WO2021193750A1 - Monoclonal antibody, bacteria detection method, bacteria detection kit, and hybridoma - Google Patents

Abstract

Provided are: a monoclonal antibody which reacts with bacteria having the 16S rRNA gene containing a base sequence that is at least 98.2% identical to the base sequence represented by SEQ ID NO: 1; and a monoclonal antibody produced from a hybridoma identified by accession number NITE BP-03165, NITE BP-03166, NITE BP-03167, or NITE BP-03168.

Description

Monoclonal antibodies, bacterial detection methods, bacterial detection kits and hybridomas

The present invention relates to a monoclonal antibody, a method for detecting bacteria, a kit for detecting bacteria, and a hybridoma.

When sewage is purified by the activated sludge method, the removed organic matter becomes flocs containing microorganisms (bacteria), and sludge called excess sludge is generated. Excess sludge discharged from wastewater treatment facilities accounts for more than 20% of industrial waste. In general, excess sludge is incinerated after being dehydrated and dried (Non-Patent Document 1: FY2018 Business, Industrial Waste Discharge / Treatment Status Survey Report, FY2016 Results (Summary Version), Non-Patent Documents 2: Masayuki Yamamoto, "On Combustion Technology of Sewage", Journal of the Combustion Society of Japan, Combustion Society of Japan, 2011, Vol. 53, No. 164, pp. 91-96).

"FY2018 Business, Industrial Waste Discharge / Treatment Status Survey Report, FY2016 Results (Summary Version)" [online], March 2019, Ministry of the Environment, Environmental Rehabilitation and Resource Recycling Bureau, Waste Regulation Division [Ordinance] Searched on March 17, 2nd year of Japan], Internet <URL: https://www.e-stat.go.jp/stat-search/file-download?statInfId=000031887949&fileKind=2> Masayuki Yamamoto, "About Combustion Technology of Sewage", Journal of the Combustion Society of Japan, Combustion Society of Japan, 2011, Vol. 53, No. 164, pp. 91-96

However, since greenhouse gases are generated when the surplus sludge is incinerated, it is required to reduce the volume of the surplus sludge in consideration of the environment. As a method for reducing the volume of excess sludge, a method for decomposing microorganisms constituting the excess sludge is proposed. The present inventors have found a bacterium having a 16S rRNA gene containing a nucleotide sequence having 98.2% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 as a bacterium capable of degrading microorganisms constituting excess sludge. ing.

An object of the present invention is to provide a novel antibody that specifically detects the above-mentioned bacteria, a method for detecting bacteria using the antibody, and a detection kit.

The present invention relates to [1] to [8] exemplified below.
[1] A monoclonal antibody that reacts with a bacterium having a 16S rRNA gene containing a base sequence having 98.2% or more identity with the base sequence shown in SEQ ID NO: 1.
[2] Tumebacillus sp. A monoclonal antibody that reacts with NITE BP-02779.
[3] A monoclonal antibody produced from a hybridoma whose accession number is NITE BP-03165, NITE BP-03166, NITE BP-03167 or NITE BP-03168.
[4] A bacterium having a 16S rRNA gene containing a nucleotide sequence having 98.2% or more identity with the nucleotide sequence set forth in SEQ ID NO: 1 using the monoclonal antibody according to any one of [1] to [3]. Detection method.
[5] The bacterium is Tumebacillus sp. The detection method according to [4], which is NITE BP-02779.
[6] A bacterium having a 16S rRNA gene containing a monoclonal antibody according to any one of [1] to [3] and containing a nucleotide sequence having 98.2% or more identity with the nucleotide sequence set forth in SEQ ID NO: 1. Detection kit.
[7] The bacterium is Tumebacillus sp. The detection kit according to [6], which is NITE BP-02779.
[8] A hybridoma whose accession number is NITE BP-03165, NITE BP-03166, NITE BP-03167 or NITE BP-03168.

According to the present invention, a novel antibody capable of specifically detecting a bacterium having a 16S rRNA gene containing a base sequence having 98.2% or more identity with the base sequence shown in SEQ ID NO: 1, and a bacterium using the same. Detection methods and detection kits are provided.

In Experiment 4, Tumebacillus sp. It is a figure which showed the dry weight of the surplus sludge after the addition of NITE BP-02779. It is a figure which shows typically the manufacturing method of the monoclonal antibody in Experiment 5. It is the cross-reactivity evaluation result of the culture supernatant of 3A5-1H1 (accession number NITE BP-03165) in Experiment 11. It is the cross-reactivity evaluation result of the culture supernatant of 4G4-1A9 (accession number NITE BP-03166) in Experiment 11. It is the cross-reactivity evaluation result of the culture supernatant of 6D9-2G6 (accession number NITE BP-03167) in Experiment 11. It is the cross-reactivity evaluation result of the culture supernatant of 7D4-1G9 (accession number NITE BP-03168) in Experiment 11.

Hereinafter, a mode for carrying out the present invention will be described in detail. The present invention is not limited to the following embodiments.

[antibody]
One embodiment of the antibody according to the present invention is a bacterium having a 16S rRNA gene containing a base sequence having 98.2% or more identity with the base sequence shown in SEQ ID NO: 1 (hereinafter, referred to as "bacteria a"). Is a monoclonal antibody that reacts with). Examples of the bacterium a include bacteria belonging to the genus Tumebacillus. Bacteria a preferably have target microbial resolution. Bacteria a may be the whole or a part of the bacterium a.

Bacteria a have an identity of 98.5% or more, 98.8% or more, 99.0% or more, 99.3% or more, 99.5% or more or 99 as compared with the nucleotide sequence shown in SEQ ID NO: 1. It may have a 16S rRNA gene containing a base sequence of .8% or more. Bacteria a are described in Tumebacillus sp. It may be NITE BP-02779.

Bacteria a may have a 16S rRNA gene containing a base sequence in which one or several bases have been substituted, deleted or added as compared with the base sequence shown in SEQ ID NO: 1. The single base or several bases can be, for example, 1 to 25 bases, preferably 1 to 10 bases, and more preferably 1 to 5 bases. The above mutation is a mutation in which the expression and function of 16S rRNA are not lost.

The bacterium a contains about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and further preferably about 18 to about 50 bases contained in the base sequence shown in SEQ ID NO: 1. It may have a 16S rRNA gene containing a base sequence that hybridizes to a contiguous sequence or a complementary sequence thereof under stringent conditions. The stringent condition means a condition in which a non-specific hybrid is not formed, for example, in 60 ° C., 1 × SSC, 0.1% SDS, preferably 68 ° C., 0.1 × SSC, 0.1% SDS. The conditions for washing at least once are mentioned.

The base sequence of the 16S rRNA gene can be analyzed by, for example, the following method. First, genomic DNA is extracted from the target microorganism using a known method, and the 16S rRNA gene is amplified. The method for amplifying the 16S rRNA gene is not particularly limited, and examples thereof include a PCR method using a universal primer usually used by those skilled in the art. The amplification product obtained by the PCR method can be purified as needed and subjected to a DNA sequencer or the like to determine the base sequence. The obtained base sequence is compared with the sequence shown in SEQ ID NO: 1.

Whether or not the bacterium a has microbial resolution can be confirmed by a method usually used by those skilled in the art. Examples of such a confirmation method include a method of reacting the bacterium a with the target microorganism in an appropriate medium or buffer for a certain period of time, and then detecting the decomposition of the target microorganism in the medium or buffer. The method for detecting the degradation of the target microorganism is not particularly limited, but for example, a method for measuring the turbidity of the target microorganism, a method for detecting the target microorganism with an SLP reagent, a method for detecting the DNA of the target microorganism by PCR, and a method for detecting the DNA of the target microorganism. Method for measuring dry cell weight, high-speed liquid chromatography (HPLC); mass spectrometry (MS); thin layer chromatography (TLC); nuclear magnetic resonance (NMR); targeting using gas chromatography (GC), etc. Examples thereof include a method for detecting a decomposition product derived from a microorganism.

When examining the decomposition of the target microorganism by turbidity, having the target microbial resolution means that the turbidity after the reaction is significantly lower than the turbidity before the reaction. For example, the turbidity after the reaction is the turbidity before the reaction. It is 80% or less of the turbidity, preferably 50% or less, and more preferably 30% or less. When examining the decomposition of the target microorganism by the dry cell weight, having the target microorganism resolution means that, for example, the dry cell weight after the reaction is significantly lower than that before the reaction, for example, the dry cell weight after the reaction is , 95% or less before the reaction, preferably 90% or less.

One embodiment of the antibody according to the present invention is Tumebacillus sp. It is a monoclonal antibody that reacts with NITE BP-02779 (hereinafter, may be referred to as "NITE BP-02779").

NITE BP-02779 was internationally deposited on September 11, 2018 at the National Institute of Technology and Evaluation Patent Microbial Deposit Center (Room 122, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818). It is a bacterium for which a certificate of acceptance for the original deposit and a certificate of survival were issued on September 26, 2018. NITE BP-02779 is considered to be a new species of bacteria belonging to the genus Tumebacillus. NITE BP-02779 is useful for treating excess sludge because it can decompose various target microorganisms such as Bacillus, Micrococcus, Staphylococcus and excess sludge. The mycological properties of NITE BP-02779 are shown in Tables 1 to 4 of Examples described later. NITE BP-02779 is a bacterium having a 16S rRNA gene containing the nucleotide sequence shown in SEQ ID NO: 1.

The bacterium a with which the antibody reacts may be a live bacterium or a dead bacterium. Viable bacteria can be obtained by culturing the bacterium a in a suitable medium. As the medium, various media containing a carbon source, a nitrogen source, an organic or inorganic salt, etc., which are usually used for culturing microorganisms, can be used. Specifically, ATCC802 medium, R2A medium and the like can be mentioned. A suitable buffer or drainage may be used instead of the medium. Killed bacteria can be obtained, for example, by denaturing live bacteria. Examples of the denaturing treatment include treatment with a denaturing agent such as SDS, physical crushing treatment, heat treatment and the like. The sample containing the bacterium a may be a mixture of live and dead bacteria of the bacterium a.

"Antibody reacts" may mean that the antibody and the antigen bind to each other or the antibody recognizes the antigen by the antigen-antibody reaction. "Antibody reacts with a bacterium" may mean that the bacterium and the antibody bind to each other or that the antibody recognizes the bacterium. Examples of a method for examining whether or not a bacterium reacts with an antibody include ELISA, flow cytometer, immunostaining, and Western blotting. The "antibody that reacts with bacteria" can also be called "antibody against bacteria".

For example, when examining the reaction between an antibody and a bacterium by ELISA, the reaction with the bacterium means that the detected value (absorbance) of the bacterium when the antibody is added is significantly higher than the detected value (absorbance) of the negative control, for example, with a positive antigen. The difference in ABS450 value from the negative antigen is 0.05 or more, and the ABS450 value of the negative antigen is 0.05 or less.

An "antibody" is an immunoglobulin that specifically binds to a certain antigen, and a "monoclonal antibody" is an antibody obtained from a clone derived from a single antibody-producing cell. The monoclonal antibody is obtained as a secretion in the culture medium, for example, by culturing a cloned hybridoma (hereinafter, may be simply referred to as “clone”). The "cloned hybridoma" is, for example, a hybridoma in which antibody-producing cells and myeloma cells (myeloma cells) are fused to produce a hybridoma, and a hybridoma that produces an antibody having the desired antigen specificity is selected and cloned. Obtained by

The antibody according to the present invention can be obtained by a method including a step of immunizing an animal with a bacterium a. As a step of immunizing an animal with the bacterium a, a step of immunizing an animal in a known method for producing a monoclonal antibody can be used. Examples of the method for producing a monoclonal antibody include a mouse spleen method and a mouse iliac lymph node method (see JP-A-2007-02547). From the viewpoint of obtaining highly specific antibody, the mouse iliac lymph node method is preferable.

The animal to be immunized is not particularly limited, and may be appropriately selected from non-human animals according to the production method of the monoclonal antibody. Specifically, when the mouse iliac lymph node method is used as a method for producing a monoclonal antibody, the animal may be immunized according to the method described in the above literature.

After immunizing an animal, a hybridoma can be prepared, sorted, or the like according to a known method to obtain an antibody according to the present invention. In the selection of hybridomas, bacteria a, for example, NITE BP-02779, are used as positive antigens, and BSA, Escherichia, Rhodococcus loti, Xanthomonas, Sphingomonas, Brevibacterium, Pseudomonas, Pseudomonas, etc. are used as negative antigens. good.

The criteria for selecting hybridomas is, for example, when ELISA is used, the difference in ABS450 value between the positive antigen and the negative antigen is 0.05 or more and the ABS450 value of the negative antigen is 0 for the produced monoclonal antibody. It is less than 0.05.

The isotype of the antibody according to the present invention is not particularly limited. The antibody according to the present invention may be an immunoglobulin molecule, or may be a partial fragment of an antibody having an antigen-binding activity, for example, F (ab') 2, Fab', Fab, CDR, or the like.

The antibody according to the present invention may be an artificially synthesized antibody. The antibody according to the present invention is preferably an isolated or purified antibody, for example, an antibody purified from the serum of an immunized animal, ascites, etc. or the culture supernatant of the hybridoma. The antibody may be purified by a known method, and examples thereof include sulfur fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography for protein A or protein G, gel filtration chromatography, and isoelectric point precipitation. .. One of these methods may be used alone, or two or more thereof may be used in combination as appropriate. The purification of the antibody can be performed to the desired degree.

The antibody according to the present invention may be labeled with an enzyme such as alkaline phosphatase (ALP) and horseradish peroxidase (HRP), or a labeling substance such as biotin, a fluorescent substance, a luminescent substance, or a radioactive isotope.

The antibody according to the present invention preferably does not substantially react with microorganisms other than the bacterium a. The antibody according to the present invention preferably does not substantially react with any one or more bacteria selected from group (I), and more preferably substantially with any one or more bacteria selected from group (II). Not substantially reacting, and more preferably substantially not reacting with any one or more bacteria selected from group (III).
Group (I): Tumebacillus, Escherichia, Rhizobium, Xanthomonas, Sphingomonas, Brevibacterium, Pseudomonas, Rhodococcus, Novosphingobium and Sphingomonas excluding bacteria a
Group (II): Tumebacillus, Escherichia, Rhizobium, Xanthomonas, Brevibacterium, Pseudomonas, Rhodococcus, Novosphingobium and Sphingobium excluding bacteria a
Group (III): Escherichia, Xanthomonas, Pseudomonas, Novosphingobium and Sphingobium

Here, "substantially no reaction" means that the number of antibodies that bind to the target bacterium is smaller than the number of antibodies that bind to the bacterium a by the antigen-antibody reaction. "Substantially not reacting" means that, for example, when evaluated by a solid-state ELISA, the ratio (%) of the absorbance when the target bacterium is used as an antigen to the absorbance when bacterium a is used as an antigen is 0% or more and 50%. It means that it is preferably 0% or more and 40% or less, more preferably 0% or more and 30% or less, and particularly preferably 0% or more and 20% or less.

An embodiment of the antibody according to the present invention includes a monoclonal antibody produced from the cloned hybridomas 3A5-1H1, 4G4-1A9, 6D9-2G6 or 7D4-1G9 described in Examples described later. These monoclonal antibodies show high reactivity to bacteria a, especially NITE BP-02779, excluding bacteria a, Tumebacillus, Escherichia, Rhodococcus, Xanthomonas, Sphingomonas, Brevibacterium and Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas. ..

3A5-1H1 has the accession number NITE BP-03165 (consignment date: March 3, 2020), 4G4-1A9 has the accession number NITE BP-03166 (consignment date: March 3, 2020), and 6D9-2G6 has. , Commission number NITE BP-03167 (consignment date: March 3, 2020) and 7D4-1G9 are designated as trust number NITE BP-03168 (consignment date: March 3, 2020), respectively. It has been internationally deposited at the National Institute of Technology Patented Microbial Depositary Center (NPMD, address: 〒292-0818, 2-5-8, Kazusakamatari, Kisarazu City, Chiba Prefecture, Room 122) based on the Budapest Treaty.

The antibody according to the present invention is an antibody having high specificity for bacteria a, and is useful for detecting bacteria a in a sample containing, for example, a plurality of types of microorganisms. Examples of samples that can contain a plurality of types of microorganisms include activated sludge, excess sludge, soil, wastewater, and water. Since the bacterium a having the target microbial resolution, for example, NITE BP-02779, can decompose the microorganisms constituting the surplus sludge, it is not possible to confirm the presence of the bacterium a in the environment containing the surplus sludge. , Useful for reducing the volume of excess sludge.

[Bacterial detection method]
The detection method according to the present invention detects bacteria a using the antibody according to the present invention. According to the detection method according to the present invention, the bacterium a in the sample can be specifically detected, and for example, the bacterium a can be detected even when a plurality of types of microorganisms are present in the sample. .. The bacterium detected in the detection method according to the present invention is, for example, NITE BP-02779. One aspect of the present invention is the use of the antibody to detect bacteria a.

As a method for detecting the bacterium a, a known immunological measurement method can be used. Examples of immunological measurement methods include immobilized ELISA, competitive ELISA, sandwich ELISA, immune complex transfer measurement method, immunoturbidimetric method, immunochromatography method, latex agglutination method and the like. As an example of a method for detecting a bacterium, a case where the bacterium a in a sample is detected by solid-phase ELISA will be described below.

First, a complex containing the antibody according to the present invention and the bacterium a is formed. The complex can be formed by mixing the antibody according to the present invention with a sample containing the bacterium a. The complex is then immobilized on the solid phase by contacting the solution containing the complex with a solid phase capable of capturing the bacterium a. As the solid phase, a solid phase in which a sample containing bacteria a is fixed in advance may be used. That is, the above complex can be formed on the solid phase by contacting the sample fixed on the solid phase with the antibody according to the present invention. The sample may or may not be subjected to pretreatment such as centrifugation, denaturation treatment, washing, etc. before the detection of the bacterium a.

The mode of fixing the sample to the solid phase is not particularly limited, and for example, the sample and the solid phase may be directly bound, or the capture antibody and the solid phase may be indirectly bound via another substance. good. Examples of the method of direct bonding include physical adsorption and the like.

The material of the solid phase is not particularly limited, and can be selected from, for example, an organic polymer compound, an inorganic compound, a biopolymer and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene and the like. Examples of the inorganic compound include magnetic substances (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, and the like. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose and the like. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include particles, membranes, microplates, microtubes, and test tubes.

Add the antibody according to the present invention to the solid phase on which the complex is immobilized, and wash. After washing, the presence or absence of the bacterium a in the sample can be determined and the amount (number of bacteria) thereof can be measured by detecting the antibody bound to the solid phase by a method known in the art. For example, when an antibody labeled with a labeling substance is used as the antibody according to the present invention, the presence or absence of bacteria a in the sample and the amount thereof can be measured by detecting the signal generated by the labeling substance. When an unlabeled antibody is used as the antibody according to the present invention, a labeled secondary antibody against the antibody according to the present invention is added to the solid phase, and the signal generated by the labeled substance is detected to detect the bacteria in the sample. The presence or absence of a and its amount can be measured.

"Detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection means to indicate the signal intensity stepwise, such as "no signal generated", "weak", "medium", "strong", and the like. In this embodiment, it is preferable to detect the signal intensity quantitatively or semi-quantitatively.

The labeling substance is not particularly limited as long as a detectable signal is generated. For example, it may be a substance that generates a signal by itself (hereinafter, also referred to as a “signal generating substance”), or may be a substance that catalyzes the reaction of another substance to generate a signal. Examples of the signal generating substance include a fluorescent substance, a radioisotope and the like. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of the radioactive isotope include 125I, 14C, 32P and the like. Examples of substances that catalyze the reaction of other substances to generate signals include enzymes. Examples of the enzyme include ALP, HRP, β-galactosidase, luciferase and the like. As the labeling substance, an enzyme is preferable, and ALP or HRP is particularly preferable.

The method itself for detecting the signal is known in the art, and the detection method according to the type of the signal derived from the labeling substance may be appropriately selected. For example, when the labeling substance is an enzyme, signals such as light and color generated by reacting a substrate with the enzyme may be detected using a known device such as a spectrophotometer.

The substrate of the enzyme can be appropriately selected from known substrates according to the type of enzyme. For example, when ALP is used as the enzyme, the substrate is CDP-Star® (4-chloro3- (methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) trixilo] [3. 3.1.13,7] decane] -4-yl) disodium phenylphosphate), CSPD® (3- (4-methoxyspiro [1,2-dioxetane-3,2- (5'-) Chemiluminescent substrate such as chloro) tricyclo [3.3.1.13,7] decane] -4-yl) disodium phenylphosphate; 5-bromo-4-chloro-3-indrill phosphate (BCIP) , 5-Bromo-6-chloro-indrill phosphate disodium, p-nitrophenyl phosphate and other chromogenic substrates can be mentioned. When HRP is used as the enzyme, the substrate is a chemiluminescent substrate such as luminol and its derivatives; 2,2'-azinobis (3-ethylbenzothiazolin-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine. (OPD), 3,3', 5,5'-tetramethylbenzidine (TMB) and other color-developing substrates can be mentioned.

When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of the fluorescent substance used.

[Bacterial detection kit]
The antibody according to the present invention can be used in a kit for detecting bacteria a. The kit according to the present invention contains the antibody according to the present invention, and also contains a developing solvent, a buffer solution, a washing solution, a blocking reagent, an enzyme substrate, a coloring reagent, a solid phase (including latex particles), and a standard for bacteria a. Reagents such as samples and / or devices or instruments such as containers, reactors, fluorescent readers, etc. may be included. The kit may be an immobilized ELISA kit containing the antibody according to the present invention, a sandwich ELISA kit, a latex agglutination method kit, an immunochromatography kit, or the like. The kit is preferably a latex agglutination kit because it can be measured on-site. The bacterium a detected by the detection kit according to the present invention is, for example, NITE BP-02779. The kit may be the kit used for the above-mentioned detection method. One aspect of the present invention is the use of the above antibody for the production of a kit for detecting bacteria a.

Hereinafter, this kit will be described in more detail by taking the latex agglutination method kit as an example. The latex particles used in the latex agglutination method kit preferably have a specific gravity of 0.8 to 1.2 mg / ml, and more preferably about 1.05 mg / ml. The average particle size of the latex particles is, for example, about 0.1 to 4.0 μm, preferably about 0.5 to 1.5 μm, and more preferably about 1.0 μm on average. Colored latex particles are preferably used as the latex particles. Examples of such latex particles include Bact Latex 0.81 (manufactured by DIFCO, average particle size 0.81 μm, specific density 1.0 g / ml), Polybeads # 15713 (manufactured by Polyscience, average particle size 1.0 μm). Commercially available products such as (specific gravity 1.05 g / ml) can be exemplified. The latex particles are not limited to these, and any type of latex particles having the same effect as these can be used.

It is preferable that the latex particles have the antibody according to the present invention adsorbed on them. The method for producing the latex particles of the carrier and the sensitized latex particles for adsorbing the specific antibody is not particularly limited, and can be prepared according to, for example, International Publication No. 2017/138608. As a method for sensitizing the antibody to the latex particles, both a physical adsorption method and a chemical bond method can be preferably used.

The sensitizing antibody is preferably a monoclonal antibody produced by any one or more hybridomas selected from group (A), and more preferably produced by any one or more hybridomas selected from group (B). It is a monoclonal antibody, more preferably a monoclonal antibody produced by any one or more hybridomas selected from the group (C). For these monoclonal antibodies, one type of monoclonal antibody may be sensitized to latex particles (latex beads) alone, or several types of monoclonal antibodies may be mixed and sensitized to latex particles. Further, latex particles sensitized with different monoclonal antibodies may be mixed and used.
Group (A): The accession numbers are NITE BP-03165, NITE BP-03166, NITE BP-03167 and NITE BP-03168.
Group (B): The accession numbers are NITE BP-03166, NITE BP-03167 and NITE BP-03168.
Group (C): The accession numbers are NITE BP-03166 and NITE BP-03168.

The step of sensitizing the latex particles to the antibody is preferably performed in a buffer solution. Specifically, a suspension of latex particles diluted to an appropriate concentration and a solution of an antibody are mixed, left for a while, and then washed to produce sensitized latex particles. As the buffer solution used for dilution, a buffer solution having an ionic strength and pH that does not inhibit the reaction of the carried antibody with the antigen, which is the substance to be measured, is generally selected. As the buffer solution, for example, TBS, PBS, GBS, phosphate buffer solution and borate buffer solution can be used. Among them, a phosphate buffer solution, a borate buffer solution and GBS are preferably used as a buffer solution because of the cohesiveness of the sensitized latex particles and the difficulty of self-aggregation. The buffer solution is generally selected in the range of pH 5 to 10, and particularly preferably in the range of pH 6 to 9.

A suitable concentration of latex particles at the time of sensitization is 0.01 to 0.5 (w / v)%, and particularly good sensitized latex particles are obtained at about 0.05 to 0.25 (w / v)%. can get. Further, when the antibody is sensitized, the antibody protein concentration of the antibody solution is preferably 1.0 to 1000 μg, and in other cases, it is difficult to judge positive or negative due to the decrease in sensitivity and the self-aggregation of the sensitized latex particles. Become. The temperature at the time of the sensitization reaction is in the range of 0 to 60 ° C., but when the reaction is carried out at room temperature or a temperature slightly higher than room temperature (up to 40 ° C.), highly sensitive latex particles can be obtained.

In order to detect bacteria a and measure the number of bacteria using these sensitized latex particles, the reverse passive latex agglutination method is preferable. The reverse passive latex agglutination method includes a liquid reagent containing immunological agglutination reaction particles in which a substance (antibody, etc.) having a property of specifically binding to the test substance is bound to the latex particles, and the test substance. This is a method of mixing a sample and detecting or quantifying the test substance. More specifically, the sample diluted at an appropriate step and the reagent containing the antibody-sensitized latex particles are mixed, and the degree of dilution at which the agglutination image is observed is confirmed. On the other hand, a test in which a standard sample containing a known number of bacteria and a reagent containing antibody-sensitized latex particles are mixed is performed, and the number of bacteria may be calculated from the comparison with the results.

More specifically, a sample containing an antigen diluted stepwise with a buffer solution or a standard sample is added to a U-shaped microtiter plate. After dispensing a reagent containing an equal amount of antibody-sensitized latex particles into each hole, the mixture is allowed to stand at room temperature for 4 to 15 hours. After that, the presence or absence of the antigen is determined and the concentration (number of bacteria) is measured by observing the agglutinated image using the naked eye or a loupe of about 10 times. The agglutination image in the case of only the buffer solution is negative, the agglutination image of each hole is judged, and the number of bacteria is calculated from the dilution ratio of the test hole showing the positive agglutination image and the result of the agglutination test using the standard antigen. can do.

Other latex agglutination kits include a method of mixing latex particles and a test sample on a slide glass and determining the success or failure of the agglutination image with an optical microscope (microscopic latex method), immunochromatography, etc. It may be a kit using various qualitative and quantification methods using agglutination reaction particles.

According to the kit according to the present invention, bacteria a contained in a sample can be easily detected. The kit according to the present invention can also be used to detect bacteria a from samples such as excess sludge, soil, and wastewater.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Experiment 1. Isolation of microbially degrading bacteria]
(Method)
By culturing a group of microorganisms existing in the environment (water) using a medium containing Micrococcus bacteria as a carbon source, microorganisms degrading Micrococcus bacteria were accumulated and cultured. Next, several strains with good growth were isolated from the group of microorganisms that had been enriched and cultured.

(result)
When the resolution of Micrococcus bacteria was examined for each isolated strain, the resolution of Micrococcus bacteria was observed in one strain. Hereinafter, a strain in which the resolution of Micrococcus bacteria is recognized may be referred to as "strain A".

[Experiment 2. Identification of strain A]
(material)
-Cloning forward primer (27F: SEQ ID NO: 2)
-Reverse primer for cloning (1492R: SEQ ID NO: 3)
-Primers for sequence analysis (339F: SEQ ID NO: 4, 536R: SEQ ID NO: 5, 907F: SEQ ID NO: 6)

(Method)
Strain A was identified by 16S rRNA gene analysis, morphological observation and physiological / biochemical property tests.

16S rRNA gene analysis was performed according to the following procedure. Genomic DNA was extracted from strain A, and the obtained genomic DNA was used as a template for PCR amplification of the 16S rRNA gene using a cloning forward primer (27F) and a cloning reverse primer (1492R). PCR amplification was performed using KOD FX (manufactured by Toyobo Co., Ltd.), and the amplified product after PCR was purified.

A cycle sequence reaction was performed using the purified amplification product after PCR. The cycle sequence reaction was carried out using BigDye Terminator v3.1 Cycle Sequencing Kit. The obtained reaction solution was purified, and the purified solution was subjected to DNA sequence analysis (3730 xl DNA Analyzer) to determine the base sequence of the 16S rRNA gene of the template DNA extracted from the strain A.

Morphological observation and physiological / biochemical property tests are performed by morphological observation using an optical microscope, and the method of BARROW et al. , Lyon, France).

(result)
The obtained nucleotide sequence of the 16S rRNA gene (SEQ ID NO: 1) was subjected to homology analysis against the international nucleotide sequence database (DDBJ / ENA (EMBL) / GenBank). Among the reference strains, 98.1% identity was shown with respect to the nucleotide sequence of the 16S rRNA gene of Tumebacillus permanentifrigoris Eur_9.5. However, no microorganism had a 16S rRNA gene that completely matched the obtained nucleotide sequence. In addition, strain A did not grow at 10 ° C., but Tumebacillus permanentifrigoris Eur_9.5, which has the highest homology of the 16S rRNA gene, did not show such a feature. Therefore, it was suggested that the strain A is a new species different from the conventional Tumebacillus. Strain A was referred to as Tumebacillus sp. Internationally deposited as NITE BP-02779.

Tables 1 to 4 show the morphological observation and physiological / biochemical property test results of NITE BP-02779.

[Experiment 3. Resolution evaluation of NITE BP-02779 for excess sludge (killed bacteria) -1]
(material)
-R2A medium: Medium in which R2A Broth, DAIGO (manufactured by Nippon Pharmaceutical Co., Ltd.) was dissolved in 1000 mL of ultrapure water at a ratio of 3.2 g and sterilized by high pressure steam.-Inorganic medium containing excess sludge (killed bacteria): 986 mL substrate solution, 3.0 mL A solution, 3.0 mL B solution, 3.0 mL C solution, 3.0 mL D solution, and 1.8 mL 1% phosphoric acid mixed medium as the substrate solution and solutions A to D , The following was used.
Substrate solution: After washing excess sludge, it was mixed with 986 mL of ultrapure water so that the turbidity (OD660) was 0.2, and high-pressure steam sterilization was performed. Solution A: 4.35 g Dipotassium hydrogen phosphate, 1.70 g monopotassium dihydrogen phosphate, 8.92 g disodium hydrogen phosphate dodecahydrate, 0.34 g ammonium chloride was dissolved in ultrapure water, prepared in 200 mL, and subjected to high-pressure steam sterilization. Solution B : 4.50 g Magnesium sulfate heptahydrate dissolved in ultrapure water, prepared to 200 mL, and subjected to high-pressure steam sterilization Solution C: 5.50 g Calcium chloride anhydride dissolved in ultrapure water to 200 mL Prepared and high-pressure steam sterilized solution D solution: 0.05 g Solution of iron chloride hexahydrate dissolved in ultrapure water, prepared to 200 mL, and filtered and sterilized with a 0.2 μm syringe filter.

(Method)
NITE BP-02779 was inoculated on R2A medium and cultured at 25 ° C. for 24 to 48 hours. After completion of the culture, 50 μL of NITE BP-02779 culture solution and 5.0 mL of an inorganic medium containing excess sludge (killed bacteria) were added to a test tube and reacted at 25 ° C. and 200 rpm, and the turbidity of the test tube over time. (OD660) was measured with a simple turbidity meter (simple OD monitor miniphoto 518R, manufactured by TAITEC). The number of days elapsed when the turbidity (OD660) of the excess sludge (killed bacteria) -containing inorganic medium was 50% of the turbidity (OD660) of the negative control was calculated. As a negative control, the turbidity (OD660) of the target bacterium (killed bacterium) -containing inorganic medium without NITE BP-02779 was used.

(result)
By adding NITE BP-02779, a decrease in the turbidity of excess sludge (killed bacteria) was observed, and it became 50% of the turbidity of the negative control in 3.2 days. Therefore, NITE BP-02779 was able to decompose excess sludge (killed bacteria).

[Experiment 4. Resolution evaluation of NITE BP-02779 for excess sludge (killed bacteria)-2]
(material)
The same as in Experiment 3 was used.

(Method)
The reaction was carried out in the same manner as in Experiment 3. The reaction was carried out in 5 stations, and the entire amount of excess sludge remaining in the test tube was recovered on the 4th day after the start of the reaction. After the recovered excess sludge was dried, the weight was measured, and a significant difference test (t test, one side) was performed between the negative control group and the NITE BP-02779 addition group.

(result)
The results are shown in FIG. A significant decrease in the dry weight of excess sludge (p <0.01) was observed in the NITE BP-02779-added group as compared with the negative control group. Therefore, it was possible to reduce excess sludge by adding NITE BP-02779.

[Experiment 5. Establishment of hybridoma]
(material)
-Antigen: NITE BP-02779 heat-treated and the treated cells mixed with Freund Complete adjuvant (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)-Immune animals: Mouse (B6D2F1 / Slc) 8 weeks old, female, 5 Animals ・ Myeloma cells: SP2

(Method)
A hybridoma was prepared according to the procedure for producing the hybridoma and the evaluation procedure shown in FIG. First, the antigen (approximately 1.0 × ¹⁰ 9 cells / animal cell count) were immunized mice ridges. After 17 days, booster immunization was performed with the same amount of antigen, and 21 days later, lymphocytes were collected from the iliac lymph nodes of the mice immunized with the antigen. Lymphocytes and myeloma cells were fused to form hybridomas, which were seeded and cultured in about 800 wells. Hybridoma culture supernatant was collected from each well.

[Experiment 6. Selection of hybridomas]
(material)
-802 medium: 10 g polypeptone, 2 g yeast extract, 1 g magnesium sulfate heptahydrate dissolved in ultra-pure water, adjusted to pH 7.0, prepared to 1000 mL, and pressure-steam sterilized medium-1% BSA -PBS: A solution in which 250 mg of BSA (manufactured by Fujifilm Wako Junyaku Co., Ltd.) was dissolved in 25 mL of PBS: 1 x PBS-Primary antibody solution: Solution of hybridoma culture supernatant diluted to 1/10-Secondary antibody solution: HRP-labeled Donkey anti-Mouse IgG (H + L) (manufactured by Jackson ImmunoResearch)
HRP-labeled Goat anti-Mouse IgG (Light Chain Special) (manufactured by Jackson ImmunoResearch)
-Coloring substrate: TMBZ (manufactured by Thermo Scientific)

(Method)
Solid-phase ELISA was performed according to the following procedure, and the antigen recognition ability of the antibody in the hybridoma culture supernatant was evaluated.
NITE BP-02779 was cultured in 802 medium, collected, washed with PBS, and adjusted to a predetermined concentration. The bacterial cell fluid was inoculated on an immunoplate (Nunc-Immuno Plate I, manufactured by Nunc) at 50 μL / well, and allowed to stand at 37 ° C. for 1 hour. After washing with PBS, 100 μL / well of 1% BSA-PBS was added, and blocking was performed at room temperature for 30 minutes. After completion of blocking, 50 μL / well of the primary antibody solution was added, and the mixture was reacted at room temperature for 1 hour. After the reaction, the cells were washed 3 times with PBS, 50 μL / well of the secondary antibody solution was added, and the mixture was reacted at room temperature for 30 minutes. After the reaction, the mixture was washed 3 times with PBS, a color-developing substrate solution was added at 100 μL / well, and the reaction was carried out at room temperature for several minutes. The reaction was stopped by adding 100 μL / well of 1 M sulfuric acid. The absorbance at 450 nm was measured with a microplate reader (manufactured by Molecular Device). Based on the results of the solid phase ELISA, five hybridomas (3A5, 4G4, 7D4, 4D9 and 6D9) shown in Table 5 were selected.

[Experiment 7. Hybridoma cloning]
(Method)
The five hybridomas selected above were each diluted by an ultra-dilution method, re-seeded in 200 wells, and cultured. It was confirmed by microscopic observation that it was a single colony. Clone culture supernatant was obtained from each well.

[Experiment 8. Clone selection]
(Method)
The antigen recognition ability of the antibody in the clone culture supernatant was evaluated by the same method as in Experiment 6. As a result of the solid-phase ELISA, four clones (3A5-1H1, 4G4-1A9, 6D9-2G6 and 7D4-1G9) shown in Table 6 were selected. The obtained clones were internationally deposited with a depositary institution as shown in Table 7.

[Experiment 9. Evaluation of clone culture supernatant for airborne cells]
(material)
The same material as in Experiment 6 was used except that the clone culture supernatant (1/10 dilution) obtained in Experiment 8 was used as the primary antibody solution.

(Method)
Competitive ELISA was performed according to the following procedure to evaluate the antigen recognition ability of the antibody in the clone culture supernatant.
The cell solution of NITE BP-02779 prepared to a predetermined concentration and the primary antibody solution were mixed and allowed to stand at room temperature for 1 hour. 50 μL / well of the above mixture was added to the immunoplates that had been immobilized and blocked with NITE BP-02779, and the mixture was reacted at room temperature for 1 hour. After the reaction, the cells were washed 3 times with PBS, 50 μL / well of the secondary antibody solution was added, and the mixture was reacted at room temperature for 30 minutes. After the reaction, the mixture was washed 3 times with PBS, a chromogenic substrate solution was added at 100 μL / well, and the reaction was carried out at room temperature for 5 minutes. The reaction was stopped by adding 100 μL / well of 1 M sulfuric acid. The absorbance at 450 nm was measured with a microplate reader (manufactured by Molecular Device).

The competing cell concentration was calculated when ABS 450 nm was 50%, assuming that ABS 450 nm was 100% when the competing cell concentration was 0, and clones were evaluated according to the following criteria.
A: 1.25 x 10 ⁸ cells / mL or less B: 1.25 x 10 ⁸ cells / mL or more and ^{less than 2.5 x 10 8} cells / mL C: 2.5 x 10 ⁸ cells / mL or more

(result)
The results are shown in Table 8. All clone culture supernatants showed a reaction to NITE BP-02779 (suspended cells).

[Experiment 10. Evaluation of clone culture supernatant for solid-phase cells]
(material)
The same material as in Experiment 6 was used except that the clone culture supernatant (1/10 dilution) obtained in Experiment 8 was used as the primary antibody solution.

(Method)
The antigen recognition ability of the antibody in the clone culture supernatant was evaluated by the same method as in Experiment 6.
As 100% ABS450nm when immobilized cell concentration is ^{5.0 × 10 8 cells / mL,} ABS450nm calculates the immobilized cell concentration when showing 50% of the clones with the following criteria evaluated.
A: 1.0 × 10 ⁸ cells / mL or less B: 1.0 × 10 ⁸ cells / mL or more and ^{less than 1.5 × 10 8} cells / mL C: 1.5 × 10 ⁸ cells / mL or more 2.0 × Less than 10 ⁸ cells / mL D: 2.0 × 10 ⁸ cells / mL or more

(result)
The results of solid-phase ELISA are shown in Table 9. Both clone culture supernatants showed a reaction to NITE BP-02779 (immobilized cells).

[Experiment 11. Evaluation of cross-reactivity of clone culture supernatant]
(material)
Same as Experiment 6 except that the clone culture supernatant (1/10 dilution) obtained in Experiment 8 was used as the primary antibody solution and the bacteria shown in Table 10 were used as the immobilized bacterial cells. The material was used.

(Method)
The antigen cross-reactivity of the antibody in the clone culture supernatant was evaluated by the same method as in Experiment 6.
When ABS 450 nm when NITE BP-02779 was used as the immobilized bacterial cell was set as 100%, the cross-reactivity (%) with respect to the bacterial cell shown in Table 10 was calculated, and the bacterial cell having the highest cross-reactivity (%) was calculated. The values were adopted and the clones were evaluated according to the following criteria.
A: 0% or more and less than 20% B: 20% or more and less than 30% C: 30% or more and less than 40% D: 40% or more

(result)
For the four clones, the cross-reactivity (%) with respect to the cells shown in Table 10 is shown in FIGS. 3 to 6. The evaluation of crossability is shown in Table 11. All clone culture supernatants had high reactivity with NITE BP-02779, but low reactivity with other antigens. All four clones were found to produce antibodies with high antigen specificity.

[Experiment 12. Purification of monoclonal antibody]
(material)
Clone: 4G4-1A9, 6D9-2G6 or 7D4-1G9
-Medium: Serum-free medium Hybridoma-SFM (manufactured by GIBCO) to which Human IL-6 (manufactured by R & D Systems) was added so as to be 1 ng / mL.-Cation buffer solution for cation exchange chromatography: 15.7 mM Sodium phosphate buffer (pH 6.3)
Elution buffer for cation exchange chromatography: 1 x PBS

(Method)
The clones were cultured in 100 mL of medium and the culture supernatant was concentrated with ammonium sulfate precipitate. Monoclonal antibodies were purified by fractionating the concentrate by cation exchange chromatography (column: HiTrap SP HP).

[Experiment 13. Sensitization of antibodies to latex beads]
(material)
Monoclonal antibody: Monoclonal antibody (hereinafter referred to as 4G4-1A9 mAb, 6D9-2G6 mAb and 7D4-1G9 mAb, respectively, purified from the culture supernatant of the clone (4G4-1A9, 6D9-2G6 or 7D4-1G9) in Experiment 12 .)
-Latex beads: # 15713 (manufactured by Polyscience, grain size 1.0 μm)
-Borate buffer: 6.18 g A buffer prepared by dissolving boric acid in ultra-pure water, adjusting the pH to 8.5, and then adjusting to 1000 mL.-1% BSA-borate buffer: borate buffer 25 mL A solution in which 250 mg of BSA was dissolved.-Preservative solution: A solution in which 1 g of BSA, 0.83 g of sodium chloride, and 5 g of glycerol were dissolved in 100 mL of 100 mM phosphate buffer.

(Method)
The operation of adding 1 mL of boric acid buffer to 0.5 mL of latex beads and washing was repeated 3 times. To 1 mL of the washed latex beads, 0.3 mL of a 1 mg / mL monoclonal antibody solution was added, and the mixture was inverted and mixed overnight at room temperature. After inversion mixing, the supernatant was removed by centrifugation and blocked with 1% BSA-boric acid buffer for 30 minutes. After removing the blocking solution by centrifugation, the operation of adding 1% BSA-boric acid buffer and blocking was repeated twice, and 1 mL of the preservation solution was added and refrigerated as antibody-sensitized latex beads. The binding of the monoclonal antibody to the latex beads was confirmed by measuring the absorbance (A280 nm) of the antibody solution before and after sensitization.

(result)
Table 12 shows the absorbance (A280 nm) of the antibody solution before and after antibody sensitization. Since the absorbance of each antibody solution decreased before and after sensitization, antibody sensitization to latex beads could be confirmed.

[Experiment 14. Latex agglutination test]
(material)
-Antibody-sensitized latex beads prepared in Experiment 13-The same materials as in Experiments 11 to 13 were used as other materials.

(Method)
The operation of adding 0.5 mL borate buffer to 0.1 mL of antibody-sensitized latex beads and washing was repeated twice, and finally suspended in 4 mL borate buffer. Next, NITE BP-02779 cultured in 802 medium was serially diluted with borate buffer to a predetermined concentration. The serially diluted bacterial cell solution and the antibody-sensitized latex beads suspended in borate buffer were added to the U-plate at 50 μL / well (100 μL / well in total), respectively, and allowed to stand overnight at room temperature.

The agglutination image in the case of only the buffer solution was regarded as negative, the agglutination image of each hole was judged, and the agglutination image was classified into the following A to D based on the bacterial concentration of the test hole showing the positive agglutination image.
A: 2.0 × 10 ⁶ cells / mL or more B: 2.0 × 10 ⁶ cells / mL or more and ^{less than 2.0 × 10 7} cells / mL C: 2.0 × 10 ⁷ cells / mL or more 2.0 × Less than 10 ⁸ cells / mL D: 2.0 × 10 ⁸ cells / mL or more

(result)
The results are shown in Table 13. All antibody-sensitized latex beads reacted with NITE BP-02779 (floating cells), and an agglutinated image could be confirmed.

Claims (8)

16S containing a base sequence having 98.2% or more identity with the base sequence shown in SEQ ID NO: 1.
A monoclonal antibody that reacts with bacteria that carry the rRNA gene. Tumebacillus sp. A monoclonal antibody that reacts with NITE BP-02779. A monoclonal antibody produced from a hybridoma whose accession number is NITE BP-03165, NITE BP-03166, NITE BP-03167 or NITE BP-03168. A method for detecting a bacterium having a 16S rRNA gene containing a base sequence having 98.2% or more identity with the base sequence shown in SEQ ID NO: 1 using the monoclonal antibody according to any one of claims 1 to 3. .. The bacterium is Tumebacillus sp. The detection method according to claim 4, which is NITE BP-02779. A kit for detecting a bacterium having a 16S rRNA gene containing a nucleotide sequence having 98.2% or more identity with the nucleotide sequence of SEQ ID NO: 1, which comprises the monoclonal antibody according to any one of claims 1 to 3. .. The bacterium is Tumebacillus sp. The detection kit according to claim 6, which is NITE BP-02779. A hybridoma whose accession number is NITE BP-03165, NITE BP-03166, NITE BP-03167 or NITE BP-03168.

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