JPH1132764A - Bifidobacterium breve monoclonal antibody - Google Patents

Abstract

(57) [Summary] [Problem] Bifidobacterium breve ( Bifidobacterium breve) does not react with bacteria belonging to different genera, species and strains.
breve ) Obtain a monoclonal antibody that specifically binds to the YIT4065 strain, and detect it simply and quickly. SOLUTION: Bifidobacterium breve ( Bifid
(Bacterium breve ) A monoclonal antibody that specifically recognizes YIT4065 strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a Bifidobacterium breve YIT406.
Bifidobacterium specific for 5 strains and related strains
The present invention relates to a monoclonal antibody for breve, a fused cell producing the monoclonal antibody, and an antibody-sensitized colloidal gold.

[0002]

2. Description of the Related Art Bifidobacteriu
m ) Bacteria (so-called bifidobacteria) are useful microorganisms used in various fields such as fermented milk products such as yogurt and lactic acid bacteria beverages and pharmaceuticals. In addition, it has been conventionally recognized that Bifidobacterium has an intestinal action and an intestinal putrefaction-preventing action. In recent years, it has been found that various physiological activities such as an antiulcer action and an immunostimulatory action are present.

However, it has been confirmed that among the bifidobacteria, there is a difference in the physiological activity imparted by the bacterial cell component depending on the species and strain. For example, in the same Bifidobacterium breve strain, FERM BP-2824 strain is ATCC 156
Stronger immunostimulatory activity than 98 strains. This is thought to be due to differences in the composition of proteins, sugar chains and the like on the cell surface and in the cell components such as enzymes.

On the other hand, the Bifidobacterium breve strain YIT4065, which is owned by the present applicant, is excellent among Bifidobacteria in terms of acid resistance, bile acid resistance and the like as a strain for producing foods and drinks, but it is effective in preventing cancer. It is a strain that has particularly strong various physiological effects in vivo such as an action, an immunostimulatory action, a blood cholesterol lowering action, and an intestinal action, and is expected to be used in a wide range of applications such as foods, pharmaceuticals, and cosmetics.

[0005] For this reason, a wide variety of studies have been conducted on this strain, including confirmation of trends and actions in vivo. In this case, it is necessary to distinguish the strain from other strains of the same or similar species from the bacterial flora in the culture medium or feces and identify the strain at the strain level. At present, strains are identified by selecting on a selective medium and examining phenotypic traits, ie, glycolytic properties, fermentation products, and the like. In addition, judgment based on DNA-DNA homology is also performed. However, these analyzes are complicated and time-consuming, and some operations require the skill of the tester. Therefore,
There is a need for a more rapid and simple method for detecting the strain.

[0006]

Among the microorganisms, bacteria of the same species and different strains have very similar phenotypes, DNA base sequences and the like, and it is difficult to detect them at the strain level. In addition, even when performing detection at the strain level, a group of closely related strains (hereinafter referred to as `` related strains '') that have resulted in a slight difference in properties due to subculture must be recognized as the same strain. . This is because the strains belonging to the related strain groups have the same physiological activity.

[0007] As described above, a screening method using a monoclonal antibody was considered to be effective as a detection method that satisfies the condition that all the related strain groups are detected and other strains are not detected. Therefore, the present inventors studied with the aim of producing a monoclonal antibody that does not react with bacteria belonging to other genera, species, and strains and specifically binds to Bifidobacterium breve strain YIT4065 and related strains. Was done.

[0008]

Means for Solving the Problems In view of such a situation, the present inventor conducted intensive research and found that Bifidobacterium
We succeeded in obtaining a hybridoma that produces a monoclonal antibody that specifically recognizes the Breve YIT4065 strain and its related strains, and found that by culturing the hybridoma, it was possible to obtain the desired monoclonal antibody. Thus, the present invention was completed.

That is, the monoclonal antibody for Bifidobacterium breve according to the first aspect of the present invention is a Bifidobacterium breve ( Bifidobacterium breve) monoclonal antibody.
breve ) It specifically recognizes the YIT4065 strain and its related strains.

The fused cells (hybridomas) according to the second aspect of the present invention produce the monoclonal antibodies. In one embodiment of the present invention,
Bifidobacterium breve
ve ) A monoclonal antibody that specifically recognizes the YIT4065 strain and its related strains is produced and deposited as FERM P-16296.

The antibody-sensitized colloidal gold according to the third aspect of the present invention is obtained by adsorbing colloidal gold particles to a monoclonal antibody that specifically recognizes Bifidobacterium breve strain YIT4065 and related strains. It is a thing.

According to a fourth aspect of the present invention, there is provided a method for detecting an antigen, comprising preparing the colloidal gold according to the third aspect, reacting the antibody-sensitized colloidal gold with a group of antigens to be tested, and then washing the colloidal gold. Then, the red test antigen to which the antibody-sensitized colloidal gold is fixed is visually detected.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The Bifidobacterium of the present invention
Breve YIT4065 shares (hereinafter referred to as “YIT4065 shares”)
Is the strain owned by the applicant and has accession number FE
Deposited as RM P-15488 strain with the National Institute of Biotechnology and Industrial Technology. This strain has an intestinal action,
It has various physiological activities such as immunostimulatory activity and is one of the most useful strains used as foods, pharmaceuticals and the like.

On the other hand, the related strain of the present invention is a group of strains originally belonging to the same strain. That is, due to the mutation accompanying subculture, etc., the surface shape was slightly changed,
It refers to a group of strains in a so-called "parent-child relationship", which has a very slight difference in phenotypic traits, but does not substantially change the activity given to the human body. Specifically, YIT4
Starting with the parent strain YIT4010 of 065,
The sister strain YIT4052 strain and the like can be mentioned.

In order to prepare monoclonal antibodies specific to the YIT4065 strain and related strains, first,
Immunize with the cells of the strain. The immunized animal is then dissected to obtain cells producing the strain-specific antibody (hereinafter abbreviated as antibody-producing cells). Next, the obtained antibody-producing cells are fused with myeloma cells (myeloma cells) to obtain hybridomas (fused cells), and these hybridomas are cultured. Furthermore, a hybridoma producing an antibody is cloned by using the specificity of the antibody obtained in the culture against the YIT4065 strain as an index, and a hybridoma producing a monoclonal antibody specific to the YIT4065 strain and a related strain is obtained by culturing the clone. can do.

The above-mentioned hybridoma is formed by fusing antibody-producing cells obtained from an animal immunized with cells of strain YIT4065 with myeloma cells. As the antibody-producing cells to be used, spleen cells of an animal immunized with (dead) cells of the YIT4065 strain are preferable. As the antibody-producing cells and myeloma cells, it is not necessary to specify the type of animal that is a source as long as they can be fused, but from the viewpoint of fusion efficiency and stability of antibody production, animals derived from animals of the same species It is desirable to use those.

Among the hybridomas described above, a preferred hybridoma is obtained by fusing spleen cells of a mouse immunized with killed cells of the YIT4065 strain and myeloma cells of the mouse. A fused hybridoma of an anti-YIT4065 strain antibody-producing cell of a BALB / C mouse immunized with a killed cell and a myeloma cell of a BALB / C mouse is exemplified.

Immediately after cell fusion, hybridomas producing other kinds of antibodies are mixed. So, YIT406
It is necessary to select (screen) cells that produce antibodies exhibiting specificity for the antigen-determining sites of the five strains and their related strains to obtain the desired antibody-producing cells. Vo for antibody detection
The enzyme immunoassay (ELISA) reported by Ller et al. [Bull. WHO., 53, 55-56, (1976)] is preferably used.

More specifically, the screening will be described. To the YIT4065 strain antigen immobilized on the hole wall of a tissue culture plate, for example, a 96-well microtiter plate, ascites of a hybridoma culture solution or a mouse inoculated with the hybridoma is added. After adding an anti-mouse antibody labeled with peroxidase, the peroxidase activity is assayed by the color development of orthophenylenediamine (OPD). This color development of the OPD indicates the presence of the antibody. The titer of the anti-YIT4065 strain antibody of the hybridoma culture solution and its concentrated solution, or the ascites fluid inoculated with the hybridoma was OPD.
Is determined by the final dilution factor of the sample that gives a color development (measured value at an absorbance of 492 nm) of 0.5 or 1.0. Culture medium of BALB / C mouse myeloma cells and BALB / C mice
The serum and ascites obtained by inoculating / C mice were YIT406
No antibody activity against 5 strains.

The production of the monoclonal antibody against the YIT4065 strain is carried out by maintaining and growing the above-mentioned antibody-producing clone (hybridoma) in a medium or in a histocompatible animal or an immunodeficient animal such as a nude mouse. Or, it is recovered from the serum or ascites of the animal. [For the recovery method, for example, Tatsuo Iwasaki,
Monoclonal antibodies, pp. 88-94 (1983)].

Next, using the YIT4065 strain-specific monoclonal antibody thus obtained, the carrier of the monoclonal antibody necessary for more easily detecting the YIT4065 strain was examined.
Latex particles and colloidal gold particles were used as carriers to be adsorbed, and sensitized latex and sensitized colloidal gold were prepared. Sensitization of antibodies to latex particles is Ts
uda, S. et al. (Tsuda, S. et al., Plant Diseas
e, 76, 466-469, (1992)). In addition, colloidal gold particles were self-produced and sensitized with an antibody using the method described later.

The latex particles used for preparing the sensitized latex have a specific gravity of 1.0 to 1.5 g / cc, preferably 1.5 g / c
The high specific gravity latex particles around c are used as suitable ones, and further, the average particle size is about 0.1 to 2.0 μm, preferably 1.
Latex particles of around 0 um are used as suitable.

Examples of such latex particles include, for example, Bacto latex 0.81 (manufactured by Difco, average particle size 0.81).
um, specific gravity 1.0 g / cc), and H0901, H0902, H2002 (manufactured by Nippon Synthetic Rubber Co., Ltd., average particle size and specific gravity are 0.94 um1.5 g / cc, 0
・ 98um ・ 1.5g / cc, 2.22um ・ 1.5g / cc), etc., are not limited to this, but can be used irrespective of their type as long as they have the same effect. It is.

The colloidal gold particles are red associated colloids in which gold particles are colloidally associated. The average particle diameter of the colloidal gold used for producing the sensitized colloidal gold is preferably about 0.01 μm to 0.02 μm. However, even if it is out of this range, it can be used as long as the gold particles can form a colloid.

Using the YIT4065 strain-specific monoclonal antibody obtained as described above and latex or colloidal gold sensitized with the antibody, YIT4
When the 065 strain was identified, there were advantages and disadvantages,
A system using sensitized colloidal gold appeared most suitable. The use of a monoclonal antibody that does not adsorb the carrier takes too much work time, and the use of sensitized latex has the advantage that self-aggregation is unlikely to occur and detection can be performed in a short time, but washing is Sometimes the carrier was separated from the solid phase. In contrast,
When the sensitized colloidal gold was used, the bag was cleaner than the latex, the antigen-antibody complex was stable, and the operation was simple. In addition, since colloidal gold can be easily produced, use of self-made colloidal gold is lower in cost than latex or the like. Since latex particles having various colors can be used, when it is convenient to use a combination of colors such as analysis of bacterial flora, sensitized latex is preferable.

The monoclonal antibody prepared in the present invention can be used for strain identification and quantification by the ELISA method or the like. In addition, if the sensitized latex of the present invention is used, the sensitized latex and a comparative sample are mixed on a slide glass, and various identification methods such as a method of judging the correctness of the aggregation image under an optical microscope (microscope latex method), etc. A quantitative method can be performed.

A simple method (colloidal gold method) for detecting strains using sensitized colloidal gold used in the present invention is as follows. That is, when the YIT4065 strain and other strains (of the same species) are spotted on a nitrocellulose membrane or the like, and then washed with the sensitized colloidal gold of the present invention, only the portion spotted with the YIT4065 strain is stained. The red color of colloidal gold is observed. At the time of spotting, the strain can be specifically detected by directly using a solution containing other kinds of bacteria and components such as fermented milk. According to such a detection method, it is possible to detect strains simply, quickly and inexpensively, and it does not require much skill of an operator. Also,
The sensitized colloidal gold can also be suitably used in other detection methods and the like conventionally performed with sensitized latex and the like.

[0028]

【Example】

Embodiment 1 FIG. Preparation of Hybridoma and Production of Ascites Antibodies Purified Bifidobacterium breve ( Bifidob
Acterium breve ) YIT4065 strain was washed with physiological saline, and then suspended in physiological saline so that the absorbance at 660 nm became 0.5. 0.2 ml of the obtained suspension is applied to BALB
A / C mouse was injected into the tail vein, and 10 days later, an additional 0.2 ml of a suspension prepared in the same manner was additionally injected. In addition, a total of 4-5 injections of the suspension were made every 10 days. Before sacrifice of the mice, 0.2 ml of the suspension prepared similarly was injected for final immunization. Four days after the final immunization, the mouse was killed and the spleen was excised, and the spleen cells were suspended in an RPMI1640 medium (manufactured by Sigma). The spleen cell suspension is treated with a 0.82% aqueous solution of ammonium chloride at room temperature for 10 minutes, and then centrifuged in a serum-free medium (2
(× 50 × g) and then suspended in serum-free medium.

On the other hand, myeloma cells were transformed with 15% fetal bovine serum.
After growth in the contained RPMI 1640 medium, the cells were washed twice by centrifugation in a serum-free medium, and then suspended in a serum-free medium. After mixing 4.0 × 10 ⁷ spleen cells in suspension and 1.0 × 10 ⁷ myeloma cells, centrifugation was performed for sedimentation, and 1 ml of a serum-free medium containing 45% polyethylene glycol 6000 was added thereto, followed by 6 minutes. The cells were fused at 37 ° C. After washing the obtained cell mixture by centrifugation in a serum-free medium, RPMI1640 containing 15% fetal calf serum was used.
Suspension to 1.0 × 10 ⁵ pancreatic cells / ml in the medium,
0.1 ml thereof was dispensed into a 96-well plastic microtiter plate, and cultured in an incubator in the presence of 7% carbon dioxide.

On the first day of the culture, 0.1 ml of HAT medium was added, and on the second, fourth, seventh and tenth days, half of the culture was aspirated, and HAT medium was added instead. An average of 1 to 2 hybridoma colonies per well grew in a culture well of a 96-well plastic microtiter plate of 60 to 90%. At the time when the hybridomas were sufficiently grown (about two weeks after the start of culture), the antibody titer of the culture supernatant against the YIT4065 strain was assayed by ELISA. Antibodies to the YIT4065 strain were observed in the hybridoma culture supernatant in 139 of the 309 wells. The cloning operation by limiting dilution was repeated twice for the hybridoma in each well producing the antibody. Thus, clone 1 that stably produces antibodies
The strain was isolated and named 4A. For this clone, a YIT4065 strain-specific monoclonal antibody was obtained from the culture solution, ascites, and ammonium sulfate precipitate fraction. Incidentally, the isotype of this clone was IgM. This clone 4A has accession number FERM P
-16296 was deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on July 1, 1997.

Embodiment 2 FIG. Confirmation of Specificity of Monoclonal Antibody (2-1) Monoclonal Antibody As a mouse monoclonal antibody, 4A specific to Bifidobacterium breve strain YIT4065 and related strains was used. The cell culture supernatant of the hybridoma clone or a partially purified one was used as a monoclonal antibody sample in the experiment.

(2-2) Bacterial cell antigen Bifidobacterium breve
ve ) YIT4065 strain (FERM P-15488), its parent strain YIT4010 strain, and a standard strain to which the strain belongs or does not belong were used. Specifically, Bifidobacterium breve YIT4014 (ATCC 15700) strain, YIT40
15 (ATCC 15698) strain, YIT4049 (ATCC 15701) strain,
Aa-50 strain, La-1 strain, Na-25 strain, Bifidobacterium bifidum ( B. bifidum ) YIT4007 (FERM
BP-791) strain, Bifidobacterium adressentis
( B. adolescentis ) YIT4011 (ATCC 15703) strain, Bifidobacterium infantis ( B. infantis ) YI
T4018 (ATCC 15697) strain, Bifidobacterium longum ( B. longum ) YIT4021 (ATCC 15707) strain, YI
T4037 (ATCC 15708) strain, Bifidobacterium bifidum ( B. bifidum ) YIT4039 (IFO 14252) strain, Bifidobacterium.・Animalis ( B.animalis ) YIT
4044 (JCM 1190) strain, Lactobacillus casei ( Lacto
bacillus casei ) YIT9029 (FERM BP-1366) strain.

(2-3) ELISA method Pure culture of each of the above cell antigens was performed at 1.0
It was diluted to about × 10 ⁹ / ml, and 0.1 ml thereof was dispensed into each well of a 96-well microtiter plate, and coated at 37 ° C. for 2 hours. Next, each well on the plate was washed three times with a washing solution for ELISA (PBS containing 0.05% of Triton X-100), and a blocking agent (Blocking Ace, manufactured by Dainippon Pharmaceutical Co., Ltd.) was added to the wells. The unadsorbed portion was coated. The coating treatment was performed at 37 ° C. for 1 hour. This is ELISA
After washing three times with a washing solution for use, antibody 4A as a primary antibody was added to each plate and reacted at 37 ° C. for 1.5 hours. After washing three times, an enzyme (peroxidase) -labeled anti-mouse immunoglobulin antibody (Cappel) was used as a secondary antibody.
The reaction was carried out at a temperature of 1.5 hours. After washing three times, color development (light)
Add the substrate (orthophenylenediamine (OPD))
From the degree of color development, the reactivity between each antigen and 4A was confirmed.

The strains used in the experiments are reference strains and known strains subcultured in our company, and it is known that these strains are related strains of YIT4065 strain or strains of another strain. Then, based on this finding, when the results of the ELISA method were examined, the same results were obtained, and clone 4A was specific to the YIT4065 strain and its related strains. The results are shown in Table 1 below. In Table 1, “+” indicates that there is an antibody binding, and “−” indicates that there is no antibody binding.

[0035]

[Table 1]

Embodiment 3 FIG. Antibody sensitization to colored carrier fine particles (3-1) Preparation of sensitized latex FIG. 1 is an explanatory view showing a preparation process of an antibody-sensitized colored latex and a bacterial cell detection process using the same. As the colored latex, latex particles colored in pink (IMMUT
EX, G0304R, 0.30 μm, Nippon Synthetic Rubber) was used. Sensitization of antibodies to latex particles was performed according to the method of Tsuda, S. et al.
As shown in FIG. 1, 0.02 M Tris buffered saline (pH 7.2, 0.15 M
NaCl, hereinafter referred to as “TBS”) is mixed with an equal amount of a latex solution adjusted to 1% with a monoclonal antibody solution adjusted to 0.5 mg / ml in TBS, and allowed to stand at room temperature for 2 hours with occasional stirring. did. The latex particles were washed by centrifugation (three times) with TBS to which 1% of bovine serum albumin (hereinafter, referred to as "BSA") was added, and then the latex particles were suspended in the same solution to 1%. This was used as an antibody-sensitized colored latex, TB
Dilute with S to 1/20 to 1/40 (latex concentration: 0.025 to 0.05%) before use.

(3-2) Preparation of sensitized colloidal gold Colloidal gold for immunoreaction was self-produced. First, pure water (milli-Q water filtered with a 0.22 μm filter) was prepared, and all instruments used were thoroughly washed. A 0.01% solution of chloroauric acid (manufactured by Soegawa Rikagaku Co., Ltd.) was prepared in pure water, placed in an Erlenmeyer flask, and heated with a gas burner. When the solution boils, 1 /
Twenty volumes of 1% trisodium citrate solution (prepared with pure water) was added and heating was continued for 5 minutes. Another 1/20 volume of 1% trisodium citrate solution was added, and the mixture was ripened for another 10 minutes to stop the fire. The resulting colloidal gold was reddish-brown. The colloidal gold solution was sealed and stored at 5 ° C. until use without adding a preservative or the like.

FIG. 2 is an explanatory view showing a step of preparing colloidal gold sensitized with an antibody and a step of detecting bacterial cells using the same. As shown in FIG. 2, the sensitization of the antibody to the colloidal gold particles was performed using the colloidal gold solution prepared above and a monoclonal antibody solution prepared with PBS (filtered with a 0.22 μm filter) at 0.1 to 1 mg / ml. Were mixed in equal amounts and left at room temperature for 2 hours or longer. After adding 1/20 volume of 10% BSA solution, centrifuge (12,000 rpm)
pm, 30 minutes) to collect colloidal gold particles as precipitate. 0.1% BSA, 0.2% polyethylene glycol and 0.
A suspension of colloidal gold particles in PBS containing 1% sodium azide (0.01%: the concentration of the original colloidal gold solution) was used in the experiment as antibody-sensitized colloidal gold.

Embodiment 4 FIG. YIT4 using sensitized colloidal gold
Simple detection of 065 strain (4-1) Monoclonal antibody The 4A-sensitized colloidal gold prepared in Example 3 was used.

(4-2) Cell antigen Lactobacillus casei YIT9029 (FERM BP casei YIT0209 (NCDO151) strain, Bifidobacterium
Breve YIT4065 (FERM P-15488) strain, Bifidobacterium breve YIT4014 (ATCC 15700)
Strain, Bifidobacterium bifidum YIT4007 (F
ERM BP-791) strain, Bifidobacterium bifidum Y
The IT4039 (IFO 14252) strain was used.

(4-3) Detection by Colloidal Gold Method FIG. 3 is an explanatory view schematically showing a detection operation by the colloidal gold method, FIG. 3A shows spots of the strain on a nitrocellulose membrane, and FIG. It is explanatory drawing which shows a state after. As shown in FIG. 3a, the six strains were spotted on a nitrocellulose membrane (40). In the figure, it is surrounded by a dotted line
(41) is Lactobacillus casei YIT9029 strain, (42)
Is Bifidobacterium breve YIT4065 strain,
(43) is Bifidobacterium bifidum YIT4007
(44) is Lactobacillus casei YIT0209 strain,
(45) is Bifidobacterium breve YIT4014
(46) is Bifidobacterium bifidum YIT4
039 spots are shown.

After blocking the nitrocellulose filter, it was washed once with water, and each spot was reacted with 4A-sensitized colloidal gold. After washing this with water once,
As shown in FIG. 3B, only the spot (42) of Bifidobacterium breve YIT4065 specific to 4A was stained.

Embodiment 5 FIG. Detection of YIT4065 strain in dairy products using sensitized colloidal gold Investigating whether it is possible to detect YIT4065 strain using 4A sensitized colloidal gold even in a solution containing components other than bacterial cells such as carbohydrates and proteins In order to do this, the following experiment was performed.

First, Bifidobacterium breve Y
The the IT4065 strain 10 ⁸ / ml containing) were serially diluted every 10 times,
These are spotted on a nitrocellulose filter. This is reacted with 4A sensitized colloidal gold for 10 minutes at room temperature,
Washing was performed once with water. FIG. 4 is an explanatory diagram showing the detection results of the YIT4065 strain when the dilution ratio was changed. In the figure, (50) is a nitrocellulose filter, (51) is a spot diluted 10-fold, and (52) is 100-fold. Dilution spot, (53) is 100
The spot at 0-fold dilution is shown. When the presence or absence of color development by colloidal gold was examined, it was detectable by 100-fold dilution (10 ⁶ / ml as the number of bacteria). It should be noted that treatment such as the solubility ratio of a milk component with an alkali was considered to be useful in order to make detection possible even with higher dilution.

Embodiment 6 FIG. Detection of YIT4010 Strain in Feces Using Sensitized Colloidal Gold The following experiment was performed to examine whether the YIT4065 strain-related strain could be detected in feces using 4A sensitized colloidal gold.

The super-premature babies (3) were administered YIT4010 strain at 5 × 10 ⁸ / day, and the feces were collected. Further, feces of the non-administration group (3 persons) were also collected. A solution obtained by diluting each stool with PBS and YIT401 as a positive control
The 0 dilution was spotted on a nitrocellulose filter. This was reacted with 4A-sensitized colloidal gold for 10 minutes at room temperature, and washed once with water. Table 2 shows the results of confirming the presence or absence of color development by colloidal gold. As shown in Table 2, YIT40 was collected from the feces of the administration group and the positive control.
Ten strains were detected.

[0047]

[Table 2]

[0048]

The use of the monoclonal antibody of the present invention makes it possible to easily and quickly detect the YIT4065 strain and its related strains. The YIT4065 strain and its related strains are useful microorganisms used in foods, pharmaceuticals, and the like. Therefore, if their detection is simple and rapid, various studies such as confirmation of their effects on living organisms can be assisted.

The monoclonal antibody can be easily produced by using the monoclonal antibody-producing fusion cells of the present invention. Furthermore, if sensitized colloidal gold sensitized with a monoclonal antibody or the like is used, microorganisms can be detected more simply, more quickly, and at lower cost than conventional immunological techniques. Using the YIT4065 strain-specific monoclonal antibody, Y
Only IT4065 and its related strains can be detected.

[Brief description of the drawings]

FIG. 1 is an explanatory view showing a step of preparing an antibody-sensitized colored latex and a step of detecting bacterial cells using the same.

FIG. 2 is an explanatory view showing a preparation step of an antibody-sensitized colloid bacterium and a cell detection step using the same.

FIG. 3 is an explanatory view schematically showing a detection operation by a colloidal gold method. FIG. 3A is an explanatory view showing spots of a strain on a nitrocellulose membrane, and FIG. 3B is an explanatory view showing a state after staining.

FIG. 4 is an explanatory diagram showing detection results of the YIT4065 strain when the dilution ratio is changed.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/553 G01N 33/569 F 33/569 C12N 5/00 B (72) Inventor Makoto Owaki 1-chome, Higashi-Shimbashi, Minato-ku, Tokyo No. 1-19 Yakult Honsha

Claims (4)

[Claims] 1. Bifidobacterium breve ( Bifid)
Bacterium breve ) A monoclonal antibody for Bifidobacterium breve that specifically recognizes YIT4065 strain and its related strains. 2. The Bifidobacterium according to claim 1,
A fused cell (hybridoma) producing a monoclonal antibody for breve. 3. The Bifidobacterium according to claim 1,
Antibody-sensitized colloidal gold characterized in that colloidal gold particles are adsorbed to a breve monoclonal antibody. 4. After preparing the colloidal gold according to claim 3, reacting the antibody-sensitized colloidal gold with the antigen group to be tested, the colloidal gold is washed, and the red test antigen to which the antibody-sensitized colloidal gold is fixed is visually observed. A method for detecting an antigen, comprising: