DE3413339A1 - ANTIBODIES AGAINST MUSHROOMS OF THE GENE CANDIDA - Google Patents

Description

The present invention relates to the technical field of cell fusion; in particular, the invention relates to a Antibody that is produced by a fused cell, which in turn comes from a cell that produces the antibody capable, and such a cell, which can reproduce permanently by subculture under in vitro conditions, emerged is. This cell or the resulting cell association formed by cell fusion is hereinafter referred to as "hybridoma cell clone" designated. More particularly, this invention relates to an antibody raised against the surface antigen of a the fungus belonging to the genus Candida, which antibody has a high specific (monoclonal) effectiveness having. The invention is further directed to a hybridoma cell clone which produces this antibody; Farther the invention is directed to the production and use of this antibody.

More recently, with the development of preventive medicine and the spread of antibiotics, those of bacteria have been reduced infectious diseases caused are reduced. On the other hand, the diseases caused by fungi, i.e. the mycoses worldwide an increasing tendency. Lots of these mycoses Causing fungi are usually present in the usual environment, e.g. in the mouth, in the digestive tract, in the throat area, on the skin or in the vagina of the healthy body. Among these mycoses is particularly about endocarditis, Pneumonia, uropathy, meningitis, osteopathy, arthritis, dermatosis and the like have been reported. It is also known that Mycoses associated with serious chronic diseases like tuberculosis and cancer can occur and the symptoms of these Significantly aggravate diseases.

There are currently a few known remedies effective for curing mycoses, but some with serious side effects are connected. Furthermore, antibiotics with an antifungal effect, such as amphotericin B and 5-fluorocytosine, have been used so far been used to heal mycoses. However, their effect on fungi is insufficient, and when using these agents side effects occur.

Treatment of these diseases with an antibody has also already been attempted, namely, for example, by administration a human gamma globulin preparation against the infection of a patient. However, this preparation obviously contains only a small amount of an antibody which is specifically directed against the fungus causing the infection because, for example, in the manufacturing process of this preparation no immunization step is provided, which altogether somewhat restricts the activities. Regarding a Treatment with an antibody that has an increased titer due to an immunization step is already that Known serotherapy, which is used in acute poisoning, for example by snake venom. Although such a Therapy shows some notable effects, an immune reaction by the foreign antigen can and may occur allergic reactions continue to occur because the serum is a foreign serum, for example comes from the horse and next to the actual specific antibodies are a number of accompanying substances contains. Such serotherapy has so far not been used to treat mycoses.

A quick detection and determination, as well as the exact identification such fungi is therefore important for a clinical diagnosis; there is still a need for an effective healing method for the treatment of mycoses. With regard to other areas of application, such as fermentation processes and brewing is the detection and separation of a ferment yeast from

- 12 -

harmful fungi are also of particular concern. Fungi of the genus Candida, which is an essential aspect of the Present invention represent a typical example of such mushrooms.

To date, the classification and identification of the Mushrooms of the genus Candida for their morphological and biochemical properties. These procedures take a long time laborious experimental analyzes and are therefore useful for practical Usage unsuitable. Furthermore, a serological method for identification is known that uses serum factors works by immunizing an animal, such as a rabbit, and then absorbing it with a cell, different from the cell used for immunization treatment. The serum factor forms a Mixture of different antibodies that have different specificities, hence the specificity or the titer necessarily fluctuates to some extent between the individual approaches or lots. Furthermore, a Unexpected side reactions caused by accompanying substances occasionally occur. On the other hand, is the titer of the serum factor obtained mostly relatively low, because the fungi belonging to the same genus differ in terms of their antigenic properties are quite similar. Furthermore, such immunization is associated with rather laborious steps and measures. That's why are these methods of classification and / or identification limited to a certain extent in terms of reliability and complexity of fungi of the genus Candida and limited.

The inventors named for the present invention have investigated methods for producing a monoclonal antibody, which is directed or effective against a fungus of the genus Candida, and in this way have such an antibody found with high specificity, high titer and a low content of accompanying substances obtained from a hybridoma cell clone which cell clone in turn can be derived from a cell,

- 13 -

the antibodies directed against a fungus of the genus Candida (this cell is hereinafter sometimes referred to as "an anti-Candida antibody-producing cell" and a cell of this type which can be permanently subcultured in an in vitro (this cell is hereinafter referred to as briefly sometimes referred to as "a subcultured cell"). The inventors named for the present invention have also found that this antibody can be used to classify and / or identify a genus Candida belonging fungus is useful. The antibody can continue to be successful in curing the fungus caused by this Infections are used.

It is already known in the specialist field that by cell fusion of a cell which is able to produce an antibody with a myeloma cell, a hybridoma cell clone which is able to produce a specific antibody can be obtained; For example, see Köhler et al, Nature, 256 , 495-497 (1975) and Eur .J. Immunol. , j>, 511-519 (1976). Until the present invention, however, a hybridoma cell clone which is capable of generating an antibody directed against a fungus of the genus Candida has not become known.

An essential object of the present invention is therefore to provide a method for producing an antibody indicate that is specifically directed or effective against the surface antigen of a fungus belonging to the genus Candida is.

Another object of the present invention is to provide a highly specific antibody directed against Candida and one based on cell fusion measures To specify the method of production of this antibody.

It is still another object of the present invention to provide a hybridoma cell clone that contains the same Able to generate antibodies.

Another object of the present invention is to provide a method for classifying and / or identifying a Specify fungus of the genus Candida with the help of this antibody.

Furthermore, it is an object of the present invention to provide a remedy for candidiasis which contains this antibody as an active ingredient.

Finally, there is another object of the present invention in providing one or more derivatives of the antibody useful for classification and / or identification of fungi of the genus Candida are useful and / or which are useful in the treatment of diseases caused by Fungi of the genus Candida are caused.

Further objects, objectives and special features of the present invention will become apparent to those skilled in the art from studying the the following detailed description of, non-restrictive, particular embodiments.

The inventive method for the preparation of the against Candida directed antibody is a hybridoma cell clone produced from a cell producing the antibodies directed against Candida and from one kept in subculture Cell, in particular a myeloma cell, and the antibody from the secretion excreted by the hybridoma cells isolate.

The process of the present invention is described in more detail below described.

- 15 A. Preparation of an antibody producing cell

In the context of the present invention, two different cells are required to obtain a hybridoma cell clone, which is able to generate the antibodies directed against Candida and to multiply permanently in an in vitro subculture able. In the case of these different cells it is a question of one cell, which is against one of the genus Candida belonging to the fungus is able to produce and continues to produce antibodies directed against it can hold an in vitro subculture.

The cell which is able to produce the antibody directed against Candida can be obtained from any animal species as well as received from man. Immunization of the animal is not necessary, although such previous immunization can markedly improve the formation and yield of the desired hybridoma cell clone.

If such a cell is of human origin, any person with a history of candidiasis can: or which has a high titer of the serum indicative of a fungus of the genus Candida. For an alternative Proposal, this cell can be taken from a living body that has been immunized with an immunogen. The immunogen can be a viable cell or a cell that has been treated with glutaraldehyde, mitomycin, or has been subjected to a heat treatment so that this cell cannot multiply, or it may be the surface antigen act, which is separated from a cell by means of an appropriate treatment, for example by means of an enzyme and has then been cleaned.

Any species from the genus Candida can be used in the context of the present invention, and any species can be used for this purpose morphological shape acceptable, for example mushrooms, yeast, spores with thick membrane (so-called chlamydospores),

- 16 -

and the like. Examples of such species include Candida albicans Serotype A, Candida albicans Serotype B, Candida tropicalis, Candida guilliermondii, Candida krusei, Candida parapsilosis, or Candida pseudotropicalis.

The immunogen used for the immunization can be with an adjuvant, such as Freund ¹ S complete Adju / ants or with a non-complete adjuvant mixed werden.Das immunogen may be administered by any conventional means, such as by subcutaneous, intraperitoneal, intravenous, intradermal and intramuscular Injections. Subcutaneous or intraperitoneal injection is preferably used. Immunization treatment may be sufficient, although immunization can be repeated, expediently providing an appropriate time interval of, for example, 1 to 5 weeks between successive immunization treatments. By measuring the antibody titer in the serum of the immunized animal, those animals can be selected whose titer is sufficiently high; If the antibody-producing cell is removed from such animals, an improvement in the yield and effectiveness of the following process steps can be achieved. Preferably, the cell is removed from the animal on the third to fifth day after the final immunization treatment. This cell producing the antibody forms a plasmocyte or a lymphocyte, which are to be regarded as a precursor for the cell producing the antibody; the cell can be taken from any part of the body, mostly from the spleen, lymph nodes, peripheral blood or a combination of these starting materials.

- 17 B. Cell fusion

The cell that is permanently in an in vitro subculture can hold can be any suitable cell that can be fused to the antibody producing cell to to yield a hybridoma cell clone which in turn is capable of producing the desired antibody. To a preferred species for such cells include the leukemia cells, such as myeloma cells. Anyone can do such a cell suitable species are taken, such as humans, rats, mice and the like. Preferably, those Used cells that are deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT) or to thymidine kinase (TK) because such stem cells are in the selected medium cannot grow.

Examples of preferred cell lines include GM-1500, 6TG-A1-2 or RPMI8228 of human origin or P3-X63-Ag8, P3-NSI / 1-Ag4-1, Sp2 / 0-Agl4, X63-Ag8.653 and the like from mouse.

It is preferably provided that both the antibody producing cell as the subcultured cell are of the same species, although that with regard to the Ability to fuse cells, in terms of the stability of the properties of the fused cell and in terms of is not a necessary prerequisite for carrying out the in vivo cultivation. Especially when the subculture durable cell of the cell lines P3-X63-Ag8, P3-NSI / 1-AG4-1, Sp2 / 0-Agl4 or X63-Ag8.653 is then used as a species preferably an inbred Balb / c mouse or a hybrid mouse of which provided.

An accelerator can be provided to carry out the fusion reaction; belong to suitable accelerators the Sendai virus (HVJ) and polyethylene glycol; Preferential

in particular, polyethylene glycol 1000, 1540, 2000, 4000 or 6000 is used. The cell fusion reaction can be carried out in a solution containing approximately 30 to 55% of such polyethylene glycol. Furthermore, in this Solution may contain dimethyl sulfoxide.

C. Selection of the hybridoma cell clone

After the cell fusion has been carried out, the remaining cells are in the solution in addition to the fused cells Antibody-producing stem cells and subculture stem cells are present. The former can be used in the following in vitro culture does not survive, while the latter) together with the cells of the target hybridoma cell clone, can multiply.

It is therefore preferably provided or even necessary in individual cases to remove the cells which can be kept in subculture from the solution to remove which contains the cell mixture. For this purpose it is preferably used as a stem cell for the subculture Cell a cell with a deficiency of HGPRT or TK inserted, and the cell mixture that also contains these stem cells, is cultivated in a selected medium that contains hypoxanthine, aminopterin and thymidine after cell fusion, which only allow the selected growth of the targeted hybridoma cells. According to an alternative proposal the stem cells for the cells that can be kept in subculture, provided they are not deficient in HGPRT or TK, can be used before Cell fusion can be treated with emetine and actinomycin D. After such treatment, the stem cell cannot grow proliferate longer, and therefore the target hybridoma cells can be easily isolated and separated from the mixture of cells will.

Λ mm *

- 19 -

The hybridoma cell clones obtained in this way contain mostly two or more clones which do not match with regard to the totality of their properties. For separation cloning into individual individual clones is required and even desirable if a monoclonal antibody is desired will. Cloning is also useful to prevent population change, which often occurs in a Long term culture can occur as long as a number of clones are mixed in the system. Cloning can be done on different Manner, such as in a limited dilution culture, in a soft agar culture, or in a fibrin culture. Furthermore, an apparatus for sorting out by means of fluorescence markers activated Cells can be used to separate the cells after cloning. Cloning can still be useful, to separate possible cell variants that occur in the course of the Long-term culture may occur to ensure that the cells have the same properties as the original ones Hybrid idoma cells.

The hybridoma cell clone obtained in this way can be maintained in an in vitro subculture or in an in vivo subculture or stored in freeze-dried form by any known method.

D. Generation and isolation of the antibody

To generate the antibody, the hybridoma cell clone is cultivated in vitro or in vivo.

A suitable nutrient broth for the hybridoma cell clone according to the invention is selected for in vitro culture; for example, the nutrient medium RPMI 1640 can be used with a

Content of 10% by volume of fetal bovine serum, with 5 χ 10 M ß-mercaptoethanol, 1 mM sodium pyruvate and with antibiotics;

or instead of RPMI 1640 the Dulbecco modified nutrient medium Eagle's MEM (hereinafter referred to as Dulbecco ¹ s MEM for short) can be provided, with a content of 4.5 g / L glucose. A suitable initial cell concentration for multiplication can usually be approximately 10 cells / ml, although this value can vary depending on the particular hybridoma cell clone; In the course of the cultivation, the cell concentration can preferably be increased up to a value of about 2 × 10 cells / ml.

To carry out the in vivo culture , the hybridoma cell clone is transplanted into a living body and grows there in solid form or in the ascites fluid. Body fluid, preferably serum or ascites fluid, is then removed from the living body in order to isolate the antibody secreted by the hybridoma cells. The then obtained raw solution of the antibody can contain various substances as impurities (accompanying substances) / which come from the living host body, * nevertheless this process deserves greater attention because of the higher concentration of the target antibody compared to the antibody solution obtained in vitro. If the hybridoma cells are transplanted intraperitoneally, pristane (2,6,10,14-tetramethylpentadecane) can be administered intraperitoneally before the transplantation, preferably 3 to 9 weeks before this transplantation. This treatment can increase the yield of crude antibody solution, although it is not a necessary requirement. An animal of the same species and the same inbred line as the animal from which the stem cell or stem cells originate can preferably serve as the host; in such a case, the hybridoma cells can grow in the animal even if no special treatment has been given. If the serotypes of the histological compatibility of the antigen between the hybridoma cells and the host cells do not match with one another.

34-3339

men, then it is necessary to pre-treat the host to be subjected, for example by administration of anti-lymphocyte antibodies or by x-ray irradiation. The cells begin to increase 1 to 3 weeks after the transplant grow.

The antibodies obtained include those antibodies which react with Candida albicans; also those antibodies which react with other strains of the genus Candida, such as Candida tropicalis, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida pseudotropicalis and with Candida albicans; Furthermore, antibodies can also occur that react with other fungi.

For example, it has been found that such antibodies produced by absorption of Candida albicans immunized rabbits obtained antiserum with Candida tropicalis, both with Candida albicans as react with the other strains of the genus Candida (such as Candida parapsilosis). It has also been determined that such antibodies produced by absorption of the antiserum by Candida tropicalis, by Candida guilliermondii, by Candida krusei, by Candida parapsilosis or by Candida pseudotropicalis, a very low one Have titer and rarely react with Candida albicans.

The antibodies according to the invention can as they are in the raw antibody solution are contained, or these antibodies can be purified, including any methods suitable for immunoglobulin are useful, for example fractionation with ammonium sulfate or ion exchange chromatography or affinity chromatography with protein A or with an antigen. Such purified antibodies can be used independently, or a mixture of such purified antibodies can be used will.

- 22 -

Thanks to the method according to the invention for the production of the antibody directed against Candida, the disadvantages inherent in the known methods can essentially be overcome. Thus, the hybridoma cell clone according to the invention can be kept in subculture so that the hybridoma cells multiply essentially permanently both in vitro and in vivo. Furthermore, the hybridoma cell clone can produce an antibody which represents a specific determinant which acts as an antigen. The antibody produced can therefore have monoclonal specificity and forms an antibody which is directed or effective against a fungus of the genus Candida and consists essentially of a single molecular species. The method according to the invention allows the generation of a necessary amount of the antibody depending on the requirement and avoids fluctuations between individual batches and batches; Furthermore, the method according to the invention allows a solution to be obtained which contains the antibody in a high titer. In addition, the method does not require laborious absorption steps , which were inevitable in the known methods. On the other hand, the unpurified antigen such as the fungus belonging to the genus Candida can be used as such, and a highly specific antibody can also be obtained in such a case. This high reactivity of the antibody with an antigen enables a fungus belonging to the genus Candida to be identified quickly with high reliability, without the known laborious process measures. In addition, the purity of the antibody is so high that allergic reactions rarely occur due to the accompanying substances inevitably contained in conventional preparations, so that corresponding preparations can be used as remedies for candidiasis.

The inventive method for classification and / or Identification can be applied to a subject from any origin. For example, the fungal cells from clinical

- 23 -

see materials such as sputum, urine and vaginal secretions separated or isolated from tissue obtained from a patient, which from a medical point of view may be suffers from candidiasis; appropriate fungal cells are then cultivated. Furthermore, the fungal cells can be made from brewer's yeast originate, and the inventive method for classification and / or identification is then applied to harmful To distinguish mushrooms from the yeast used in fermentation processes or in a brewery.

For identification, a reagent that the invention Contains antibodies brought into contact with a subject who in turn is cells of fungi of the genus Candida, contains. A simple antibody-antigen reaction (such as testing for agglomeration and the like) is commonly used; Another suitable test method is immunofluorescence microscopy, immunoelectron microscopy, a radioactive combination assay, an enzyme immunoassay, a complement fixation test and the like. When working directly with the immunofluorescence microscope, the antibody according to the invention expediently used after being marked with a fluorescent dye such as fluorescein or rhodamine has been. According to another, alternative proposal, the antibody according to the invention can be used after it has been labeled with a marker for immunoelectro-microscopy such as ferritin, or the antibody can be used for the radioactive combination assay can be labeled with a radioisotope such as iodine-125 or iodine-131, or the antibody can be labeled with a Enzyme such as peroxidase, alkaline phosphatase or ß-galactosidase marked to perform an enzyme immunoassay. An indirect method can also be used A secondary antibody or a combination product can be used as a substitute, e.g. a with Biotin-labeled antibodies directed against Candida can be used together with avidin as a secondary antibody. Furthermore, instead of using the antibody per se, only one component of the antibody can be used

- 24 -

obtained by restrictive cleavage after treatment with a chemical substance and / or by means of an enzyme such as F (ab ') ₂ . Such various derivatives and restriction products are also useful for carrying out the classification and / or identification method according to the invention and are therefore also intended to be encompassed by the present invention.

If necessary, the antibody, its derivatives and restriction products can be mixed for use will. Furthermore, the reagent provided for performing the classification and / or identification can be a Contain carrier or a common solvent.

The present invention further relates to a remedy for candidiasis of the urinary tract, lung, kidney and Like. Which is due to an infection with a fungus of the genus Candida, such as Candida albicans, Candida tropicalis, Candida guilliermondii, Candida parapsilosis, Candida pseudotropicalis, Candida krusei, and the like.

The medicinal effect of the medicament according to the invention can be assessed and adjusted to protect against fatal infection with a fungus of the genus Candida, as described below in the examples, it is believed that the mechanism of protective action is thereon is based on an acceleration of a complement fixation reaction and an attack by macrophages and / or others. Immunicytes to the fungus of the genus Candida takes place after the administered antibody to the surface antigen of the Mushroom is bound. These reactions can be confirmed even under in vitro conditions.

- 25 -

The antibodies according to the invention can be administered as such independently of one another, or a mixture of these antibodies according to the invention can be administered. If the antibody is chemically combined with a germicidal agent or with an antifungal agent, for example with amphotericin B, with 5-fluorocytosine, with nystatin or with griseofulvin, further advantages can be achieved. Furthermore, instead of the antibody itself, a component of this antibody can be used as the medicinal or medicament according to the invention, which has been obtained from the antibody by restrictive cleavage by treatment with a chemical substance or with an enzyme, for example F (ab ') ₂ . In this case, the risk of the host tissue being impaired or even damaged by a non-specific complement fixation reaction or the like can be avoided. These derivatives (as well as combination products with a germicidal agent or with an antifungal agent) and the corresponding cleavage products can either be used independently of one another as such, or they can be used as a mixture of different derivatives and / or cleavage products in order to produce the medicament according to the invention.

To estimate the acute toxicity of the antibodies, their derivatives or restriction products, the Medicines administered to three groups (each consisting of 10 animals) of ICR mice; the first group became the remedy administered orally in an amount of 2 g / kg; the second group was given the remedy in the amount of 400 mg / kg administered intraperitoneally, and the third group was given the medicament in an amount of 200 mg / kg intravenously; in no case was death found within 14 days. Based on these results, the inventive Remedies are considered to be safe cures or medicines to treat infectious diseases caused by Fungi of the genus Candida are caused.

The medicament according to the invention for the treatment of candidiasis in humans and animals can be administered subcutaneously, intramuscularly or intravenously; preferably the subcutaneous or intramuscular injection provided. The drug can continue to be administered orally as part of the administered Can be absorbed through the digestive tract and still retain its structure as an antibody, as confirmed by corresponding studies by the inventors named for the present invention. An injection preparation can for example be obtained by distilling 10 mg of the antibody or its derivative in 10 ml Water is dissolved or suspended, followed by usual sterilization treatment; then in ampoules filled with 2 ml each and freeze-dried. For use, the product obtained is dissolved in saline solution. Such an injection preparation In addition to the antibody, a carrier, a diluent, a buffer substance, a stabilizer, contain an isotonic agent and the like? these agents and additives are known to those skilled in the art. The injection preparation can be made up for subcutaneous, intramuscular or intravenous injection. An orally administrable agent can be prepared according to the known process for the preparation of preparations soluble in the intestine. The dose of the invention Remedies can be selected mainly depending on the symptoms and are mostly in the range of 0.001 mg to 10 g per day and per kg of body weight in an animal such as a mouse, and is suitable for treating humans approximately in the range from 0.01 to 3,000 mg per day and kg of body weight.

The following examples serve to further illustrate the invention without restricting it.

- 27 example 1; (1) Production of the immunogen

Candida albicans ATCC 752 was inoculated into a slant agar culture containing Sabouraud's medium and inoculated for 3 days incubated at 37 ° C. After adding phosphate-buffered saline (PBS, pH 7.2) to the medium, the cells became dispersed by pipetting and centrifuged at 1000 χ g at 4 C for 10 minutes to obtain a cell pellet. This washing procedure was repeated three times, then 1.0% glutaraldehyde was added to the cell pellet and the cells fixed at 0 C for 30 min. The cells were then washed five times with PBS. The cell concentration was

by means of PBS adjusted to 2 10 cells / ml to make a suspension of the immunogen to obtain which suspension was used for the subsequent experiments.

(2) Preparation of antibody-producing cell

Female 8 week old Balb / c mice (Nihon Charles Liver, Japan), the immunogen suspension was intraperitoneally administered in an amount of 0.2 ml to immunize it. The immunization was repeated every 10 days? on the 100th day after the start of the immunization treatment a "booster" immunization by intravenous administration of 0.2 ml of immunogenic suspension. 3 days after this "booster" immunization the spleen was placed in a clean bank (Hitachi Ltd., Japan) of the mice that were put to death by blood deprivation removed under aseptic conditions. The spleen was placed in a petri dish containing RPMI 1640 medium, and cut into fine particles with a pair of tweezers; these were gently pipetted and filtered through a stainless sieve to obtain a suspension of the spleen cells. Following a centrifugation treatment (10 min at 500 χ g) were

W w W · · ·

- 28 -

1.7 mM tris-HCl buffer solution (pH 7.65) containing 0.747% ammonium chloride was added to suspend the cell pellet and to lyse and remove erythrocytes. The remaining suspension of the spleen cells was centrifuged again for 3 min at 500 χ g. After the cell pellet was washed three times with RPMI 1640, the cell concentration became
set.

concentration by adding RPMI 1640 to 10 cells / ml

(3) Cell fusion to form the hybridoma cell clone

The cell suspension produced according to (2) (1 × 10 cells) was mixed with 1 × 10 mouse myeloma cells of the Zeil line Sp2 / 0-Agl4 mixed which have previously been cultured in vitro. After the cell mixture has been at 500 χ g had been centrifuged, the supernatant was discarded, to get a cell pellet. After the cell pellet is loosened by carefully wetting the bottom of the vessel 1 ml of RPMI 1640, which contained 50% by volume of polyethylene glycol 4000 (at 37 ° C.), was added to the vessel; thereupon the batch was allowed to stand for 1 minute. The vessel was then placed in a thermostated, kept at 37 ° C Brought to oven and rotated gently for 1 min to add the polyethylene glycol solution to mix with the cell pellet. A total of 10 ml of RPMI 1640 held at 37 ° C. was added to the mixture (1 ml in each case in 30 seconds) and the resulting mixture centrifuged at 500 χ g for 5 min. The supernatant was discarded and then the cell pellet was resuspended in RPMI 1640 and centrifuged at 500 χ g for 5 min. This washing procedure was repeated once more. The cell pellet formed was then placed in 20 ml of HAT medium (that is, RPMI 1640 medium With a content of 20% fetal bovine serum, 2 mM glutamine,

1 mM pyruvic acid, 4.5 g / L glucose, 5 χ 10 M

-4 -7

ß-mercaptoethanol, 1 χ 10 M hypoxanthine, 4 χ 10 M aminopterin, 1.6 10 M thymidine and 50 mg / L kanamycin sulfate).

«A · nm m

m «

- 29 -

The cell suspension formed is distributed on a culture plate with 96 wells (Nunc 167008, from Nunc Inc., Denmark), in each well. 100 µl reached; then at 37 ° C. in a CO 2 incubator containing 5% carbon dioxide incubated. 24 hours after the start of the incubation, 100 μl of HAT medium were added to each well. The incubation was continued, with 100 μl of medium removed from each well after every 2 or 3 days and 100 μl of fresh HAT have been replaced; the hybridoma cell clones capable of replicating in the HAT were then selected.

Two weeks after the start of the incubation, the growth of the hybridoma cells and the formation of the antibodies were observed the supernatant of each well was tested according to the procedure (4) described below.

(4) Selecting and cloning the antibody-producing hybridoma cells

Enzyme immunoassay was used to determine the presence of antibodies in the supernatant of each well. In detail, 2 × 10 cells of Candida albicans, which ^{had been pretreated for 2.5 hours at 100 °} C., were injected intravenously into a rabbit 5 times in order to immunize it. The rabbit serum was salted out with ammonium sulfate to obtain an IgG fraction. This fraction (the antibodies directed against Candida albicans) was adjusted to a content of 30 pg protein / ml using 0.1 M sodium bicarbonate solution. 50 µl of the resulting solution was poured into each well of the tissue culture plate; the batch was then left to rest at 4 ° C. for 24 hours. Each well was then carefully washed with distilled water, and then 30 μl of a cell suspension of Candida albicans ATCC 752 (2 × 10 cells / ml) was added to each well; then one left

React at room temperature. The well plate was then dried for 3 hours at 70 ° C. and then until the next Use stored at -20 C. 50 µl of PBS containing 0.5% glutaraldehyde was added to each well of the plate and left Stand at room temperature for 30 minutes. After washing each well three times with PBS containing 0.05% Tween 20 100 µl of the subject (i.e., the supernatant of each well) was added to each well and allowed to react at 37 ° C for 1 hour. After each well was washed three times with PBS containing 0.05% Tween 20, 50 µl of a solution became of anti-mouse immunoglobulin antibody bound to peroxidase isolated from horseradish (Cappel Corp., USA) was given in 1000-fold diluted horse serum in each well; allowed to react at 37 ° C. for 1 hour. After this reaction, each well was washed five times with PBS containing 0.05% Tween 20; then one gave in each well 100 μl 0.1 M citrate buffer solution (pH 4.5), the 1 mg / ml o-phenylenediamine and 0.04% by volume of a 31% aqueous hydrogen peroxide solution and allowed to react for 30 minutes at room temperature. By adding 50 ul 12.5% sulfuric acid in each well was the enzyme reaction stopped, and the measurement of the absorbance at 492 nm confirmed antibody formation in 22 wells of the total 192 pans.

The hybridoma cells were then placed in such wells for which antibody formation was found, cloned. In detail, spleen cells serving as nutrient cells were in in the same way as described above under point (2), the spleen of 8 weeks old, untreated (as a control serving) female Balb / c mice and the cell concentration adjusted to 5 × 10 cells / ml with HAT. The hybridoma cells identified above became the obtained spleen cell suspension added in a proportion of 2 cells / ml and

carefully stirred. Thereupon 100 µl of the cell mixture inoculated into each well of a 96-well culture plate (Nunc 167008, Nunc Inc., Denmark). There after 24 h 100 years HAT in each well and incubated at 37 ° C in an incubator containing 5% carbon dioxide.

After cloning for 2 weeks, the hybridoma cells were grown and the presence of antibodies in the supernatant of each well was determined in the above manner checked. About 2 to 80 antibody-producing clones were obtained from each well by this cloning. From these clones became stable clones that were able to produce a large amount of antibodies and had a high rate of reproduction, selected, and again in the manner indicated above cloned to obtain the antibody-producing hybridoma cell clones designated CD-I, CD-2 and CD-3.

(5) Generation of the antibody (in an in vitro culture)

The hybridoma cell clones CD-I, CD-2 or CD-3 were suspended in RPMI 1640 medium, the 20% fetal bovine serum, 2 mM glutamine, 1 mM pyruvic acid, 4.5 g / L glucose, 5 10 M ß -Mercaptoethanol and. Contained 50 mg / L kanamycin sulfate; a cell concentration of 1 10 cells / ml was set. The suspension obtained (25 ml) was placed in a 75 ml culture flask (Corning Glass Works, USA) and incubated _{at 37 ° C. in a CO 2} incubator containing _{5% CO 2.} On the 4th day, the supernatant from the flask in which the cultivation was carried out in the stationary state was collected. The number of cultured cells was approximately 2 × 10 / ml, and the antibody content of the supernatant was 3.0 μg / ml (for CD-1), 2.3 μg / ml (for CD-2) and 2.8 jig / ml (for CD-3).

(5 ') Generation of the antibody in in vivo culture

Balb / c mice were given pristan (2,6,10,14-tetramethylpentadecane) injected intraperitoneally in an amount of 0.5 ml; on the 10th to 30th day after this injection, these mice 5 x 10 cells of the hybridoma cell CD-I, CD-2, respectively CD-3 grown in in vitro cultures were injected intraperitoneally. After 2 to 3 weeks The ascites fluid of the mice was collected and centrifuged at 1000 χ g at 4 C for 15 min, leaving an ascites supernatant was obtained. Approximately 30 ml of ascites supernatant was obtained from 10 mice per hybridoma cell clone Antibody content 1.5 mg / ml (for CD-I) or 2.3 mg / ml (for CD-2) and 1.8 mg / ml (for CD-3 was.

(6) Specificity and properties of the antibody

Specificity:

Candida albicans ATCC 752, (as another Strain of the same species) Candida albicans IFO 0588, 1385, 1389, 1594 and 1269, (as other species belonging to the same genus) Candida tropicalis ATCC 750, Candida guilliermondii IFO 0679, Candida krusei IFO 1395, Candida parapsilosis IFO 1396, and Candida pseudotropicalis IFO 0432. Each strain was cultivated according to the method described above under item (1), treated with formalin, and with PBS, containing 0.05% Tween 20 to one cell concentration

8th
set from 1 χ 10 cells / ml. The cell suspension was in a portion of 0.3 ml in silicone-treated rea-. Glasses (diameter 1.2 cm) were poured and centrifuged at 1000 χ g for 5 min. The supernatant formed was discarded, and then 0.5 ml of the supernatant was added to each test tube, which was obtained in accordance with the method under item (5) for the in vitro culture of the hybridoma cell clones

- 33 -

CD-I, CD-2 or CD-3 was obtained; then left for 1 h react at 37 ° C for a long time. After this reaction, each test tube was washed three times with PBS (containing 0.05% Tween 20); thereupon 0.5 ml of a solution of anti-mouse immunoglobulin antibody, which was added to the test tube, was added to each test tube Peroxidase obtained from horseradish was bound (Cappel Corp. USA), in 1000-fold diluted horse serum given; it was then allowed to react at 37 ° C. for 1 hour. After washing five times with PBS (containing 0.05% Tween 20) 1 ml of 0.1 M citrate buffer solution was added to each test tube, which contained 1 mg / ml of o-phenylenediamine and 0.04% by volume of 31% strength aqueous hydrogen perodix solution; thereupon was kept at room temperature for 30 minutes. The reaction was stopped by adding 0.5 ml of 12.5% strength sulfuric acid; the absorbance at 492 nm was then measured to identify the antibody formed. The received Results are shown in Table 1 below.

Characteristics:

50 μl of a solution of anti-mouse IgG antibodies, anti-mouse IgA antibodies and anti-mouse IgM antibodies (Miles, USA) were then diluted 100-fold with 0.1 M sodium bicarbonate solution placed in each well of a 96-well, flat-bottomed culture plate (Nunc 167008, Nunc, Denmark). It was then left to rest at 4 ° C. for 24 hours. Each well was then carefully washed with PBS (which contained 0.05% Tween 20), and then 100 μl of the supernatant from the hybridoma cultures CD-I, CD-2 and CD-3 were added to each well had received according to the above process step (5); it was then incubated at 37 ° C. for 1 hour. After the reaction was complete, each well was washed three times with PBS (containing 0.05% Tween 20); then 50 μl of a solution of anti-mouse immunoglobulin antibodies bound to peroxidase obtained from horseradish (Cappel Corp., USA),

given in 1000-fold diluted horse serum

and incubated at 37 ° C for 1 hour. After washing each well five times with PBS (containing 0.05% Tween 20) 100 μl of 0.1 M citrate buffer solution were added to each well (pH 4.5), the 1 mg / ml o-phenylenediamine and 0.04% by volume of a 31% aqueous hydrogen peroxide solution contained; it was then incubated for 30 minutes at room temperature. By adding 12.5% sulfuric acid The enzyme reaction was stopped in each well; then the absorbance at 492 nm was measured to determine the Identify antibodies. The results obtained are listed in Table 1 below.

Example 2;
(1) Production of the immunogen

Candida albicans IFO 1594 was based on a, Sabouraud's Medium "Slant agar culture containing" transferred and incubated in an incubator for 3 days at 37 ° C. Then PBS (pH 7.2) added, the cells distributed with the pipette, and centrifuged at 4 ° C. for 10 min at 1000 χ g; thereafter a cell pellet was obtained. This washing procedure was repeated three times, and then the cell concentration with PBS adjusted to 5 × 10 cells / ml; the immunogen suspension obtained was used for the further process steps.

(2) Preparation of the antibody-producing cell

In each case 0.2 ml of this immunogen suspension were used for 8 weeks administered to old female CDF ^ mice (Nippon Krea, Japan), to immunize them. After 2 weeks each time, the mice were immunized again. On the 70th day after the start of the immunization treatment there was one

"Booster" immunization by intravenous injection of 0.2 ml of immunogenic suspension. On the 3rd day after the "booster" immunization the spleen was placed on a clean bench (Hitachi Ltd., Japan) of the mice that were put to death by blood deprivation removed under aseptic conditions. The spleen was transferred to a petri dish containing Dulbecco's MEM medium contained; analogous to the information in Example 1 (2)

a spleen cell suspension at a concentration of 1 10 cells / ml is generated.

(3) Cell fusion to produce a hybridoma cell clone

Mouse myeloma cells from the Zeil line P3-X63-Ag8 (1 χ 10) which had previously been cultured in vitro, were mixed with the spleen cell suspension (1 × 10), the cell mixture Centrifuged at 500 χ g for 5 min and discarded the supernatant to obtain a cell pellet. After the pellet had been dissolved by carefully wetting the bottom of the vessel, the temperature was set to 37.degree. in the Over the course of approximately 1 minute, 1 ml of Dulbecco's MEM medium, containing 45% polyethylene glycol 4000 (37 ° C), was gradually added. contained. It was held at 37 ° C for 7 minutes and then an additional 15 ml of Dulbecco's over approximately 5 minutes MEM (37 ° C) added; the addition was made very carefully by slowly turning the vessel and moving the medium along the Let the vessel walls taper. Approximately 25 ml of Dulbecco's MEM were then added again for 5 minutes centrifuged at 500 χ g and the supernatant discarded.

Dulbecco's MEM, which contained 10% fetal bovine serum (37 ° C.), was added to the cell pellet formed; after on a cell concentration of 1 10 cells / ml had been set, was gently mixed in a pipette, and 1 × 10 cells in each case are placed in each well of a culture plate comprising 24 wells (Nunclon, Nunc, Denmark) and at 37 ° C in an incubator containing 5% carbon dioxide

incubated. 24 hours after the start of the incubation, 1 ml of HAT was added to each well. In the course of cultivation 1 ml of medium was removed from each well every second or third day and replaced with 1 ml of fresh HAT. It was selected a hybridoma cell clone that was found in HAT medium could multiply.

After a period of 2 weeks after the start of the incubation, growth of the hybridoma cell clones could be determined, and the antibodies contained in the supernatant of each well were prepared according to the procedure given in Example 1 (4) examined. The formation of antibodies was confirmed in 10 of the 48 cells.

(4) Detection of the antibody-producing hybridoma cell clone

The antibody-forming hybridoma cell clones were cloned from 10 wells using an agar slant. In detail, 30 ml of 2.5% agar (Difco, Germany) and 3 ml of Dulbecco's MEM of 10 times the concentration were mixed with one another at 45 ° C., and 117 ml of Dulbecco's MEM (also of 45 ° C.) were added to this mixture . The spleen cells of non-treated (used for control), 8-week-old, female CDF ₁ mice were added to this agar solution in an amount of 5 × 10 cells / ml as nutrient cells. In each case a Petri dish (diameter 10 cm, Falcon 3003, Becton-Dickinson, USA) was placed in each case 10 ml of this agar solution; then allowed to stand for 15 minutes at room temperature to form a gel. About 2 ml of hybridoma cell suspension from each well, which had shown positive antibody formation, and the same amount of Dulbecco's MEM, which contained 0.5% agar, were mixed together, and 2 ml of each of this mixture were applied to the gel obtained according to the above procedure given, the cells were evenly distributed on the gel layer. Incubation took place at 37 ° C in a 5% CO ₂ separator

- 37 -

holding incubator. On the 10th day after the start of the incubation, the colonies growing on the agar slant were collected using a Pasteur pipette and distributed to the 96 flat-bottomed wells of a culture plate; 0.2 ml of Dulbecco's MEM were placed in each well and then incubated at 37 ° C. in an incubator containing _{5% CO 2.} The growth of the hybridoma cell clones was monitored, and the presence of antibody in the supernatant of each well was examined simultaneously according to the method given in Example 1 (4).

Persistent among the positive hybridoma cell clones Selected clones that produced high levels of antibody and that multiplied well; these persistent clones became again cloned in the manner indicated above, and then the antibody-forming hybridoma cell clones with the designations CD-4 and CD-5 received.

(5) Formation of the antibodies in vitro culture

The hybridoma cell clones CD-4 and CD-5 were cultured according to the method given in Example 1 (5) to obtain corresponding To get supernatants.

in vivo culture

According to the procedure given in Example 1 (5), the hybridoma cell clones CD-4 and CD-5 were intraperitoneally transplanted onto CDF, mice, ascites fluid obtained from the mice after 2 to 3 weeks and transferred to the ascitic supernatant processed. Approximately 30 ml of this supernatant was obtained from 10 mice.

- 38 -

(6) Specificity and properties of the antibody

Specificities and immunoglobulin classes of antibodies, those in the CD-4 or. CD-5 hybridoma supernatants included were examined according to the procedure of Example 1 (6). The results obtained are in Table 1 below.

Example 3;
(1) Production of the immunogen

Candida albicans IFO 0588 was transferred to an agar slant containing Sabouraud's medium and incubated at 37 ° C for 3 days. After the incubation, the cells were collected by means of a platinum wire loop, distributed in PBS (pH 7.2), and this suspension was centrifuged at 1000 μg at 4 ° C. for 10 minutes to obtain a cell pellet. This washing process was repeated 3 times, and then the remaining cells were ^{heated to 100 °} C. for 2.5 hours.

After these heat-treated cells were washed 3 times with PBS, the cell concentration was increased to 2 × 10 with PBS Cells / ml set, and the immunogen suspension obtained used for the further process steps.

(2) Preparation of the antibody-producing cell

In each case an amount of 0.2 ml was this immunogenic suspension 8 week old female Balb / c mice (Nihon Charles Liver, Japan) were injected intraperitoneally to this to immunize. After every 2 weeks this immuno-

ization repeated; 10 weeks after the first immunization a "booster" immunization was carried out by intravenous administration of 0.2 ml of this immunogenic suspension. On the third Days after the "booster" immunization, the blood-deprived mice had their spleen on a clean Bank (Hitachi Ltd., Japan) removed under aseptic conditions. The spleen was immersed in a medium containing RPMI 1640 Given a Petri dish, cut it up and, according to the method of Example 1 (2), a spleen cell suspension in one concentration prepared from 5 10 cells / ml.

(3) Cell fusion to create the hybridoma cell clones

Mouse myeloma cells of the cell line P3-NSI / l-Ag4-l (0.5 ⁷ cells) which had previously been cultured in vitro were mixed with this spleen cell suspension (5 10 cells), the cell mixture for 5 min centrifuged at 500 χ g, the supernatant removed to obtain a cell pellet. After this cell pellet had been dissolved by carefully wetting the bottom of the vessel, 0.5 ml of RPMI 1640, which was kept at 37 ° C. and which contained 42.5% polyethylene glycol 1540 and 15% dimethyl sulfoxide, was added; then allowed to react for 1 minute. In the course of the reaction, the vessel was carefully rotated in order to carefully and carefully mix the polyethylene glycol solution with the cell pellet. One minute after this mixing, 10 ml of RPMI 1640 kept at 37 ° C. was added at a rate of 1 ml / 30 seconds while the vessel was still rotating uniformly; the mixture was then centrifuged at 500 χ g for 5 minutes. After the supernatant was removed, the cell pellet was resuspended in RPMI 1640 and centrifuged at 500 χ g for 5 minutes; the supernatant was removed and the remaining cell pellet washed again in the same way; then 10 ml of RPMI 1640 were added as a culture medium to the cell pellet

- 40 -

10% f tales bovine serum, 2 mM glutamine, 1 mM pyruvic acid, 4.5 g / L glucose, 5 χ 10 M ß-mercaptoethanol and 50 mg / L kanamycin sulfate. In this culture medium kept at 37 ° C., the cells were well distributed; 200 μl each of the cell suspension obtained were placed in each well of a 96-well culture plate (Nunc 167008, Nunc, Denmark) and _{incubated at 37 ° C. in a CO 2} incubator. After 24 hours had elapsed, half of the supernatant was discarded and 100 μl of the above HAT culture medium kept at 37 ° C., which additionally contained 1 × 10 −6 M hypoxanthine, 4 × 10 "M aminopterin and 1.6 × 10 M thymidine, was added After every 2nd or 3rd day, 100 μl of this medium were removed from each well and replaced with 100 μl of fresh HAT. Those hybridoma cell clones were selected which were able to multiply in HAT medium.

Two weeks after the start of the cultivation, growth of the hybridoma cell clones could be determined; simultaneously the presence of the antibodies in the supernatant of each well was determined according to the procedure given in Example 1 (4) examined. In this example, cells from Candida albicans IFO 0588 were identified as those bound to the plate Cells used. The formation of the antibody could be determined in 3 of a total of 48 cells.

(4) Detection of the antibody-producing hybridoma cell clone

According to the procedure given in Example 1 (4), the hybridoma cell clones from the 3 ^ antibody-producing Wells cloned, the antibody-producing hybridoma cell clones with the designations CD-6 and CD-7 were obtained.

By using HT medium (that is the one referred to above

-4 culture medium after adding 1 χ 10 M hypoxanthine and 1.6 10 M thymidine) instead of HAT medium was the Culture medium gradually exchanged for HT. after the Cultivation was carried out in HT for 2 weeks, the HT was again exchanged for culture medium that does not contain hypoxanthine, Contained aminopterin and / or thymidine; in this way it was made possible that the hybridoma cell clones also propagated in a common culture medium instead of the selected medium.

(5) Formation of the antibodies in vitro culture

According to the method according to Example 1 (5), the hybridoma cell clones with the designations CD-6 and CD-7 grown, and obtained appropriate supernatants. In this example the initial cell concentration was 2 × 10 cells / ml and the antibody levels in the resulting supernatants were 2.5 jig / ml (for CD-6) and 3.0 jig / ml (for CD-7).

in vivo culture

In accordance with the method given in Example 1 (5), 1 × 10 cells of the hybridoma cell clones CD-6 were in each case and CD-7 Balb / c mice were injected intraperitoneally into these mice 2 to 3 weeks after the transfer of ascites fluid removed, and the corresponding ascitic supernatant generated. Approximately 30 ml of supernatant was obtained from 10 mice obtain.

- 42 (6) Specificity and properties of the antibody

Following the procedure of Example 1 (6), the specificities and classes of the immunoglobulin became the antibodies in the CD-6 or. CD-7 hybridoma supernatants examined. The results obtained are shown in Table 1 below.

Example 4:
(1) Production of the immunogen

Candida albicans ATCC 752 was transferred to an agar plate containing maize flour (Nissui Seiyaku, Japan) and 10 Incubated at 25 C for days. After this incubation, a portion rich in fungal mycelium and chlamydospores was added collected in a spoon, distributed in PBS (pH 7.2), the slurry homogenized and finally at 4 ° C for 10 min centrifuged at 1000 χ g for a long time to obtain a cell pellet. The washing procedure was repeated 3 times, and then PBS added to adjust to a concentration of 1% by weight or volume; the received immunogenic suspension was used for the following experiments.

(2) Preparation of the antibody-producing cell

In each case 0.2 ml of this immunogenic suspension were female, 8-week-old Balb / c mice (Nihon Charles Liver, Japan) administered intraperitoneally to immunize these mice. After each 14 days, the mice were immunized again. On the 70th day after the start of the immunization, a “booster” immunization was carried out by intravenous administration

- 43 -

of 0.2 ml of immunogenic suspension. 3 days later the Bleeding from dead mice, spleen and mesenteric lymph nodes on a clean bench (Hitachi Ltd., Japan) removed under aseptic conditions. According to the conditions given in Example 1 (2), the spleen and

8 the lymph nodes to a cell suspension with 10 cells / ml

processed.

(3) Cell fusion to produce the hybridoma cell clones 1 χ 10 ⁷ mouse myeloma cells of the Zeil line X63-Ag8.653,

previously grown in vitro. and 1 χ 10 Cells from the above cell suspension were mixed with one another and the cell fusion according to the example 2 (3) to generate hybridoma cell clones. In 10 cells from a total of 48 cells the formation of the antibody could be determined.

(4) Detection of the antibody-producing hybridoma cell clone

The antibody-producing hybridoma cell clones were cloned in a fibrin gel culture. In detail, 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 contained, evenly distributed on the bottom of a Petri dish (Falcon 3002, Beckton-Dickinson, USA); thereupon 4 ml of Dulbecco's MEM medium were added, which was 10 mU / ml Contained thrombin (Miles, USA) and 20% fetal bovine serum; The mixture was kept at 37 ° C. for 1 hour to form a gel.

4 100 μl of the hybridoma supernatant (with 1 × 10 hybridoma cells / ml) were placed on the gel and distributed evenly; it was then incubated at 37 ° C. in an incubator containing _{5% CO 2.} After 10 days or more, the colonies growing on the gel were collected with a Pasteur pipette and placed in the wells of a 96

Wells with flat bottom culture plate transferred; 0.2 ml of Dulbecco's MEM were placed in each well and incubated at 37 ° C. in an incubator containing _{5% CO 2.} The growth of the hybridoma cells was monitored, and the presence of antibodies in the supernatant of each well was examined according to the method of Example 1 (4). From the hybridoma cell clones positive for the production of antibodies, persistent clones were selected which produced a large amount of antibodies and multiplied well; these selected hybridoma cell clones were cloned again in the manner indicated, in order then to obtain the antibody-forming hybridoma cell clones with the designations CD-8 and CD-9.

(5) Formation of the antibody

The hybridoma cell clones CD-8 and CD-9 were both in vitro culture and in vivo culture according to the method grown according to Example 1 (5). The corresponding supernatant was used in the in vitro culture and the corresponding in the in vivo culture Obtain ascites fluid.

(6) Specificity and properties of the antibodies

The specificities of the antibodies and the classes of those in the Supernatants containing the CD-8 or CD-9 hybridoma supernatants Cultures were examined according to the procedure of Example 1 (6). The results obtained are listed in Table 1 below.

Tabel

Specificities and immunoglobulin classes of the antibodies directed against Candida according to the invention

Hybridoma cell clone producing antibodies

Zl-

fity

CD-I CD-2 CD-3 CD-4 CD-5 CD-6 CD-7 CD-8 Cü-9

same
species

same
genus

other
mushrooms

properties

Candida albicans ATCC752 Candida albicans IFO0588 Candida albicans IPO1385 Candida albicans IPO1389 Candida albicans IFO1594 Candida albicans IPO1269

Candida tropicalis ATCC750
Candida guilliermondii IPO0679
Candida krusei IFO1395
Candida parapsilosis IFO1396
Candida pseudotropicalis IFO0432

Toruopsis glabrata IPO0622
Saccharomyces cereviseae IAM4207
Hansenula anolama IAM4213
Debaryomyces hansenii IAM4356

Immunoglobulin classes

IgM IgM IgM IgM IgM IgG IgG IgG IgG

+; positive, -; negative

CO Ca »CO

"- 34Ί3339

Example 5:

The antibodies obtained according to Examples 1 to 4 were administered to ICR mice; each experimental group comprised 10 mice; in the individual groups, 2 g / kg of antibody orally, or 400 mg / kg of antibody intraperitoneally or 200 mg / kg Antibodies administered intravenously; the mice were observed for 14 days. Occurred within the observation period there was no death following the administration of the antibodies.

The shape of the cells of the hybridoma cell clones CD-I to CD-9 was approximately spherical; the dimensions ranged from 10 to 20 µm, and were mostly about 15 tim; the cells of the hybridoma cell clones CD-I to CD-9 swam in the corresponding Culture media and showed only weak adhesion to the vessel wall.

Example 6:

Identification of a fungus of the genus Candida by means of an antibody

(a) subject!

The subject was sputum, urine, or vaginal secretions collected from five patients who had doubts about it had a disease of candidiasis. This material was smeared into a petri dish, the Sabouraud's Glucose agar medium contained. It was incubated at 37 ° C. for 5 days. The colonies that appear on the agar plate served as the subject.

(b) Identification:

For identification, the agglomerations were made on a

Microscope slide, and determination with fluorescent antibodies used; the starting materials used in each case were antibodies which were contained in the CD-I precipitated ascitic supernatant were included.

Specifically, in aggregation test I, drops of the antibody solution were placed on the slide, a small amount of the cells removed from the subject colonies by a platinum wire loop added, and the aggregation followed with the naked eye or under the microscope .

In the test with fluorescent antibodies, a small amount of the cell removed from the colonies to be examined with a platinum wire loop was smeared onto a microscope slide and fixed there with 1.0% formalin solution; after being kept at 4 ° C. overnight, washing was carried out with PBS (pH 7.2), and then 1 drop of the above 100-fold diluted antibody solution was added, followed by incubation at 37 ° C. for 1 hour. After carefully washing with PBS (pH 7.2), unreacted antibodies were removed; 1 drop of a solution containing fluorescein-bound anti-mouse immunoglobulin (Cappel, USA) in a 100-fold dilution was then added and the mixture was incubated at 37 ° C. for 1 hour. After removing unreacted anti-mouse immunoglobulin bound to fluorescein by washing with PBS (pH 7.2), fluorescence was examined by means of a fluorescence microscope (Olympus Vanox, Olympus, Japan) on each subject cell on the slide .

At the same time, the subjects were examined morphologically and biochemically. In detail, the chlamydospores in the slide culture, which had used cornmeal agar as the medium, were morphologically examined; With regard to the biochemical investigation, the consumption of glucose, sucrose, maltose and lactose was determined.

(c) results;

The identification of the antibodies according to the invention The results obtained are fully in agreement with the results of the morphological and biochemical study. These results are shown in Table 2 below.

Tabel

subject

Identify by antibodies from CD-I

Co-fluorescent agglomeration antibodies Morphological and biochemical examination Result:

Education consumption of sugar (identified by ChIa- glucose sucrose maltose lactose strain)
mydospores

sputum
(Patient A)

urine
(Patient B)

Vaginal secretions (patient C)

urine
(Patient D)

sputum
(Patient E) Candida albicans Candida albicans Candida albicans Candida albicans

Saccharomyces cerevisiae

Ca> CO CO CD

Example 7;

(1) Identification of the antibodies secreted by the hybridoma cell clones CD-I, CD-4, CD-6 and CD-8 with peroxidase

In 1 ml of distilled water were 4 mg of horseradish peroxidase (Sigma Type VI, Sigma, USA) solved; 60 μl of a 0.1 M NaJO. solution were added to this solution, which immediately had been prepared beforehand; the solutions were mixed at room temperature for 20 minutes. The resulting solution was overnight against 1 mM sodium acetate buffer solution (pH 4.4) dialyzed; then 20 ml of 0.2 M sodium carbonate solution were added added. Soon thereafter, the antibodies secreted by the hybridoma cell clones CD-I, CD-4, CD-6, or CD-8 became added; Specifically, these antibodies were in the form of the purified ascitic supernatants with a protein content of 10 mg added, which in 1 ml of 0.01 M carbonate buffer solution (pH 9.5) were dissolved; the mixture was stirred at room temperature for 2 hours. After this implementation were 0.1 ml of freshly prepared aqueous NaBH. Solution (4 mg of NaBH. Were dissolved in 1 ml of distilled water) were added; man allowed the reaction to stand at 4 ° C. for 2 hours and then dialyzed against PBS overnight.

The mixed solution obtained thereafter was applied to a column filled with SephadeX G-100 (Pharmacia, Sweden) and eluted with PBS. The first fraction whose absorptions at 280 nm and 403 nm corresponded to each other was collected. To 1 ml of this, the enzyme / antibody coupling product-containing solution was added 10 mg of rabbit serum albumin and dissolved, and the mixture then stored at -70 ⁰ C until use.

(2) Identification of the hybridoma cell clones CD-I, CD-4,

CD-6 and CD-8 secreted antibodies with alkaline phosphatase

5 mg alkaline phosphatase of type VII (Sigma, USA) previously against

PBS had been dialyzed to complete the amino sulphate content and 17 mg of that from the hybridoma cell clones Antibodies excreted from CD-I, CD-4, CD-6 or CD-8 (in the form of the purified ascitic supernatant) were in PBS dissolved and brought to a total volume of 1 ml. To the resulting solution, 10 µl of 20% glutaraldehyde solution was added added and the mixture stirred at room temperature for 2 hours. After the reaction, the reaction mixture was reduced to a Sephadex G-200 (Pharmacia, Sweden) filled column, equilibrated with Tris-HCl buffer solution (pH 7.6) and then eluted with the same buffer. A high molecular fraction was collected, which elutes from the beginning to the IgG point became; this fraction was mixed with 5% bovine serum albumin, sterile filtered (0.22 μm Millipore filter, Millipore, USA) and stored in the dark at 4 ° C until further use.

(3) Identification of the hybridoma cell clones CD-I, CD-4,

CD-6 and CD-8 secreted antibodies with ß-galactosidase

In accordance with the method given above under (2), the hybridoma cell clones CD-I, CD-4, CD-6 and CD-8 secreted antibodies labeled with ß-galactosidase, ß-galactosidase was used for this label Sigma, Type IV (Sigma, USA) used.

(4) Labeling of the antibodies secreted by the hybridoma cell clones CD-I, CD-4, CD-6 and CD-8 with iodine-125

125 to 10 μl solution of 100 mCi / ml Na I (carrier-free, Amersham, USA), 50 μl of antibody solution, secreted by the hybridoma cell clones CD-I, CD-4, CD-6 or CD-8, was added had been; in detail it was the purified ascitic supernatant with a protein content of 1.0 mg / ml;

furthermore 30 µl of 0.5 M phosphate buffer solution (pH 7.2) containing 0.30 mg / ml chloramine T was added; the components were mixed well together. After 15 seconds , 100 μl PBS were added which was saturated with L-tyrosine; the components were immediately mixed with one another, and the mixture formed was chromatographed on a column filled with Amberlite IRA 400; it was eluted with PBS containing 1% bovine serum albumin. The eluted fraction was collected and stored at 4 C until used. The specific activity of the labeled product was 1.0 iiCi / mg antibody protein.

(5) Labeling of the antibodies secreted by the hybridoma cell clones CD-I, CD-4, CD-6 and CD-8 with fluorescein

1 ml solution of the hybridoma cell clones CD-I, CD-4, CD-6 or CD-8 secreted antibodies (in the form of the purified ascitic supernatant) with a protein concentration of 10 mg / ml were 0.1 ml of 0.5 M carbonate buffer solution (pH 9.3) added; furthermore 0.1 mg of fluorescein isothiocyanate powder were added and for 6 hours at 4 ° C. without formation of bubbles touched. Immediately after the reaction, the reaction mixture was filled with a Sephadex G-25 (Pharmacia, Sweden) Column given to remove the unreacted, low molecular weight substances. The high molecular weight fraction was collected and stored in the dark at 4 ° C until further use.

(6) Labeling of the antibodies secreted by the hybridoma cell clones CD-I, CD-4, CD-6 and CD-8 with tetramethylrhodamine

To 1 ml of a solution of the CD-I from the hybridoma cell clones, CD-4, CD-6 or CD-8 secreted antibodies (in the form of the purified ascitic supernatant with a protein content

• · ♦ «*

- 53 -

of 10 mg / ml) were 0.1 ml of 0.5 M carbonate buffer solution (pH 9.3) added; furthermore 0.2 nig tetrahydrofuran wasothiocyanate powder added, and the mixture was stirred for 20 h at 4 ° C without bubbling. Following the implementation the reaction product was immediately applied to a column filled with Sephadex G-25 (Pharmacia, Sweden); the unresponsive Any low molecular weight substances were removed, and the high molecular weight fraction was collected. The product was stored in the dark at 4 ° C.

(7) Labeling of the antibodies secreted by the hybridoma cell clones CD-I, CD-4, CD-6 and CD-8 with biotin

1 mM d-biotin (= 244 mg, Wako Pure Chemical Industries, Ltd., Japan) and 1.5 mM N-hydroxysuccinimide (= 173 mg, Eastman Kodak, USA) were added in a mixture of 8 ml of dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.) and 5 ml of 1,2-dimethoxyethane (Nakadai Chemical, Japan). A solution of 1 mM Ν, Ν'-dicyclohexylcarbodiimide (= = 206 mg, Kanto Chemical, Japan) in 0.5 ml of 1,2-dimethoxyethane was added to the resulting solution; the reaction mixture was then left to react at 4 ° C. overnight. The precipitate formed was filtered off and the filtrate collected for further use. The solvent contained in the filtrate was removed in vacuo, and the resulting oily residue was dissolved in 10 ml of dichloromethane (Wako Pure Chemical Industries, Ltd., Japan) and cooled to 4 ° C. Then 10 ml of 0.1 M NaHCO ₃ solution (4 C) was added and the whole was shaken well to mix. The dichloromethane phase formed was separated off, 10 ml of 0.1 M NaHCO-1 solution and then 10 ml of distilled water (both at 4 ° C.) were added; these process steps were repeated again. The combined dichloromethane phases were dehydrated with anhydrous sodium sulfate powder (Koizumi Chemical, Japan). The powder was filtered off, and

• W · W

- 54 -

η-hexane was added to the collected filtrate until it became cloudy. Then the mixture was cooled to -20 ⁰ C, separated the precipitated crystals and placed into a dryer to dry to remove the solvent, and to the crystalline material. The 3iotin-N-hydroxysuccinimide ester was obtained as the product.

This biotin-N-hydroxysuccinimide ester was made in dimethyl sulfoxide dissolved, and adjusted to a concentration of 1 mg / ml. 60 μl of this solution were mixed with 1 ml of the solution of excreted the hybridoma cell clones CD-I, CD-4, CD-6 or CD-8 Antibodies (in the form of the purified ascitic supernatant with a protein content of 1 mg / ml) added; then left to react for 4 hours at room temperature. Following the reaction, the solution was left for 3 days Dialyzed at 4 C against PBS. During the course of dialysis, the dialysis fluid was exchanged 3 times. The dialysate, i.e. the 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 were obtained from a Sabouraud's Transfer slant culture containing medium and incubate for 3 days at 37 ° C. The cells were by means of a Platinum wire loop was collected and used as the subject fungus for the following investigations.

(2) Identification by means of the fluorescent antibody

A small amount of each subject fungus was placed on a microscope slide lubricated, there with 1.0% formalin solution at 4 ° C

• β

■ # * Λ

m Λ **

- 55 -

fixed overnight and washed with PBS (pH 7.2). One drop of the 10-fold diluted solution of the corresponding example 7 (5) labeled with fluorescein, from the hybridoma cell clone CD-I, Antibody deposited with CD-4, CD-6 or CD-8 was added; the reaction was then allowed to take place at 37 ° C. for 1 hour. By careful Washing with PBS removed the unreacted antibody. The fluorescence of the slide was up examined under a fluorescence microscope (Olympus Vanox, Olympus, Japan). As a result, fluorescence was related to Candida albicans ATCC 752 detected.

An analogous investigation was carried out with, in accordance with example 7 (6) performed with antibodies labeled with rhodamine. Again, fluorescence for Candida albicans ATCC 752 was detected.

Furthermore, each of the biotin-labeled antibodies obtained according to Example 7 (7) was applied to the subject fungus bound and carefully washed out with PBS; a drop of 5 µg / ml solution of fluorescein-bound solution was then added Avidin (Funakoshi Yakuhin, Japan) was added and allowed to react at 37 ° C. for 1 hour. After careful washing with PBS it was fluorescence detected on the slide from Candida albicans ATCC 752 under the fluorescence microscope.

(3) Identification by the enzyme immunoassay

0.3 ml of a cell suspension of the subject fungus were in brought a silicone-treated test tube (diameter 1.2 cm) and centrifuged at 1000 χ g for 5 min. To Removal of the supernatant was 0.5 ml of a solution of the 100-fold diluted horse serum with peroxidase, alkaline phosphatase or ß-galactosidase labeled antibodies (obtained according to Example 7 (1), (2) or (3)) added;

it was then allowed to react at 37 ° C. for 1 hour. After completion of the reaction, the reaction mixture was 5 times with PBS, the Containing 0.05% Tween 20, washed; then 1 ml of a substrate solution was added.

In detail, the antibody labeled with peroxidase was given 1 ml of 0.1 M citrate buffer solution (pH 4.5) containing 1 mg / ml o-phenylenediamine and 0.04% by volume of 31% aqueous hydrogen peroxide solution contained, added; the reaction mixture was allowed to react for 30 minutes at room temperature, after which 0.5 ml was added Add 12.5% sulfuric acid to stop the reaction; the absorption at 492 nm was then determined.

In the case of the antibody labeled with alkali phosphatase, 1 ml of a substrate solution (pH 9.8) containing 1 mg / ml of p-nitrophenyl phosphate (Sigma, USA), 9.7% by volume of diethanolamine and 0.01 Contained% MgCl ₂ * 6H ₂ O; allowed to react for 30 minutes at room temperature, then 0.5 ml of 5 M sodium hydroxide was added to stop the reaction; the absorption at 405 nm was then determined.

In the case of the antibody labeled with ß-galactosidase, 1 ml of borate buffer solution (pH 8.5) containing 1 mg / ml of 4-methylumbelliferyl-ß-galactoside was added; was allowed to react for 10 min at 30 ⁰ C and added subsequently 0.5 ml of 0.1 M glycine-sodium hydroxide buffer solution (pH 10.3) was added to stop the reaction; the amount of released 4-methylumbelliferone was measured with a HITACHI spectrofluorometer (at an excitation wavelength of 3 60 nm and at an emission wavelength of 450 nm).

The measurement results confirmed that the inventive, Enzyme-labeled antibodies had reacted with Candida albicans ATCC 752.

(4) Identification by radio-immunoassay

The subject fungus was grown in PBS containing 0.05% Tween 20
suspended. One ml of this suspension was placed in a silicone-treated test tube (diameter 1.2 cm) and
Centrifuged at 1000 χ 9 for 10 min. The cell pellet formed was washed twice with PBS containing 0.05% Tween 20; then 50 μl of the according to example 7 (4)
obtained antibody labeled with iodine-125 (specific activity: 1 μCi / mg antibody) added; it was then allowed to react at 37 ° C. for 1 hour. After washing 5 times with PBS containing 0.05% Tween 20, the radioactivity bound to the subject fungus was measured using a counter tube (Beckman Gamma 8500, Beckman, USA).

The higher radioactivity was found on Candida albicans ATCC, as shown in Table 3 in the case of the invention, with iodine-125 labeled antibody is detectable.

Tabel

Subject mushroom

radioactivity bound to the cell (imp./min)

125

125

125

125

J-CD-I ^J - ' ^J J-CD-4 ^Μ "3- <ρ-6 " ^J J-CD-8 ""' J-marked
Antibodies Antibodies Antibodies Antibodies Mouse immunoglobin

Candida albicans

ATCC 752 8900

Candida trcpicalis

ATCC 750 760

Candida guilliermondii

IPO 0679 530

10400 890 916

13600

672

778

11000

700

855

CO
IT »

«4

- 59 -

Example 9;

Urine from three patients who may have had urinary tract candidiasis and candida albicans ATCC 752 were each transferred to Sabouraud's glucose agar and incubated at 37 ° C. for 5 days. Which on The yeast-like colony developing from the agar plate was removed using a platinum wire loop, in 3 ml PBS, containing 0.05% Tween 20, suspended and homogenized; the resulting cell suspension was used for the following Investigations used as the subject.

(1) Identification by the enzyme immunoassay

0.3 ml of the cell suspension was placed in a silicone-treated test tube (diameter 1.2 cm) and kept for 5 minutes Centrifuged 1000 χ g. After removing the supernatant, 0.5 ml of a solution containing peroxidase, alkaline phosphatase or ß-galactosidase labeled antibody (prepared according to Example 7 (1), (2), or (3) in 100-fold dilution Horse serum added; it was then allowed to react at 37 ° C. for 1 hour. The reaction mixture was 5 times with 0.05% Tween washed containing PBS; then 1 ml of substrate solution was added.

In detail, 1 ml of 0.1 M citrate buffer solution (pH 4.5) was added to the antibody labeled with peroxidase, which contained 1 mg / 1 ml of o-phenylenediamine and 0.04% by volume of a 31% strength aqueous hydrogen peroxide solution; left for 30 min react at room temperature for a long time and then added 0.5 ml of 12.5% sulfuric acid to stop the reaction; the absorption at 492 nm was then determined.

In the case of the antibody labeled with alkaline phosphatase, 1 ml of substrate solution (pH 9.8) was added 1 mg / ml p-nitropheny! Phosphate (Sigma, USA), 9.7% by volume di-

contained ethanolamine and 0.01% MgCl ₂ · 6H-O; allowed to react for 30 minutes at room temperature, then 0.5 ml of 5 M sodium hydroxide was added to stop the reaction; the absorption at 405 nm was then determined.

Finally, in the case of the antibody labeled with ß-galactosidase, 1 ml of borate buffer solution (pH 8.5) was added, which contained 1 mg / ml of 4-methylumbelliferyl-ß-galactoside; the reaction was allowed to take place for 10 minutes at 30 ^° C., and 0.5 ml of 0.1 M glycine-sodium hydroxide buffer solution (pH 10.3) was added in order to stop the reaction; the content of free 4-methylumbelliferone was then determined with the HITACHI spectrofluorometer (at the excitation wavelength 360 nm, and at the emission wavelength 450 nm).

The results obtained are shown in Table 4 below.

(2) Identification by the fluorescent antibody

The cell suspension was smeared on a slide, fixed there overnight with 1.0% formalin solution and with PBS washed. Then 1 drop of a solution was labeled with fluorescein or rhodamine or biotin Antibody (prepared according to Example 7 (5), (6) or (7)) in 100-fold diluted horse serum added; allowed to react for 1 hour at 37 ° C and washed carefully with PBS to avoid unreacted Remove antibodies.

In the case of the one labeled with fluorescein or rhodamine Antibody, the presence of fluorescence on the subject fungus was determined with a fluorescence microscope (Olympus Vanox, Olympus, Japan) on the slide.

In the case of the antibody marked with biotin, 1 drop of a solution of 5 ug / ml of fluorocin • perL'Undont •: Avidin D (Funakoshi Yakuhin, Japan) added; thereupon allowed to react for 1 hour at room temperature and washed thoroughly with PBS. The presence of fluorescence on the slide was determined with the fluorescence microscope.

The results obtained are shown in Table 4 below.

The colonies formed from the urine of patients F and G were identified as Candida albicans on the basis of simultaneously performed morphological and biochemical examinations . This result was completely in agreement with the results of the tests with the labeled antibodies. Furthermore, the colony formed from the urine of patient H was identified as Saccharomyces cerevisiae.

Tabel

• t
» • * · procedure used it's reagent CD-6 Patient P S u b j e k t Candida albicans Marking Origin of CD-8 urine Patient G. Patient H. ΑΊΧΧ 752 ϊ
ι »I> »» J
1
) middle Antibody CD-I + (492nm) urine urine + (492nra) > 1
>)
> ι »» » CD-I CD-4 4- (492nm) + (492nm) - (492nm) + (492nm) i> »*> I. Peroxidase CD-4 CD-6 + (492nm) + (492nm) - (492nm) + (492nm) I) CD-6 CD-8 ■ M492nm) + (492nm) - (492nm) + (492nm) * * 9 O CD-8 + (405nm) -K492nm) - (492nm) + (405nm) I. CD-I CD-I H- (405nm) 4 (405nm) - (405nm) + (405nm) Alkali- CD-4 CD-4 • »- (405nm) + (405ran) - (405nm) + (405nm) Enzyme- Phosphatase CD-6 CD-6 + (405nm) + (405nm) - (405nm) + (405nm) Immuno- CD-8 CD-8 + (405nm) - (405nm) Assay CD-I + CD-I CD-4 + + - + ß-Galactos idase CD-4 CD-6 + ■ + - + CD-8 +. - + + + - + + + + Fluorescein + + _ + + + _ + with + _ fluoresc. + + Anti + + _ + body Rhodamine + + - + + + - + + + - + + + + Biotin + + ■ _ + + + _ + _

+: positive absorption or fluorescence? -: negative absorption or fluorescence.

CO

** "34T3339

- 63 -

Example 10 :

20 ml urine from two patients who may have had one Urinary tract candidiasis, and a small amount of Candida albicans ATCC 752 was placed in PBS (the 0.05% Tween 20 contained) suspended. 1 ml of this suspension was placed in a silicone-treated test tube (diameter 1.2 cm) and centrifuged at 1500 χ g for 20 min to obtain a cell pellet.

After the cell pellet had been washed twice with PBS containing 0.05% Tween 20, 50 μl of the antibody labeled with iodine-125 according to Example 7 (4) (with a specific activity of 1 μCi / mg antibody) or iodine -125 labeled mouse immunoglobulin (specific activity of 1.5 jjCi / mg protein) added; then left to react at 37 ° C. for 1 hour. After washing 5 times with PBS containing 0.05% Tween 20, the radioactivity was determined by means of a y ^ radiation counter (Beckman Gamma 8500, Beckman, USA). At the same time, the subject cell was transferred to Sabouraud's dextrose agar and incubated at 37 ° C for 5 days. The colony formed was examined morphologically and biochemically.

The results obtained are shown in Table 5 below.

As can be seen from Table 5, the subject cells were bound to the antibodies labeled with iodine-125, which was indicated indicates that these cells corresponded to Candida albicans. The morphological and biochemical carried out simultaneously Studies also confirmed that the subject cells were Candida albicans.

- 64 Table 5

Subject antibody used

Radioactivity (imp./min)

J-labeled J-labeled

CD-1 antibody mouse immunoglobulin

Urine des

Patient F 313 155

Urine des

Patients G 422 147

Candida albicans

ATCC 752 844 172

Example 11; Medicinal effect of the antibody against candidiasis

Candida albicans ATCC 752 (5 ΙΟ ⁶ CFU) was administered intraperitoneally to each group of 15 female, 8 week old Balb / c mice (Nihon Charles Liver, Japan). After 3 weeks, 0.2 ml of the solution of the antibody from the hybridoma cell clones CD-I, CD-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8 or CD -9 (aggregation titer 1: 512) administered intravenously. After a further 2 hours, a lethal dose of Candida albicans ATCC 752 (5 10 ⁷ CFÜ) was injected intravenously, and possible deaths were recorded within the following 14 days.

The results obtained are shown in Table 6 below.

- 65 -

As can be seen from Table 6, all those animals to which the antibody was not administered died ; on the other hand, in the group to which the antibody was administered , some mice survived, indicating the remarkable curative effect of the antibody on infectious diseases caused by fungi belonging to the genus Candida.

Tabel Total number of survivors Control group (not treated)

treated with the antibody from the hybridoma cell clone CD-I

CD-2 CD-3 CD-4 CD-5 CD-6 CD-7 CD-8 CD-9

0/15

5/15 6/15 7/15 4/15 5/15 6/15 4/15 8/15 6/15

* Number of animals which survived the following 14 days following injection of a lethal dose of Candida albicans survived.

34V3339

Example 12;

Enhancement of the phagocyte activity of intraperitoneal macrophages by the antibodies

Female 8 week old Balb / c mice (Nihon Charles Liver, Japan) 1 ml of 0.1% glycogen solution (Kanto Chemical, Japan) was administered intraperitoneally. 4 days later the mice were slaughtered. The cells seeping into the abdomen were treated with RPMI 1640 medium containing 10% fetal bovine serum, washed, collected and centrifuged at 500 χ g for 5 min. The cells were in RPMI 1640, which contained 10% fetal bovine serum, was suspended, and a cell concentration of 1 × 10 cells / ml became set. In each case 1 ml of this suspension was introduced into the wells of a cooling chamber (Lab-Tek Inc., USA). It was left there for 30 minutes at 37 ° C., so that the macrophages in the oozed abdominal cells on the Could stick to the chamber floor. The chamber was then washed with RPMI 1640 to remove non-adherent cells remove. Then 1 ml of RPMI 1640, which also contained 10% fetal bovine serum and Candida albicans ATCC 752 in a cell concentration of 1 χ 10 cells / ml was added; the reaction was then allowed to take place at 37 ° C. for 2 hours. At the same time, the antibodies were added that were derived from the hybridoma cell clones CD-I, CD-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8, or CD-9 were obtained. After the chamber was washed with PBS (pH 7.2), the Cells fixed with 1.0% formalin solution. The number of cell-killing macrophages was measured using the May-Grünwald staining determined.

The results obtained are listed in Table 7 below. Under "cell-destroying ratio" the ratio of the cell-destroying macrophages to the total number of macrophages is understood.

As can be seen from Table 7, the addition of the antibodies approximated the cell killing ratio 14 to 23% increased.

Table 7

"cell-killing ratio" (%)

Control group (not treated) 25

Treated with the antibody out

the hybridoma cell clone CD-I 42

CD-2 45

CD-3 40

CD-4 42

CD-5 48

CD-6 41

CD-7 42

CD-8 39

CD-9 43

Example 13;

The antibody content was reduced by adding distilled water to the purified ascitic supernatant the hybridoma cell clones CD-I, CD-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8, or CD-9 adjusted to 10.4 mg / ml; the solution obtained thereafter was 13.0 mg / ml amphoterizine B-dimethyl sulfoxide solution and hydrochloric acid added with stirring to adjust the pH of the solution to 4.75; were at the same time 3.7 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride added. After a reaction time of 1, 3 or 4 hours

Tabel

Anti-fungal agents antibody Coupling ratio 3 h (jig / mq) 1 h 2.0 4 h from CD-I IfO 1.8 2.5 from CD-2 0.8 2.0 2.8 from CD-3 1.0 1.5 2.5 Amphotericin B from CD-4 1.0 2.3 2.3 from CD-5 1.0 1.5 3.0 from CD-6 1.0 1.5 2.5 from CD-7 1.3 2.0 2.8 from CD-8 1.0 2.0 2.5 from CD-9 1.0 2.5

5-fluorocytosine

from CD-I 0.8 1.8 2.5 from CD-2 1.0 2.0 2.8 from CD-3 1.0 2.0 3.3 from CD-4 1.3 1.5 2.8 from CD-5 1.0 1.8 2.5 from CD-6 1.0 1.5 2.5 from CD-7 1.0 1.5 2.5 from CD-8 1.0 1.5 2.3 from CD-9 0.8 1.3 2.5

Example 14:

It was the healing effect of the coupling products obtained according to Example 13 from anti-fungal agents and antibodies against Infectious diseases checked. For this purpose, 5 × 10 cells of Candida albicans ATCC 752 female, 8 weeks old Balb / c mice (Nihon Charles Liver, Japan) (15 animals per test group) administered intravenously. 1, 24 or 48 h

the reaction was stopped by adding 2 ml of acetate buffer solution (pH 4.70). The reaction mixture obtained was then dialyzed against 5 L of distilled water at 4 ° C. for 72 hours, the dialysis solution being exchanged 3 times. After the dialysate had been concentrated, the reaction mixture was applied to a column (diameter 1.5 cm, height 55 cm, filled with Sephadex G-25 (Pharmacia, Sweden)) and the low molecular weight substances were completely removed. The remaining solution was freeze-dried at -20 ⁰ C, to obtain a amphotericin B antibody coupling product. The proportions of amphotericin B contained in the coupling product per mg of antibody are listed in Table 8 below.

The coupling products of 5-fluorocytosine with those mentioned Antibodies were made in the same way. The proportions of 5-fluorocytosine contained in the coupling products per mg of antibody are also listed in Table 8.

The coupling products of these antifungal agents with the antibodies were given to ICR mice (10 animals per test group) administered; Specifically, the animals in a test group were administered 2 g / kg orally; the animals of the others Experimental group was given 400 mg / kg intraperitoneally; and the animals of the third experimental group were given 200 mg / kg administered intravenously. No death was found in any of the test groups within the following 14 days.

After this injection, the amphotericin B antibody coupling product obtained according to the example after a reaction time of 4 hours was given to the animals administered intravenously or the S-fluorocytosine-antibody coupling product intraperitoneally administered. For comparison purposes, the compounds amphotericin B and 5-fluorocytosine were administered intravenously and orally, respectively administered. Each animal received the coupling product in an amount of 5 µg in terms of amphotericin or of 20 pg based on 5-fluorocytosine.

The results obtained are shown in Table 9 below.

As can be seen from Table 9, show the invention Coupling products of anti-fungal agents and antibodies have a clear, pronounced healing effect on Candida albicans caused infectious diseases.

3V13339

Table 9

Survivors * / total number

O / 4 / 8th / 7 / 9 / 10 / 8th / 9 / 7 / 8th / 8th / '15 '15 '15 '15 '15 ^f 10 '15 '15 '15 '15 '15

Control group (untreated)

Amphotericin B 5 pg (i.V.)

Coupling product

Amphotericin B + antibodies from CD-I (i.v.)

from CD-2 from CD-3 from CD-4 from CD-5 from CD-6 from CD-7 from CD-8 from CD-9

5-fluorocytosine 2000 µg (p.o.) 3/15

Coupling product

5-fluorocytosine + antibody from CD-I (i.p.) 7/15

from CD-2 6/15

from CD-3 5/15

from CD-4 7/15

from CD-5 8/15

from CD-6 6/15

from CD-7 6/15

from CD-8 7/15

from CD-9 6/15

* Number of animals which the following 14 days

survived the injection of Candida albicans.

Claims (1)

Antibodies directed against fungi of the genus Candida Patent claims: A method of making an antigen against the surface antigen of a fungus belonging to the genus Candida, directed against an antibody, wherein a hybridoma cell clone is created between cells that are able to produce the antibody and those cells that can be can keep permanently in an in vitro subculture, and the antibody from that secreted by the hybridoma cell clone Secretion is isolated. 2 - The method according to claim 1, characterized in that the cell producing the antibody is selected from spleen cells, from lymph node cells, from leukocyte cells from peripheral cells Blood or a mixture of these cells. 3. The method according to claim 2, characterized in that the cells are from a mouse. 4. The method of claim 3,
characterized in that
a Balb / c mouse or a hybrid mouse thereof serves as the mouse. 5. The method according to claim 1, characterized in that that cell that is permanently in an in vitro subculture can hold is a myeloma cell. 6. The method according to claim 1, characterized in that that cell that can be kept permanently in an in vitro subculture is deficient in hypoxanthine guanine phosphoribosyltransferase or on thymidine kinase. 7. The method according to claim 5 or 6, characterized in that the cell is from a mouse. 8. The method according to claim 7, characterized in that the cell is derived from one of the following cell lines, namely P3-X63-Ag8, P3-NSI / 1-Ag4-1, 3p2 / 0-Agl4 or X63-Ag8.653. 9. The method according to claim 1, characterized in that the cell producing the antibody comes from an animal that has been immunized with the fungus. 10. The method according to claim 1, characterized in that the hybridoma cell clone is generated in the presence of polyethylene glycol will. 11. The method according to claim 1, characterized in that the hybridoma cell clone is cultivated in a medium containing hypoxanthine, aminopterin and thymidine for selection. 12. The method according to claim 1, characterized in that the hybridoma cell clone has been cloned to obtain a single clone. 13. The method according to claim 12, characterized in that the cloning has been carried out by cultivation in limited dilution, by cultivation on soft agar or by cultivation on fibrin gel. 14. The method according to claim 1, characterized in that the hybridoma cell clone is cultivated in vitro in order to secrete the antibody. 15. The method according to claim 1, characterized in that the hybridoma cell clone is transplanted onto or into an animal and cultivated in vivo, and the excreted antibody from the body fluid of the Animal is isolated. 16. The method according to claim 15, characterized in that serum or ascites fluid is used as the body fluid. 17. The method according to claim 15 or 16, characterized in that a mouse is used as the animal. 18. The method according to claim 17, characterized in that a Balb / c mouse or a hybrid mouse thereof is used as the mouse. 19. The method according to any one of claims 1 to 18, characterized in that a strain of Candida albicans is used as the fungus. 20. A hybridoma cell clone that produces an antibody that against the surface antigen of a belonging to the genus Candida Fungus is directed, which hybridoma cell clone is obtainable from a cell that able to form the antibody and from such a cell that can be kept permanently in an in vitro subculture. 21. The hybridoma cell clone according to claim 20, characterized by the designation CD-I, CD-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8 or CD-9. 22. An antibody raised against the surface antigen of a The fungus belonging to the genus Candida, which antibody is obtainable by the method according to claim 1 is. 23. The antibody according to claim 22, characterized in that the antibody from one of the following hybridoma cell clones, namely CD-I, CD-2, CD-3, CD-4, CD-5, CD-6, CD-7, CD-8 or CD-9 has been eliminated. 24. The antibody directed against the surface antigen of the Candida albicans strain,
characterized in that this antibody by one of the following hybridoma cell clones, namely CD-I, CD-4, CD-6 or CD-8 has been eliminated. 25. The one against the surface antigen of a strain Candida albicans, Candida tropicalis, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida pseudotropicalis-directed antibodies, characterized in that this antibody by one of the following hybridoma cell clones, namely CD-2, CD-5, CD-7 or CD-9 has been eliminated. 26. A reagent for the classification and / or identification of a fungus belonging to the genus Candida,
characterized in that this reagent contains one or more antibodies according to claim 22. 27. The reagent according to claim 26, characterized in that this reagent is also a carrier or a diluent contains. - ο - 28. A reagent used to classify and / or identify a strain of Candida albicans,
characterized in that this reagent contains one or more antibodies according to claim 24. 29. The reagent according to claim 26 or 28, characterized in that this reagent for the identification of candidiasis in humans or animal can be used. 30. A derivative or a restriction product of the antibody according to claim 22. 31. The derivative of claim 30, characterized in that to the antibody a radioactive substance, a fluorescent dye, an enzyme, a marker for electron microscopy, or a residual group is chemically bonded, which contains a structure for the secondary combination of the substances mentioned. 32. The restriction product according to claim 30, characterized in that this product by restrictive cleavage of the antibody by the action of a chemical substance or by treatment with enzymes has been received. 33. The derivative according to claim 31, characterized in that the radioactive substance is iodine-125. 34. The derivative of claim 31,
characterized in that
the fluorescent dye is fluorescein or rhodamine. 35. The derivative according to claim 31, characterized in that the enzyme peroxidase, alkaline phosphatase or ß-galactosidase is. 36. The derivative according to claim 31, characterized in that
the structure is biotin. 37. A reagent for the classification and / or identification of a fungus belonging to the genus Candida, characterized in that the reagent contains one or more derivative (s) or restriction product (s) according to claim 30. 38. The reagent according to claim 37, characterized in that this reagent is also a carrier or a diluent contains. 39. A method for the classification and / or identification of a fungus belonging to the genus Candida, characterized by the use of the reagent according to claim 26 or 37. 40. The method according to claim 39, characterized in that the reagent is brought into contact with a subject containing the fungus, and the agglomeration is checked. 41. The method according to claim 39, characterized in that the reagent is brought into contact with a subject containing the fungus, and the antigen-antibody reaction is applied. 42. The method of claim 40 or 41, characterized in that the subject is from a human or an animal. 43. An effective remedy for candidiasis, characterized in that it contains one or more antibodies according to claim 22 as active ingredient. 44. The remedy according to claim 43, characterized in that it further contains a carrier or a diluent. 45. A combination preparation, characterized in that it contains one or more antibodies according to claim 22 and an antifungal agent. 46. The combination preparation according to claim 45, characterized in that the antifungal agent is amphotericin B or 5-fluorocytosine. 47. An effective remedy for candidiasis, characterized in that it contains one or more combination preparations according to claim 45 as active ingredient.