JP2004258024A - Dermatophyte detection method, reagent for dermatophyte detection, and antigenicity activation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new means for diagnosing and identifying dermatophytes in a dermatophyte detection method and an antigenicity activating method. <P>SOLUTION: In the dermatophyte detection method, an antibody, having reactivity with three or more types of dermatophytes, such as Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, and Epidermophyton floccosum. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a method for detecting dermatophytes and a method for activating antigens, and can provide a novel means for diagnosing and identifying dermatophytes using the present method.

  Athlete's foot is a disease caused by infection / proliferation of dermatophytes collectively referred to as Trichophyton in the keratinized layer of the skin. Dermatophytes are mainly a generic term for the genus Trichophyton. For example, it has been reported that Trichophyton rubrum (T. rubrum, hereinafter T. rubrum) and Trichophyton mentagrophytes (T. mentagrophytes, below T. mentagrophytes) account for about 99% of invasive athlete's patients. See Non-Patent Document 1.) At present, there are about 10 types of dermatophytes causing athlete's foot in Japan. rubrum, T.R. In addition to mentagrophytes, dermatophytes other than the genus Trichophyton such as Microsporum canis (hereinafter referred to as M. canis) and Epidermophyton floccosum (hereinafter referred to as E. floccosum) are also included. In addition, M.P. canis, T .; In recent years, the number of cases of ringworm caused by mentagrophytes infection has been increasing.

  Athlete's foot causes inflammation and severe itching in infected areas. About 10% of dermatological outpatients are occupied by athlete's foot. The infected part of the bacterium is mainly between the toes, but it also infects the genital area and body, and is called tinea pubis or tinea corporis. As for frequency, the foot accounts for 80% of the whole.

  Diagnosis of athlete's foot is conventionally performed by dissolving a keratinized layer collected from a patient's skin with a 10 to 20% potassium hydroxide solution and confirming mycelia under a microscope. For further identification, morphological observation is performed after culturing at room temperature or 30 ° C. for 2 to 3 weeks on an appropriate medium, for example, chloramphenicol-added Sabouraud glucose agar medium.

  In the field of testing, many testing methods, testing means, and enrichment methods have been devised and used by applying the property of an antibody to specifically bind to an antigen. For example, (1) a method in which an antigen-specific antibody is allowed to act on a tissue section fixed with formalin or the like, and the bound antibody is detected to examine antigen localization in a tissue (immunohistological staining) (for example, Non-Patent Document 2, Non-Patent Document 2) (Refer to Reference 5.) (2) A method in which a specific antibody is immobilized on blood cells or latex particles and the presence or absence of an antigen in a sample is determined by the presence or absence of aggregation (agglutination method) (for example, Non-Patent Document 6), and (3) a specific antibody The immobilized insoluble carrier is allowed to react with a liquid sample containing an antigen for a certain period of time, B / F-separated, and further reacted with another labeled antibody for a certain period of time to detect a formed labeled antibody-antigen-immobilized antibody complex (Sandwich immunoassay) (for example, refer to Non-Patent Document 3) (4) Transferring a measurement object-labeled antibody complex reacted with a labeled antibody on a movable and expandable porous carrier to the porous carrier Development For detecting a labeled antibody-antigen-immobilized antibody complex formed and captured by the immobilized antibody layer provided in (Immunochromatography method) (for example, see Patent Document 1) (5) Specific to test sample One that reacts with the antibody-immobilized particle carrier to concentrate and capture bacteria that bind to the antibody at one position on the particle carrier (for example, see Patent Document 2). (6) Test sample and specific antibody-immobilized particle carrier After concentrating and capturing the bacteria that bind to the antibody at a position on the particle carrier, separating the insoluble solid support from the liquid phase, isolating the gene from the pathogenic microorganism captured by the insoluble solid support A method for detecting a pathogenic fungus in which the gene is amplified and then the amplified gene is detected (for example, see Patent Document 7) is known.

  In addition, various antibodies against fungi such as Candida, Aspergillus, and Saccharomyces used for the analysis have been produced (for example, see Patent Documents 3 to 5). Antibodies that specifically recognize (1 → 3) -β-D-glucan expressed on the fungal cell wall of Candida and Aspergillus have also been produced (for example, see Patent Document 6).

  A monoclonal antibody relating to dermatophytes has been disclosed (for example, see Non-Patent Document 3). According to this, a monoclonal antibody was prepared using Trichophyton tonsurans allergen I as an antigen, and an inhibition immunoassay and a sandwich immunoassay using two different monoclonal antibodies were examined. Not only Trichophyton tonsurans (hereinafter, T. tonsurans) but also T. tonsurans. rubrum and T. It is described that cross-reaction with mentagrophytes was observed. Similarly, T.S. A monoclonal antibody was prepared using Protein IV purified from the C. tonsurans extract as an antigen. tonsurans as well as T.T. rubrum and T. It is disclosed that a protein IV component that reacts in the medium filtrate of Mentagrophytes was found, but was not found in skin test extracts such as Candida albicans and Aspergillus mix (for example, See Non-Patent Document 4.).

JP-A-2000-35429 U.S. Pat. No. 5,695,946 JP-A-59-186925 JP 59-186926 A JP 07-250676 A Japanese Patent Publication No. 08-806858 JP-A-11-142409 The Epidemiological Investigation Committee of the Japanese Society for Medical Mycology, “Results of Epidemiological Survey of Dermatomycosis 1992”, Journal of the Japanese Society for Medical Mycology, 1995, Vol. 87-95 Fukui Saburo ed., "Monoclonal Antibody" (Issue, March 10, 1986, first edition), Kodansha Scientific, p. 78 Deuell et al., "J. Immunol.", 1991, Vol. 147, p. 96-101 Woodfold et al., "J. Immunol.", 1996, vol. 156, p. 1695-1701 Edited by Hiroshi Nakura et al., "Revised 4th Edition Watanabe and Nakane Enzyme Antibody Method", (published February 18, 2002), Interdisciplinary project Makoto Uchikawa, "2. Agglutination (1) Hemagglutination, passive (indirect) agglutination", Inspection and Technology, 1994, Vol. 22, (5) Extra Number, 1994, pp. 38-41.

It is considered that patients often use commercially available drugs, judging that they are athlete's foot due to the pruritus between the toes and the formation of blisters. However, athlete's foot is not the only disease that causes such pruritus or inflammation between the toes. For example, atopic dermatitis also causes symptoms similar to athlete's foot. Importantly, the treatment of such diseases is quite different from the treatment of athlete's foot. For this reason, inflammation continues due to treatment based on the wrong self-diagnosis, and in many cases it is difficult to cure.
Although a method that can easily distinguish athlete's foot or other diseases is desired, there is no good method for simple detection of tinea causative bacteria, despite the fact that tinea is extremely ill in patients with dermatology as described above. . The conventional method of dissolving the keratinized layer collected from the patient's skin with a potassium hydroxide solution and checking the mycelium under a microscope is a method that can be inspected at a facility equipped with a microscope and a heating alcohol lamp However, it requires skill and sometimes mistakenly discriminates mycelium from dust and misses the bacteria by microscopic examination. The culture test method is a method that is rarely overlooked, but has a drawback that it takes one to four weeks until a result is obtained. As a method for detecting athlete's foot, Japanese Patent Application Laid-Open No. 2001-187750 describes a method for detecting a site of infection with athlete's foot using a copper sulfate solution.
An object of the present invention is to provide a simple, highly sensitive and simple means for detecting dermatophytes.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, immunized mice with Trichophyton rubrum allergen as an antigen, fused spleen cells with a mouse myeloma cell line to prepare hybridomas, and used them as immunogens. Screening for antibodies that reacted and various dermatophytes, rubrum, T.R. mentagrophytes as well as M. mentagrophytes. canis, E .; A monoclonal antibody having reactivity with flocsum was also obtained, and its presence was found for the first time. More surprisingly, the present inventors have found that the monoclonal antibody can be applied not only to the immunostaining method but also to a single type of sandwich immunoassay, and can detect dermatophytes with high sensitivity in a short period of time.

That is, the present invention relates to the following (1) to (11).
(1) A method for detecting dermatophytes, which comprises using an antibody reactive with three or more dermatophytes shown below.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum
(2) A method for detecting dermatophytes, which comprises using an antibody reactive with the following four dermatophytes.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum
(3) The antibody (1) or (2), wherein the antibody used is one kind of anti-dermatophyte monoclonal antibody or a combination of two or more kinds of anti-dermatophyte monoclonal antibodies having the same antigen recognition site. )).
(4) The method for detecting a dermatophyte according to any one of the above (1) to (3), wherein the antibody is an antibody produced by a hybridoma deposited under accession number FERM P-19057.
(5) A reagent for detecting dermatophytes, which comprises using an antibody reactive with three or more dermatophytes shown below.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum
(6) The reagent for detecting a dermatophyte according to the above (5), wherein an antibody having reactivity with the following four dermatophytes is used.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum
(7) The antibody (5) or (6), wherein the antibody used is one kind of anti-dermatophyte monoclonal antibody or a combination of two or more kinds of anti-dermatophyte monoclonal antibodies having the same antigen recognition site. ). A reagent for detecting dermatophytes according to the above).
(8) The reagent for detecting a dermatophyte according to any one of the above (5) to (7), wherein the antibody is an antibody produced by a hybridoma deposited under accession number FERM P-19057.
(9) A method for activating antigenicity, which comprises subjecting a sample to enzyme treatment or heat treatment in advance in the detection of dermatophytes using an antibody.
(10) The method for activating antigenicity according to the above (9), wherein the antibody is an antibody produced by a hybridoma deposited under accession number FERM P-19057.
(11) The method for activating antigenicity according to any one of (9) and (10), wherein the dermatophyte detection method is an immunostaining method.

The method for detecting a dermatophyte according to the present invention uses a novel antibody that reacts with various dermatophytes. It took several weeks for existing Trichophyton cultivation and separation, but the separation method of the present invention enables specific enrichment of Trichophyton-causing bacteria in a sample. Higher sensitivity detection is possible by combining the enrichment means and each detection method.
In addition, the method for detecting dermatophytes of the present invention has wide specificity to a wide variety of tinea causative bacteria, so that bacteria can be detected only by detecting the presence or absence of a substance that binds to the antibody. Shorter detection is possible compared to the isolation culture test that took several weeks to form a colony for morphological confirmation.
Furthermore, the method for detecting dermatophytes of the present invention enables bacterial-specific visualization detection. Further, the antigenicity activation method of the present invention makes it possible to enhance the presence of bacteria as a clearer signal and to detect it. The present invention provides a method useful for studying the infection process of tinea-causing bacteria to the skin and methods of preventing infection, diagnosing tinea in humans as well as animals, and detecting dermatophytes in living environments and clothing.

  The dermatophytes described in the present invention are the causative bacteria of ringworm. The form of the bacterium may be any form, including some spores, hyphae, conidia and other bacterial fragments, as well as fungal proteins, polysaccharides, and water-soluble suspensions. Species, for example Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Trichophyton violaceum, Trichophyton equinum, Trichophyton glabrum, Trichophyton shoenleinii, Microsporum canis, Microsporum gypseum, Epidermophyton floccosum, Arthroderma vanbreuseghemii, Arthroderma simii, like Arthroderma benhamiae.

  The dermatophytes T. described in the present invention. rubrum, T.R. mentagrophytes, M .; canis, E .; Flocosum may be separated and identified from patients and living environment, or may be obtained from public institutions. Public institutions include, for example, the American Type Culture Collection (ATCC) and the National Institute of Technology and Evaluation / Biological Resource Center. To increase these bacteria, culture them in an appropriate medium. The medium used for the culture may be a Sabouraud-glucose agar medium generally used for dermatophytes, or may be a medium described in a culture method disclosed by a public institution. The cultivation temperature is 10 to 40 ° C, preferably 20 to 30 ° C, and it is desirable that the cultivation is carried out by allowing the mixture to stand in an aerobic environment. Bacterial species can be identified by known separation and identification methods, for example, mold separation, culture and identification. Shunichi Udagawa, Tetsuo Muroi / Translation 1983 Medical and Dental Medicine, Genetic Diagnosis of Mycosis Koichi Makimura / Ed. Medical Sense 1997, Toda Shinbacteria Gaku Amano Kazunobu / Minamijima Yoichi / ed. Nanzando 1997, Journal of Clinical Microbiology, Vol. 36, No. 9, p. 2629-2633 Makimura Koichi et al., 1998.

  The antibody described in the present invention may be a polyclonal antibody or a monoclonal antibody, but it is preferable to use a monoclonal antibody because a lot difference or the like hardly occurs. As the monoclonal antibody, for example, an antibody produced by hybridoma 0014 deposited as FERM P-19057 at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary can be used.

  The immunizing antigen and the screening antigen used for preparing the antibody can be obtained by preparing an aqueous suspension using the cells obtained by culturing the above strain. The liquid used for suspension is not particularly limited, but a buffer having a pH of 5 to 9 is preferable. Before or after suspension, an appropriate amount of a stabilizer such as glycerol or phenol may be added. Suspension may be performed by physically shaking the cells in an appropriate test tube to disperse them.However, the cells are forcibly forced by a method using a homogenizer or a conventionally known method such as ultrasonic crushing or bead crushing. It may be crushed. The obtained suspension may be subjected to immunization as it is, or a suspension prepared by filtration with an appropriate mesh filter or centrifugation may be used. A commercially available product such as T. cerevisiae may be used if it is an aqueous suspension of the above bacteria obtained by the same preparation method. rubrum allergen and T. rubrum. Mentagrophytes allergens may be used as antigens.

  The antibody used in the detection / separation method of the present invention can be prepared by a known antibody preparation method. Known monoclonal antibody preparation methods for monoclonal antibodies For example, monoclonal antibody experiment manual Sakuni Tomita and Tamie Ando / Ed. Kodansha Scientific 1987, Handbook of Immune Research Methods Daimi Fujiwara and Junji Yodoi / Ed. Chugai Medical Company 1996, Tissue Culture Technology [3rd Edition] Application Edition Japanese Society for Tissue Culture / Edited by Asakura Shoten 1999 can be prepared according to the method described in more detail. More specifically, it can be prepared by the following method and procedure. .

  As a immunized animal used for immunization of an animal, a mammal used for a known hybridoma production method can be used. Specifically, for example, mice, rats, goats, sheep, cows, horses and the like are used. However, from the viewpoint of availability of myeloma cells to be fused with the extracted antibody-producing cells, mice and rats are preferably used as immunized animals. The strains of mice and rats actually used are not particularly limited. In the case of mice, for example, each strain A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C57BR, C57L, DBA, FL, HTH, HT-1, LP, NZB, NZW, RF, RIII, SJL, SWR, WB, 129, etc., and in the case of rats, for example, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer Etc. can be used. Among them, considering the applicability of fusion with myeloma cells described later, the BALB / c strain in mice and the low strain in rats are particularly desirable as animals to be immunized. The age at the time of immunization of these mice or rats is preferably 5 to 12 weeks.

  The animal can be immunized by administering the bacterial suspension, which is an immunogen, to the animal intradermally, intraperitoneally, or in vivo (in vivo immunization). The administration schedule varies depending on the type of the immunized animal and individual differences, but in general, the antigen administration frequency is preferably 2 to 6 times, and the administration interval is preferably 1 to 2 weeks. The dose of the antigen varies depending on the type of animal, individual differences, and the like. It is generally said that the dose is about 10 to 100 μg / animal / time, but it is also possible to carry out immunization by changing the dose and select an animal to be immunized with the highest antibody titer in serum or plasma. When administering, it may be administered together with an immune activator called an adjuvant. For example, adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, CpG DNA, muramyl dipeptide, lipopolysaccharide, and the like. In addition, as an animal immunization method, a method called in vitro immunization instead of the above-mentioned method, specifically, after aseptically removing spleen cells or lymph cells in advance, adjuvant substance, immunogen together with animal culture medium It may be carried out by culturing in the presence of carbon dioxide for 2 to 7 days.

  At the time of cell fusion, in the case of in vivo immunization, spleen cells or lymph cells containing antibody-producing cells are aseptically removed from the immunized animal 1 to 5 days after the last immunization day of the above antigen administration schedule. Isolation of antibody-producing cells from these spleen cells or lymph cells can be performed according to a known method. In the case of in vitro immunization, cells cultured with the immunogen can be subsequently used.

  The above antibody-producing cells and myeloma cells are used for cell fusion. The myeloma cells are not particularly limited, and can be appropriately selected from known cell lines and used. However, in view of the convenience in selecting hybridomas from the fused cells, it is preferable to use a HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) -deficient strain for which the selection procedure has been established. That is, mouse-derived X63-Ag8 (X63), NS1-Ag4 / 1 (NS-1), P3X63-Ag8. U1 (P3U1), X63-Ag8.653 (X633.653), SP2 / 0-Ag14 (SP2 / 0), MPC11-45.6TG1.7 (45.6TG), F0, S149 / 5XX0, BU. 210. Rat-derived 210. RSY3. Ag. Human-derived U266AR (SK0-007), GM1500 · GTG-A12 (GM1500), UC729-6, LICR-LOW-Hmy2 (Hmy2), 8226AR / NIP4-1 (NP41) such as 1.2.3 (Y3) And so on.

  The fusion of the antibody-producing cell and the myeloma cell can be appropriately performed according to a known method under conditions that do not extremely reduce the cell viability. As such a method, for example, a chemical method of mixing antibody-producing cells and myeloma cells in a high-concentration polymer solution such as polyethylene glycol or a physical method using electrical stimulation can be used.

  It is preferable to select a fused cell and a non-fused cell by, for example, a known HAT (hypoxanthine / aminopterin / thymidine) selection method. This method is effective when obtaining fusion cells using myeloma cells of an HGPRT-deficient strain that cannot survive in the presence of aminopterin. That is, by culturing unfused cells and fused cells in a HAT medium, only fused cells having aminopterin resistance can be selectively left and grown. As the HAT medium or a medium used for cell culture such as cloning described below, a known medium may be used, and for example, RPMI1640, DMEM, eRDF, IMDM and the like can be used. At the same time, proteins such as animal serum, growth factors, conditioned media, antibiotics and the like may be added, but when cloning by limiting dilution or the like, it is preferable to add them in combination. For example, 1-20% fetal bovine serum may be added for animal serum, and for growth factors, for example, IL-6, insulin, ethanolamine, selenium, 2 mercaptoethanol, pyruvic acid, and non-essential amino acids may be added. It may be added, if it is a condition medium, for example, 5% to 20% of the culture supernatant after thymocyte culture, or if it is an antibiotic, for example, gentamicin, kanamycin, streptomycin, penicillin. Alternatively, if it is a protein, bovine serum albumin, casein, and transferrin may be added. The temperature at which the cells are cultured may be any temperature at which the cells proliferate, and may be, for example, 37 ° C. When sodium bicarbonate is added to the medium, it is preferable to perform cultivation in the presence of carbon dioxide gas, for example, at 5% to 10% carbon dioxide gas, which makes the pH of the medium neutral.

  Screening of hybridoma cells producing the desired monoclonal antibody can be performed by known enzyme immunoassays (EIA: Enzyme Immunoassay), radioimmunoassays (RIA: Radio Immunoassay), fluorescent antibody method, immunostaining method, and the like. it can.

  When screening is performed by EIA or RIA, an antigen used for screening may be an aqueous suspension of bacteria used as an immunogen or an allergen solution. The immobilization of the antigen on the solid phase is carried out according to a known method. For example, a bacterial suspension diluted with Dulbecco's phosphate physiological buffer (PBS (-)) or 0.05 M carbonate buffer is used at a temperature of 2 to 40 ° C. For 30 minutes to overnight, or may be covalently bonded to a functional group on the surface of the solid phase using a crosslinking reagent such as glutaraldehyde or carbodiimide. Known materials can be used for the solid phase, and examples thereof include polystyrene, glass, silica, cellulose, polyvinyl, nylon, nitrocellulose, and PVDF. After binding the antigen, it is preferable to carry out a blocking step of bringing a solution containing another protein, a surfactant, a polymer, or serum into contact with the solid phase in order to suppress nonspecific protein adsorption. Examples of proteins used for blocking include bovine serum albumin, casein, gelatin, and ovalbumin. A 0.1 to 10% solution can be prepared and used. Examples of surfactants and polymers used for blocking include sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate (TWEEN20), and polyvinyl alcohol. A 0.01 to 0.5% solution is prepared and used. it can. Serum used for blocking includes, for example, fetal calf serum, newborn calf serum, horse serum, goat serum, rabbit serum, rat serum, guinea pig serum, porcine serum, and mouse serum, and a 0.5% to 100% solution is used. can do. When using serum or protein for blocking, it is preferable to select and use in consideration of the cross-specificity of the immunized animal and the secondary antibody for specific antibody detection used for screening, for example, when screening a mouse monoclonal antibody-producing hybridoma. Is preferably performed by blocking with a protein-containing solution not derived from a mouse, such as a bovine serum albumin solution, and detecting using a secondary antibody showing no or little cross-reactivity with the bovine antibody. The blocking can be performed, for example, in the range of 2 ° C to 40 ° C. The time can be, for example, from 30 minutes to 1 day, but an appropriate preservative, for example, 0.05% sodium azide may be added to the solution used for blocking, and the solution may be stored as it is after blocking.

  Specimens used for screening by the fluorescent antibody method or the immunostaining method can be slide glasses in which bacteria are smeared in advance, fixed, washed with water, and further blocked and washed as necessary. Alternatively, the cultured bacterial mass can be embedded in folimarin-fixed paraffin without applying the bacteria, and a tissue specimen section can be prepared by a known method.

  Screening of antibody-producing hybridomas having reactivity to a plurality of types of bacteria can be performed by detecting the presence or absence of production of an antibody that reacts with each of the bacteria to be confirmed.

  For example, if screening is performed by RIA or EIA, T.I. rubrum, T.R. mentagrophytes, M .; canis, E .; A solid phase in which various bacterial antigens of floccosum were immobilized alone was prepared, the same hybridoma culture supernatant sample appropriately diluted was reacted with the same solid phase, and after B / F separation, a labeled secondary antibody was further reacted. Alternatively, a labeled secondary antibody remaining on the solid phase after B / F separation may be detected, the reactivity between the antigens may be compared, and those having confirmed reactivity with three or more types of bacteria may be selected as candidates. . Also, for example, T.I. rubrum, T.R. mentagrophytes, M .; canis, E .; An antigen prepared by immobilizing one or more species antigens of Floccosum is prepared, the same hybridoma culture supernatant sample appropriately diluted is reacted with the same solid phase, and after B / F separation, further labeled secondary The antibody is reacted, and the labeled secondary antibody remaining on the solid phase after B / F separation is detected. Those whose reactivity has been confirmed are selected as first screening candidates, and the culture supernatant of the selected population is then used as the first screening candidate. T. was used as a sample by a method such as EIA, RIA, fluorescent antibody method or immunostaining method. rubrum, T.R. mentagrophytes, M .; canis, E .; The secondary screening may be performed by examining the reactivity with floccosum. The primary screening and the secondary screening may be performed successively, or the cloning may be performed after the primary screening, and the secondary screening may be performed after forming a single population of cells.

  For example, if screening is performed by an immunostaining method, T.I. rubrum, T.R. mentagrophytes, M .; canis, E .; Specimens prepared by immobilizing various bacteria of floccosum alone were prepared, and the same hybridoma culture supernatant sample appropriately diluted was reacted with the same sample for a certain period of time. After washing, the enzyme-labeled secondary antibody was further reacted, and further washed. After that, the presence or absence of the labeled secondary antibody remaining on the specimen is compared with the stainability on the specimen by contacting the enzyme substrate, which is a chromogen, on the specimen. Those that have been confirmed may be selected as candidates.

  The hybridoma cells selected by the screening are cloned by a known method such as a methylcellulose method, a soft agarose method, a limiting dilution method, and used for antibody production.

  The hybridoma cells obtained by the above method can be stored in a frozen state in liquid nitrogen or a freezer at -80 ° C or lower. The cell concentration during cell freezing is preferably in the range of 1 × 10 6 / ml to 1 × 10 7 / ml, and 5 to 10% (v / v) dimethyl sulfoxide may be added to the medium as a freezing stabilizer.

  The desired monoclonal antibody can be obtained by culturing the hybridoma cells prepared by the above-mentioned method by a known method as the antibody used in the detection / separation method of the present invention. For example, in the case of a hybridoma prepared by immunizing a mouse, if the cells are transplanted into the peritoneal cavity of a mouse to which mineral oil such as pristane has been administered in advance, ascites containing a monoclonal antibody can be obtained in 1 to 3 weeks. For example, when the hybridoma is cultured in a medium, the monoclonal antibody is secreted from the hybridoma, and a culture supernatant containing the antibody is obtained. If necessary, a monoclonal antibody with higher purity can be obtained from the ascites fluid or culture supernatant containing the antibody by a known antibody purification technique.

Further, the antibody used in the detection / separation method of the present invention is a trait obtained by obtaining a gene or a gene fragment encoding the antibody expressed in the above-described hybridoma by a known technique, and incorporating an expression vector by a known gene recombination technique. By culturing the transformant, a desired monoclonal antibody can be obtained. When the antibody is accumulated in the transformant due to the production of the antibody, the transformant is recovered and crushed by a known method to obtain a crushed liquid containing the antibody. When the antibody is secreted into the medium in which the transformant is cultured by the production of the antibody, a culture supernatant containing the antibody is obtained by culturing the transformant. If necessary, a crushed solution or culture supernatant containing the antibody can be used to obtain a monoclonal antibody with higher purity by a known antibody purification method.
In addition, the antibody used for the detection / separation method obtains a gene or a gene fragment encoding the antibody expressed in the above-described hybridoma by a known technique, and encodes a protein portion having a binding activity from the antibody gene or the gene fragment. Messenger RNA can be synthesized by a known method, and an antibody can be produced by a known cell-free protein synthesis method. By producing the antibody, a protein synthesis reaction solution containing the antibody is obtained. If necessary, a monoclonal antibody having a higher purity can be obtained from the protein synthesis reaction solution containing the antibody by a known antibody purification method.

  Examples of the antibody purification method include ammonium sulfate salting out and column chromatography. Examples of resin carriers that can be used for column chromatography include anion-exchange resins, protein A, protein G, affinity chromatography resins such as Protein L, hydroxyapatite resins, hydrophobic adsorbing resins such as hydrophobic chromatography resins, cross-linked dextran resins and agarose resins. For example, a gel filtration carrier utilizing the fact that the mobility in a resin differs depending on the molecular weight is exemplified. Before performing these purification operations, it is desirable to examine the subtype of the monoclonal antibody and select an appropriate purification means. The purified antibody is preferably subjected to buffer exchange with a pH-neutral buffer by a method such as dialysis. As the pH-neutral buffer, for example, a phosphate buffer solution (PBS (-)) having a physiological salt concentration or physiological saline is used. Tris buffer (TBS) with a target salt concentration can be used. The pH of the buffer is preferably between 5.5 and 8.5.

  The monoclonal antibody thus obtained can be stored in a solution state or a frozen state. When stored in a liquid state, it is preferable to add a preservative, or to filter the solution with a sterile filter having a mesh of 0.22 μm or the like, and then store it in a sterilized container. As a preservative, for example, 0.05% sodium azide can be added. The container may be sterilized by a known method, and examples thereof include γ-ray irradiation sterilization, UV irradiation sterilization, autoclave sterilization, and ethylene oxide gas sterilization. As the storage container, one using an inert material that does not react with the antibody during storage can be used, and examples thereof include glass, polyethylene, and polypropylene. If the method does not impair the antibody binding activity, a container which has been subjected to a surface treatment so as not to be adsorbed on the surface of the storage container may be used. The storage temperature of the liquid may be any temperature that does not freeze, but is preferably as low as possible to suppress protein denaturation, and more preferably 2 to 10 ° C. When an anti-freezing agent is added at a concentration that can prevent freezing, it can be stored at a lower temperature. For example, when glycerol having a final concentration of 50% is added, it can be stored in a liquid state without freezing even at −20 ° C. . When stored in a frozen state, it is preferable to store at -20 ° C or lower, more preferably at -30 ° C or lower. The antibody concentration during storage can be set at a concentration that does not cause precipitation, but is preferably 0.1 to 5 mg / ml. In addition, proteins, water-soluble polymer polymers, surfactants, saccharides, and sugar alcohols can be added as stabilizers. Examples of the stabilizing protein include bovine serum albumin and gelatin, which can be added at a concentration that does not cause precipitation during storage. The concentration range is more preferably 0.1 to 5 mg / ml.

  The monoclonal antibody used in the present invention may be used with some modification, or may be used without modification. Modification refers to the binding of another substance to the monoclonal antibody molecule. The bond includes a covalent bond, an ionic bond, a physical bond, a coordination bond, and a hydrogen bond. A known labeling substance can be used as the substance to be bound, and the substance can be bound by a known method. It is also possible to introduce a new functional group or active group into the antibody, and then perform modification. Examples of the substance to be bound include a fluorescent substance, a luminescent substance, a protein, and a compound. Examples of the fluorescent substance include fluorescein-4-isothiocyanate (CAS No. 3326-32-7). Examples of the luminescent substance include acridinium esters. Proteins include, for example, avidin, streptavidin, anti-mouse immunoglobulin G antibody, protein A, protein G, protein L, horseradish peroxidase, alkaline phosphatase, β-galactosidase, amylase, luciferase, glucose oxidase, glucose dehydrogenase, catalase, green fluoroescent Protein (GFP), a photoprotein derived from Oan jellyfish (aequorin) and the like. The compound is, for example, an N-hydroxysuccinimide ester compound (NHS ester compound: for example, biotin-NHS ester (CAS No. 35013-72-0), biotin-sulfosuccinimide ester (CAS No. 105248-43-9), 5 (6) carboxyfluorescein-NHS ester, digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-NHS ester). In order to introduce a functional group into an antibody, for example, when introducing a sulfhydryl group, 2-iminothiolane can be introduced by reacting with an amino group on the antibody. When introducing an active group into an antibody, a divalent crosslinking agent can be used. For example, when introducing a maleimide group, N- (6-maleimidocaproyloxy) succinimide (CAS No. 55750-63-5) is used. ), N- (6-maleimidocaproyloxy) sulfosuccinimide (CAS No. 103848-61-9) can be used. When used without modification, it may be used after adjusting to an appropriate concentration in a buffer having a known composition, or may be used as it is in the state of the above-mentioned storage solution.

Further, the monoclonal antibody used in the present invention can be used in a form bound to a carrier. As the carrier, known materials can be used, and examples thereof include polystyrene, glass, silica, cellulose, polyvinyl, nylon, nitrocellulose, PVDF, magnetite, gold, platinum, quartz, lipid, protein, and latex. The antibody is immobilized according to a known method, for example, by diluting an antibody solution diluted with Dulbecco's phosphate buffer (PBS (-)) or 0.05 M carbonate buffer at 2 to 40 ° C for 30 minutes to overnight. It may be brought into contact with a solid phase, or may be covalently bonded to a functional group on the surface of the solid phase using a crosslinking reagent such as glutaraldehyde or carbodiimide. Known carrier shapes can be used. For example, 96-well ELISA plates, silica beads, polystyrene beads, membranes for immunochromatography, glass fibers, porous membranes, magnetic beads, latex particles, dextran particles, agarose particles, colloidal gold, blood cells Cell, virus, liposome, quartz oscillator, gold thin film and the like. These carriers may be colored if necessary.
After binding the antibody to these carriers, a blocking step of bringing a solution containing another protein, a surfactant, a polymer, serum, or the like into contact with the solid phase may be performed in order to suppress nonspecific protein adsorption. Examples of proteins used for blocking include bovine serum albumin, casein, gelatin, and ovalbumin. A 0.1 to 10% solution can be prepared and used. Examples of surfactants and polymers used for blocking include sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate (TWEEN20), and polyvinyl alcohol. A 0.01 to 0.5% solution is prepared and used. it can. Serum used for blocking includes, for example, fetal calf serum, newborn calf serum, horse serum, goat serum, rabbit serum, rat serum, guinea pig serum, porcine serum, and mouse serum, and a 0.5% to 100% solution is used. can do.

  The detection of the dermatophyte of the present invention can be carried out by using an antibody reactive with all of Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, and Epidermophyton floccosum in combination with a known method.

  As the sample used in the method for detecting dermatophytes described in the present invention, a solid sample or a liquid sample can be used as long as the activity of the antibody used is not significantly inhibited. For example, a liquid sample is a sample containing 50% (V / V) or more of water and showing a liquid form at 20 ° C., and a sample of less than 50% can be used after being diluted with an appropriate diluent. Even when the water content is less than 50% (V / V), it can be used for detection in some cases, but generally, the activity of the antibody is often not retained, which is not preferable. On the other hand, a solid sample is a sample that exhibits a solid shape at a normal temperature of 20 ° C. In the case of a solid sample, the antibody can directly act, but it can be used as a liquid sample by immersing or suspending it in a liquid, or by collecting the liquid after applying the liquid and extracting and suspending the bacteria in the liquid. Further, a treatment for denaturing and degrading the sample can be performed by a method that does not significantly reduce the binding between the antibody and the bacterium after another solid is brought into contact with the test sample. The denaturation / decomposition treatment includes, for example, physical crushing using a glass bead homogenizer, sonication, freeze crushing, heat treatment, cell wall lytic enzyme treatment, proteolytic enzyme treatment, surfactant treatment, organic solvent treatment, etc. And some of them can be used in combination. Alternatively, the sample may be added to the separation medium, cultured for a certain period of time under known conditions, and then a liquid medium or an agar medium may be used as the test sample. Examples of solid samples include nails, skin pieces, scales, scalp, dandruff, hair, body hair, animal nails, body hair, skin, food, dust, dust, shoes, insoles, slippers, sandals, clogs, boots, gauze, bandages , Tweezers, combs, scissors, mats for wiping feet, towels, handkerchiefs, hats, socks, tabi, underwear, gloves, stockings, duvets, cushions, cushions, sheets, blankets, rags, snowboards, floors, tatami mats, carpets, vacuum cleaners Dust collected, clothes washing machine washing tub, clothes dryer drum filter debris, bath tub, wash basin, porous membrane trapped in air by air sampler, porous membrane trapped in liquid by water sampler And a filter after sample filtration. Examples of the liquid sample include body fluids such as sweat and blood, shampoo washing solution after scalp washing, bath water, washing water, rag washing solution after rag cleaning, and washing solution after foot washing. Alternatively, the solid sample or the liquid sample may be preliminarily brought into contact with a carrier to which no antibody is bound, and after holding some or all of the sample components, the carrier may be sampled. As the carrier, for example, a slide specimen in which bacteria grown on an agar medium are spread on a slide glass, a slide specimen in which a horimarin-fixed paraffin-embedded specimen is sliced and fixed on the slide glass, and a liquid sample is subjected to nitrocellulose membrane or glass A porous membrane in which bacteria are retained by passing through a porous membrane such as a fiber, a well in which a liquid sample is dispensed and adsorbed on an unused ELISA plate well in which proteins and the like are not adsorbed, a sock in contact with a ringworm affected area, Gauze that wipes the affected area of tinea.

In one embodiment of the method for detecting dermatophytes described in the present invention, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Epidermophyton floccum, and the dermatophytes in the test sample are all conjugated to an antibody reactive with all of the dermatophytes in the test sample. And separating from the test sample. When the test sample is brought into contact with the antibody, the reaction is performed in a liquid. The pH in the liquid may be any pH that does not significantly hinder the reaction between the antibody and the bacterium, and is more preferably in the range of 5 to 9. The temperature in the liquid may be any temperature that does not significantly hinder the reaction between the antibody and the bacterium, and is preferably 4 to 45 ° C. The antibody used for the separation may be brought into contact with the liquid sample in a state where the antibody is bound to the carrier in advance. For example, a device having a configuration equivalent to the immunoimmunochromatographic device described in JP-A-2001-208756 can be used, and is specific to a sample electrophoresis body having at least a sample introduction part and a dermatophyte capture part and a dermatophyte to be detected. As long as it contains an antibody capable of binding to as a component, it can be used. In addition, for example, beads in which an antibody that specifically binds to dermatophytes is bound to magnetic beads can be used.After contacting with a liquid sample for a sufficient time, magnetic beads are collected using a magnet, and the liquid sample and bacteria are separated. can do. For example, a 96-well ELISA plate well made of polystyrene to which an antibody that specifically binds to a dermatophyte can be used. After contacting with a liquid sample for a sufficient time, bacteria bound to a carrier can be recovered from the carrier by removing the liquid sample from the carrier. It can be removed and separated. The antibody to be used for separation may be allowed to bind to a carrier after the sample and the antibody are brought into contact for a sufficient time, and in this case, a carrier having affinity for the antibody is used. For example, when a mouse monoclonal antibody (subtype IgG1) against dermatophytes is used, a carrier to which an anti-mouse IgG antibody is bound can be used. For example, when a mouse monoclonal antibody against dermatophytes to which biotin is bound is used, a carrier to which a protein capable of binding biotin (eg, avidin, streptavidin, anti-biotin antibody) can be used. When using these methods, the ratio of the anti-dermatophyte monoclonal antibody to the carrier to be used and the amount of the carrier, the capture time, and the reaction temperature are preliminarily examined by experiments and the like. It is desirable to set the reaction conditions so that the binding to the carrier is preferably 90% or more.
For subsequent detection after separation, known detection methods can be used, and examples include culture tests, immunoassays, detection of bacterial nucleic acids, amplification of bacterial nucleic acids, and identification by sequencing. As a detection means, for example, cultivation is carried out by a known separation medium culture method, and the presence / form of the formed bacterial colony or the metabolite of the bacterial can be detected to detect the presence of the dermatophyte in the sample. As a method for observing the presence or form of the bacterium, it can be determined, for example, by visual observation or microscopic observation. Alternatively, the culture image can be collected by a digital image collection device such as a CCD camera and determined by known numerical processing. As the detecting means, for example, a method described in JP-A-11-142409 can be used. Using a nucleic acid extraction from a sample obtained by culturing the isolated bacterium directly or by enriching the isolated bacterium by a known culture method, a nucleic acid portion having a known dermatophyte nucleic acid sequence in the nucleic acid extracted sample is subjected to a known nucleic acid amplification method. The dermatophytes can be identified and detected from the homology of the nucleic acid sequences by analyzing the nucleic acid sequence of the nucleic acid fragment that has been amplified and detected or the presence or absence of the amplified nucleic acid fragment is compared with a known database. It is desired that the medium used for these separation cultures does not proliferate other than the dermatophytes derived from the sample during the culture. For example, it is preferable to use the medium after previously performing a known sterilization or aseptic treatment. More preferably, a known antibiotic or antibacterial agent that is suitable for separation in a medium used for separation culture, and more specifically does not affect the growth of dermatophytes but is known to inhibit the growth of bacteria. Is used at an appropriate concentration. Examples of the antibiotic include chloramphenicol, cycloheximide, chlortetracycline, gentamicin, streptomycin, penicillin and the like. Examples of the antibacterial agent include an isothiazoline compound. One or more antibiotics and antibacterial agents can be added to the medium as needed. The addition amount can be set with reference to a known minimum inhibitory concentration of bacteria, and is preferably set within a range of 1/5 to 10 times the minimum inhibitory concentration in consideration of drug solubility and drug stability. Further, it is desirable to use an antibody-immobilized carrier used for these separations, a buffer solution used for washing the carrier after the separation, and an antibody that has been sterilized or sterilized. As a sterilization method, a known method can be used as long as the antibody and the carrier are not denatured and deteriorated or their amount is not reduced. In the case of a carrier, γ-ray sterilization, electron beam sterilization and the like are preferable. It is preferable to use a buffer solution after removing microorganisms by filter filtration or autoclave.

  In one embodiment of the method for detecting a dermatophyte described in the present invention, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Epidermophyton flocsum, and an antibody having a reactivity with all of the dermatophytes in the test sample, Detecting the presence or absence of the antibody bound to the antibody.

  When the test sample is brought into contact with the antibody, the reaction is performed in a liquid. The pH in the liquid may be any pH that does not significantly hinder the binding reaction between the antibody and the bacterium, and is more preferably in the range of 5 to 9. The reaction temperature may be any temperature that does not significantly hinder the binding reaction between the antibody and the bacterium, and is preferably 4 to 45 ° C. The reaction time is desirably preliminarily examined and set in accordance with the method to be used and the target detection sensitivity. In consideration of workability, the examination reaction time zone may be set within the range of 1 minute to 1500 minutes. In order to obtain reproducible results repeatedly for each detection, it is desirable to carry out the reaction in an environment in which the pH of the reaction solution, the reaction temperature, and the reaction time are controlled to be the same. After contacting the test sample with the antibody, the antibody that has not bound to the bacteria can be separated as needed from the bound antibody. Known immunoassay methods can be used to detect the presence or absence of the antibody bound to the bacterium, for example, immunostaining, fluorescent antibody, flow cytometer, counting immunoassay, sandwich immunoassay, competition, immunochromatography , Coagulation method, quartz crystal microbalance method, surface plasmon resonance method and the like.

  When the presence of bacteria in the liquid sample is detected by the sandwich 2 antibody method, the detection can be performed by a known sandwich 2 antibody method, for example, the first antibody is immobilized on a carrier, and the liquid sample is contacted with the carrier. To separate the dermatophytes, remove the unreacted sample by washing, contact the labeled antibody-bound antibody as a second antibody with the carrier for a certain period of time, and then wash the unreacted second antibody. And directly or indirectly detect the amount of the labeling substance corresponding to the amount of the generated carrier-antibody-bacterium-labeled antibody complex, and finally determine the presence or absence of the bacterium. For example, if the labeling substance is a fluorescent substance, the fluorescence intensity generated by the excitation light may be measured, if the labeling substance is a luminescent substance, the emission intensity may be measured, and if the labeling substance is radioactive iodine, the radioactivity may be measured. If the labeling substance is an enzyme, the amount of the product obtained by the action of the enzyme substrate may be detected. If the labeling substance does not have a signal by itself, a carrier-antibody- is prepared using a second labeling substance in which another labeling substance capable of generating a signal is bound to a substance capable of specifically binding to the labeling substance. A bacterium-labeled antibody-second labeling substance complex may be generated, and the signal of the second labeling substance may be detected. In the detection method according to the present invention, the carrier-side antibody and the label-side antibody used in the sandwich 2 antibody method may be the same anti-dermatophyte antibody or a combination of two or more anti-dermatophyte antibodies having different antigen recognition sites. It may be a combination of two or more anti-dermatophyte antibodies having the same antigen recognition site. Two or more antibodies having the same antigen-recognition site are combinations of antibodies with different primary structures that prevent one antibody from binding to the other antibody when these antibodies simultaneously act on an antigenic substance. For example, a combination of antibodies that recognize the same antigen site in antibodies produced from different hybridoma clones corresponds to the above. When the detection is performed by the competitive method, the detection can be performed by a known competitive measurement method.For example, the first antibody is immobilized on a carrier, and the bacterial antigen in the sample and the antigen bound to the labeling substance are simultaneously or sequentially contacted with the antibody-bound carrier. Then, the unbound sample or the antigen bound to the labeling substance is removed by washing, and the amount of the labeling substance corresponding to the amount of the generated carrier-antibody-labeled antigen complex is directly or indirectly measured. Can be determined.

  For example, the detection of bacteria on or in a solid such as skin scale tissue, the test solid as it is, or after enrichment of the solid by a known separation medium culture method, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Epidermophyton floc It can be detected by a known immunostaining method using an antibody having the property. Depending on the sample, immunostaining can be performed after fixing by a known fixing method that does not affect the binding between the antibody and the bacterium, such as formalin treatment. It is preferable to carry out the sample activation treatment since the result of the staining becomes clear if it is carried out before the immunostaining.

  Further, for example, detection of bacteria on a solid such as skin scale tissue or in a solid is performed simultaneously with or after a treatment of denaturing or degrading the solid, and bacteria are extracted from the sample into a liquid, and the liquid is subjected to Trichophyton rubrum, Trichophyton mentagrophytes, It can be detected by an immunoassay using an antibody reactive with all of Microsporum canis and Epidermophyton floccosum. The treatment for denaturing / decomposing a solid is, for example, a known method that can be applied to the skin if it is a method that destroys the tissue but does not significantly reduce the subsequent binding of antibodies to bacteria, such as a glass bead homogenizer. Physical crushing used, ultrasonic crushing, French press treatment, freeze crushing, heat treatment, cell wall lysing enzyme treatment, proteolytic enzyme treatment, surfactant treatment, organic solvent treatment, crushing treatment using osmotic pressure change, etc. Yes, and some can be used in combination. The sample may be used for detection after culture enrichment by a known separation medium culture method in advance.

  By using the dermatophyte detection method using the antibody of the present invention, a detection reagent for detecting dermatophytes can be produced, and such a detection reagent is also one of the present invention. . As such a detection reagent, for example, an agglutination detection reagent containing gelatin particles or latex particles bound with a monoclonal antibody used in the detection method of the present invention can be produced by a commonly known method. it can. Similarly, an enzyme immunoassay (EIA) reagent, an ELISA reagent, and a radioimmunoassay (RIA) reagent are used as solid phases, for example, polymers such as polystyrene, glass beads, magnetic particles, microplates, and immunochromatography. By using a filter paper, a glass filter, or the like, it can be produced by an ordinary known method.

  In the method of activating antigenicity in the detection of dermatophytes using the antibody of the present invention, a sample is subjected to an enzyme treatment or a heat treatment in advance. Enzymes used in the enzyme treatment are proteolytic enzymes and filamentous fungus cell wall lysing enzymes, and examples thereof include Pronase TM, trypsin, proteinase K, and yatalase TM (a filamentous fungus lysing enzyme having chitinase and chitobiase activities). The enzyme treatment time is changed by changing the conditions depending on the concentration of the enzyme used, the pH of the reaction solution, and the reaction temperature. For example, when a nail sample is stained by immunostaining, 0.1% (W / V) of trypsin is used for 30 minutes at a trypsin concentration of 0.05 M Tris-HCl buffer, pH 7.6 at 37 ° C., and 2% (for yatalase). (W / V) Yalase concentration 0.05M Maleate buffer pH 5.5 Performing the treatment at 37 ° C. for 30 minutes allows the stained image to be clearer than the untreated one. The buffer used for the enzyme treatment is not limited to the above-mentioned conditions, and can be carried out using PBS (-) or TBS. A known heating apparatus can be used for the heat treatment, and examples thereof include a microwave oven (microwave irradiation), an autoclave, a pressure cooker, a water bath, a steamer, and a hot plate. At the time of heating, it is preferable to perform the treatment in a buffer so as not to dry the sample. For example, a known buffer such as 0.01 M citrate buffer pH 6.0 or 0.01 M citrate buffer pH 7.0 may be used. it can. The microwave irradiation in a microwave oven is performed while adding a buffer solution on the way so as not to dry the sample. For example, irradiation can be performed at 500 W output for 10 minutes while adding a buffer solution every 5 minutes.

Hereinafter, the present invention will be described in detail and specifically with reference to examples, but the present invention is not limited to these examples.
Example 1: Detection of dermatophytes by immunostaining method (1) Culture of dermatophytes: Trichophyton rubrum (IFO No. 9185, 32409) and Trichophyton mentagrophytes (IFO No.) distributed by the Fermentation Research Institute. 6202, 32410), Microsporum canis (IFO No. 32463), and Epidermophyton floccosum (IFO No. 32461) were restored from ampoules by a predetermined method, inoculated on Sabouraud Dextrose Agar Slant (Becton Dickinson), and cultured at room temperature for one week.
(2) Preparation of antigen solution: Trichophyton rubrum allergen (20,000 PNU / ml: 1 PNU (PROTEIN NITROGEN UNIT) refers to proteinaceous nitrogen that produces 1.0 × 10 −6 g of phosphotungstic acid precipitates; Greener Laboratories, Inc.) It was diluted with an acid-buffered saline (hereinafter PBS (-)) to prepare two kinds of 10,000 PNU / ml antigen solution (hereinafter antigen solution H) and 1000 PNU / ml antigen solution (hereinafter antigen solution L).
(3) Immunization of mouse: 500 μL of Freund's complete adjuvant was mixed with 500 μL of each of the above two antigen solutions to form an emulsion, and 200 μL / animal of each BALB / cA ♀ 5 weeks old, 2 mice in total, 4 mice intraperitoneally Injected. (This day is referred to as day 0.) On days 14, 21, and 28, 400 μL of each antigen solution and 400 μL of Freund's incomplete adjuvant were mixed to form an emulsion in the same manner as described above, and 100 μL of the mixture was used as a mouse peritoneal cavity. Was injected.
(4) Acquisition of mouse serum: mice alive on day 32 (one mouse individual (H- # 1) intraperitoneally injected with the emulsion prepared with antigen solution H, intraperitoneally injected with the emulsion prepared from antigen solution L Blood was collected from the tail of two mouse individuals (L- # 1, L- # 2). After leaving the blood at room temperature for 30 minutes, the coagulated clot was separated by centrifugation at 3000 RPM for 10 minutes to obtain serum.
(5) Confirmation of serum antibody titer by ELISA: The collected serum was diluted with PBS (-) from 1000-fold dilution to 128000-fold dilution and used as a sample. Trichophyton rubrum allergen was diluted with PBS (-) to a concentration of 200 PNU / ml, dispensed 50 μL into each well of a 96-well ELISA plate (# 9018 Corning), allowed to stand at room temperature for 1 hour, and immobilized antigen on the plate. It has become. After removing the liquid in the wells, 300 μL of Block Ace (Snow Brand Milk Products) diluted 4 times with distilled water was dispensed into each well and left at room temperature for 1 hour to perform blocking. The liquid in the wells was removed, and then 300 μL of PBS containing 0.05% (w / v) TWEEN 20 (hereinafter referred to as “washing solution”) was dispensed and discarded three times. After washing each well, 50 μL of the sample was dispensed into each well. The reaction with the solid phase antigen was performed for 1 hour at room temperature. The liquid in the well was removed, and 300 μL of a washing solution was repeatedly dispensed and discarded three times to wash each well, and then horseradish peroxidase-labeled anti-mouse immunoglobulin diluted 2000-fold with Block Ace diluted 10-fold with distilled water. Rabbit antibody (DAKO) was dispensed into wells at 50 μL / well and allowed to react at room temperature for 1 hour. The liquid in the wells is removed, and 300 μL of a washing solution is repeatedly dispensed and discarded three times to wash each well, and then a horseradish peroxidase substrate solution containing 3,3′-5,5′-tetramethylbenzidine (hereinafter, TMB). 50 μL of TMB + (DAKO) was dispensed into each well, and the reaction was performed at room temperature under light shielding. Ten minutes after the reaction, 1N sulfuric acid was dispensed into each well in an amount of 50 μL / well to stop the enzymatic reaction, and the absorbance of each well was measured with a microplate reader at a main wavelength of 450 nm and a subwavelength of 650 nm. As a control, the same operation was performed after blocking on a plate on which the antigen was not immobilized. Table 1 shows the measured absorbance.

An antibody titer significantly higher than that of L- # 1 and L- # 2 was observed in the individual H- # 1 injected at 2000 PNU-1000 PNU / time, and cell fusion was performed using the mouse individual H- # 1. . On days 41, 55, 56, and 57, 100 μL of Trichophyton rubrum allergen diluted 2-fold with PBS (−) was injected intraperitoneally to prepare for cell fusion.
(6) Cell fusion: Myeloma cells Sp2 / 0-Ag14 were cultured and passaged in an e-RDF medium (Kyokuto Pharmaceutical) containing 10% (v / v) fetal bovine serum at 37 ° C under 5% carbon dioxide gas concentration. . On the 58th day, the spleen was aseptically removed from the individual H- # 1, and the organ was thoroughly washed in an e-RDF medium. Place the spleen in a 10 cm Petri dish containing 20 ml of e-RDF medium, cut in the tip of the 22-gauge injection needle with the end fixed to the spleen with sterile tweezers in a clean bench, and further remove the spleen cells from the spleen as much as possible. It was extruded aseptically, and the cell mass was well loosened by pipetting. The cell suspension was transferred to a 50 ml centrifuge tube equipped with a cell strainer at the top, followed by centrifugation at 200 × g for 10 minutes, and the supernatant was discarded. 20 ml of e-RDF medium was added, and the precipitated cells were suspended by pipetting. Further, centrifugation, supernatant discard, and cell suspension in the medium were performed once more. When the number of cells was counted using a hemocytometer, it was 1.1 × 108. The cells were washed with e-RDF medium in order to add 1.1 × 107 myeloma cells Sp2 / 0-Ag14, which is 1/10 the amount of the obtained spleen cells. That is, only cells were separated by centrifugation of 20 ml of a 7 × 105 / ml Sp2 / 0-Ag14 cell culture solution cultured in e-RDF medium (Far East Pharmaceutical) containing 10% (v / v) fetal bovine serum at 150 × g for 5 minutes, The supernatant was discarded, 20 ml of e-RDF medium was added, the cells were suspended by pipetting, and the operations of centrifugation, supernatant discard, and cell suspension in the medium were repeated twice, and finally e-RDF medium Was obtained, and Sp2 / 0-Ag14 was obtained. 1.1 × 10 7 Sp2 / 0-Ag14 were added to a 50 ml centrifuge tube containing spleen cells, mixed well by pipetting, and centrifuged at 200 × g for 10 minutes. After discarding the supernatant, tapping the entire tube to loosen the cell mass, then adding 1 ml of 50% polyethylene glycol 1500 solution (Roche) at 37 ° C. and mixing with the cells, and then adding 1 ml, 3 ml, and 10 ml of e-RDF medium. Were added in this order. After centrifugation at 150 × g for 5 minutes, the mixture was allowed to stand at 37 ° C. for 5 minutes, and then the supernatant was discarded. Using a Pasteur pipette, transfer the total amount of cells to 192 ml of selective culture medium (e-RDF medium containing 1/100 volume of 100 × HAT supplement (GIBCO), 1/10 volume of Condimed H1 (Roche), 1/10 volume of fetal calf serum). The suspension was aseptically dispensed into 10 wells of a 96-well floating cell culture plate (manufactured by Sumitomo Bakelite Co., Ltd.) in a volume of 200 μL per well. HAT selective culture was performed for 9 days at 37 ° C. and 5% carbon dioxide.
(7) Primary screening of antibody-producing hybridomas by ELISA: 50 μL of each well culture supernatant after selective culture was diluted with 200 μL of PBS (−) and used as a sample. Trichophyton rubrum allergen was diluted with PBS (-) to a concentration of 200 PNU / ml, dispensed 50 μL into each well of a 96-well ELISA plate (# 9018 Corning), allowed to stand at room temperature for 1 hour, and immobilized antigen on the plate. It has become. Also, Trichiphyton mentagrophytes allergen (20,000 PNU / ml: GREER LABORATORIES, INC.) Was diluted with PBS (−) to 200 PNU / ml, and 50 μL was dispensed into each well of a 96-well ELISA plate (# 9018 Corning). The mixture was allowed to stand at room temperature for 1 hour to immobilize the antigen on the plate. After removing the liquid in the wells, 300 μL of Block Ace (Snow Brand Milk Products) diluted 4 times with distilled water was dispensed into each well and left at room temperature for 1 hour to perform blocking. The liquid in the well was removed, and then 300 μL of the washing solution was repeatedly dispensed and discarded three times to wash each well. After that, the sample was dispensed into wells 50 μL each and reacted with the solid phase antigen for 1 hour at room temperature. The liquid in the wells was removed, and 300 μL of the washing solution was repeatedly dispensed and discarded three times to wash each well, and then horseradish peroxidase-labeled anti-mouse immunoglobulin diluted 2000-fold with Block Ace diluted 10-fold with distilled water. Rabbit antibody (DAKO) was dispensed into wells at 50 μL / well and allowed to react at room temperature for 1 hour. The liquid in the wells was removed, and then 300 μL of a washing solution was repeatedly dispensed and discarded three times to wash each well. Then, horseradish peroxidase substrate solution TMB + (DAKO) containing TMB was dispensed in 50 μL each well, and light was shielded from light at room temperature. The reaction was carried out below. Ten minutes after the reaction, 1N sulfuric acid was dispensed into each well in an amount of 50 μL / well to stop the enzymatic reaction, and the absorbance of each well was measured with a microplate reader at a main wavelength of 450 nm and a subwavelength of 650 nm. As a control, the same operation was performed after blocking on a plate on which the antigen was not immobilized. The dilution of the culture supernatant in 960 wells was measured, and the reactivity with the antigen solid phase was confirmed in 5 wells. Table 2 shows the absorbance by ELISA.

T. wells 0011 and 0014 rubrum antigen; Reactivity was observed with both Mentagrophytes antigens. The two wells were selected as a primary screening, and cloning was subsequently performed.
Cloning of hybridoma: Cloning was performed by the limiting dilution method. That is, the cells in each of the selective culture wells selected by screening were aseptically sampled so as to have 100 cells, and the cloning medium (Condimed H1 (Roche), 1/10 volume, e-RDF medium containing 1/10 volume of fetal calf serum, respectively) was used. ) The suspension was suspended at 5 cells / ml with respect to 20 ml, and aseptically dispensed in a 96-well 96-well floating cell culture plate (manufactured by Sumitomo Bakelite Co., Ltd.) into a 96-well plate. Cultivation was performed at 37 ° C. under 5% carbon dioxide for 10 days. After cloning culture of each well, 50 μL of the supernatant was sampled, diluted with 200 μL of PBS (−) to prepare an ELISA sample, and Trichophyton rubrum allergen, Trichiphyton mentagrophytes allergen, and three solid phases without solid phase antigen were used as in the primary screening. ELISA was performed using rubrum antigen; One well was selected from each of the wells from which the culture supernatant in which reactivity with the Mentagrophytes antigen was recognized was sampled, and hybridoma cells after primary cloning were obtained. Subsequently, the same method was repeated to carry out secondary cloning and tertiary cloning, thereby finally obtaining antibody-producing hybridomas 0011 and 0014. The hybridoma 0014 has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST) as FERM P-19057.
(8) The obtained hybridoma was cultured and subcultured in an e-RDF medium containing 10% (v / v) fetal bovine serum at 37 ° C. under 5% carbon dioxide gas concentration, and the obtained cells were finally 10% ( v / v) 5 × 10 6 cells were aseptically suspended in 1 ml of e-RDF medium supplemented with dimethyl sulfoxide and 10% (v / v) fetal calf serum, and placed in a 2 ml serum tube (Sumitomo Bakelite) for 5 minutes on ice. It was stored at -20 ° C for 50 minutes, at -80 ° C for 12 hours, and finally in liquid nitrogen.
(9) Preparation of each hybridoma culture supernatant: The tube containing hybridomas 0011 and 0014 which had been frozen and stored in liquid nitrogen was stored at -80 ° C for 1 hour, and then quickly thawed in hot water at 37 ° C. The whole amount was put into a centrifuge tube containing 10 ml of e-RDF medium containing 10% (v / v) fetal calf serum, and the cells were suspended by pipetting. After centrifugation at 150 × g for 5 minutes and discarding the supernatant, the cells were suspended by pipetting in 5 ml of e-RDF medium containing 10% (v / v) fetal bovine serum, and each was inoculated in a 25 cm 2 bottom cell suspension cell culture flask. did. After culturing at 37 ° C. under 5% carbon dioxide concentration for 2 passages, the cells were suspended in 15 ml of medium and cultured for 7 days in a flask for floating cell culture with a bottom area of 75 cm 2 to sufficiently secrete the antibody into the medium. The cell culture solution obtained by the culture was transferred to a 50 ml centrifuge tube and centrifuged at 700 × g for 10 minutes to obtain a hybridoma culture supernatant. A 5% (w / v) aqueous solution of sodium azide was added to 1/100 volume of each culture supernatant, filtered through a 0.22 μm filter, dispensed into γ-ray sterilized polypropylene tubes, and frozen at −20 ° C. saved.
(10) Subtyping of monoclonal antibody: Using a commercially available subtyping reagent (Iostrip: Roche) using immunochromatography, 150 μL of culture supernatant dilution diluted 10-fold with PBS (−) was analyzed as a sample. The subtype of the monoclonal antibody contained in the culture supernatant of hybridoma 0011 was IgM κ, and the subtype of the monoclonal antibody contained in the culture supernatant of hybridoma 0014 was IgG1 κ.
(11) Preparation of dermatophyte formalin specimens and smears used for secondary screening: Dermatophyte formalin specimens were prepared as follows. After fixing the bacterial masses of various dermatophytes grown on Sabouraud dextrose agar slant together with the medium for 1 day in a 10% neutral buffered formalin solution, alcohol 70%, 80%, 90%, 100%, 100%, It was immersed in 100% and 100% alcohol tanks for 2 hours each for dehydration, and then immersed in 3 xylene tanks for 2 hours each for clearing. Then, each of the four paraffin tanks was immersed in each of the tanks for 2 hours to allow the paraffin to penetrate, and the lumps of the bacteria and the medium were placed in an embedding dish containing the dissolved paraffin. Then, the slice was cut into 4 μm, and a paraffin specimen fixed with a dermatophyte formalin was prepared on a slide glass.
A smear was prepared as follows. Collect the various dermatophytes grown on the Sabouraud dextrose agar slant with a clean cotton swab slightly moistened with sterile physiological saline to scrape, press against the slide glass, and roll the friction surface with cotton to squeeze it out. It was prepared by spreading and immediately fixing in a 95% ethanol solution for 30 minutes.
(12) Secondary screening of monoclonal antibodies by immunostaining: Formalin-fixed paraffin specimens of dermatophytes were deparaffinized in advance and immunostained. After immersing in 0.3% hydrogen peroxide-added methanol for 30 minutes and washing with water, PBS supplemented with 10% (v / v) normal rabbit serum was dropped on the section and left at room temperature for 30 minutes. After washing with PBS, the hybridoma culture supernatants 0011 and 0014 were diluted 50-fold with PBS and reacted for 1 hour in a room humidity room. After washing with PBS, ENVISION + (Dako) was dropped on the specimen, and reacted for 1 hour in a room humidity room. After washing with PBS, the mixture was washed with PBS, containing 0.02% (w / v) 3,3′-diaminobenzidine (hereinafter referred to as DAB) and 0.003% (w / v) hydrogen peroxide, with occasional microscopic examination. The color was developed separately. After washing with water, dehydration with ethanol and xylene penetration were performed in this order, and sealing was performed using a hydrophobic sealing agent (biolite). Microscopic observation was performed at 10 × 20 × and 10 × 40 ×, and it was determined that a site stained brown or yellow brown was positive, a slightly stained site was weakly positive, and a case where staining was not confirmed was negative. . Tables 3 and 4 show the determination results.

The hybridoma culture supernatants of both 0011 and 0014 were weakly positive and positive in all of the formalin and smear samples examined, and the monoclonal antibodies produced from these hybridomas were Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, and Epidermophycophyte of Episporum sp. All were reactive and detectable by immunostaining.
Example 2: Detection using periodic acid-treated Trichophyton rubrum allergen and Trichiphyton mentagrophytes allergen immobilized on an ELISA plate using monoclonal antibodies Example 1 (6) "Primary screening of antibody-producing hybridomas by ELISA" To the wells of the same Trichophyton rubrum allergen-adsorbed EIA plate or Trichiphyton mentagrophytes allergen-adsorbed EIA plate, 50 μl of a 0.05 M aqueous sodium metaperiodate solution was added, and the mixture was reacted overnight at 4 ° C. to oxidize the sugar contained in the antigen. After washing with PBS, adding Block Ace diluted 4 times with distilled water and performing blocking at room temperature for 1 hour, the same procedure as described in Example 1 (6) "Primary screening of antibody-producing hybridomas by ELISA" was applied. Then, the binding property of each monoclonal antibody-containing culture supernatant was examined (FIGS. 1 and 2). As a result, it was possible to detect the allergen immobilized on the ELISA plate in any of the clones without antigen treatment. However, when the antigen was treated with periodate, the monoclonal antibody 0011 showed reduced binding even when the antigen was treated with periodate. However, the binding was reduced and the detection power of monoclonal antibody 0014 was reduced.

Example 3: Detection of proteolytic enzyme-treated Trichophyton rubrum allergen and Trichiphyton mentagrophytes allergen immobilized on an ELISA plate using monoclonal antibodies. Example 1 (6) "Primary screening of antibody-producing hybridomas by ELISA" 50 μl of 100 μg / ml pronase (Pronase Calbiochem, diluted with 71,000 PUK / g, PBS) was added to the same Trichophyton rubrum allergen or Trichiphyton mentagrophytes allergen well, and reacted at 37 ° C. for 2 hours to decompose the antigen protein. After washing with PBS and adding Block Ace diluted 4 times with distilled water and performing blocking at room temperature for 1 hour, the method described in Example 1 (6) "Primary screening of antibody-producing hybridoma by ELISA" was described. The binding property of each monoclonal antibody-containing culture supernatant was examined in the same manner as described above (FIGS. 3 and 4). As a result, there was no change in the binding of both monoclonal antibodies 0011 and 0014 due to the pronase treatment of the antigen, and the allergen immobilized on the ELISA plate could be detected.

Example 4: Detection of bacteria by sandwich EIA method (1) Preparation of bacterial sample: Dermatophytes Trichophyton rubrum (Tr. Abbreviated IFO No. 9185, 32409), Trichophyton mentagrophytes (TM. Abbreviation IFO No. 6202; 32410), Microsporum canis (abbreviated as Mc, IFO number 32463), Epidermophyton floccosum (abbreviated as Ef IFO number 32461), and Candida Albicans (abbreviated as AT number from ATCC number 90028). The cells were reconstituted, inoculated on Sabouraud dextrose agar slant (Becton Dickinson), and cultured at room temperature for 1 week. Escherichia coli (abbreviated as Ic, IFO number 13500), Streptococcus faecalis (abbreviated as Sf, IFO number 12968), and Bacillus subtilis (abbreviated as IFO number 3026) are L-dried by a predetermined method. Reconstituted from the ampoule, LB agar culture dish (10 g of polypeptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder) dissolved in 1 L of purified water, sterilized by high pressure, and aseptically dispensed and gelled in a 10 cmφ sterilized petri dish. The cells were cultured at 30 ° C. for 1 day.
Each of the obtained bacteria was collected by scraping with a sterilized 10 μL disposable loop, and sufficiently suspended in 5 ml of Block Ace (Snow Brand Milk Products) diluted 10 times with distilled water. A 125,625-fold dilution was used as an EIA sample. T. A sample obtained by diluting the rubrum allergen to 100 PNU / ml in the same manner as above was used in a 5, 25, 125, or 625-fold dilution.
(2) Preparation of 0014 culture supernatant: The tube containing the hybridoma 0014 which had been frozen and stored in liquid nitrogen was stored at -80 ° C for 1 hour, and then immediately thawed in 37 ° C hot water. The whole amount was put into a centrifuge tube containing 10 ml of e-RDF medium containing 10% (v / v) fetal calf serum, and the cells were suspended by pipetting. After centrifugation at 150 × g for 5 minutes and discarding the supernatant, the cells were suspended by pipetting in 5 ml of e-RDF medium containing 10% (v / v) fetal bovine serum, and each was inoculated in a 25 cm 2 bottom cell suspension cell culture flask. Then, the cells were cultured at 37 ° C. under 5% carbon dioxide gas concentration. After culturing in the same culture medium for 2 passages, the medium was changed to CD-Hybridoma Medium (Invitrogen) containing 1.17 g / L of L-glutamine, the medium was changed to acclimation, and further cultured in the same culture medium for 3 passages. After that, the cells were suspended in 15 ml of the medium and subcultured in a flask with a bottom area of 75 cm2 for floating cell culture, then subcultured in 45 ml of medium and subcultured in a flask with a bottom area of 225 cm2, and suspended in 200 ml of the final medium. The cells were cultured in four suspension cell culture flasks having an area of 225 cm 2 for 7 days, and the antibody was sufficiently secreted into the medium. The cell culture solution obtained by the culture was transferred to four 50 ml centrifuge tubes and centrifuged at 700 × g for 10 minutes to obtain 200 ml of a hybridoma culture supernatant.
(3) Preparation of 0014 Purified Antibody: The obtained culture supernatant was dissolved and mixed with 35 g of NaCl and 10 ml of 1M borate buffer, pH 9, and filtered through a 0.8 μm filter. 1 ml of rProtein A Sepharose FastFlow (manufactured by Amersham Biosystems) equilibrated in advance with a 50 mM borate buffer (pH 9: binding buffer) containing 3 M NaCl was packed into the column, and the entire culture supernatant was flowed at a flow rate of 1 ml / min. The solution was passed through the column. Next, the column was washed with 5 ml of the binding buffer, and then 0.1 M citrate buffer (pH 3) was passed at the same flow rate to elute the antibody from the column to obtain 2 ml of an eluate containing the antibody. Immediately, 0.4 ml of 1M Tris buffer (pH 9) was added, and dialysis was performed against 1 L of PBS containing 0.05% (w / v) sodium azide. As a result of calculating the amount of protein by absorption at OD 280 nm, the purified 0014 antibody obtained was 7.2 mg.
(4) Electrophoretic analysis of purified antibody: 2-mercaptoethanol 1 volume 0.1% in 10 volumes of 0.156M Tris-HCl buffer (pH 6.8) containing 5% SDS and 25% (v / v) glycerol (W / v) One volume of bromophenol blue aqueous solution was added to prepare a buffer for sample electrophoresis. 9 μL of purified water and 10 μL of a sample electrophoresis buffer were added to 1 μl of the purified antibody, treated at 95 ° C. for 5 minutes, and then electrophoresed in a 10-20% polyacrylamide gradient end gel. The gel after electrophoresis was subjected to Coomassie staining to visualize proteins in the gel. As a result, only bands corresponding to the heavy and light chains of the antibody were observed, and it was found that other bands due to impurities were not included.
(5) Preparation of biotinylated 0014 antibody: 50 ml of a 1 M sodium bicarbonate solution was added to 0.5 ml of a 2.8 mg / ml antibody solution, mixed, and then dissolved in distilled water to a concentration of 6.7 mg / 100 ml of Biotin-XX. -Sulfosuccinimidyl ester (Dojindo) was added in an amount of 1.5 μL, and the mixture was reacted at room temperature for 4 hours with gentle stirring. The whole amount was loaded on a NAP-5 column (Amersham Bioscience) pre-equilibrated with PBS, and the sample was eluted with 1 ml of PBS to obtain 1.2 mg of biotinylated 0014 antibody.
(6) Measurement of bacterial suspension sample by sandwich EIA: [0014] The purified antibody was diluted to 10 µg / ml with 50 mM carbonate buffer (pH 9.6) and added to each well of a 96-well ELISA plate (# 9018 Corning). , And left at room temperature for 1 hour to immobilize the antibody on the plate. After removing the liquid in the wells, 300 μL of Block Ace (Snow Brand Milk Products) diluted 4 times with distilled water was dispensed into each well and left at room temperature for 1 hour to perform blocking. After the liquid in the well was removed, 300 μL of a washing solution was repeatedly dispensed and discarded three times to wash each well, and then 50 μL of the sample was dispensed into each well and allowed to react with the solid phase antibody for 1 hour at room temperature. The liquid in the well was removed, and then 300 μL of the washing solution was repeatedly dispensed and discarded three times to wash each well, and then 50 μL of biotinylated 0014 antibody diluted to 1 μg / ml with Block Ace diluted 10-fold with distilled water. Each well was dispensed and reacted at room temperature for 1 hour. The liquid in the wells was removed, and then 300 μL of a washing solution was repeatedly dispensed and discarded three times to wash each well, and then, horseradish peroxidase-labeled streptavidin (KPL) diluted 5000-fold with Block Ace diluted 10-fold with distilled water. Was dispensed into wells at 50 μL each and reacted at room temperature for 1 hour. The liquid in the wells is removed, and 300 μL of a washing solution is repeatedly dispensed and discarded four times to wash each well, and then a horseradish peroxidase substrate solution containing 3,3′-5,5′-tetramethylbenzidine (hereinafter, TMB). 50 μL of TMB + (DAKO) was dispensed into each well, and the reaction was performed at room temperature under light shielding. Ten minutes after the reaction, 1N sulfuric acid was dispensed into each well in an amount of 50 μL / well to stop the enzymatic reaction, and the absorbance of each well was measured with a microplate reader at a main wavelength of 450 nm and a subwavelength of 650 nm. The results are shown in Tables 5 and 6.

  Dermatophytes and T. r. When allergen was used as a measurement sample, a response was observed in the dilution series, but not in other bacteria. That is, immunoassays using one kind of anti-dermatophyte monoclonal antibody were able to detect not only Trichophyton rubrum and Trichophyton mentagrophytes, but also dermatophytes of four species, Microsporum canis and Epidermophyton floccosum.

Example 5: Immunostaining of tinea unguium on nails of patients with tinea unguium Nail collected from a patient diagnosed as tinea unguium based on clinical findings is fixed in formalin, embedded in paraffin according to a standard method, sliced into 3 μm, and sliced on a slide glass to prepare tinea unguium paraffin on a slide glass. Specimens were prepared. Immunostaining was carried out by the method described in Example 1 (11), using the purified antibody prepared in Example 4 (3) instead of the hybridoma culture supernatant in 0.1% (w / v) bovine serum albumin, 0.05%. % (W / v) antibody solution prepared to be 100 μg / ml with PBS (−) containing sodium azide was diluted 5, 10, 25, 50, 100, 200, 500, 1000 times with PBS and used. The stained image of the bacterium was compared with other backgrounds. The results are shown in Table 7. (-) Indicates no staining, (±) indicates slight coloring, and (+) <(++) <(++) according to staining intensity, depending on staining intensity. did. It was possible to distinguish the bacteria from the background at an antibody concentration of 1 to 20 μg / ml.

Example 6: Immunostaining after antigen activation treatment using tinea unguium patient nails The tinea unguium paraffin specimen used in Example 5 was deparaffinized, and then 2% (W) in 0.05M maleic acid buffer pH 5.5. / V) in Yatalase TM (Ozeki Shuzo) dissolved at a concentration of 37 ° C. for 30 minutes. After washing with PBS, 100 μg of the purified 0014 antibody was added to PBS (−) containing 0.1% (w / v) bovine serum albumin and 0.05% (w / v) sodium azide in the same manner as in Example 5. The antibody solution prepared so as to be / ml was diluted 10, 25, 50, 100, 200, 500, and 1000 times with PBS, and the stained image of the bacterium was compared with the other background. The results are shown in Table 8. Compared with Example 5, the staining became clear by performing the yatalase treatment at an antibody concentration of 4 to 10 μg / ml.

Example 7: Effect of antigen activation treatment using nails of tinea unguium The paraffin specimen of tinea unguium used in Example 5 was deparaffinized, subjected to the following pretreatment, and then purified by the same method as in Example 5. The antibody was diluted to 4 μg / ml and immunostaining was performed. (1) No pretreatment (2) Immersion in 1% (W / V) sodium metaperiodate solution at room temperature for 10 minutes, followed by washing with PBS (periodic acid treatment) (3) 0.05M Tris-HCl buffer pH7 6. Trypsin (SIGMA) dissolved in 0.1% (W / V) at 37 ° C. for 30 minutes and then washed with PBS (trypsin treatment) (4) 0.01M citrate buffer pre-heated While immersed in pH 6.0, irradiation is performed for a total of 10 minutes while adding a buffer solution every 5 minutes at a power of 500 W in a microwave oven, and then left at room temperature for 30 minutes (microwave treatment).
The results are shown in Table 9. Staining became clear in trypsin treatment and microwave treatment as compared with no treatment.

Example 8: Detection of bacteria by sandwich EIA using scale samples Samples collected from patients diagnosed as tinea pedis from clinical findings (parts where the epidermis has peeled off) and skin pieces collected from feet of healthy subjects One piece of each was placed in a 0.5 ml polypropylene tube, 200 μL of a trypsin solution previously dissolved in PBS and adjusted to 2% (W / V) was added, and the mixture was stirred until the scales were sufficiently contacted with the trypsin solution. It was left at 210 ° C. for enzyme treatment. The tube was then heated in boiling water for 15 minutes, while simultaneously inactivating the enzyme. The tube was centrifuged at 2000 × g for 1 minute to recover 160 μL of the supernatant, which was used as a scale extract. On the other hand, 200 μL of PBS was added to the tube in which the residue after centrifugation remained, and the mixture was stirred well. Then, the tube was centrifuged at 2000 × g for 1 minute to collect 180 μL of the supernatant, which was used as a scale washing solution. Using these four types as samples, analysis was performed according to the sandwich EIA method described in Example 4. Table 10 shows the results of measuring the wavelength of 450 nm with a microplate reader after stopping the enzyme reaction. Signals were observed in the scale extract and scale wash of tinea pedis patients, while only the signals equivalent to the background were observed in scale extract and scale wash of healthy subjects. Conventionally, it was possible to distinguish between tinea pedis patients and healthy subjects using scale samples, which could not be achieved without the use of a microscope, using an immunoassay technique that does not use a microscope.

Example 9: Bacteria detection by sandwich EIA using tinea pedis patient's socks as a sample (1) Preparation of sock washing solution Unused for patient volunteers diagnosed as tinea pedis from clinical findings (3 days after start of antifungal cream treatment) Wear antibacterial socks (cotton, acrylic, nylon, polyurethane material, Biosil TM processing (Approval No. 73292 of the Sanitary and Textile Processing Association), size 24-26 cm), and wear the shoes that patients use everyday. He was engaged in light work including rest, movement, and desk work for 6 hours, and then collected socks. One foot of the sock was immersed in 100 ml of a washing solution (PBS containing 0.05% TWEEN 20), and stirred and washed at room temperature for 10 minutes. After the washing, the sock and the washing liquid were separated to obtain 25 ml of the sock washing liquid. On the other hand, an unused antimicrobial sock was immersed in 100 ml of the washing liquid for one foot, and a non-wearing sock cleaning liquid was prepared by the same operation as the wearing sock.
(2) Preparation of Anti-Skin Filamentous Antibody-Binding Magnetic Beads Activated magnetic beads DynaBeads M-280 Tosylacticated (manufactured by Dinal) 2 × 109 / ml 10 ml were thoroughly stirred, then transferred to a polypropylene 15 ml centrifuge tube, and the centrifuge tube was covered. And set it on a magnetic stand (MagicalTrapper ™ manufactured by Toyobo Co., Ltd.) and let stand for 2 minutes. After confirming that the magnetic beads had aggregated on the side of the centrifuge tube, the supernatant was discarded. Next, 10 ml of 0.1 M phosphate buffer pH 7.4 was dispensed into a centrifuge tube, and the magnetic beads were dispersed by stirring. Then, 6 mg of purified antibody was added, and the centrifuge tube was capped and further stirred. . Subsequently, the mixture was set on an inclined rotating machine installed in a 37 ° C. incubator and left standing for 24 hours while continuously inverting and mixing at low speed together with the centrifuge tube to allow the antibody to bind to the beads. Next, the centrifuge tube was set on a magnetic stand and left for 2 minutes. After confirming that the magnetic beads had aggregated on the side of the centrifuge tube, the supernatant was discarded. 10 ml of 0.1 M phosphate buffer pH 7.4 containing 0.1% (W / V) bovine serum albumin was dispensed into a centrifuge tube, and the magnetic beads were dispersed by stirring. Then, the centrifuge tube is set on a magnetic stand and left for 2 minutes. After confirming that the magnetic beads have aggregated on the side of the centrifuge tube, the supernatant is discarded and further contains 0.1% (W / V) bovine serum albumin. 10 ml of 0.1 M phosphate buffer pH 7.4 was dispensed into a centrifuge tube, and the supernatant was discarded by the same operation. After dispensing 10 ml of 0.2 M Tris buffer pH 8.5 containing 0.1% (W / V) bovine serum albumin and thoroughly stirring, the mixture was set on a tilting rotary machine installed in a 37 ° C. incubator, and the centrifuge tube was set at a low speed. The mixture was allowed to stand for 4 hours while being continuously mixed by inversion to block unreacted tosyl groups on the magnetic beads. Next, the centrifuge tube was set on a magnetic stand and left for 2 minutes. After confirming that the magnetic beads had aggregated on the side of the centrifuge tube, the supernatant was discarded. Finally, 10 ml of 0.1 M phosphate buffer (pH 7.4) containing 0.1% (W / V) bovine serum albumin and 0.05% (W / V) sodium azide was dispensed into a centrifuge tube, and the mixture was stirred. The magnetic beads were dispersed to obtain beads binding to the anti-dermatophyte antibody.
(3) Dispense 10 ml of the sock washing solution prepared in (1) into a 15 ml centrifuge tube for dermatophyte isolation using the sock washing solution as a sample, and then prepare the antibody prepared in (2) so as to obtain anti-dermatophyte antibody binding beads 2 × 108. Bound beads were added. The centrifuge tube was capped and set on an inclined rotating machine, and the centrifuge tube was left at room temperature for 1 hour while continuously mixing by inversion at a low speed. Next, the centrifuge tube was set on a magnetic stand and left for 2 minutes. After confirming that the magnetic beads had aggregated on the side of the centrifuge tube, the supernatant was discarded. 1 ml of the washing solution was dispensed into a centrifuge tube and stirred to disperse the magnetic beads and wash the beads. The obtained suspension of magnetic beads was transferred to a 1.5 ml polypropylene tube, and the tube was set on a magnetic stand and left for 2 minutes. After confirming that the magnetic beads had aggregated on the side of the tube, the supernatant was discarded. 1 ml was dispensed into a tube and stirred to wash the beads. The same washing operation was further repeated once to obtain separated anti-dermatophyte antibody-bound beads suspended in 1 ml of the washing solution. At the same time, the unattached sock washing solution and PBS were used as samples, and separation operations were respectively performed using magnetic beads.
(4) The tube containing the anti-dermatophyte antibody-bound bead suspension that had been subjected to bacterial detection and separation treatment with biotinylated 0014 antibody was set on a magnetic stand and left for 2 minutes, after which the magnetic beads aggregated on the side of the centrifuge tube. Was confirmed and the supernatant was discarded. Then, 500 μL of each biotinylated 0014 antibody diluted to 2 μg / ml with Block Ace diluted 10-fold with distilled water was dispensed, covered and sufficiently stirred to disperse. The mixture was allowed to stand at room temperature for 30 minutes while being continuously inverted at low speed. Thereafter, as a washing operation, the tube was set on a magnetic stand and allowed to stand for 2 minutes. After confirming that the magnetic beads had aggregated on the side of the tube, the supernatant was discarded. Further, 1 ml of a washing solution was dispensed into the tube and stirred to wash the beads. . The same washing operation is repeated twice to wash the beads, the magnetic beads and the supernatant are separated and discarded using a magnetic stand, and horseradish peroxidase-labeled streptose diluted 1000-fold with Block Ace diluted 10-fold with distilled water. 500 μL of avidin (KPL) was dispensed, and the mixture was covered with a lid and sufficiently stirred to disperse the mixture. Then, the mixture was set on an inclined rotating machine and left at room temperature for 30 minutes while continuously inverting and mixing the whole tube at low speed. Thereafter, as a washing operation, the tube was set on a magnetic stand and allowed to stand for 2 minutes. After confirming that the magnetic beads had aggregated on the side of the tube, the supernatant was discarded. Further, 1 ml of a washing solution was dispensed into the tube and stirred to wash the beads. . The beads were washed by repeating the same washing operation three times, and the magnetic beads and the supernatant were separated and discarded using a magnetic stand, and then horseradish peroxidase substrate solution TMB + (DAKO) containing TMB was dispensed at 100 μL each, After sufficiently stirring, the mixture was reacted under light shielding at room temperature. After 10 minutes from the reaction, 100 μL of 1N sulfuric acid was dispensed to stop the enzyme reaction. Next, the tube was set on a magnetic stand and allowed to stand for 2 minutes. After confirming that the magnetic beads had aggregated on the side surface of the tube, the supernatant was recovered and dispensed into unused wells of a polystyrene ELISA plate. The absorbance of each well was measured with a microplate reader at a main wavelength of 450 nm and a subwavelength of 650 nm. Table 11 shows the measured absorbance. Absorbance twice as high as that of the patient's sock washing solution was observed as compared with PBS as a control and no sock washing solution, and dermatophytes in the washing solution could be detected. It was considered that the absorbance was also observed in the PBS sample due to the peroxidase-like activity of the magnetic beads themselves, and it was considered that the S / N ratio could be detected with a higher S / N ratio by using another enzyme detection system such as alkaline phosphatase or β-galactosidase.

Example 10: ELISA analysis after liquid culture of socks washing solution with tinea pedis patient, socks washing solution without wearing, and washing solution (1) Preparation of anti-dermatophyte antibody-bound magnetic beads sample Cystine tinea by the method described in Example 6 (3) The magnetic beads are contacted with 10 ml of the sock washing solution for the patient's socks and non-wearing socks and separated, and the separated beads are washed and suspended in 100 μL of PBS. And
(2) Culture using a liquid medium using each sample 2 ml of sterilized Sabouraud dextrose liquid medium was dispensed into five sterilized 25 cm2 flasks for culture in a clean bench using a liquid medium, and then a sock washing solution for tinea pedis patients was added. 0.05 ml of each of the sock washing solution attached, PBS, the bead washing solution separation beads attached to a tinea pedis patient, and the unmounted sock washing solution separation beads were added, and left at 25 ° C. for 6 days to culture.
(3) Analysis by sandwich EIA Socks washing solution with tinea pedis patient attached, unsocked sock washing solution, culture solution with sock washing solution attached with tinea pedis patient after liquid culture, culture solution without sock washing solution without wearing, culture solution with PBS, wearing tinea pedis patient Using a total of seven types of culture medium containing socks for washing liquid separation beads and a culture medium containing no socks for washing liquid separation beads as samples, and diluting it twice with Block Ace diluted 10 times with distilled water, the sandwich EIA method described in Example 4 was used. The analysis work was performed according to the above. Table 12 shows the results obtained by measuring the main wavelength at 450 nm and the sub-wavelength at 650 nm using a microplate reader after stopping the enzyme reaction. Although the sock washing solution attached to a tinea pedis patient did not show much difference from the non-wearing condition as measured by the sandwich EIA as it was, the signal was enhanced by culturing the sample in a liquid medium. Further, in the case of culturing after separating bacteria from the washing solution sample with the anti-dermatophyte antibody-bound magnetic beads, the difference from the sample not attached was further increased and detected as a clearer difference.

Example 11: Detection of bacteria by sandwich EIA using scale samples whose bacterial mass has been stratified by caustic potassium microscopy A scale keratinized layer collected from a lesion of a patient with tinea pedis was dissolved with a 10% potassium hydroxide solution and examined under a microscope. A scale sample (approximately 10 to 30 mg in weight) collected from a patient whose stratum was small, medium, or large by a method of confirming hyphae (caustic potassium microscopy) was used. As a control, scales of skin lesions in which no bacteria were detected by caustic potassium microscopy were used. According to the method described in Example 8, the scale was treated to prepare a scale extract. That is, one piece of the scale is put into a 1.5 ml polypropylene tube with an O-ring with a screw hole, and 200 μL of a trypsin solution previously dissolved in PBS and adjusted to 2% (W / V) is added. After stirring until the solution came into contact, the mixture was allowed to stand at 37 ° C. for 180 minutes to perform enzyme treatment. The sample tube was then heated in boiling water for 15 minutes, while simultaneously inactivating the enzyme. Then, the tube was centrifuged at 2000 × g for 1 minute, and 160 μL of the supernatant obtained was collected as a scale extract. Using these scale extracts as samples, analysis was performed according to the sandwich EIA method described in Example 4. FIG. 8 shows the results of measurement using a microplate reader after stopping the enzyme reaction with 650 nm as a sub wavelength and 450 nm as a main wavelength. The average OD value increased according to the amount of bacteria observed by microscopic observation, and a correlation was observed. In addition, although specimens showing a lower bacterial density and a lower OD value than the control in the middle classification were observed, although the scale itself was collected from the same patient, it was different from the caustic potassium-scaled scale, so that the scale without chance was used. The possibility of measurement by ELISA was also considered. It was thought that if a plurality of scales were collected and several pieces were used to prepare a scale extract and the same measurement was performed, the detection ability of bacteria could be further improved.

Example 12: Detection of Bacteria by Sandwich EIA Using Nail Samples Stratified by Caustic Potassium Microscopy Part of nail pieces obtained from 10 patients with tinea unguium were nailed with 10% potassium hydroxide solution, respectively. The pieces were dissolved, and the number of bacteria in the nails was stratified into 3 cases, 3 medium cases, and 4 large cases by a method of confirming mycelia under a microscope (caustic potash microscopy) and used as nail sample. . As controls, three cases of healthy human nails in which no bacteria were detected by caustic potash microscopy were used. According to the method described in Example 8, nail pieces were treated to prepare a nail extract. That is, each nail piece is put into a 1.5 ml polypropylene tube with an O-ring with a screw mouth for each patient (21 mg to 125 mg per sample as nail weight), dissolved in PBS in advance to 2% (W / V). After adding 200 μL of the adjusted trypsin solution, stirring was performed until the trypsin solution sufficiently contacted the nail pieces, the mixture was allowed to stand at 37 ° C. for 180 minutes to perform enzyme treatment. The sample tube was then heated in boiling water for 15 minutes, while simultaneously inactivating trypsin. Then, the tube was centrifuged at 2000 × g for 1 minute to collect 160 μL of the supernatant, which was used as a nail extract. Using these nail extracts as samples, analysis was performed according to the sandwich EIA method described in Example 4. FIG. 9 shows the results of measurement using a microplate reader after stopping the enzyme reaction with 650 nm as a sub wavelength and 450 nm as a main wavelength. Compared with the control example, the average of the OD values of the patient groups was higher in all of the low, medium and high bacterial cell types, and the presence or absence of bacteria in the nail sample could be detected by using this sandwich EIA method.

Example 13: Comparison of sandwich EIA analysis in the case of using various enzyme treatments and heat treatment in combination with the same scales of tinea pedis patients, scales collected from patients whose bacterial mass was greatly stratified by caustic potash microscopy The sample was minced with a razor blade to prepare fine scales. Fine scales were put into a 1.5 ml polypropylene tube with an O-ring at the screw opening to a weight of about 10 to 20 mg, and the scale weight was recorded. The enzyme solution used for the enzyme treatment was 1) a pronase solution dissolved in PBS to 2% (W / V) 2) a tissue-staining proteinase K solution (manufactured by DAKO, with an enzyme concentration of 0.4 mg / ml). is there. In accordance with the method described in Example 8, each scale was treated with various enzymes to prepare a scale extract. That is, 200 μL of the enzyme solution was added to the scales placed in a polypropylene tube, stirred until the scales were sufficiently contacted with the enzyme solution, and then left at 37 ° C. for 180 minutes to perform the enzyme treatment. The sample tube was then heated in boiling water for 15 minutes, while simultaneously inactivating the enzyme. Then, the tube was centrifuged at 2000 × g for 1 minute, and 160 μL of the supernatant obtained was collected as a scale extract. Under control conditions where no enzyme treatment was performed, PBS containing no enzyme was added instead of the enzyme solution, and the same operation was performed. Using these scale extracts as samples, analysis was performed according to the sandwich EIA method described in Example 4. Table 13 shows the results of measurement using a microplate reader at 450 nm as the main wavelength after stopping the enzyme reaction. In the case of detection by the sandwich EIA, a sample that had been physically crushed such as being minced with a razor blade could be detected by the sandwich EIA with high sensitivity only by heating regardless of the presence or absence of the enzyme treatment.

Example 14: Antibody staining using slide culture specimens of various Trichophyton fungi Seven species of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton violaceum, Trichophyton viscorum, Trichophyton ponics, Microchips, Microchips, Microchips That is, the plate Sabouraud agar medium was cut into 8 mm pieces, the medium pieces were mounted on a previously sterilized slide glass, and each bacterium was inoculated into the four corners of the medium pieces. The cells were covered with a sterilized cover glass, left in a humidified box, and cultured at room temperature for one week. The slide glass on which the slide culture was performed was fixed with 99.5% ethanol. Further, the agar was fixed in formalin and then embedded in paraffin to prepare a thin section. In the same manner as in Example 5, immunostaining was performed by diluting the purified 0014 antibody to 4 μg / ml. Table 14 shows the staining results. Staining was observed in all of the seven types of Trichophyton used and in ethanol-fixed formalin-fixed methods, and both the hyphae and conidia observed in the slide culture were stained. The 0014 antibody was considered useful for detection of a wide variety of Trichophyton.

  The method for detecting a dermatophyte of the present invention, a reagent for detection and a method for activating antigenicity are described in the field of clinical examination, in which dermatophytes such as tinea unguium and tinea pedis could only be diagnosed using a microscope. (Athlete's foot) is provided with a specific and novel detection method. By this method, information on the presence / absence and quantity of athlete's foot bacteria is obtained, which enables more accurate grasp of the disease state and selection of a treatment method. In addition, development and provision of a test for athlete's foot can be performed, and a large number of samples can be efficiently tested.

The reactivity of a culture supernatant containing a 0011 monoclonal antibody with a periodate-treated antigen-immobilized plate and an untreated antigen-immobilized plate was examined. The reactivity of a culture supernatant containing 0014 monoclonal antibody with a periodate-treated antigen-immobilized plate and an antigen-immobilized plate without treatment was examined. The reactivity of a 0011 monoclonal antibody-containing culture supernatant with a pronase-treated antigen-immobilized plate and an untreated antigen-immobilized plate was examined. The reactivity of a 0011 monoclonal antibody-containing culture supernatant with a pronase-treated antigen-immobilized plate and an untreated antigen-immobilized plate was examined. [Fig. 2] Fig. 2 is an image obtained by immunostaining a smear specimen of Trichophyton rubrum culture bacterium separated from clinical material with a culture supernatant containing 0014 monoclonal antibody, and observing it at 10x40 magnification. FIG. 2 is an image obtained by performing immunostaining on a nail formalin-fixed specimen prepared from a tinea unguium patient-treated culture supernatant with yatalase as an antigen activation treatment using a culture supernatant containing a 0014 monoclonal antibody and observing the sample at 20 × 3 magnification. FIG. 3 is an image obtained by immunostaining a tinea unguium patient's nail formalin-fixed specimen with a culture supernatant containing a 0014 monoclonal antibody without antigen activation treatment and observing the sample at 20 × 3 magnification. 0014 A scale sample extract of a ringworm patient or a non-ringworm patient is measured by a sandwich EIA method using a monoclonal antibody, and the obtained ELISA OD is stratified according to the amount of bacteria in the scale, and the distribution and average value are graphed. . 0014 The nail extract of tinea or non-tinea tinea patients was measured by the sandwich EIA method using a monoclonal antibody, and the obtained ELISA OD was stratified by the amount of bacteria in the nail and the distribution and average were graphed. is there.

Claims (11)

A method for detecting dermatophytes, comprising using an antibody reactive with three or more dermatophytes shown below.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum The method for detecting a dermatophyte according to claim 1, wherein an antibody having reactivity with all four dermatophytes shown below is used.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum   The dermatophyte according to claim 1 or 2, wherein the antibody used is one kind of antidermatophyte antibody or a combination of two or more antidermatophyte monoclonal antibodies having the same antigen recognition site. Detection method.   The method for detecting a dermatophyte according to any one of claims 1 to 3, wherein the antibody is an antibody produced by a hybridoma deposited under accession number FERM P-19057. A reagent for detecting dermatophytes, which comprises an antibody reactive with three or more dermatophytes shown below.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum 6. The reagent for detecting dermatophytes according to claim 5, comprising an antibody reactive with all four dermatophytes shown below.
Trichophyton rubrum
Trichophyton mentagrophytes
Microsporum canis
Epidermophyton floccosum   7. The dermatophyte according to claim 5, wherein the antibody used is one kind of anti-dermatophyte antibody or a combination of two or more anti-dermatophyte monoclonal antibodies having the same antigen recognition site. Reagent for detection.   The reagent for detecting dermatophytes according to any one of claims 5 to 7, wherein the antibody is an antibody produced by a hybridoma deposited under accession number FERM P-19057.   A method for activating antigenicity, comprising subjecting a sample to enzyme treatment or heat treatment in advance in detecting dermatophytes using an antibody.   The method for activating antigenicity according to claim 9, wherein the antibody is an antibody produced by a hybridoma deposited under accession number FERM P-19057.   11. The method for activating antigenicity according to claim 9, wherein the dermatophyte detection method is an immunostaining method.