomas. Antibody production was confirmed in 10 wells among 48 wells.

(4) ESTABLISHMENT OF ANTIBODY PRODUCING HYBRIDOMA The hybridomas producing antibodies were then closed by a fibrin gel culture. Namely, 1 ml of a solution containing 2.5 mg/ml fibrinogen (Miles, USA), 8 mg/ml sodium chloride, 0.5 mg/ml potassium chloride and sodium citrate was placed in a petri dish (Falcon 3002, Beckton Dickinson, USA) and spreaded uniformly over the bottom of the petri dish, and 4 ml of Dulbecco's MEM medium containing 10 mU/ml thrombin (Miles, USA) and 20 % fetal bovine serum was added and allowed to stand at 37"C for one hour to gel. 100 it1 of the hybridoma (1 x t04 per ml) was added to the gel and uniformly distributed, then incubated at 37"C in a 5% CO2 incubator.After ten or more days, colonies growing on the gel were collected by a Pasteur's pipette, transferred into 96 well tissue culture flat-bottomed plate, and 0.2 ml of Dulbecco's MEM medium was added and incubated at 37'C in a 5% CO2 incubator. The growth of the hybridoma was observed and the presence of the antibodies in the supernatant of each well was investigated by the method of Example 1 (4). Among the hybridomas positive in the production of antibodies, stable clones which produced a high amount of antibodies and could well proliferate were chosen and cloned again in the same manner as aforementioned to establish the antibody producing hybridomas CD-8 and CD-9.

(5) PRODUCTION OF ANTIBODY The hybridomas CD-8 and CD-9 were cultured in vitro and in vivo by the method of Example 1 (5) to obtained the supernatant of the in vitro culture and the ascites (in vivo).

(6) SPECIFICITY AND PROPERTIES OF ANTIBODIES) The specificities of the antibodies and the classes of the immunoglobulins contained in the supernatants of the cultures of the hybridomas of CD-8 and CD-9 were estimated according to the method of Example 1 (6). The results are shown in Table 1 below.

TABLE 1: SPECIFICITIES AND IMMUNOGLOBULIN CLASSES OF ANTI-CANDIDA ANTIBODIES OF THE INVENTION ANTIBODY PRODUCING HYBRIDOMA CD-1 CD-2 CD-3 CD-4 CD-5 CD-6 CD-7 CD-8 CD-9 SAME Candida albicans ATCC752 + + + + + + + + + SPE- Candida albicans IF00588 + + + + + + + + + CIES Candida albicans IF01385 + + + + + + + + + SPE- Candida albicans IF01389 + + + + + + + + + Cl- Candida albicans IF01594 + + + + + + + + + Fl- Candida albicans IF01269 + + + + + + + + + Cl- SAME Candida tropicalis ATCC750 + + + - + - + - + TY GE- Candida guilliermondii IF00679 - + + - + - + - + NUS Candida krusei IF01395 - + + - + - + - + Candida parapsilosis IF01396 - + + - + - + - + Candida pseudotropicalis IF00432 - - + - + - + - + OTHER Torulopsis glabrata IF00622 - - + - - - - - FUNGI Saccharomyces cerevisiae IAM4207 - - + - - - - - Hansenula anolama IAM4213 - - + - - - - - Debaryomyces hansenii IAM4356 - - - - - - PROPERTY IMMUNOGLOBULIN CLASS IgM IgM IgM IgM IgM IgG IgG IgG IgG + ; positive, - ; negative EXAMPLE 5 The antibodies obtained in Examples 1-4 were administered to ICR mice with a group of ten mice in an amount of 2 g/'kg perorally, 400 mg/kg intraperitoneally or 200 mg/kg intravenously and the mice were observed for 14 days. No death due to the antibodies was observed at all.

On the other hand, the shapes of the hybridomas CD-l to CD-9 were approximately spherical, the dimensions thereof were 10-20 gel with almost being approximately 1 5 it and the hybridomas CD-1 to CD-9 were floating and weak in adhesion to vessel wall.

EXAMPLE 6: IDENTIFICATION OF FUNGUS OF CANDIDA BY ANTIBODY (i) SUBJECT: Sputum, urine or Vaginal secretion was taken out from five patients who were thought to suffer from candidiasis, smeared in a petri dish containing Sabouraud's glucose agar medium and incubated at 37"C for 5 days. The colonies appeared in the agar plate were used as a subject.

(ii) IDENTIFICATION: The identifications were carried out by a slide agglutination and a fluorescent antibody technique using antibodies (ascitic supernatant) secreted by the hybridoma CD-1.

Namely, in the slide agglutination method, one drop of the antibody solution was placed on a glass slide, a small amount of the cells obtained from the subject colonies by a platinum loop was added and the agglutination was observed by naked eye or microsope.

In the fluorescent antibody technique, a small amount of the cells obtained by a platinum loop from the colonies to be tested was smeared on a slide glass, fixed by 1.0 % formalin at 4"C overnight, washed with PBS (pH 7.2), one drop of the 100-fold dilution of the aforementioned antibody solution was then added and incubated at 37'C for one hour. After washing thoroughly with PBS (pH 7.2) to remove unreacted antibodies, one drop of the 100-fold diluted fluorescein-bound anti-mouse immunoglobulin (Cappel, USA) was added and incubated at 37"C for one hour. After removing unreacted fluorescein-bound anti-mouse immunoglobulin by washing with PBS (pH 7.2), fluorescence was observed by a fluorescence microscope (Olympus Vanox, Olympus, Japan) on each subject cell of the slide.

Simultaneously the subjects were morphologically and biochemically observed. Namely, chlamydospores in the slide culture using the corn meal agar medium was morphologically investigated and utilization of glucose, saccharose, maltose and lactose were biochemically investigated.

(iii) RESULTS (Table 2): As seen from Table 2, the identification results by the antibodies of the present invention completely coincided with the results of the morphological and biochemical methods.

TABLE 2 method identification by morphological-biochemical results antibody from method (identified CD-1 strain) chlamydo- utilization of sugar spores aggluti- fluores- formation glu- saccha- mal- lac nation cent cose rose tose tose subject antibody suputum (pa- Candida tient A) + + + + + + - albicans urine (pa- Candida tient B) + + + + + + - albicans vaginal secretion Candida (patient C) + + + + + + - albicans uring (pa- Candida tient D) + + + + + + - albicans sputum (pa- Saccharomyces tient E) - - - + + + - cerevisiae EXAMPLE 7 (1) PEROXIDASE LABELLING OF ANTIBODIES SECRETED BY HYBRIDOMAS CD-l. CD-4, CD-6 AND CD-8 Four mg of horseradish peroxidase (Sigma Type VI, Sigma, USA) was dissolved in 1 ml distilled water and 60 ml of 0.1 M NalO4 solution, prepared immediately before use, was added and mixed at room temperature for 20 minutes. The resultant solution was dialysed overnight against 1 mM sodium acetate buffer (pH 4.4) and 20 ml of 0.2 M solution of sodium carbonate was added. Soon after, the antibody secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8 (purified ascitic supernatant, protein content of 10 mg) dissolved in 1 ml of 0.01 M carbonate buffer (pH 9.5) was added and maintained at room temperature for two hours while stirring.

After reaction, 0.1 ml of freshly prepared aqueous NaBH4 solution (4 mg NaBH4 dissolved in 1 ml distilled water) was added, allowed to stand at 4"C for two hours and dialysed overnight to PBS.

The resultant mixed solution was applied to a column of Sephadex G-1008 (Pharmacia, Sweden), eluted with PBS and first fraction of which absorptions at 280 nm and 403 nm correspond to each other was collected. To 1 ml of the fraction (enzyme-antibody binding product) was added 10 mg of rabbit serum albumin to dissolve and stored at - 70"C until use.

(2) ALKALINE PHOSPHATASE LABELLING OF ANTIBODIES SECRETED BY HYBRIDOMAS CD 1, CD-4, CD-6 AND CD-8 Five mg of alkaline phosphatase Type VII (Sigma, USA), preliminarily dialysed to PBS to completely remove ammonium sulfate, and 17 mg of antibody secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8 (purified ascitic supernatant) were dissolved in PBS to a total volume of 1 ml. To the resultant solution was added 10 l of 20 % glutaraldehyde solution and stirred at room temperature for two hours. After reaction, the reaction mixture was applied to a Sephadex G-200(D column (Pharmacia, Sweden) equilibrated with Tris-HCI buffer (pH 7.6), and eluted with the same buffer.A high molecular weight fraction from void volume to the IgG-eluted point was collected, bovine serum albumin was added to 5 % (W/V), sterilized by passing a Millipore filter (0.22 , Millipore, USA) and stored at 4"C in dark until use.

(3) ssGALACTOSlDASE LABELLING OF ANTIBODIES SECRETED BY HYBRIDOMAS CD-1, CD4, CD-6 AND CD-8 The antibodies secreted by the hybridomas CD-1, CD-4, CD-6 and CD-8 were labelled with ss- galactosidase according to the method of (2) above. The ssgalactosidase used was Sigma grade IV (Sigma, USA).

(4)1251 LABELLING OF ANTIBODIES SECRETED BY HYBRIDOMAS CD-1, CD-4, CD-6 AND CD8 To 10 pal solution of 100 mCi/ml Na'251 (carrier-free, Amersham, USA) were added 50 jul of the antibody solution secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8 (purified ascitic supernatant, protein content of 1.0 mg/ml) and 30 iL1 of 0.5 M phosphate buffer (pH 7.2) containing 0.30 mg/ml chloramine T, and well mixed. After 15 seconds, 100 coil PBS saturated with L-tyrosine was added and immediately mixed. The resultant mixture was chromatographed on a column charged 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 activity of the labelled product was 1.0 iLCi per mg antibody protein.

(5) FLUORESCEIN LABELLING OF ANTIBODIES SECRETED BY HYBRIDOMAS CD-i, CD-4, CD6 AND CD-8 To 1 ml solution of the antibody secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8 (purified ascitic supernatant) in a concentration of 10 mg/ml, 0.1 ml of 0.5 M carbonate buffer (pH 9.3) was added, 0.1 mg of fluorescein isothiocyanate powder was further added and stirred at 4"C for 6 hours without bubbling. Immediately after reaction, the reaction mixture was applied to a Sephadex G-25 column (Pharmacia, Sweden) to remove unreacted low molecular weight substances and the high molecular weight fraction to be desired was obtained. The obtained product was stored at 4"C in dark until use.

(6) TETRAMETHYLRHODAMINE LABELLING OF ANTIBODIES SECRETED BY HYBRIDOMAS CD-l, CD-4, CD-6 AND CD-8 To 1 ml of 10 mg/ml solution of the antibody secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8 (purified ascitic supernatant), 0.1 ml of 0.5 M carbonate buffer (pH 9.3) was added, 0.2 mg tetramethylrhodamine-isothiocyanate powder was further added and stirred at 4"C for 20 hours without bubbling. After reaction, the reaction mixture was immediately applied to a Sephadex G-25(g) column (Pharmacia, Sweden) to remove unreacted low molecular weight substances and the high molecular weight fraction to be desired was obtained. The product was stored at 4"C in dark.

(7) BIOTIN LABELLING OF ANTIBODIES SECRETED BY HYBRIDOMAS CD-i, CD-4, CD-6 AND CD-8 One mM d-biotin (244 mg) (Wako Pure Chemical Industries, Ltd., Japan) and 1.5 mM Nhydroxysuccinimide (173 mg) (Eastman Kodak, USA) were dissolved in a mixture of 8 ml dimethylsulfoxide (Wako Pure Chemical Industries, Ltd.) and 5 ml 1,2-dimethoxyethane (Nakadai Chemical,~Japan). To the resultant solution was added a solution of 206 mg (1 mM) N,N'dicyclohexylcarbodimide (Kanto Chemical, Japan) in 0.5 ml 1,2-dimethoxyethane and reacted at 4"C overnight. The resultant precipitate was filtered out to obtain a filtrate. The solvent in the filtrate was removed under vacuum and the residual oily material was dissolved in 10 ml dichloromethane (Wako Pure Chemical Industries, Ltd., Japan) and cooled at 4'C.Ten ml of 0.1 M NaHCO3 solution (4"C) was added and shaked to well mix. The resultant dichloromethane phase was removed, 10 ml of 0.1 M NaHCO3 and then 10 ml of distilled water, 4 C, were added and this procedure was again repeated. The resultant dichloromethane phase was added with anhydrous powder of sodium sulfate (Koizumi Chemical, Japan) to dehydrate. The powder was filtered out and n-hexane was added to the filtrate to be turbid. The solution was cooled to - 20"C, the precipitated crystal was then placed in a desiccator to remove the solvent and dry.

The product, biotin-N-hydroxysuccinimide ester was obtained.

The biotin-N-hydroxysuccinimide was dissolved in dimethylsulfoxide, and the concentration was adjusted to 1 mg/ml. The solution (60it1) was mixed with 1 ml solution of the antibody secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8 (purified ascitic supernatant, protein content of 1 mg/ml) and reacted at room temperature for 4 hours. After reaction, the solution was dialysed to PBS at 4"C for three days. For this the dialysing liquid was exchanged three times. The dialysate (solution in the dialysis tube) was stored at 4"C until use.

EXAMPLE 8 (1) SUBJECT FUNGUS Candida albicans ATCC 752, Candida tropicalis ATCC 750 or Candida guilliermondii IFO 0679 was inoculated in a slant agar containing Sabouraud's medium and incubated at 37"C for three days. The cells was collected by a platinum loop and used as a subject fungus.

(2) IDENTIFICATION BY FLUORESCENT ANTIBODY METHOD A small amount of each subject fungus was smeared on a slide glass, fixed with 1.0 % formalin at 4"C overnight and washed with PBS (pH 7.2). A drop of a 10-fold diluted solution of fluorescein-labelled antibody secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8, prepared in Example 7 (5), was added and reacted at 37"C for one hour. By thoroughly washing with PBS was removed the unreacted antibody. Fluorescence on the slide was observed by a fluorescence microscope (Olympus Vanox, Olympus, Japan). As the result, fluorescence was obseved on Candida albicans ATCC 752.

Similar experiment was carried out on the rhodamin-labelled antibodies prepared in Example 7 (6) and fluorescence was observed on Candida albicans ATCC 752.

On the other hand, each biotin-labelled antibody prepared in Example 7 (7) was bound to the subject fungus, thoroughly washed with PBS, one drop of 5,ug/ml solution of fluorescein-bound avidin (Funakoshi Yakuhin, Japan) was added and reacted at 37"C for one hour. After thoroughly washing with PBS, fluorescence was observed by the fluorescence microscope on the slide of Candida albicans ATCC 752.

(3) IDENTIFICATION BY ENZYME IMMUNOASSAY A cell suspension (0.3 ml) of the subject fungus was inoculated into a silicon-treated test tube of 1.2 cm in diameter and centrifuged at 1000 x g, 5 minutes. After removing the supernatant, 0.5 ml of solution, 100-fold diluted with horse serum, of the peroxidase-, alkaline phosphataseor ss-galactosidase-labelled antibody prepared in Example 7 (1), (2) or (3) was added and reacted at 37"C for one hour. After reaction, the reaction mixture was five times washed with PBS containing 0.5 % Tween 20(E) and a substrate solution was added in an amount of 1 ml.

Namely, in the peroxidase-labelled antibody, 1 ml of 0.1 M citrate buffer (pH 4.5) containing 1 mg/ml o-phenylenediamine and 0.04 % (V/V) of 31 % aqueous hydrogen peroxide was added, reacted at room temperaure for 30 minutes, 0.5 ml of 12.5 % sulfuric acid was then added to stop the reaction and absorption was measured at 492 nm.

In the alkaline phosphatase-labelled antibody, 1 ml of a substrate solution (pH 9.8) containing 1 mg/ml p-nitrophenyl phosphate (Sigma, USA), 9.7 % (V/V) diethanolamine and 0.01 % MgCI2.6H20 was added, reacted at room temperature for 30 minutes, 0.5 ml of 5 M sodium hydroxide was then added to stop the reaction and absorption was measured at 405 nm.

In the ss-galactosidase-labelled antibody, 1 ml of borate buffer (pH 8.5) containing 1 mg/ml 4-methylumbelliferyl-ss-galactoside was added, reacted at 30"C for 10 minutes, 0.5 ml of 0.1 M glycine-sodium hydroxide buffer (pH 10.3) was then added as the reaction stopper, and an amount of freed 4-methylumbelliferone was measured by the HITACHI spectrofluorometer (360 nm as excitation wavelength and 450 nm as emission wavelength, HITACHI, JAPAN).

The enzyme-labelled antibodies of the present invention were reacted with Candida albicans ATCC 752.

(4) IDENTIFICATION BY RADIOIMMUNOASSAY The subject fungus was suspended into PBS containing 0.05 % Tween 20. One ml of the suspension was inoculated into a silicon-treated test tube of 1.2 cm in diameter and centrifuged 1000 x g, 10 minutes to obtain a cell pellet. After washing twice the cell pellet with PBS containing 0.05 % Tween 20(E), 50 it1 of the '251-labelled antibody prepared in Example 7 (4) (specific activity of 1,uCi per mg antibody) was added and reacted at 37"C for one hour. After washing five times with PBS containing 0.05 % Tween 20(E), radio-activity bound to the subject fungus was measured by y-counter (Beckman Gamma 8500, Beckman, USA).

Higher radioactivity was observed on Candida albicans ATCC 752 as seen from Table 3 in the case of '251-labelled antibody of the invention.

TABLE 3 labelled antibody radioactivity bound to cell (c.p.m.) 125l-CD-1 125I-CD-4 125l-CD-6 125l-CD-8 '251-labelled antibody antibody antibody antibody mouse immunosubject fungus globulin Candida albicans ATCC 752 8900 10400 13600 11000 630 CandidatropicalisATCC750 760 890 672 700 790 Candida guilliermon dii IFO 0679 530 916 778 855 575 EXAMPLE 9 Urines of three patients who possibly suffered from candidiasis in urinary tract and Candida albicansATCC 752 were each inoculated on Sabouraud's glucose agar medium and incubated at 37"C for five days.Yeast-like colony appearing on the agar plate was taken up by a platinum loop, suspended into 3 ml of PBS containing 0.05 % Tween 20(B) and homogenized in a homogenizer, and the resultant cell suspension was used as a subject.

(1) IDENTIFICATION BY ENZYME IMMUNOASSAY The cell suspension (0.3 ml) was placed into a silicon-treated test tube of 1.2 cm in diameter and centrifuged (1000 x g, 5 minutes). After removing the supernatant, 0.5 ml of a solution, 100-fold diluted with horse serum, of the peroxidase-, alkaline phosphatase- or ssgalactosidase- labelled antibody prepared in Example 7 (1), (2) or (3) was added and reacted at 37"C for one hour. The reaction mixture was washed five times with PBS containing 0.05 % Tween 208.

One ml of a substrate solution was then added.

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

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

In the ss-galactosidase-labelled antibody, 1 ml of borate buffer (pH 8.5) containing 1 mg/ml I 4-methylumbelliferyl-ss-galactoside was added and reacted at 30"C for 10 minutes, 0.5 ml of 0.1 M glycine-sodium hydroxide buffer (pH 10.3) was then added as a reaction stopper and an amount of free 4-methylumbelliferon was measured by the HITACHI spectrofluorometer (360 nm as excitation wavelength and 450 nm as emission wavelength, HITACHI, JAPAN).

The results are shown in Table 4.

(2) IDENTIFICATION BY FLUORESCENT ANTIBODY METHOD The cell suspension was smeared on a slide glass, fixed with 1.0 % formalin overnight and washed with PBS. One drop of a solution, diluted 100-fold with horse serum, of fluorescein-, rhodamine- or biotin-labelled antibody prepared in Example 7 (5), (6) or (7) was added, reacted at 37'C for one hour and washed thoroughly with PBS to remove unreacted antibodies.

In the fluorescein- or rhodamine-labelled antibody, the presence of fluorescence on the subject fungus was investigated by a fluorescence microscope (Olympus Vanox, Olympus, Japan) on the slide.

In the biotin-labelled antibody, one drop of 5 ju g/ml fluorescein-bound avidin D (Funakoshi Yakuhin, Japan) was added, reacted at room temperaure for one hour and thoroughly washed with PBS. The presence of fluorescence on the slide was observed by the fluorescence microscope.

The results are shown in Table 4.

On the other hand, the colonies derived from urine of patients F and G were identified as Candida albicans in the morphological and biochemical test simultaneously carried out, completely corresponding with the results by the labelled antibodies. Furthermore, the colony derived from urine of patient H was identified as Saccharomyces cerevisiae.

TABLE 4 Method Reagent used subject Source of Patient Patient Patient Candida albicans Label antibody F urine G urine H urine ATCC 752 enzyme peroxidase CD-1 +(492nm) +(492nm) -(492nm) +(492nm) immuno CD-4 +(492nm) +(492nm) -(492nm) +(492nm) assay CD-6 +(492nm) +(492nm) -(492nm) +(492nm) CD-8 +(492nm) +(492nm) -(492nm) +(492nm) alkaline CD-1 +(405nm) +(405nm) -(405nm) +(405nm) phosphatase CD-4 +(405nm) +(405nm) -(405nm) +(405nm) CD-6 +(405nm) +(405nm) -(405nm) +(405nm) CD-8 +(405nm) +(405nm) -(405nm) +(405nm) ss-galactosidase CD-1 + + - + CD-4 + + - + CD-6 + + - + CD-8 + + - + fluore- fluorescein CD-1 + + - + scent CD-4 + + - + anti- CD-6 + + - + body CD-8 + + - + tech- rhodamine CD-1 + + - + nique CD-4 + + - + CD-6 + + - + CD-8 + + - + biotin CD-1 + + - + CD-4 + + - + CD-6 + + - + CD-8 + + - + + : positive absorption or fluorescence, - : negative absorption or fluorescence EXAMPLE 10 Twenty ml of urine of two patients, who possibly suffered from urinary tract candidiasis, and a small amount of Candida albicansATCC 752 were suspended in PBS containing 0.05 96 Tween 208. One ml of the suspension was poured into a silicon-treated test tube of 1.2 cm in diameter and subjected to centrifugation (1500 X g, 20 minutes) to obtain a cell pellet.

After washing the cell pellet twice with PBS containing 0.05 % Tween 20(E), 50 l of 1251 labelled antibody prepared in Example 7 (4) (specific activity of 1 llCi per mg antibody) or 1251.

labelled mouse immunoglobulin (specific activity of 1.5 juCi per mg protein) was added and reacted at 37"C for one hour. After washing five times with PBS containing 0.05 % Tween 20, radioactivity was measured by a y-counter (Beckman Gamma 8500, Beckman, USA).

Simultaneously, the subject cell was inoculated on Sabouraud's dextrose agar medium and incubated at 37"C for five days, and the resultant colony was morphologically and biochemically examined.

The results are shown in Table 5.

As seen from Table 5, the subects were bound to '251-lebelled antibodies indicating Candida albicans contained therein. On the other hand, the simultaneous morphological and biochemical tests showed that the subjects contained Candida albicans.

TABLE 5 Antibody used Radioactivity (c.p.m.) '251-labelled '251-labelled mouse CD-l-derived immunoglobulin Subject antibody Patient F urine 313 155 Patient G urine 422 147 Candida albicans ATCC 752 844 172 EXAMPLE 11: CURING EFFECT OF ANTIBODY ON CANDIDIASIS Candida albicans ATCC 752 (5 x 106 CFU) was intraperitoneally inoculated to each group of 15 female BALB/c mice of 8 weeks old (NIHON CHARLES LIVER, JAPAN). After three weeks, 0.2 ml solution of the antibody secreted by the hybridoma CD-1, CD-2, CD-3, CD-4, CD-5, CD6, CD-7, CD-8 or CD-9 (agglutination titer 1 : 512) was intravenously administered.After two hours, a lethal amount of Candida albicans ATCC 752 (5 X 107 CFU) was intravenously injected and death due to infection was observed for 14 days.

The results are shown in Table 6.

As seen from table 6, all animals to which the antibody was not administered died, while in the group administered with the antibody some mice survived, indicating the significant curing effect of the antibody on the infectious diseases with fungi of Candida.

TABLE 6 Survival'/Total animal Control (non-treated) 0/1 5 Treated with antibody from hybridoma CD-1 5/15 from hybridoma CD-2 6/15 from hybridoma CD-3 7/15 from hybridoma CD-4 4/15 from hybridoma CD-5 5/15 from hybridoma CD-6 6/15 from hybridoma CD-7 4/15 from hybridoma CD-8 8/15 from hybridoma CD-9 6/15 Number of animals survived for 14 days after inoculation of lethal amount of Candida albicans.

EXAMPLE 12: ENHANCEMENT OF PHAGOCYTOSIS OF INTRAPERITONEAL MACROPHAGE BY ANTIBODY One ml of 0.1 % glycogen (Kanto Chemical, Japan)'was intraperitoneally administered to female BALB/c mice of 8 weeks old (NI HON CHARLES LIVER, JAPAN). After four days, mice were slaughtered. Intraperitoneally percolated cells were washed out by RPMI 1 640 medium containing 10 % fetal bovine serum and collected by centrifugation'(500 X g, 5 minutes).The cells were suspended in RPMI 1 640 medium containing 10 % fetal bovine serum, adjusted to 1 X 106, placed into a tissue culture chamber (Lab-Tek Inc., USA irì each 1 ml and allowed to stand at 37"C for 30 minutes, so that macrophage in the intraperitoneally percolated cells was adhered to the chamber bottom. The chamber was washed with RPMI 1 640 medium to remove non-adhered cells and 1 mi of 10 % fetal bovine serum-containing RPMI 1640 medium containing Candida albicans ATCC 752 in a cell concentration of 1 x 106 per ml was added and reacted at 37"C for two hours. Simultaneously, the antibody obtained from the hybridoma CD-1, CD-2, CD-4, CD-5, CD-6, CD-7, CD-8 or CD-9 was added.After washing the chamber with PBS (pH 7.2), the cells were fixed by 1.0 % formalin. A number of macrophage phagocytosing was measured by May-Grunwald staining.

The results are shown in Table 7. The phagocytosis ratio is shown in the ratio of pliagocytosing macrophage to total macrophage.

As seen from Table 7, the phagocytosis ratio was enhanced by approximately 14-23 % due addition of the antibody.

TABLE 7 Phagocytosis ratio (%) Control (non-treated) 25 Treated with antibody from hybridoma CD-1 42 Treated with antibody from hybridoma CD-2 45 Treated with antibody from hybridoma CD-3 40 Treated with antibody from hybridoma CD-4 42 Treated with antibody from hybridoma CD-5 48 Treated with antibody from hybridoma CD-6 41 Treated with antibody from hybridoma CD-7 '42 Treated with antibody from hybridoma CD-8 39 Treated with antibody from hybridoma CD-9 43 EXAMPLE 13 After adjusting to 10.4 mg/ml by adding distilled water to the antibody (purified ascitic supernatant) from the hybridoma CD-1, CD-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8 or CD-9, 13.0 mg/ml of amphotericin B-dimethylsulfoxide solution was added, and then hydrochloric acid was added while stirring to adjust pH of the solution to 4.75 and simultaneously 3.7 mg 1ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was added. After reacting for 1, 3 or 4 hours, the reaction was stopped by adding 2 ml acetate buffer (pH 4.70). The reaction mixture was then dialysed to 5 L distilled water at 4"C for 72 hours while exchanging the dialysing solution three times. After concentrating the dialysate, the reaction mixture was applied to a column of 1.5 cm in diameter and 55 cm in height charged with Sephadex G-258 (Pharmacia, Sweden) to completely remove low molecular weight substances. The resultant solution was freeze-dried at - 20"C to obtain the amphotericin B-antibody binding product. The binding amounts of amphotericin B in the products per mg of the antibody are shown in Table 8.

The binding products of 5-fluorocytosine with the antibodies were obtained in the same manner as aforementioned. The binding amounts of 5-fluorocytosine in the products per mg of the antibody are also shown in Table 8.

On the other hand, these binding products of antifungal agent with the antibody were administered to ICR of 10 animals in each group in a dose rate of 2 g/kg perorally, 400 mg/kg intraperitoneally or 200 mg/kg intravenously. No death was observed for 14 days.

TABLE 8 binding ratio ( g/mg) antifungal agent antibody 1 hour 3 hours 4 hours amphotericin B CD-l-derived 1.0 2.0 2.5 CD-2-derived 0.8 1.8 2.8 CD-3-derived 1.0 2.0 2.5 CDA-den.ved 1.0 1.5 2.3 CD-5-derived 1.0 2.3 3.0 CD-6-derived 1.0 1.5 2.5 CD-7-derived 1.3 1.5 2.8 CD-8-derived 1.0 2.0 2.5 CD-9-derived 1.0 2.0 2.5 5-fluorocytosine CD-l-derived 0.8 1.8 2.5 CD-2-derived 1.0 2.0 2.8 CD-3-derived 1.0 2.0 3.3 CD-4-derived 1.3 1.5 2.8 CD-5-derived 1.0 1.8 2.5 CD-6-derived 1.0 1.5 2.5 CD-7-derived 1.0 1.5 2.5 CD-8-derived 1.0 1.5 2.3 CD-9-derived 0.8 1.3 2.5 EXAMPLE 14 The curing effect on the infectious diseases of the antifungal agent-antibody binding products prepared in Example 1 3 was examined.Namely, Candida albicans ATCC 752 (5 X 107) was intravenously inoculated to female BALB/c mice of 8 weeks old (NIHON CHARLES LIVER, JAPAN) of 1 5 animals in each group, and the antifungal agent-antibody binding product prepared in Example 1 3 by 4 hour reaction was intravenously (amphotericin B-binding products) or intraperitoneally (5-fluorocytosine-binding products) administered after 1, 24 of 48 hours from the infection. On the other hand, the comparison compounds, amphotericin B and 5fluorocytosine were administered intravenously and perorally, respectively. The dose amounts of the binding products of the invention were 5pg based on amphotericin B and 20 iLg based on 5fluorocytosine, respectively, in each animal.

The results are shown in Table 9.

As seen from Table 9, the antifungal agent-binding products of the present invention showed apparently the significant curing effect on the infectious disease caused by Candida albicans.

TABLE 9 survival'/total control 0/15 amphotericin B 5,ug (i.v.) 4/15 amphotericin B CD-1-derived antibody binding product (i.v.) 8/15 amphotericin B CD-2-derived antibody binding product (i.v.) 7/15 amphotericin B CD-3-derived antibody binding product (i.v.) 9/1 5 amphotericin B CD-4-derived antibody binding product (i.v.) 10/10 amphotericin B CD-5-derived antibody binding product (i.v.) 8/15 amphotericin B CD-6-derived antibody binding product (i.v.) 9/15 amphotericin B CD-7-derived antibody binding product (i.v.) 7/15 amphotericin B CD-8-derived antibody binding product (i.v.) 8/15 amphotericin B CD-9-derived antibody binding product (i.v.) 8/15 5-fluorocytosine 2000 ill (p.o.) 3/15 5-flyorocytosine CD-l-derived antibody binding product (i.p.) 7/15 5-fluorocytosine CD-l-derived antibody binding product (i.p.) 6/15 5-fluorocytosine CD-3-derived antibody binding product (i.p.) 5/15 5-fluorocytosine CD-4-derived antibody binding product (i.p.) 7/15 5-fluorocytosine CD-5-derived antibody binding product (i.p.) 8/15 5-fluorocytosine CD-6-derived antibody binding product (i.p.) 6/15 5-fluorocytosine CD-7-derived antibody binding product (i.p.) 6/16 5-fluorocytosine CD-8-derived antibody binding product (i.p.) 7/15 5-fluorocytosine CD-9-derived antibody binding product (i.p.) 6/15 Number of survived animals after 14 days from inoculation of Candida albicans

Claims (45)

1. A method for preparing an antibody to a surface antigen of a fungus belonging to the genus Candida, which method comprises preparing a hybridoma between a cell which produces a said antibody and a cell which can be permanently maintained in vitro by subculture and recovering the said antibody secreted by the hybridoma.

2. A method according to claim 1, wherein the said cell which produces antibody is a spleen cell, lymph node cell, peripheral blood leukocyte or a mixture of two or more thereof.

3. A method according to claim 2, wherein the cell is derived from a mouse.

4. A method according to claim 3, wherein the mouse is a BALB/c mouse or a hybrid mouse thereof.

5. A method according to any one of the preceding claims, wherein the cell which can be permanently maintained in vitro by subculture is a myeloma cell.

6. A method according to any one of the preceding claims, wherein the cell which can be permanently maintained by in vitro subculture is deficient in hypoxanthine-guanine phosphoribosyltransferase or thymidine kinase.

7. A method according to claim 5 of 6, wherein the cell is derived from a mouse.

8. A method according to claim 7, wherein the cell is derived from the cell line P3-X63 Ag8, P3-NSI/1 -Ag4-1, Sp2/0-Ag14 or X63-Ag8.653.

9. A method according to any one of the preceding claims, wherein the said cell which produces antibody is derived from an animal immunized with a said fungus.

10. A method according to to any one of the preceding claims, wherein the hybridoma is prepared in the presence of polyethylene glycol.

11. A method according to any one of the preceding claims, wherein the hybridoma is cultured in a medium containing hypoxanthine, aminopterin and thymidine.

1 2. A method according to any one of the preceding claims, wherein the hybridoma is cloned to obtain a single clone.

13. A method according to claim 12, wherein the cloning is performed by limiting dilution culture, soft agar culture or fibrin gel culture.

14. A method according to any one of the preceding claims, wherein the hybridoma is cultured in vitro for secretion of the antibody.

1 5. A method according to any one of claims 1 to 13, wherein the hybridoma is transplanted into an animal and cultured in vivo and the secreted antibody is separated from body fluid of the animal.

1 6. A method according to claim 15, wherein the body fluid is serum or ascites.

17. A method according to claim 15 or 16, wherein the animal is a mouse.

1 8. A method according to claim 17, wherein the mouse is a BALB/c mouse or a hybrid mouse thereof.

1 9. A method according to any one of the preceding claims, wherein the said fungus is a strain of Candida albicans.

20. A method for preparing an antibody to a surface antigen of a fungus belonging to the genus Candida by the preparation and in vitro culture of a hybridoma secreting a said antibody, said method being substantially as herein before described in any one of Examples 1 to 4.

21. A method for preparing an antibody-to surface antigen of a fungus belonging to the genus Candida by the preparation and in vivo culture of a hybridoma secreting a said antibody, said method being substantially as hereinbefore described in any one of Examples 1 to 4.

22. A hybridoma which secretes an antibody to a surface antigen of a fungus belonging to the genus Candida.

23. A hybridoma according to claim 22, which is any one of hybridomas CD-1, CD-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8 and CD-9 hereinbefore described.

24. An antibody to a surface antigen of a fungus belonging to the genus Candida, prepared by a method as claimed in any one of claims 1 to 21.

25. An antibody to a surface antigen of a fungus belonging to the genus Candida and which has been produced by a hybridoma as claimed in claim 22.

26. The antibody to a surface antigen of a strain of Candida albicans, which is secreted by the hybridoma CD-1, CD-4, CD-6 or CD-8 hereinbefore described.

27. The antibody to a surface antigen of a strain of Candida albicans, Candida tropicalis, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida pseudotropicalis, which is secreted by the hybridoma CD-2, CD-5, CD-7 or CD-9 hereinbefore described.

28. A derivative or restriction product or an antibody as claimed in any one of claims 24 to 27.

29. A restriction product according to claim 28, which has been obtained by restrictive cleavage of the antibody by a chemical or enzyme treatment.

30. A derivative according to claim 28 comprising the antibody to which is bound a radioactive label, a fluorescent dye, an enzyme, a marker for electron microscopy or a residual group containing a structure to which a further substance is capable of binding.

31. A derivative according to claim 30, in which the radioactive label is 1251.

32. A derivative according to claim 30, in which the fluorescent dye is fluorescein or rhodamine.

33. A derivative according to claim 30, in which the enzyme is a peroxidase, alkaline phosphatase or ssgalactosidase.

34. A derivative according to claim 30, in which biotin is bound to the antibody.

35. A derivative according to claim 30 substantially as hereinbefore described in any one of sections (1) to (7) of Example 7.

36. A reagent suitable for use in the classification and/or identification of a fungus belonging to the genus Candida, which reagent comprises an antibody as claimed in any one of claims 24 to 27 or a derivative or restriction product as claimed in any one of claims 28 to 35, together with a carrier or a diluent therefor.

37. A method of classification and/or identification of a fungus belonging to the genus Candida in which an antibody as claimed in any one of claims 24 to 27 or a derivative or restriction product as claimed in any one of claims 38 to 35 is used.

38. A method according to claim 37, wherein a sample containing the fungus is brought into contact with the antibody, derivative or restriction product and the agglutination reaction, if any, which occurs is utilized to classify or identify the fungus.

39. A method according to claim 37, wheren a sample containing the fungus is brought into contact with the antibody, derivative or restriction product and the antigen-antibody reaction, if any, which occurs other than the agglutination reaction is utilized to classify or identify the fungus.

40. A method according to claim 38 or 39, wherein the sample is derived from human or animal.

41. A method for the identification of a fungus belonging to the genus Candida, said method being substantially as herein before described in any one of Examples 6, 8(2), 8(3), 8(4), 9(1), 9(2) or 10.

42. An antibody as claimed in any one of claims 24 to 27 to which an antifungal agent is bound.

43. An antibody according to claim 42, in which the antifungal agent is amphotericin B or 5-fluorocytosine.

44. An antibody according to claim 42, which is any one of the binding products hereinbefore described in Example 1 3.

45. A pharmaceutical composition, suitable for use in the treatment of candidiasis, which composition comprises as active ingredient an antibody as claimed in any one of claims 24 to 27 or of claims 42 to 44 together with a pharmaceutically acceptable carrier or diluent.