JPH1017598A - Monoclonal antibody to nitrobacter agilis and measurement thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new monoclonal antibody, capable of specifically recognizing Nitrobacter agilis and specifically identifying active nitrifying bacteria in an activated sludge, water, a soil, a microorganism-immobilized specimen, etc., and rapidly and properly controlling the number thereof. SOLUTION: This monoclonal antibody is a new anti-Nitrobacter agilis monoclonal antibody capable of specifically recognizing the Nitrobacter agilis and useful for specifically detecting and identifying nitrifying bacteria in an active sludge, water, a soil, an immobilized carrier, etc., and rapidly and properly controlling the number thereof. The monoclonal antibody is obtained by washing the Nitrobacter agilis, suspending the washed Nitrobacter agilis in a physioloigical saline solution, injecting the suspension into a BALB/c mouse for immunization, collecting a cell of the spleen after the final immunization, fusing the resultant cell of the spleen to a cell of a myeloma, then culturing the fused cell in a hypoxanthine, aminopterin and thymidine (HAT) culture medium, providing a hybdridoma, screening the prepared hybridoma, selecting a strain capable of producing the antibody, cloning the selected strain according to a limiting dilution method, etc., and culturing the resultant monocloned hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a nitrobacter
Monoclonal antibody showing high specificity to Agilis (referred to as nitrite-oxidizing bacteria) and a method for easily detecting Nitrobacter agilis present in activated sludge, water, soil, or microorganism-immobilized carrier The present invention relates to a monoclonal antibody-sensitized latex for detection of Nitrobacter agilis used in the above, and a method for detecting nitrifying bacteria using the monoclonal antibody-sensitized latex. More specifically, the present invention relates to a nitrifying bacterium (Nitr) having nitrite oxidizing activity, such as in activated sludge or soil, or in a microorganism-immobilized carrier.
The present invention provides a monoclonal antibody against P. ifying Bacteria), and is produced by culturing a clone of a fusion cell (so-called hybridoma) obtained by fusing antibody-producing cells and tumor cells from an animal immunized with Nitrobacter agiris. A monoclonal antibody against Nitrobacter agilis that detects active target microorganisms in activated sludge, soil, or a microorganism-immobilized carrier;
A novel monoclonal antibody capable of establishing a method for accurately quantifying the number of bacteria, a monoclonal antibody-sensitized latex for detecting Nitrobacter agilis using the antibody, and a method for detecting and quantifying the same It is about.

[0002]

2. Description of the Related Art Conventionally, as a method for easily detecting nitrite-oxidizing bacteria in activated sludge or soil, generally, a sample is collected, and after a culture period of about one to two months, A method for indirectly measuring nitrites based on the presence or absence of nitrous acid in the soil was known (see Ryusuke Kimura et al., Soil Microorganism Experiment Method, pp. 207-214 (1992)). Also,
Conventionally, by using such a measuring method, it has been practiced to control trends of active target microorganisms in activated sludge and soil. However, in this method, the detection and measurement of the target microorganism requires skill, and it takes a considerable number of days, so that it is difficult to quickly respond to manage the trend of the target microorganism from the measurement result. was there.

On the other hand, the present inventors have developed a detection method using an antibody for ammonia-oxidizing bacteria or nitrite-oxidizing bacteria, and rapidly detect ammonia-oxidizing bacteria or nitrite-oxidizing bacteria by the method. (Japanese Patent Laid-Open Publication No. 6-76336). In addition, a method has been proposed in which ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, and even denitrifying bacteria can be detected more easily by using an antibody-sensitized latex in which the antibody is adsorbed to latex particles. 8-29426. However, since the antibody is a polyclonal antibody, the method of producing the antibody differs depending on the individual animal, and it has been difficult to always obtain the same antibody with the same specificity, binding strength, and antibody titer. For this reason, it is difficult to standardize the properties of the antiserum reagent, and there is a possibility that test results may vary accordingly. In addition to the problem of individual differences between animals, there may be difficulties at the stage of antibody production, and furthermore, antibody molecules with various properties produced by countless different clones of antibody-producing cells possessed by immunized animals There were various problems such as being mixed. Thus, the present inventors have prepared a monoclonal antibody for Nitrosomonas europaea having ammonia oxidation activity and provided a method for detecting Nitrosomonas europaea using the antibody. However, the only antibody that recognized nitrite-oxidizing bacteria was a polyclonal antibody.

[0004]

SUMMARY OF THE INVENTION In view of such a situation, the present inventors have developed a method that does not rely only on immunized animals.
Application of tissue cell culture technology, production of monoclonal antibodies that make it possible to always obtain antibodies with extremely uniform specificity and binding strength based on antigen information, and nitrite-oxidizing bacteria using the monoclonal antibodies As a result of intensive studies with the aim of establishing a new detection method for nitrocellulose, Nitrobacter, a Gram-negative chemoautotrophic bacterium with nitrite removal activity,
In addition to producing an anti-Nitrobacter agilis monoclonal antibody that specifically recognizes Agilis, and succeeded in establishing a new detection method using the monoclonal antibody,
The present invention has been completed.

[0005] That is, an object of the present invention is to provide a novel monoclonal antibody to nitrobacter agiris, a nitrite-oxidizing bacterium, and further provide a method for producing the monoclonal antibody, and a monoclonal antibody. It is an object of the present invention to provide a method for detecting Nitrobacter agilis using a sensitized latex and quantitatively determining the number of the bacteria with high sensitivity.

[0006]

Means for Solving the Problems The present invention for solving the above-mentioned problems relates to an anti-Nitrobacter agiris monoclonal antibody which specifically recognizes Nitrobacter agiris, and comprises a bacterium of Nitrobacter agiris. In a preferred embodiment, the above-mentioned anti-nitrobacillus Agilis monoclonal antibody that specifically recognizes an antigenic determinant site of the body is used. The present invention also relates to a fused cell (hybridoma) producing the above-mentioned anti-Nitrobacter agilis monoclonal antibody, and a Nitrobacter characterized in that a monoclonal antibody specific to Nitrobacter agilis is adsorbed to latex particles. The present invention relates to Agilis antibody-sensitized latex, wherein the latex particles have a specific gravity of 1.5 g / cc.
The above-mentioned antibody-sensitized latex of Nitrobacter-Agilis, which is a latex particle having a high or low specific gravity, wherein the latex particle is an antibody-sensitized latex of Nitrobacter-Agilis, which is a latex particle having an average particle size of about 1.0 μm. It is an embodiment. Furthermore, the present invention relates to a method for detecting and quantifying Nitrobacter agilis, which comprises using the antibody-sensitized latex of Nitrobacter agilis described above.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The novel monoclonal antibody according to the present invention is produced as follows. That is, an animal is immunized with a gram-negative chemoautotrophic bacterium Nitrobacter agilis having nitrite removing activity, and a fused cell (hybridoma) is formed between an antibody-producing cell and a tumor cell obtained from the animal, Next, the fused cells are proliferated, cells that produce an antibody having specificity for Nitrobacter agilis are selected from the proliferated fused cells, and the antibody-producing cells are cultured to obtain anti-Nitrobacter agilis Produce monoclonal antibodies. The anti-nitrobacillus Agilis monoclonal antibody according to the present invention may be used as a reagent for quantifying the number of Nitrobacter agilis in activated sludge or soil, or a nitrifying bacterium having an ammonia oxidizing activity in a microorganism-immobilized carrier. It is extremely useful as a reagent for use in the maintenance and management of lipase.

[0008] The above-mentioned fused cells (so-called hybridomas) are cells that produce an antibody against Nitrobacter agilis obtained from an animal immunized with Nitrobacter agilis (hereinafter abbreviated as Nitrobacter agilis antibody-producing cells). ) And myeloma cells that are tumor cells. As the Nitrobacter agiris antibody-producing cells to be used, spleen cells from an animal immunized with Nitrobacter agiris are preferable. It is not necessary to specify the type of animal that is the source of the above-mentioned Nitrobacter-Agilis antibody-producing cells and myeloma cells as long as they can be fused, but from the viewpoint of fusion efficiency and stability of antibody production. It is preferable to use those derived from animals of the same species.

[0009] Among the above hybridomas, a preferred hybridoma is obtained by fusing spleen cells of a mouse immunized with Nitrobacter agilis with myeloma cells of the mouse. Anti-nitrobacter agiris antibody-producing spleen cells from BALB / c mice immunized with B. agiris
A hybridoma with a myeloma cell of an ALB / c mouse can be mentioned. After the cell fusion, the hybridoma is cloned, the clone is cultured, and a clone that produces an antibody specific to the cells of Nitrobacter agilis or its antigenic determination site is selected from the clone group.
For antibody detection, see Voller et al. (Bull. WHO.,
53, pp. 55-56, (1976)), an enzyme immunoassay (ELISA) is preferably used.

[0010] More specifically, this point will be described in more detail. A mouse obtained by inoculating a hybridoma culture solution or a hybridoma with a Nitrobacter agilis antigen immobilized on a hole wall of a tissue culture plate, for example, a 96-well microtiter plate. After adding ascites and then adding an anti-mouse immunoglobulin 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-nitrobacter agilis monoclonal antibody in the ascites fluid inoculated with the hybridoma culture solution and its concentrated solution, or the hybridoma, is the color development of OPD (measured at an absorbance of 492 nm).
Is the final dilution of the sample giving 0.5 or 1.0. BALB / c mouse myeloma cells P3-NS1-
The culture solution of 1-Ag4-1 (hereinafter abbreviated as NS-1) and the serum and ascites obtained by inoculating the cells into BALB / c mice have no antibody activity against Nitrobacter agilis.

The production of a monoclonal antibody against Nitrobacter agilis is carried out by maintaining and growing the above-mentioned antibody-producing clone in a medium or in a histocompatible animal or nude mouse. Or collected from animal serum or ascites (for the method of collection, see, for example, Tatsuo Iwasaki et al., Monoclonal Antibody, 88-9).
4 (1983)).

Next, the latex particles of the carrier for adsorbing the above-mentioned specific monoclonal antibody and the method of producing the sensitized latex can be prepared according to the method reported by the present inventors. That is, as the latex particles, high specific gravity latex particles having a specific gravity of 1.0 to 1.5 g / cc, preferably around 1.5 g / cc are used, and the average particle diameter is about 0.8 g / cc. ~ 2.
Latex particles of 2 μm, preferably around 1.0 μm on average are used as suitable ones.

Examples of such latex particles include Bacto latex 0.81 (manufactured by Difco, average particle size 0.81 μm, specific gravity 1.0 g / cc), and H0
901, H0902, H2002 (manufactured by Nippon Synthetic Rubber Co., Ltd.
The average particle size and specific gravity are 0.94 μm and 1.5 g, respectively.
/ Cc, 0.98 μm · 1.5 g / cc, and 2.22
μm · 1.5 g / cc) and the like,
However, the present invention is not limited to this, and can be used irrespective of the type as long as it has the same effect.

The antibody-sensitized latex can be obtained by mixing a suspension of latex particles diluted with an appropriate buffer with a buffer solution of the antibody. In this case, the buffer may be, for example, , Ammonium chloride buffered saline,
A PBS buffer, a glycine buffered saline and the like can be used, and among them, a glycine buffered saline is preferable from the viewpoint of the cohesiveness of the antibody-sensitized latex and the difficulty of self-aggregation.

The concentration of the antibody-sensitized latex is 0.05 to
It is good to use at 1.0%, but preferably 0.1 to 0.1%.
It is good to use at 5%, more preferably around 0.25%. The concentration of the antibody protein at the time of sensitization was 0.05
% To 0.3% is appropriate. Otherwise, the sensitivity tends to be low, and the self-aggregation of the antibody-sensitized latex tends to make it difficult to determine positive / negative. Further, as for the temperature when the sensitized latex reaction is performed, the reaction is carried out in the range of 0 ° C to 60 ° C. The resulting latex is obtained.

In order to detect nitrifying bacteria and measure the number of bacteria using these sensitized latexes, a sample solution diluted to an appropriate stage and a sensitized latex solution are mixed, and an aggregation image is observed. Then, a mixing test using a standard sample of the bacterial solution is performed, and the number of bacteria may be calculated from the results of comparison.

As a specific method, it is possible to use a microtiter method which can be easily judged with the naked eye. That is, in a U-shaped microtiter plate, a solution of an antigen or a sample serially diluted with a buffer solution is sampled, antibody-sensitized latex is dispensed into each well, and left at room temperature for 3 to 15 hours. It is possible to observe and judge using the naked eye or a loupe of about 10 times.

Specifically, the agglutination image in the case of using only the buffer solution was regarded as negative, the agglutination image in each well was determined, and the dilution ratio of the test hole showing a positive agglutination image and the agglutination test using the standard antigen were performed. From the results, calculate the number of bacteria. In the present invention, in addition to the above, a method of mixing the monoclonal antibody-sensitized latex according to the present invention with a comparative sample on a slide glass and judging the correctness of the aggregation image by an optical microscope (microscopic latex) Needless to say, the method can be used for various detection and quantification methods using a monoclonal antibody-sensitized latex to which a monoclonal antibody of nitrifying bacteria is adsorbed.

[0019]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention. (1) Preparation of Hybridoma and Production of Ascites Antibodies Purely cultured Nitrobacter agilis ( Nitrob)
acter agilis ) with physiological saline,
Then, the cells were suspended in physiological saline so that the absorbance at 660 nm became 0.5. 0.2 ml of the obtained suspension
ALB / c mice were injected into the tail vein, and 10 days later, an additional injection of 0.2 ml of a suspension prepared in the same manner. Furthermore, 1
The suspension was injected every 4 days for a total of 4-5 injections. Before sacrifice of the mice, 0.2 ml of the suspension prepared in the same manner was injected for final immunization. Four days after the final immunization, the mouse was killed, the spleen was excised, and the spleen cells were suspended in RPMI1640 medium (Sigma). After treating the spleen cell suspension with a 0.82% aqueous solution of ammonium chloride at room temperature for 10 minutes,
Washed twice by centrifugation (250 × g) in serum-free medium and then suspended in serum-free medium.

On the other hand, NS-1 cells were transformed with 15% bovine fetal serum.
After growing in RPMI1640 containing medium, the cells were washed twice by centrifugation in a serum-free medium, and then suspended in a serum-free medium. 4.0 × 10 ⁷ spleen cells in suspension and NS
After mixing 1.0 × 10 ⁷ -1 cells, the mixture was centrifuged to settle the precipitate, and polyethylene glycol 6000 was added thereto for 4 hours.
1 ml of a serum-free medium containing 5% was added, and the cells were fused at 37 ° C. for 6 minutes. After washing the obtained cell mixture by centrifugation in a serum-free medium, R containing 15% fetal bovine serum is obtained.
1.0 × 10 ⁵ spleen cells / ml in PMI1640 medium
And 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.
On the second, fourth, seventh, and tenth days, half of the culture solution was aspirated, and HAT medium was added instead. On average, 1-2 hybridoma colonies per well grew in 60-90% of the culture wells of a 96-well plastic microtiter plate. When the hybridomas were sufficiently grown (about two weeks after the start of the culture), the antibody titer of the culture supernatant to Nitrobacter agilis was assayed by enzyme immunoassay. Antibodies to Nitrobacter agilis were observed in the hybridoma culture supernatant in 55 wells out of a total of 1536 wells. The cloning operation by the limiting dilution method was repeated twice for the hybridomas in each well producing the antibody. Thus, a clone stably producing the antibody was isolated. These clones were named Nam223
It was named α. This clone Nam223α was provided to the National Institute of Bioscience and Biotechnology by the National Institute of Advanced Industrial Science and Technology.
223α, accession number FERM P-15650. The isotype of this clone is shown in Table 1.

[0022]

[Table 1]

(3) Test for Specificity of Each Monoclonal Antibody The specificity of the produced monoclonal antibody against Nitrobacter agilis against the bacteria constituting the activated sludge was determined by a color reaction using an enzyme-linked immunosorbent assay (ELISA). A comparative test was performed using. Table 2 shows the cells used for comparison.
FIG. 1 shows the results.

[0024]

[Table 2]

As the antigen, Pseudomonas sp., Alcaligenes sp., Flavobacterium sp., Actinomycetes, etc., which are often reported as dominant species of activated sludge, were used. As is clear from FIG. 1, the prepared monoclonal antibody Nam223α
Has high specificity for Nitrobacter agilis. And, these monoclonal antibodies having high specificity can always obtain the same specificity, binding strength, antibody conversion, etc., so that there is no variation in the test results, and as a reagent, It is possible to standardize the properties, and as described above, a reaction characteristic such as agglutination reaction of the antibody to each bacterium is directly used as an index, or a color development reaction correlated with the reaction characteristic is used as an index, It was found that the produced latex was useful for easily detecting nitrifying bacteria in activated sludge, water, soil and the like.

(3) Production of monoclonal antibody-sensitized latex The monoclonal antibody produced as described above was diluted with 0.1 M glycine buffered saline (pH 8.2) to an appropriate protein concentration. To 1 volume of this monoclonal antibody dilution, add 1 volume of high specific gravity latex H0902 solution,
After mixing well, the mixture was allowed to stand at 37 ° C. for 1 hour to adsorb the antibody to the latex. Thereafter, a buffer containing 1% BSA was added to stop the reaction. After removing excess antibody by centrifugation, the stock solution was suspended so that the latex concentration was 0.25%.

(4) Examination of the optimal antibody protein concentration at the time of sensitization The optimal monoclonal antibody protein concentration at the time of sensitization was examined. For each monoclonal antibody, the concentration of the antibody protein used for sensitization was changed, and the optimal antibody protein concentration was examined.

Other conditions are as follows: high specific gravity latex is H0
Sensitization temperature is 37 ° C., and sensitization time is 60
Glycine buffered saline was used as the buffer and the sensitized latex concentration was 0.25%, and the agglutination reaction time was 3 to 15 hours. The number of cells to be compared was measured under a microscope using a hemocytometer because nitrifying bacteria were autotrophic bacteria and the growth of the cells was slow. Table 3 shows the results.

[0029]

[Table 3]

As shown in Table 3, it was revealed that the number of bacteria could be quantified in the same manner as when using the polyclonal antibody or even less.

[0031]

As described above in detail, the present invention relates to a monoclonal antibody or the like specific to nitrifying bacteria exhibiting nitrite oxidizing activity. It is possible to produce an antibody having the same specificity, binding strength, and antibody titer by using the same. Further, the present invention makes it possible to standardize the properties of the antibody-sensitized latex reagent by using the above-mentioned monoclonal antibody, and to activate activated sludge, in water, in soil, in a microorganism-immobilized carrier, or the like, to activate the target microorganism. Are more specifically identified, and the number can be quickly and appropriately managed.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram relating to the antibody specificity of the monoclonal antibody Nam223α.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location G01N 33/545 G01N 33/569 F 33/569 C12N 5/00 B // C12N 15/02 9282− 4B 15/00 C

Claims (6)

[Claims] 1. An anti-Nitrobacter agiris monoclonal antibody that specifically recognizes Nitrobacter agiris. 2. The anti-nitrobacter according to claim 1,
Fusion cells (hybridomas) producing Agilis monoclonal antibodies. 3. An antibody-sensitized latex of Nitrobacter agilis, wherein a monoclonal antibody specific to Nitrobacter agilis is adsorbed to latex particles. 4. The method according to claim 1, wherein the latex particles have a specific gravity of 1.5 g /
4. The antibody-sensitized latex of Nitrobacter agilis according to claim 3, which is a latex particle having a high specific gravity of about cc. 5. The latex particles having an average particle size of 1.0 μm.
The antibody-sensitized latex of Nitrobacter agilis according to claim 3 or 4, which is latex particles having a particle size of about m. 6. A method for detecting and quantifying Nitrobacter agilis, comprising using the antibody-sensitized latex of Nitrobacter agilis according to claim 3, 4, or 5.