JPH09121887A - Monoclonal antibody against nitrosomonas-europaea and its detection and determination - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monoclonal antibody expressing a specific property to Nitrosomonas.europaea, an antibody-sensitive latex for detecting a nitrifying bacterium such as a fused cell producing the monoclonal antibody, and a method for detecting the nitrifying bacterium with the latex, etc. SOLUTION: This monoclonal antibody specific to Nitrosomonas.europaea is produced by culturing the clone of a fused cell (hybridoma) prepared by fusing a tumor cell with an antibody-producing cell obtained from an animal immunized with the Nitrosomonas.europaea having an ammonia-removing activity. The antibody-sensitive latex for detecting the nitrifying bacterium is produced by allowing latex particles to adsorb the antibody. The Nitrosomonas.europaea is detected and determined with the antibody-sensitive latex. The antibody always having the same specificity, the same binding force and the same antibody titer can be produced with the monoclonal antibody- producing bacterium specific to the nitrifying bacterium. The objective microorganisms in active sludges, etc., can be identified and determined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to nitrosomonas
Monoclonal antibody with high specificity to Europaea (ammonia-oxidizing bacteria) and a method for easily detecting Nitrosomonas europaea present in activated sludge, water, soil, or microorganism-immobilized carrier The present invention relates to a monoclonal antibody-sensitized latex used for detection of Nitrosomonas europaea, and further to a method for detecting nitrifying bacteria using the monoclonal antibody-sensitized latex. More specifically, the present invention relates to a nitrifying bacterium having an ammonia oxidizing activity in activated sludge, soil, or a microorganism-immobilized carrier.
to provide a monoclonal antibody against ia),
Nitrosomonas europaea ( Nitrosom) produced by culturing a clone of a so-called hybridoma, which is a fusion cell obtained by fusing antibody-producing cells and tumor cells from an animal immunized with Nitrosomonas europaea
onas europaea ), which enables the establishment of a method for detecting the active target microorganism in activated sludge or soil, or in a microorganism-immobilized carrier and accurately quantifying the number of bacteria. The present invention relates to a novel monoclonal antibody, a monoclonal antibody-sensitized latex for detecting Nitrosomonas europaea using the antibody, and a method for detecting and quantifying the latex.

[0002]

2. Description of the Related Art Conventionally, as a method for simply detecting ammonia-oxidizing bacteria in activated sludge or soil, generally, a sample is taken, and after a culture period of about 1 to 2 months, the sample A method for indirectly measuring nitrite based on the presence or absence of nitrite production has been known (see Kimura Ryusuke et al., Soil Microbial Experiments, pp. 207-214 (1992)). Conventionally,
By utilizing such a measuring method, the trend of active target microorganisms in activated sludge and soil has been managed. 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 antibodies for ammonia-oxidizing bacteria or nitrite-oxidizing bacteria, and rapidly detecting ammonia-oxidizing bacteria or nitrite-oxidizing bacteria by the method. A method of doing so has been proposed (JP-A-5-322896). In addition, a method for easily detecting ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, and further denitrifying bacteria by using an antibody-sensitized latex in which the antibody is adsorbed on latex particles is proposed (patent application). Hei 6-76336). However, since the antibody is a polyclonal antibody, the method of producing the antibody varies depending on the individual animal, and it has been extremely difficult to always obtain the same specificity, binding strength and antibody titer. From this, it was difficult to standardize the properties of the antiserum reagent, and there was a possibility that variations in test results could occur 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.

[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.
Applying the technology of tissue cell culture, production of a monoclonal antibody that makes it possible to always obtain an antibody having extremely uniform specificity and avidity based on antigen information, and production of an ammonia-oxidizing bacterium using the monoclonal antibody We have conducted intensive studies to establish a new detection method.

That is, an object of the present invention is to provide a novel monoclonal antibody against the ammonia-oxidizing bacterium Nitrosomonas europaea. Further, a method for producing the monoclonal antibody and a sensitization of the monoclonal antibody. The present invention provides a method for detecting Nitrosomonas europaea using latex and quantifying the number of bacteria with high sensitivity.

[0006]

Means for Solving the Problems The present invention for solving the above problems relates to an anti-nitrosomonas europaea monoclonal antibody that specifically recognizes nitrosomonas europaea, which is a bacterium of nitrosomonas europaea. The above-mentioned anti-Nitrosomonas europaea monoclonal antibody that specifically recognizes the antigen-determining site of the body is a preferred embodiment. Further, the present invention is a fused cell (hybridoma) that produces the above-mentioned anti-nitrosomonas europaea monoclonal antibody, wherein nitrosomonas characterized by adsorbing a monoclonal antibody specific for nitrosomonas europaea onto latex particles. The above is related to the antibody-sensitized latex of Europaea, wherein the latex particles are high specific gravity latex particles having a specific gravity of around 1.5 g / cc. A preferred embodiment is the antibody-sensitized latex of Nitrosomonas europaea, which is latex particles of about 1.0 μm. Further, the present invention relates to a method for detecting and quantifying Nitrosomonas europaea, which comprises using the antibody-sensitized latex of Nitrosomonas europaea.

[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 Nitrosomonas europaea, and a fused cell (hybridoma) between an antibody-producing cell and a tumor cell obtained from the animal.
Then, the fused cells are grown, and cells producing antibodies showing specificity for Nitrosomonas europaea are selected from the grown fused cells, and the antibody-producing cells are cultured to Nitrosomonas europaea monoclonal antibody is produced. The anti-Nitrosomonas europaea monoclonal antibody according to the present invention is a reagent for quantifying the number of bacteria of Nitrosomonas europaea in activated sludge or the like, or a nitrifying bacterium having an ammonia-oxidizing activity in a microorganism-immobilized carrier. It is extremely useful as a reagent for the maintenance and management of.

The above-mentioned fused cell, so-called hybridoma, is a cell that produces an antibody against the nitrosomonas europaea obtained from an animal immunized with nitrosomonas europaea (hereinafter, abbreviated as nitrosomonas europaea antibody-producing cell). , A myeloma cell that is a tumor cell. The Nitrosomonas europaea antibody-producing cells used are preferably spleen cells from an animal immunized with Nitrosomonas europaea. As the above-mentioned Nitrosomonas europaea antibody-producing cells and myeloma cells, it is not necessary to specify the type of animal that is the source as long as they can be fused, but in terms of fusion efficiency and stability of antibody production, It is preferable to use those derived from animals of the same race.

Among the above hybridomas, preferred hybridomas are those obtained by fusing spleen cells of mice immunized with Nitrosomonas europaea and mouse myeloma cells, and examples thereof include nitrosomonas. Anti-nitrosomonas europaea antibody-producing spleen cells from BALB / c mice immunized with Europaea and BAL
An example is a fusion hybridoma with myeloma cells of B / c mouse. After cell fusion, clone the hybridoma,
This clone is cultivated, and from the clone group, a clone producing an antibody showing specificity to the bacterium of Nitrosomonas europaea or its antigenic determinant site is selected. For detection of antibodies, Voller et al. [Bull. WHO., 53, 55-56,
(1976)], the enzyme-linked immunosorbent assay (ELIZA method) is preferably used.

More specifically, a tissue culture plate, for example, nitrosomonas europaea antigen immobilized on the hole wall of a 96-well microtiter plate is added with hybridoma culture solution or ascites of a hybridoma-inoculated mouse, Then, an anti-mouse immunoglobulin antibody labeled with peroxidase was added to this, and the peroxidase activity was changed to ortho-phenylenediamine (OP
Test with the color development of D). The development of OPD means the presence of antibody. The titer of anti-Nitrosomonas europaea monoclonal antibody of ascitic fluid inoculated with hybridoma culture solution or its concentrate or hybridoma was determined by the final dilution ratio of the sample giving OPD color development (measured value at absorbance 492 nm) of 0.5 or 1.0. Shown. A culture solution of myeloma cells P3-NS1-1-Ag4-1 (hereinafter abbreviated as NS-1) of BALC / c mice, and serum and ascites obtained by inoculating BALC / c mice with the cells were treated with nitroso. It does not have antibody activity against Monas europaea.

The production of a monoclonal antibody against Nitrosomonas europaea is carried out by maintaining and growing the above antibody-producing clone in a medium or in a histocompatibility animal or nude mouse. Or recovered from animal serum or ascites [For the recovery method, see, for example, Tatsuo Iwasaki et al., Monoclonal antibody, 88-
See page 94 (1983)].

Next, latex particles of a carrier for adsorbing the above-mentioned specific monoclonal antibody, a method for producing a sensitized latex, and the like have already been reported by the present inventors (Japanese Patent Application No. 6-76336). It can be prepared according to the publication).

That is, as the latex particles, high specific gravity latex particles having a specific gravity of 1.0 to 1.5 g / cc, preferably about 1.5 g / cc are preferably used, and the average particle size is about 0.8 to 2.2 μm. Latex particles having an average of around 1.0 μm are preferably used.

As such latex particles, for example, Bact latex 0.81 (manufactured by Difco, average particle size 0.81
μm, specific gravity 1.0 g / cc), and H0901, H0902, H2002
(Made by Japan Synthetic Rubber Co., Ltd., average particle size and specific gravity are 0.
94μm ・ 1.5g / cc, 0.98μm ・ 1.5g / cc, and 2.22μm
・ Commercially available products such as 1.5 g / cc) are exemplified, but the present invention is not limited to this, and any type of product having the same effect can be used regardless of the type.

The antibody-sensitized latex can be obtained by mixing a suspension of latex particles diluted with an appropriate buffer solution with a buffer solution of the antibody. In this case, the buffer solution is, for example, , Ammonium chloride buffered saline,
PBS buffer, glycine buffered saline, etc. are available,
Of these, glycine buffered saline is preferable because of the aggregability of the antibody-sensitized latex and the difficulty of self-aggregation.

The concentration of antibody-sensitized latex is 0.05 to 1.0%
However, it is preferable to use 0.1 to 0.5%, and more preferably about 0.25%. In addition, the concentration of antibody protein during sensitization is appropriately 0.05% to 0.3%, otherwise, it is difficult to determine whether it is soluble or negative due to decreased sensitivity or self-aggregation of antibody-sensitized latex. is there. Regarding the temperature for the sensitized latex reaction, the reaction is performed in the range of 0 ° C to 60 ° C. A latex produced is obtained.

In order to detect nitrifying bacteria and measure the number of bacteria using these sensitized latex, a sample solution diluted to an appropriate stage and a sensitized latex solution are mixed, and an agglutination image is observed. On the other hand, the number of bacteria may be calculated by comparing the results with the results of a mixing test using a standard sample of the bacterial solution.

As a concrete method of this, it is possible to use a microtiter method which can be easily judged visually. That is, a U-type microtiter plate was sampled with an antigen or sample solution diluted stepwise with a buffer solution, and antibody-sensitized latex was dispensed into each well and allowed to stand at room temperature for 3 to 15 hours. It is possible to observe and judge with the naked eye or a magnifying glass of about 10 times.

Specifically, the agglutination image in the case of using only the buffer solution is regarded as negative, the agglutination image of each hole is determined, and the agglutination test using the dilution ratio of the specific test hole and the standard antigen showing a positive agglutination image. Calculate the number of bacteria from the results of. In addition to the above, in the present invention, a monoclonal antibody-sensitized latex according to the present invention,
Monoclonal antibody-sensitized latex that adsorbs monoclonal antibodies of nitrifying bacteria, such as a method of determining whether the agglutination image is correct by optical microscopy by mixing with a comparative sample on a slide glass. Various detection used
It goes without saying that it can be used as a quantitative method.

[0020]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples. (1) Preparation of hybridoma and production of ascites antibody Pure-cultured Nitrosomonas europaea ( Nitr
Osomonas europaea ) was washed with physiological saline and then suspended in physiological saline so that the value at an absorbance of 660 nm was 0.5. 0.2 ml of the obtained suspension is
LB / c mice were injected into the tail vein, and 10 days later, 0.2 ml of the suspension prepared in the same manner was additionally injected. In addition, every 10 days in total
The suspension was injected 4-5 times. Before sacrificing the mouse, 0.2 ml of a suspension prepared in the same manner was injected to give the final immunization. Four days after the final immunization, the mouse was killed to remove the spleen, and the spleen cells were suspended in RPMI1640 medium (Sigma). The spleen cell suspension was treated with 0.82% ammonium chloride solution at room temperature for 10 minutes and then centrifuged in serum-free medium (250 xg
) Washed twice, and then suspended in serum-free medium.

On the other hand, RP containing NS-1 cells containing 15% fetal bovine serum
After being grown in MI1640 medium, it was washed twice with serum-free medium by centrifugation and then suspended in serum-free medium. After mixing 4.0 x 10 ⁷ spleen cells in suspension and 1.0 x 10 ⁷ NS-1 cells, centrifuge to settle and add 1 ml of serum-free medium containing 45% polyethylene glycol 6000 to this, 6
The cells were allowed to fuse at 37 ° C for min. After washing the resulting cell mixture by centrifugation in serum-free medium, it was suspended in RPMI1640 medium containing 15% fetal bovine serum at 1.0 x 10 ⁵ spleen cells / ml, and 0.1 ml of the suspension was added to 96 wells. It was dispensed to a plastic microtiter plate and cultured in an incubator in the presence of 7% carbon dioxide gas.

On the first day of the culture, 0.1 ml of HAT medium was added,
On the second, fourth, seventh, and tenth days thereafter, half of the culture solution was aspirated, and HAT medium was added instead. An average of 1 to 2 hybridoma colonies grew per well in 60 to 90% of the culture wells of a 96-well plastic microtiter plate. When the hybridomas have grown sufficiently (approximately 2 weeks after the start of culture), the nitrosomonas
The antibody titer against Europaea was assayed by enzyme immunoassay. Antibodies against Nitrosomonas europaea were detected in the hybridoma culture supernatant in 154 of the total wells.
The cloning operation by the limiting dilution method was repeated twice for the hybridoma in each well producing the antibody. Thus, the clone 2 strain which stably produces the antibody was isolated. These clones were designated as Nem7α and Nem23α, respectively. This clone Nem 7 α has a consignment number FERM
P-15277 has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology, and the clone Nem 23α has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Depository No. FERM P-15278. The antibody titers against Nitrosomonas europaea of the culture broth, the ascites fluid, and the ammonium sulfate precipitation fraction obtained for these two clones are shown in Table 1.
It was shown to. The isotypes of these two clones are shown in Table 2.

[0023]

[Table 1]

[0024]

[Table 2]

(2) Test for specificity of each monoclonal antibody The specificity of the produced two types of monoclonal antibodies against Nitrosomonas europaea against the bacteria constituting the activated sludge was determined by enzyme-linked immunosorbent assay (ELIZA method). The color reaction was compared. Table 3 shows the bacterial cells used for comparison.
Moreover, the result is shown in FIG.

[0026]

[Table 3]

As the antigen, Pseudomonas sp., Alcaligenes sp., Flavobacterium sp., Actinomycetes, etc., which are often reported as the dominant species of activated sludge, were used. As is clear from FIG. 1, the produced monoclonal antibodies Nem 7 α and Nem
23α was confirmed to have high specificity for Nitrosomonas europaea. And, these monoclonal antibodies having high specificity, it is possible to always obtain the same specificity, avidity and antibody titer, etc., without causing variations in test results,
It is possible to standardize the properties as a reagent, as described above, the reaction characteristics such as the agglutination reaction of the antibody against each bacterium as a direct index, or the color reaction that correlates with the reaction characteristics as an index, and It has been found that the detection antibody-sensitized latex is 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 subjected to ammonium sulfate precipitation and further to a protein A column kit (Amersham (Amersh)).
am)) and then diluted with 0.1 M glycine buffered saline (pH 8.2) to an appropriate protein concentration. 1 volume of the high specific gravity latex H0902 solution was added to 1 volume of this monoclonal antibody diluted solution, mixed well, and allowed to stand at 37 ° C. for 1 hour to adsorb the antibody to the latex. Then, a buffer containing 1% BSA was added to stop the reaction. After the excess antibody was washed off and removed by centrifugation, the stock solution was suspended so that the latex concentration became 0.25%.

(4) Examination of optimal antibody protein concentration during sensitization The optimal monoclonal antibody protein concentration during sensitization was examined. For each monoclonal antibody, the antibody protein concentration used for sensitization was 0.01, 0.015, 0.02, 0.04 (mg / m
l)).

Other conditions are H090 for high specific gravity latex.
2, the sensitization temperature was 37 ° C., the sensitization time was 60 minutes, the buffer solution was glycine buffered saline, the sensitization latex concentration was 0.25%, and the agglutination reaction time was 3 to 15 hours. Also,
The number of bacteria to be compared was measured under a microscope using a hemocytometer because nitrifying bacteria are autotrophic bacteria and the growth of the bacteria is slow. Table 4 shows the results.

[0031]

[Table 4]

As shown in Table 4, protein amount 0.02 mg / m
It was revealed that the number of bacteria can be quantified in the same manner as or lower than the case of using the polyclonal antibody at the concentration of 1.

[0033]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention relates to a monoclonal antibody specific for nitrifying bacteria and the like, and always uses the same monoclonal antibody-producing cells specific for nitrifying bacteria. It makes it possible to produce a monoclonal antibody having specificity, avidity, and antibody titer. Further, the present invention, by using the above monoclonal antibody, it is possible to standardize the properties of the antibody-sensitized latex reagent, activated sludge, water, in the soil, active target microorganisms such as in the immobilization carrier. It enables more specific identification and manages the number quickly and appropriately.

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[Procedure amendment]

[Submission date] February 23, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Added

[Correction contents]

[Brief description of the drawings]

FIG. 1 shows the results of examining the specificity of a purified monoclonal antibody.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location G01N 33/569 C12N 5/00 B 33/577 9162-4B 15/00 C

Claims (7)

[Claims] 1. An anti-nitrosomonas europaea monoclonal antibody that specifically recognizes nitrosomonas europaea. 2. The anti-Nitrosomonas europaea monoclonal antibody according to claim 1, which specifically recognizes an antigen-determining site of a bacterium of Nitrosomonas europaea. 3. A fused cell (hybridoma) producing the anti-Nitrosomonas europaea monoclonal antibody according to claim 1 or 2. 4. An antibody-sensitized latex of Nitrosomonas europaea, wherein a monoclonal antibody specific to Nitrosomonas europaea is adsorbed on latex particles. 5. The antibody-sensitized latex of Nitrosomonas europaea according to claim 4, wherein the latex particles are high specific gravity latex particles having a specific gravity of about 1.5 g / cc. 6. The latex particles have an average particle size of 1.0 μm.
The antibody-sensitized latex of Nitrosomonas europaea according to claim 4 or 5, which is latex particles before and after. 7. A method for detecting and quantifying Nitrosomonas europaea, which comprises using the antibody-sensitized latex of Nitrosomonas europaea according to claim 4, 5, or 6.