JPS6210100A - Monoclonal antibody against mold of genus lacto-bacillus - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody active against molds belonging to the genus Lactobacillus to cause deterioration of refined SAKE. USE:A reagent suitable for refined SAKE production, etc., for detecting molds belonging to the genus Lactobabacillus to cause deterioration of refined SAKE, for studying surface structure of cell of the molds and their classification. PREPARATION:For example, Lactobacillus homohiochii RIB8201 strain, etc., a mold to cause deterioration of refined SAKE, is administered to a mammal such as mouse, etc., etc., and the mammal is immunized against it. The lymphocyte collected from the spleen and a myeloma cell are subjected to cell fusion and cultivated in HAT medium, etc., to give a hybridoma to produce an antibody active against the mold to cause deterioration of refined SAKE. Then, the hybridoma is cloned by limiting dilution method, etc., to give a monoclone, which is cultured in a medium and the yielded monoclonal antibody is separated and purified to give a monoclonal antibody against the mold to cause deterioration of refined SAKE.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a monoclonal antibody against Hiotoshi bacteria, which causes "hiotoshi" in sake. The present invention also relates to a method for producing the antibody, a hybridoma used therein, and a method for detecting Hiotochibacterium using the antibody. The monoclonal antibody of the present invention is useful, for example, for detecting Hiotochibacterium in the process control of sake production, and can also be used for bacteriological, immunological, and chemical research on Hiotochibacterium.

The conventional technology, ``hiochi,'' is a phenomenon in which sake becomes turbid, increases in acidity, and changes in the flavor deterioration ring due to the action of bacteria during storage or after being made into a product. It is called bacterial drop.

In recent years, research on "fire-off" has progressed, effective prevention methods such as heat sterilization have been adopted, and the phenomenon of "fire-off" has become less common compared to conventional methods. However, the possibility of this occurring always exists, and recently there has been a trend towards the development of products with lower alcohol content (softened), and there has been a trend towards the development of products with lower alcohol content (softer products), which require heat sterilization, such as low-alcohol raw sake or low-alcohol sake with gas. Some products have been developed that are extremely susceptible to "heat loss", such as products that do not undergo heat sterilization at all or products that are difficult to heat sterilize. Therefore, in the production of sake, prevention of "hiochi" remains an important issue, and detection of the cause of "hiochi" bacteria is an important means of preventing "hiochi" by predicting "hiochi".

On the other hand, currently, a culture method using a Hi-Ochi bacterium detection medium (Sl medium, sold by Japan Brewing Association) is mainly used to detect Hi-Ochi bacterium. However, the detection and determination of the presence or absence of Hiochi bacteria using the culture method can take 5 days to 2 weeks at the earliest, and in some cases more than a month, depending on the bacterial species. There is no known method for detecting Hiotoshi bacteria that can reliably detect Hiotoshi bacteria in a short period of time and serve as a means of preventing Hiotoshi.

Problems to be Solved by the Invention In view of the above circumstances, the present inventors have discovered a method for detecting hiochi bacteria that can reliably detect hiochi bacteria in a short period of time and can serve as a means of preventing ``hiochi''. I did a lot of research to find out. As a result, it was found that it is appropriate to apply an antigen-antibody reaction to such a detection method, and that by applying cell fusion technology that has been established in recent years, it has been found that the detection method has specificity for Hi-Ochibacterium. It has been discovered that a new monoclonal antibody can be obtained.

That is, one object of the present invention is to provide a novel monoclonal antibody that is active against Hi-Ochibacterium.

Another object of the present invention is to provide a method for producing the monoclonal antibody.

Furthermore, another object of the present invention is to provide a novel hybridoma useful for producing the monoclonal antibody.

Furthermore, another object of the present invention is to provide a method for detecting Hiochi bacteria using the monoclonal antibody.

Another object of the present invention is to provide a reagent kit for detecting Hiochi bacteria, which contains the monoclonal antibody as an essential component.

Means for Solving the Problems The monoclonal antibodies of the present invention are monoclonal antibodies of the IgG class produced by hybridomas formed by the fusion of myeloma cells and cells derived from mammalian spleen cells that have been previously immunized against Hirochi. It is an antibody that preferentially reacts with Hiochi bacteria. The Hiotoshi bacteria in the present invention include Lactobacillus homohiothi (L a
cLobacillus shomobiochi i),
Lactobacillus heterohioji
Lactobacillus acidophilus (Lactobacillus acidophilus)
bacillus acidophilus), Lactobacillus fermentum (Lactobacillus
In addition to homozygous and heterozygous deficient lactic acid bacteria such as U.S. rcrmentum, whole or part of cells having cell surface antigens thereof are included. The antibody of the present invention is preferably inactive against lactic acid bacteria that do not have alcohol tolerance, and active against Hiotoshi bacteria.

The monoclonal antibody of the present invention can be used, for example, during tissue culture.
It is produced by culturing a hybridoma secreting the antibody formed by the fusion of myeloma cells and cells from the lung of a mammal previously immunized against Hirochi.

The hybridoma of the present invention that secretes the antibody can be prepared by known techniques. For example, the method described in Nature by Köhler and Milstein (Kol+lcr
, G., &Mi'1sLein, C., Natur.
e.

256, '495-497 (+975)). Myeloma cells useful for the hybridoma preparation include P3-X63-Ag8-Ul and P3-X63-Ag8-Ul of mouse origin.
3-X63-Ag8, P3-NS t/'1-Ag4-
1, SP210-Agl 4, MPCll-45,6,
TGI, 7, X63-Ag8-6゜5.3, FO, S
l 9415XXO, I3u, 1° 210 of rat origin
．． Kazuya CY, Ag1.2.3, 5KO-00 of human origin
Known cell lines include 7,0M15006TG-ΔIt, CEM, and the like.

Spleen cells may be obtained from any mammalian species, and mouse or rat lung cells are typically used. Lung cells are immunized against Hirochi bacteria prior to fusion with myeloma cells. This immunization can be carried out according to a conventional method, for example, by inoculating the spleen cell donor animal with Hi-Ochi bacteria.

The missing strain can be obtained from the National Tax Agency Brewing Research Institute, for example, and typical examples include the rtlB8201 strain and the RI
Examples include B8315i, and these can be used for immunization.

The obtained hybridomas are screened for those that have the ability to produce anti-deficiency antibodies, and cell clones that purify monoclonal antibodies active against Hiochi bacteria are selected by limiting dilution method or soft agar method, and the desired hybridomas are purified. obtain.

Hybridomas can be cultured to obtain the monoclonal antibodies of the present invention using known tissue culture media, and the secreted antibodies are separated from hybridoma cells by conventional methods.

The isolated monoclonal antibody can be prepared using conventional methods such as DEA.
It is purified by chromatography using E-cellulose, gel filtration using Sephadex G-100, 150, 200, etc., affinity chromatography using protein A, etc. At this time, the antibody may be concentrated in advance using ethanol precipitation or ammonium sulfate precipitation, and then purified.

The monoclonal antibody of the present invention reacts with the antigenic determinant of the cell surface antigen of Hi-Ochibacterium, and thus can be used for the detection of Hi-Ochibacterium according to known antigen-antibody reaction methods. If desired, the monoclonal antibodies of the present invention may be conventionally prepared using enzymes such as peroxidase, alkaline phosphase, β-galactosidase, etc., fluorescent dyes such as fluorescein, rhodamine, etc. useful for such antigen-antibody reactions. Labels may be affixed in accordance with the law. Furthermore, a group such as N-hydroxysuccinimide or maleimide containing a structure for secondary bonding of a marker for electron microscopy such as avidin may be chemically bonded.

The monoclonal antibody of the present invention is packaged in a container as a solution such as a buffer solution or a lyophilized product containing it,
If necessary, in combination with other reagents necessary for the antigen-antibody reaction, for example, a secondary antibody when using an unlabeled monoclonal antibody, a substrate for an enzyme when using an enzyme-labeled antibody, etc. It can be a reagent kit.

Thus, by using the monoclonal antibody of the present invention, the antigen-antibody reaction can be achieved in a very short period of time, for example, 2 to 3 days.
It is possible to reliably detect hiochi bacteria and determine their presence or absence within hours.
This makes it possible to properly control the process in sake production, and to accurately predict and prevent "fire fall". In addition, conventionally, it took a long time to detect hiochi bacteria.
In order to meet the requirement of shipping the product to the market promptly after commercialization, it is necessary to
There is a tendency to perform excessive thermal sterilization to ensure safety and meet the demand for quick shipping. However, according to the antigen-antibody reaction using the monoclonal antibody of the present invention, it is possible to quickly confirm sterility at the time of commercialization and then ship the product, making it possible to set an appropriate sterilization temperature, save energy during heat sterilization, and improve quality. It is possible to prevent deterioration and efficiently produce products free of fire-related bacteria. Furthermore, the antigen-antibody reaction using the monoclonal antibody of the present invention can be applied to research on the cell surface structure of Hi-Ochi bacteria, classification of Hi-Ochi bacteria, and other bacteriological, immunological, and chemical studies.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples. (1) Preparation of hybridomas Immunization of mice Antigen Hirochibacillus, Lactobacillus homohioji RII3820!
strain was used as an antigen. For cell culture, pl containing sake (final alcohol concentration 10%), River Digest (manufactured by Oxoid, UK, final concentration 0.5%) and yeast extract (manufactured by Difco, USA, final concentration 0°5%) -1
A liquid medium (SYL medium) of Nos. 4 and 8 was prepared. This medium was inoculated with I'RI[38201 strain, and cultured at 30°C for 2 weeks. After culturing, the bacterial cells were suspended in physiological saline by pipetting at 4°C and +0000xy for 10 days.
Centrifugation was performed for a minute to sediment the bacterial cells. After repeating this washing operation three times, the bacterial cells were heat-treated at 65° C. for 10 minutes.

This bacterial cell was suspended in physiological saline at a concentration of 3XIO' cells/rx (l) and used as an immunogen suspension in the following procedure.

Immunization method Five week old female Balb/c mice were immunized by intravenously administering the above immunogen suspension 0 and 2 ytQ.

Furthermore, this operation was repeated twice a week for 10 weeks. Three days after the last intravenous administration, the mice were sacrificed, the lungs were removed, and RPMI-1640 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) was added to the mice.
was placed in a Petri dish containing After loosening the lungs into small pieces with tweezers and gently pipetting, the resulting spleen suspension was filtered through a stainless wire mesh to prepare a lung cell suspension. This spleen cell suspension was centrifuged at 500XIF for 10 minutes, and the resulting cell pellet was added to a red blood cell hemolysis buffer of 1 mM ethylenediaminetetraacetic acid disodium salt (pl- 17,0) was added and shaken to destroy and remove red blood cells. 50% of this lung cell suspension
Cell pellets were obtained by centrifugation at 0 xy for 10 min, which were washed three times with rtP-M[-1640 medium.

Culture, Fusion, and Selection The mouse myeloma cell line P3/X63-Ag8-Ul, which had been cultured in vitro in advance, and the hypoxandinguanine phosphoribosyltransferase-deficient strain was mixed with the lung cells, and the cells were incubated at 500×9 at 1°. Centrifugation was performed for 1 minute, and the supernatant was removed to obtain a cell pellet. 50% (W/V) polyethylene glycol 4000 kept at 37°C by gently tapping the bottom of the container to loosen the pellets.
RPMI-1640 medium containing 1 mQ was added for 1 minute, and the container was gently rotated for another 1 minute to mix the polyethylene glycol and the cell pellet.

After adding 2ttrQ of rlPMI-1 (340 medium) kept at 37°C over 2 minutes and 8 hours over 3 minutes, centrifugation was performed at 500 xg for 10 minutes. After removing the clear fluid, the cell pellet was rtP
Suspend in MI-1640 medium and incubate again at 500x9 for 10
Centrifuged for minutes. This washing operation was repeated once again to obtain a cell pellet. The resulting cell pellet was heated to 37°C.
I (Δ'1゛ selective medium (10% tallow serum,
2mM L-glutamine, 1mM sodium pyruvate, 100μM hyboxandine, 0.4μM aminopterin, 16μM demidine and penicillin G potassium 1
rtl"Ml-1640 medium containing 120,000 units/IIQ) was added so that the spleen cell concentration was 10 cells/IIQ and thoroughly suspended. This cell suspension was placed in a 24-well tissue culture plate. Dispense 5% into each well of a commercial plate (Corning 2B20-24F, Corning, USA).
Carbon dioxide culture P containing carbon dioxide gas! Culture was started at 37°C in a vessel. 24 hours after the start of culture, 1-18'r medium was added one village at a time. After that, at intervals of 2 to 3 EI IIn,
Remove 1 mg of medium in each well and add fresh II A i'
Culture medium! The culture was continued, and hybridomas having the ability to grow in II A'I' medium were selected.

On the 14th day, the subjects screened for the presence of anti-deficiency bacteria antibodies using supernatants from wells containing well-grown hybridoma colonies according to the method described in section (2) below. Hybridomas secreting anti-defective bacterial antibodies were selected, and subcloning was performed by limiting dilution method to confirm the monoclonal characteristics of the secreted antibodies and the secretion stability of the antibodies. From the cloned hybridomas, those with excellent antibody secretion ability, proliferation ability, and stability were selected to obtain desired antibody-producing hybridomas.

(2) Detection of monoclonal antibodies (enzyme immunoassay) Lactobacillus homohiodii used as an immunogen
1138201 strain suspension in physiological saline for 10'
Piece 10. Adjusted to 6 degrees of ImQ. This was used as a standard antigen, and 0 and 1 m (l) were dispensed into each well of a Militite IA plate (manufactured by Millivore, USA), fixed, and 0.
05% Tween 20. Contains 0.01% Dimerosal
It was washed with a washing solution consisting of 0 mM Tris-HCl buffer (pt, t 8.0). hybridoma culture supernatant,
contact with the antigen-coated surface within the well. Monoclonal antibodies similar to the antigen were retained by the antigen immobilized in the wells, and after washing three times with the washing solution, their presence was detected using a peroxidase-labeled anti-mouse antibody (Monoclonal Antibody Detection Reagent Mono, manufactured by Zymet, USA). Δb
- screen system). After further cleaning with cleaning solution, 0
゜001% (V/V) 30% hydrogen peroxide solution and 2゜2゛-azinodi (3-ethylbenzdiazoline sulfate)
(AB'l'S) in 0, 1M citrate buffer (
ptl4,2) was added in 0.05i12 portions. The reaction was carried out at room temperature for 10 to 30 minutes, 2mM sodium azide was added as a reaction terminator, and the absorption at 420 nm was measured.

The presence of hybridomas secreting monoclonal antibodies that recognize the antigen was confirmed by color change of the reagent.

(3) Production of monoclonal antibodies Collection of hybridoma culture supernatant The hybridomas obtained above were incubated in vitro in RPMI-1+340 medium containing 10% tallow serum at 37°C until the cell density was approximately IXIO. The cells were cultured until the cell density reached about 100 cells per cellar (if the cell density was too high, the growth state would decrease to 4). The desired monoclonal antibody was secreted into the culture medium, centrifuged at 500×9 for 10 minutes at room temperature, and the supernatant was collected.

Purification of antibodies by affinity chromatography - The obtained hybridoma culture supernatant was preliminarily divided into 0.02
Affigel Protein A (manufactured by Bio-Rad, USA) equilibrated with M1-Lis-HCl buffer (pl(7,2))
column and the column was washed with the same buffer until no protein was eluted. Then, by elution with 0.1M acetic acid containing 0.15M sodium chloride,
The desired monoclonal antibody was obtained.

The resulting antibodies consisted of total 1gG, l) by using [16, 5 and 3.5 O, 1M citrate buffers, respectively, the majority IgG, the majority IgG ta and IgG* and IgG, b. Furthermore, IgM and rg8 are eluted at both pH levels during washing.

(4) Monoclonal antibody labeling Peroxidase-labeled Horseradish peroxidase 1000 units (Sigma Type ■, manufactured by Sigma, USA) was dissolved in 1 ml of distilled water,
Immediately before use, a 1M sodium metaperiodate solution GOmQ prepared by Ate 1 was added and mixed for 20 minutes at room temperature. The reaction mixture was diluted with 1mM sodium acetate buffer (ptl4,
After overnight dialysis against 4), 20 mQ of a 0.2M aqueous sodium carbonate solution was added. Immediately, the purified monoclonal antibody IOmfl dissolved in 0.01 M carbonate buffer (pH 9,5) lx& was added, and the mixture was allowed to react for 2 hours at room temperature with continuous stirring. After the reaction was completed, 0.1++σ of a freshly prepared aqueous solution of sulfur and lithium borohydride (4 shoulders 9 dissolved in 11 distilled water) was added, left at 4°C for 2 hours, and phosphate buffered saline ( +)L3S) overnight.

The resulting reaction mixture was passed through a column of Sephadex G-100 (manufactured by Pharmacia, Sweden), and P
[It was eluted with 3S, and both fractions with the same absorption at 280 nm and 403 nm were collected. Bovine serum albumin was added to this labeled antibody fraction to a final concentration of 1%, and -
Stored at 80°C.

Alkaline phosphatase-labeled 1000 units of alkaline phosphatase (manufactured by Sigma, USA, Sigma type A 20% glutaraldehyde solution IOμσ was added to this, and the mixture was reacted with stirring at room temperature for 2 hours. After completion of the reaction, the reaction mixture was transferred to Sephadex G- which had been equilibrated with Tris-HCl buffer (p+-17,6).
200 column and dissolved in 1 mQ of the same distilled water) 0.
lxQ was added, the mixture was left at 4°C for 2 hours, and further dialyzed against phosphate buffered saline (+'l3S) overnight.

The resulting reaction mixture was passed through a column of Sephadex G-100 (manufactured by Pharmacia, Sweden), and I
'13. Elute with S, absorbance at 280 nm and 403 nm
The two samples with the same absorbance were collected. Bovine serum albumin was added to this labeled antibody fraction to a final concentration of 1% and stored at -80°C.

1000 units of a purified alkaline phosphatase (alkaline phosphatase) monoclonal antibody (manufactured by Sigma, USA, Sigma type X To this was added 10 μQ of a 20% glutaraldehyde solution, and the mixture was reacted with stirring at room temperature for 2 hours. After the reaction was completed, the reaction mixture was passed through a Sephadex G-200 column previously equilibrated with Tris-HCl buffer (ptl 7,6) and eluted with the same buffer. Collect the high molecular fraction from the void volume to the antibody elution position, and add 1 portion of bovine serum albumin to both parts.
% and stored at -20°C.

β-galactosidase labeled β-galactosidase 1000 units (manufactured by Sigma, USA,
A β-galactosidase-labeled monoclonal antibody was obtained in the same manner as in the alkaline phosphatase labeling process using Sigma Grade VI).

Fluorescein labeled purified monoclonal antibody, 1/10m of 0.5M
Add carbonate buffer (p+-t9,5) and add 1/100 of the antibody protein! l! i of fluorescein isothiocyanate powder (Fli'C) was added.

The reaction was carried out at 4°C for 6 hours with stirring without bubbling [1]. After the reaction was completed, the mixture was immediately passed through a column of Micephadex G-25 (manufactured by Pharmacia, Sweden) to remove unbound fluorescein. did. Then, it was passed through a column of DE8-Sephadex A-50 (manufactured by Pharmacia, Sweden) to obtain both fractions with a [''/P molar ratio (fluorescein and protein binding molar ratio) of 1 to 2. Add 0.1% sodium azide and store at 4℃ until use.
Saved to.

Tetramethylrhodamine-labeled purified monoclonal antibody was treated with 0.5 of its I/l on.
M carbonate buffer (pt[9,5'') was added, and to this was added tetramethylrhodamine isothiocyanate powder (Trtl'l'C) at I/l00ffl of antibody protein. 20 hours at 4°C. The reaction was carried out while stirring to prevent foaming, and immediately after the reaction was completed, Sephadex G-25 was added.
column to remove unbound tetramethylrhodamine. Then, 1) 13-SephadexΔ- to IC
50 column to obtain both fractions with an I;'/P ratio (the binding molar ratio of tetramethylrhodamine and protein) of 1 to 2. 0.1% sodium azide was added and stored at 4°C until use.

Biodin-labeled 1mM d-biotin and 1.5mM N-hydroxysuccinimide were dissolved in a mixture of 1mM of dimethoxysulfur, 8m of oxidide, and 5m of ethylene glycol diethyl ether, and 1mM of N-hydroxysuccinimide was added to this solution. , N'-dicyclohexylcarbodiimide in 0.5% ethylene glycol diethyl ether was added, and the mixture was reacted overnight at 4°C. The resulting precipitate was filtered off and the filtrate was freed from solvent under reduced pressure. The remaining oil was dissolved in 10 ml of dichloromethane and cooled to 4°C. To this, O
, 10 μl of 1M sodium bicarbonate, then 10 μl of distilled water cooled to 4° C. were added, and after repeating this operation, anhydrous sodium sulfate powder was added to the resulting dichloromethane layer to dehydrate it. After filtering off the powder, , n-hexano was added to the filtrate until turbidity occurred. It was cooled to -20°C and
The precipitated crystals were collected by filtration, placed in a desiccator to remove the solvent, and dried to obtain biotin-N-human[1-oxysuccinimide ester.

This biotin-N-hydroxysuccinimide ester was dissolved in dimethoxysulfoxide and 204f! was reacted with purified monoclonal antibody of m at room temperature for 4 hours.

After the reaction was completed, the mixture was dialyzed against PBS at 4°C for 3 days. The fluid in the dialysis tube was stored at 4°C as a biodin-labeled antibody.

(5) Detection of Hi-Ochi Bacteria by Enzyme Immunoassay Using the purified monoclonal antibody and its enzyme-labeled antibody, a detection and identification test for Hi-Ochi Bacteria was conducted according to the enzyme immunoassay described in (2) above.

As a test bacterium, Lactobacillus homohiodii Rf [38
201 strain, R1138210 strain, IIL18211 strain and 111B8213 strain, Lactobacillus heterohyothirttnsats strain, 111138319 strain, It
I 13 B 330 strain and 1111313331 strain, homofermentative deficient lactic acid bacteria It I B 8450 strain,
Heterofermentative deficient lactobacillus R, IB8400 strain, Lactobacillus acidophilus 111B8001 strain, and Lactobacillus fermentum 111138090 strain were used, and these bacteria were immobilized in the wells of a Milititer 11A plate in the same manner as the standard antigen, and after washing. , diluted solutions of each antibody were added, reacted at 37° C. for f hours, and then washed.

Next, when using a purified monoclonal antibody, use a peroxidase-labeled anti-mouse antibody as a secondary antibody,
When using the peroxidase-labeled antibody in the same manner as in section (2) above, no secondary antibody was used, and o, oot
% (V/v) of 30% hydrogen peroxide and ABTS in O, 1M citrate buffer was added as a substrate solution;
Using 2mM sodium azide as a reaction terminator, 42
Absorption at 0 nm was measured.

Even when alkaline phosphatase-labeled antibodies and β-galactosidase-labeled antibodies were used, no secondary antibodies were used, and 10mM p-nitrophenylphosphate and lomMp-nitrophenylgalactopyranoside were added as substrates, respectively, and the reaction was carried out in both cases. Absorption at 405 nm was measured using 1M sodium carbonate as a stopper.

The results are shown in Table 1. In the table, 10 means that color was developed by the enzymatic reaction, that is, detection of the hiochi bacterium, and - indicates that no color was developed. Also, B7, D3, D8,
G9 and r-I G respectively refer to the hybridoma of the present invention from which the antibodies used were derived. (Manufactured by West German Eppendorf, Eppe 7 F/I/7
3810) I;-, 13SS solution (per Iff, glucose 1, 09, potassium phosphate 0.Oeg, disodium phosphate dihydrate 0.251i1, calcium chloride dihydrate 0.+8 (ig, potassium chloride 0.401?
Sodium chloride 8.09, magnesium chloride hexahydrate 0.
20g, containing 0.20g of magnesium sulfate),
Centrifugation was performed at 0000xy for 10 minutes, and the supernatant liquid was removed. This washing procedure was repeated two more times to obtain purified monoclonal antibodies and fluorescein-labeled antibodies.
Hi-Ochi bacteria were detected and identified using tetramethylrhodamine-labeled antibodies and biodine-labeled antibodies.

When a purified monoclonal antibody was used, the antibody-containing solution was brought into contact with Hiochi bacteria to bind the monoclonal antibodies of the same type, and the above-mentioned washing operation was repeated three times to remove unbound antibodies. Then, U. was reacted with FI'r C-labeled anti-mouse antibody as a secondary antibody. Fluorescence emission was observed under a fluorescence microscope.

When a fluorescein-labeled antibody and a tetramethylrhodamine-labeled antibody were used, the antibodies were bound in the same manner as above, and after washing, fluorescence emission was observed under a fluorescence microscope.

When a biodin-labeled antibody was used, the antibody was reacted in the same manner as described above, and then fluorescein-conjugated avidin was added. After the reaction was completed, fluorescence emission was observed under a fluorescence microscope.

The results are shown in Table 2. In the table, + means that fluorescence was observed, that is, detection of the Hiotoshi bacteria, and - means that no fluorescence was emitted. B7, D3, D8, G9
, H6 are the same as in Table 1.

As shown in Tables 1 and 2, by appropriately combining the monoclonal antibodies of the present invention, it is possible not only to detect Hiotochibacterium but also to identify specific Hiotochibacterium.

Patent distributor Ozeki Sake Brewery Co., Ltd.

Claims (12)

[Claims] (1) Monoclonal antibody active against Hiotachi bacteria. (2) A monoclonal antibody produced by a hybridoma formed by the fusion of myeloma cells and cells derived from mammalian spleen cells that have been previously immunized against Hiochi bacteria, and which reacts preferentially with Hiochi bacteria. Monoclonal antibody described in (1). (3) The above (1) labeled with a fluorescent dye or enzyme
Monoclonal antibody according to paragraph or paragraph (2). (4) The monoclonal antibody according to item (3) above, wherein the fluorescent dye is fluorescein or rhodamine. (5) The monoclonal antibody according to item (3) above, wherein the enzyme is peroxidase, alkaline phosphatase or β-galactosidase. (6) The above-mentioned (1) or
2) Monoclonal antibody. (7) The monoclonal antibody according to item (6) above, wherein the marker is avidin and the group containing a structure for binding the marker is N-hydroxysuccinimide. (8) formed by the fusion of myeloma cells and cells derived from mammalian spleen cells previously immunized against Hirochibacterium;
Hybridomas capable of producing monoclonal antibodies active against Hiotochibacterium are cultured, and the produced monoclonal antibodies are isolated and purified, and optionally labeled with a fluorescent dye or enzyme, or labeled with a marker for microscopic observation. A method for producing a monoclonal antibody active against Hirochibacterium, which comprises chemically bonding a group containing a structure for bonding. (9) A hybridoma having the ability to produce monoclonal antibodies active against Hiotachi bacteria. (10) formed by the fusion of myeloma cells and cells derived from mammalian spleen cells that have been previously immunized against Hirochi bacteria;
The selected hybridoma of item (9) above. (11) A method for detecting and identifying Hiotoshi bacteria, which comprises contacting the sample with a monoclonal antibody active against Hiotchi bacteria, and determining the presence or absence of Hiotchi bacteria in the specimen through an antigen-antibody reaction. (12) A reagent kit for detection and identification of Hiotobacterium, which comprises a monoclonal antibody active against Hiotobacterium as an essential component.