Monoclonal antibody, bacterial detection method, bacterial detection kit, and hybridoma

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

[Technical Field]

[0001] The present invention relates to a monoclonal antibody, a method for detecting bacteria, a kit for detecting bacteria, and a hybridoma. [Background technology]

[0002] When wastewater is purified using the activated sludge process, the removed organic matter becomes flocs containing microorganisms (bacteria), generating a sludge called excess sludge. The excess sludge discharged from wastewater treatment facilities accounts for more than 20% of industrial waste. Generally, excess sludge is dehydrated, dried, and then incinerated (Non-Patent Document 1: FY2018 Project, Industrial Waste Discharge and Treatment Status Survey Report, FY2016 Results (Summary); Non-Patent Document 2: Yamamoto Masayuki, "On Sewage Combustion Technology," Journal of the Japan Fuel Society, Combustion Society of Japan, 2011, Vol. 53, No. 164, pp. 91-96). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] "Fiscal Year 2018 Project, Industrial Waste Discharge and Treatment Status Survey Report, FY2016 Results (Summary Version)" [online], March 2019, Waste Management Division, Environmental Regeneration and Resource Recycling Bureau, Ministry of the Environment [Retrieved March 17, 2020], Internet <URL:https: / / www.e-stat.go.jp / stat-search / file-download?statInfId=000031887949&fileKind=2> [Non-patent document 2] Masayuki Yamamoto, "On Sewage Combustion Technology," Journal of the Japan Fuel Society, Combustion Society of Japan, 2011, Vol. 53, No. 164, pp. 91-96 Summary of the Invention [Problem to be solved by the invention]

[0004] However, since incineration of excess sludge generates greenhouse gases, there is a need to reduce the volume of excess sludge from environmental considerations. A method for reducing the volume of excess sludge is proposed, which involves decomposing the microorganisms that make up the excess sludge. The present inventors have discovered a bacterium capable of decomposing the microorganisms that make up excess sludge, the bacterium having a 16S rRNA gene containing a base sequence that is 98.2% or more identical to the base sequence set forth in SEQ ID NO: 1.

[0005] An object of the present invention is to provide a novel antibody that specifically detects the above-mentioned bacteria, and a method and kit for detecting the bacteria using the antibody. [Means for solving the problem]

[0006] The present invention relates to the following [1] to [8]. [1] A monoclonal antibody that reacts with bacteria having a 16S rRNA gene containing a base sequence that has 98.2% or more identity to the base sequence set forth in SEQ ID NO: 1. [2] Monoclonal antibody that reacts with Tumebacillus sp. NITE BP-02779. [3] A monoclonal antibody produced by a hybridoma having accession number NITE BP-03165, NITE BP-03166, NITE BP-03167, or NITE BP-03168. [4] A method for detecting bacteria having a 16S rRNA gene containing a base sequence having 98.2% or more identity to the base sequence set forth in SEQ ID NO: 1, using a monoclonal antibody described in any one of [1] to [3]. [5] The detection method according to [4], wherein the bacterium is Tumebacillus sp. NITE BP-02779. [6] A kit for detecting bacteria having a 16S rRNA gene containing a base sequence having 98.2% or more identity to the base sequence set forth in SEQ ID NO: 1, comprising a monoclonal antibody described in any one of [1] to [3]. [7] The detection kit according to [6], wherein the bacterium is Tumebacillus sp. NITE BP-02779. [8] Hybridomas with accession numbers NITE BP-03165, NITE BP-03166, NITE BP-03167, or NITE BP-03168. [Effects of the Invention]

[0007] According to the present invention, there are provided a novel antibody that can specifically detect bacteria having a 16S rRNA gene containing a base sequence that has 98.2% or more identity to the base sequence set forth in SEQ ID NO: 1, as well as a method and a detection kit for detecting bacteria using the antibody. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 10 is a graph showing the dry weight of excess sludge after the addition of Tumebacillus sp. NITE BP-02779 in Experiment 4. [Figure 2] FIG. 1 is a diagram showing a schematic diagram of a method for producing a monoclonal antibody in Experiment 5. [Figure 3] 11 shows the results of cross-reactivity evaluation of the culture supernatant of 3A5-1H1 (accession number NITE BP-03165) in Experiment 11. [Figure 4] 11 shows the results of cross-reactivity evaluation of the culture supernatant of 4G4-1A9 (accession number NITE BP-03166) in Experiment 11. [Figure 5] 11 shows the results of cross-reactivity evaluation of the culture supernatant of 6D9-2G6 (accession number NITE BP-03167) in Experiment 11. [Figure 6] 11 shows the results of cross-reactivity evaluation of the culture supernatant of 7D4-1G9 (accession number NITE BP-03168) in Experiment 11. DETAILED DESCRIPTION OF THE INVENTION

[0009] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following describes in detail the embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0010] [antibody] One embodiment of the antibody of the present invention is a monoclonal antibody that reacts with a bacterium (hereinafter, sometimes referred to as "bacterium a") having a 16S rRNA gene containing a nucleotide sequence that has 98.2% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1. Examples of bacterium a include bacteria of the genus Tumebacillus. Bacterium a preferably has the ability to degrade the target microorganism. Bacterium a may be the whole or a part of bacterium a.

[0011] Bacterium a may have a 16S rRNA gene containing a nucleotide sequence that has an identity of 98.5% or more, 98.8% or more, 99.0% or more, 99.3% or more, 99.5% or more, or 99.8% or more to the nucleotide sequence set forth in SEQ ID NO: 1. Bacterium a may be Tumebacillus sp. NITE BP-02779, which will be described later.

[0012] Bacterium a may have a 16S rRNA gene containing a nucleotide sequence in which one or several nucleotides have been substituted, deleted, or added compared to the nucleotide sequence set forth in SEQ ID NO: 1. The one or several nucleotides may be, for example, 1 to 25 nucleotides, preferably 1 to 10 nucleotides, and more preferably 1 to 5 nucleotides. The above mutations do not impair the expression and function of 16S rRNA.

[0013] Bacterium a may have a 16S rRNA gene containing a nucleotide sequence that hybridizes under stringent conditions to a continuous sequence of about 15 or more nucleotides, preferably about 18 to about 500 nucleotides, more preferably about 18 to about 200 nucleotides, and even more preferably about 18 to about 50 nucleotides, contained in the nucleotide sequence set forth in SEQ ID NO: 1, or to its complementary sequence. Stringent conditions refer to conditions under which nonspecific hybrids are not formed, and include, for example, conditions in which the strain is washed one or more times at 60°C, 1×SSC, 0.1% SDS, preferably at 68°C, 0.1×SSC, 0.1% SDS.

[0014] The nucleotide sequence of the 16S rRNA gene can be analyzed, for example, by 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 include PCR using universal primers commonly used by those skilled in the art. The amplified product obtained by PCR can be purified as necessary and subjected to a DNA sequencer or the like to determine the nucleotide sequence. The obtained nucleotide sequence is compared with the sequence set forth in SEQ ID NO: 1.

[0015] Whether or not Bacterium a has microbial degradation ability can be confirmed by methods commonly used by those skilled in the art. Examples of such confirmation methods include reacting Bacterium a with a target microorganism in an appropriate medium or buffer solution for a certain period of time, and then detecting the degradation of the target microorganism in the medium or buffer solution. Methods for detecting the degradation of the target microorganism are not particularly limited, and examples include measuring the turbidity of the target microorganism, detecting the target microorganism using an SLP reagent, detecting the DNA of the target microorganism by PCR, measuring the dry cell weight of the target microorganism, and detecting degradation products derived from the target microorganism using high-performance liquid chromatography (HPLC), mass spectrometry (MS), thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), gas chromatography (GC), etc.

[0016] When examining the degradation of a target microorganism by turbidity, having the ability to decompose the target microorganism means that the turbidity after the reaction is significantly lower than that before the reaction, for example, the turbidity after the reaction is 80% or less, preferably 50% or less, and more preferably 30% or less of the turbidity before the reaction.When examining the degradation of a target microorganism by dry cell weight, having the ability to decompose the target microorganism means, for example, that 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, preferably 90% or less.

[0017] One embodiment of the antibody according to the present invention is a monoclonal antibody that reacts with Tumebacillus sp. NITE BP-02779 (hereinafter, sometimes referred to as "NITE BP-02779").

[0018] NITE BP-02779 is a bacterium that was internationally deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) on September 11, 2018, and a certificate of deposit and a certificate of viability for the original deposit were issued on September 26, 2018. NITE BP-02779 is considered to be a new species of Tumebacillus bacteria. NITE BP-02779 is capable of degrading various target microorganisms, such as Bacillus, Micrococcus, and Staphylococcus, as well as excess sludge, making it useful for excess sludge treatment. The bacteriological properties of NITE BP-02779 are shown in Tables 1 to 4 in the Examples section below. NITE BP-02779 is a bacterium that has a 16S rRNA gene containing the nucleotide sequence set forth in SEQ ID NO: 1.

[0019] Bacteria a, to which the antibody reacts, may be live or killed. Live bacteria can be obtained by culturing bacteria a in an appropriate medium. As the medium, various media containing appropriate carbon sources, nitrogen sources, organic or inorganic salts, etc., commonly used in the cultivation of microorganisms can be used. Specific examples include ATCC802 medium and R2A medium. An appropriate buffer solution or wastewater may be used instead of a medium. Killed bacteria can be obtained, for example, by denaturing live bacteria. Examples of denaturing treatment include treatment with a denaturant such as SDS, physical disruption, heat treatment, etc. A sample containing bacteria a may contain a mixture of live and killed bacteria a.

[0020] "An antibody reacts" may mean that an antibody binds to an antigen through an antigen-antibody reaction, or that an antibody recognizes an antigen. "An antibody reacts with bacteria" may mean that an antibody binds to a bacterium, or that an antibody recognizes a bacterium. Methods for examining whether an antibody reacts with bacteria include, for example, ELISA, flow cytometry, immunostaining, and Western blotting. "An antibody that reacts with bacteria" can also be referred to as "an antibody against bacteria."

[0021] For example, when examining the reaction between an antibody and bacteria by ELISA, a reaction with bacteria means that the detection value (absorbance) of the bacteria when the antibody is added is significantly higher than the detection value (absorbance) of the negative control, for example, 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 0.05 or less.

[0022] 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. Monoclonal antibodies are obtained as secretions in the culture medium, for example, by culturing cloned hybridomas (hereinafter sometimes simply referred to as "clones"). A "cloned hybridoma" is obtained, for example, by fusing antibody-producing cells with myeloma cells to produce hybridomas, and then selecting and cloning hybridomas that produce antibodies with the desired antigen specificity.

[0023] The antibody according to the present invention can be obtained by a method including a step of immunizing an animal with Bacterium a. The step of immunizing an animal with Bacterium a can be the same as the step of immunizing an animal in a known method for producing a monoclonal antibody. Examples of methods for producing a monoclonal antibody include the mouse spleen method and the mouse iliac lymph node method (see JP 2007-020547 A). From the viewpoint of obtaining a highly specific antibody, the mouse iliac lymph node method is preferred.

[0024] The animal to be immunized is not particularly limited, and may be appropriately selected from non-human animals depending on the method for producing the monoclonal antibody. Specifically, when the mouse iliac lymph node method is used as a method for producing the monoclonal antibody, the animal may be immunized according to the method described in the above-mentioned literature.

[0025] After immunizing the animal, hybridomas can be produced and selected according to known methods to obtain the antibody of the present invention. When selecting hybridomas, bacteria a, such as NITE BP-02779, may be used as a positive antigen, and BSA, Escherichia, Rhizobium loti, Xanthomonas, Sphingomonas, Brevibacterium, Pseudomonas, Rhodococcus, Novosphingobium, etc. may be used as a negative antigen.

[0026] The criteria for selecting hybridomas, for example, when using ELISA, are that the difference in ABS450 value between the positive antigen and the negative antigen for the produced monoclonal antibody is 0.05 or more, and the ABS450 value of the negative antigen is 0.05 or less.

[0027] The isotype of the antibody of the present invention is not particularly limited. The antibody of the present invention may be an immunoglobulin molecule or a partial fragment of an antibody having antigen-binding activity, such as F(ab')2, Fab', Fab, or CDR.

[0028] The antibody of the present invention may be an artificially synthesized antibody. The antibody of the present invention is preferably an isolated or purified antibody, for example, an antibody purified from the serum, ascites, etc. of an immunized animal or the culture supernatant of the above-mentioned hybridoma. Antibody purification may be carried out by known methods, such as ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography for protein A or protein G, gel filtration chromatography, and isoelectric precipitation. These methods may be used alone, or two or more may be used in appropriate combination. Antibody purification can be carried out to the desired extent.

[0029] The antibody according to the present invention may be labeled with a labeling substance such as an enzyme such as alkaline phosphatase (ALP) or horseradish peroxidase (HRP), biotin, a fluorescent substance, a luminescent substance, or a radioisotope.

[0030] The antibody of the present invention preferably does not substantially react with microorganisms other than Bacterium A. The antibody of the present invention preferably does not substantially react with any one or more bacteria selected from group (I), more preferably does not substantially react with any one or more bacteria selected from group (II), and even more preferably does not substantially react with any one or more bacteria selected from group (III). (I) Group: Tumebacillus, Escherichia, Rhizobium, Xanthomonas, Sphingomonas, Brevibacterium, Pseudomonas, Rhodococcus, Novosphingobium, and Sphingobium excluding bacteria a (II) Group: Tumebacillus, Escherichia, Rhizobium, Xanthomonas, Brevibacterium, Pseudomonas, Rhodococcus, Novosphingobium, and Sphingobium, excluding bacteria a Group (III): Escherichia, Xanthomonas, Pseudomonas, Novosphingobium and Sphingobium

[0031] 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 bacterium A through an antigen-antibody reaction. "Substantially no reaction" means that, when evaluated by solid-phase ELISA, for example, the ratio (%) of absorbance when bacterium A is used as the antigen to the absorbance when the target bacterium is used as the antigen is 0% or more and 50% or less, 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.

[0032] One embodiment of the antibody of the present invention is a monoclonal antibody produced by the cloned hybridoma 3A5-1H1, 4G4-1A9, 6D9-2G6, or 7D4-1G9 described in the Examples below. These monoclonal antibodies are highly reactive with bacteria a, particularly NITE BP-02779, and are substantially non-reactive with Tumebacillus, Escherichia, Rhizobium, Xanthomonas, Sphingomonas, Brevibacterium, Pseudomonas, Rhodococcus, Novosphingobium, and Sphingobium, except for bacteria a.

[0033] 3A5-1H1 has been deposited under accession number NITE BP-03165 (deposit date: March 3, 2020), 4G4-1A9 under accession number NITE BP-03166 (deposit date: March 3, 2020), 6D9-2G6 under accession number NITE BP-03167 (deposit date: March 3, 2020), and 7D4-1G9 under accession number NITE BP-03168 (deposit date: March 3, 2020). They have been deposited internationally under the Budapest Treaty at the National Institute of Technology and Evaluation (NPMD) Patent Microorganisms Depositary (NPMD, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818).

[0034] The antibody of the present invention is highly specific to Bacterium a and is useful, for example, for detecting Bacterium a in a sample that may contain multiple types of microorganisms. Examples of samples that may contain multiple types of microorganisms include activated sludge, excess sludge, soil, wastewater, and water. Bacterium a with the ability to decompose target microorganisms, such as NITE BP-02779, can decompose the microorganisms that make up excess sludge, so confirming the presence of Bacterium a in an environment containing excess sludge is useful for reducing the volume of excess sludge.

[0035] [Bacteria detection method] The detection method of the present invention detects bacteria a using the antibody of the present invention. The detection method of the present invention can specifically detect bacteria a in a sample, and can detect bacteria a even when multiple types of microorganisms are present in the sample. In the detection method of the present invention, the bacteria to be detected is, for example, NITE BP-02779. One aspect of the present invention is the use of the antibody described above to detect bacteria a.

[0036] Known immunological assay methods can be used to detect bacteria a. Examples of immunological assay methods include solid-phase ELISA, competitive ELISA, sandwich ELISA, immune complex transfer assay, immunoturbidimetry, immunochromatography, and latex agglutination. As an example of a method for detecting bacteria, the detection of bacteria a in a sample by solid-phase ELISA will be described below.

[0037] First, a complex containing the antibody of the present invention and bacterium A is formed. The complex can be formed by mixing the antibody of the present invention with a sample containing bacterium A. Next, a solution containing the complex is brought into contact with a solid phase capable of capturing bacterium A, thereby immobilizing the complex on the solid phase. The solid phase may be one to which a sample containing bacterium A has been immobilized in advance. In other words, the above complex can be formed on the solid phase by contacting the antibody of the present invention with the sample immobilized on the solid phase. The sample may or may not be subjected to pretreatment such as centrifugation, denaturation, or washing before detection of bacterium A.

[0038] The manner in which the sample is immobilized on the solid phase is not particularly limited, and for example, the sample may be directly bound to the solid phase, or the capture antibody may be indirectly bound to the solid phase via another substance. Examples of direct binding methods include physical adsorption.

[0039] The material of the solid phase is not particularly limited and can be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, etc. Examples of organic polymer compounds include latex, polystyrene, polypropylene, etc. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, etc. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, cellulose, etc. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited and examples include particles, membranes, microplates, microtubes, test tubes, etc.

[0040] The antibody of the present invention is added to the solid phase on which the complex is immobilized, and the solid phase is washed. After washing, the antibody bound to the solid phase is detected by a method known in the art, thereby determining the presence or absence of bacteria A in the sample and measuring the amount (number of bacteria). For example, when an antibody labeled with a labeling substance is used as the antibody of the present invention, the presence or absence and amount of bacteria A in the sample can be measured by detecting the signal generated by the labeling substance. When an unlabeled antibody is used as the antibody of the present invention, the presence or absence and amount of bacteria A in the sample can be measured by adding a labeled secondary antibody against the antibody of the present invention to the solid phase and detecting the signal generated by the labeling substance.

[0041] "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 refers to indicating the signal intensity in stages such as "no signal," "weak," "medium," "strong," etc. In this embodiment, it is preferable to detect the signal intensity quantitatively or semi-quantitatively.

[0042] The labeling substance is not particularly limited as long as it generates a detectable signal. For example, it may be a substance that generates a signal by itself (hereinafter also referred to as a "signal-generating substance"), or it may be a substance that generates a signal by catalyzing the reaction of another substance. Examples of signal-generating substances include fluorescent substances and radioisotopes. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioisotopes include 125I, 14C, and 32P. Examples of substances that catalyze the reaction of another substance to generate a signal include enzymes. Examples of enzymes include ALP, HRP, β-galactosidase, and luciferase. As the labeling substance, enzymes are preferred, and ALP or HRP is particularly preferred.

[0043] The method for detecting a signal is known in the art, and a detection method may be appropriately selected depending on the type of signal derived from the labeling substance. For example, when the labeling substance is an enzyme, a signal such as light or color generated by reacting a substrate for the enzyme may be detected using a known device such as a spectrophotometer.

[0044] The enzyme substrate can be appropriately selected from known substrates depending on the type of enzyme. For example, when ALP is used as the enzyme, examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenylphosphate disodium) and CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenylphosphate disodium); and chromogenic substrates such as 5-bromo-4-chloro-3-indolylphosphate (BCIP), 5-bromo-6-chloro-indolylphosphate disodium, and p-nitrophenylphosphate. When HRP is used as the enzyme, the substrate may be a chemiluminescent substrate such as luminol or its derivatives; or a chromogenic substrate such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD), or 3,3',5,5'-tetramethylbenzidine (TMB).

[0045] 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, the fluorescence as a signal can be measured using a known device such as a fluorescence microplate reader. The excitation wavelength and fluorescence wavelength can be appropriately determined depending on the type of fluorescent substance used.

[0046] [Bacteria detection kit] The antibody of the present invention can be used in a kit for detecting Bacteria a. The kit of the present invention contains the antibody of the present invention and may also contain other reagents such as a developing solvent, a buffer solution, a washing solution, a blocking reagent, an enzyme substrate, a color-developing reagent, a solid phase (including latex particles), and a standard sample of Bacteria a, and / or devices or instruments such as a container, a reaction device, and a fluorescence reader. The kit may be a solid-phase ELISA kit, a sandwich ELISA kit, a latex agglutination kit, an immunochromatography kit, etc., containing the antibody of the present invention. A latex agglutination kit is preferred because it allows on-site measurement. Bacteria a detected by the detection kit of the present invention is, for example, NITE BP-02779. The kit may be a kit used in the above-mentioned detection method. One aspect of the present invention is the use of the above-mentioned antibody for producing a detection kit for Bacteria a.

[0047] The kit will be described in more detail below, taking a latex agglutination kit as an example. The latex particles used in the latex agglutination kit preferably have a specific gravity of 0.8 to 1.2 mg / ml, more preferably approximately 1.05 mg / ml. The average particle size of the latex particles is, for example, approximately 0.1 to 4.0 μm, preferably approximately 0.5 to 1.5 μm, and more preferably an average of approximately 1.0 μm. Colored latex particles are preferably used. Examples of such latex particles include commercially available products such as Bactratex 0.81 (manufactured by DIFCO, average particle size 0.81 μm, specific gravity 1.0 g / ml) and Polybeads #15713 (manufactured by Polysciences, average particle size 1.0 μm, specific gravity 1.05 g / ml). Latex particles are not limited to these, and any type of latex particles can be used as long as they have the same effect as these.

[0048] The latex particles preferably have the antibody of the present invention adsorbed thereto. The methods for producing the latex particles serving as carriers for adsorbing the specific antibodies and the sensitized latex particles are not particularly limited, and can be prepared, for example, according to International Publication No. 2017 / 138608. As a method for sensitizing the latex particles to the antibody, either physical adsorption or chemical binding can be suitably used.

[0049] The antibody to be sensitized is preferably a monoclonal antibody produced by one or more hybridomas selected from group (A), more preferably a monoclonal antibody produced by one or more hybridomas selected from group (B), and even more preferably a monoclonal antibody produced by one or more hybridomas selected from group (C). 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. Alternatively, a mixture of latex particles sensitized with different monoclonal antibodies may be used. (A) Group: Accession numbers NITE BP-03165, NITE BP-03166, NITE BP-03167, and NITE BP-03168 (B) Group: Accession numbers NITE BP-03166, NITE BP-03167, and NITE BP-03168 (C) Group: Accession numbers NITE BP-03166 and NITE BP-03168

[0050] The step of sensitizing latex particles with antibodies is preferably carried out in a buffer solution. Specifically, a suspension of latex particles diluted to an appropriate concentration is mixed with a solution of antibodies, allowed to stand for a while, and then washed to produce sensitized latex particles. The buffer solution used for dilution is generally selected to have an ionic strength and pH that do not inhibit the reaction of the supported antibody with the antigen, which is the substance to be measured. Examples of buffer solutions that can be used include TBS, PBS, GBS, phosphate buffer, and borate buffer. Among these, phosphate buffer, borate buffer, and GBS are preferred because of the agglutination properties of sensitized latex particles and their resistance to self-agglutination. The buffer solution is generally selected in the pH range of 5 to 10, with a pH range of 6 to 9 being particularly preferred.

[0051] The appropriate concentration of latex particles during sensitization is 0.01 to 0.5 (w / v)%, with particularly good sensitized latex particles being obtained at around 0.05 to 0.25 (w / v)%. Furthermore, when sensitizing with antibodies, the appropriate antibody protein concentration in the antibody solution is 1.0 to 1000 μg; otherwise, sensitivity decreases and the sensitized latex particles self-aggregate, making it difficult to determine whether the result is positive or negative. The temperature during the sensitization reaction should be within the range of 0 to 60°C, but latex particles with good sensitivity can be obtained by performing the reaction at room temperature or slightly higher (up to 40°C).

[0052] The reversed passive latex agglutination method is preferred for detecting bacteria a and quantifying the number of bacteria using these sensitized latex particles. The reversed passive latex agglutination method involves mixing a liquid reagent containing immunological agglutination particles, in which a substance (e.g., an antibody) that specifically binds to the test substance is bound to latex particles, with a sample containing the test substance to detect or quantify the test substance. More specifically, a sample diluted to an appropriate level is mixed with a reagent containing antibody-sensitized latex particles, and the dilution level at which an agglutination image is observed is confirmed. Meanwhile, a test is conducted in which a standard sample containing a known number of bacteria is mixed with a reagent containing antibody-sensitized latex particles, and the number of bacteria can be calculated by comparing the results with the standard sample.

[0053] More specifically, a sample containing an antigen or a standard sample, serially diluted with buffer, is added to a U-shaped microtiter plate. An equal amount of a reagent containing antibody-sensitized latex particles is dispensed into each well and allowed to stand at room temperature for 4 to 15 hours. The agglutination image is then observed with the naked eye or with a 10x magnification loupe to determine the presence or absence of the antigen and measure its concentration (number of bacteria). The agglutination image obtained with buffer alone is considered negative, and the agglutination image in each well is evaluated. The number of bacteria can be calculated from the dilution factor of the test well that showed a positive agglutination image and the results of the agglutination test using the standard antigen.

[0054] Other latex agglutination kits may also be kits that utilize various qualitative and quantitative methods using immunological agglutination reaction particles, such as a method in which latex particles and a test sample are mixed on a glass slide and the formation of an agglutination image is determined under an optical microscope (microscopic latex method), or an immunochromatography method.

[0055] The kit according to the present invention allows for the simple detection of bacteria a contained in a sample. The kit according to the present invention can also be used to detect bacteria a from samples such as excess sludge, soil, and wastewater. [Example]

[0056] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0057] [Experiment 1. Isolation of microbial decomposition bacteria] (method) Microorganisms that decompose Micrococcus bacteria were enriched by culturing environmental (aquatic) microorganisms in a medium containing Micrococcus bacteria as a carbon source. Next, several strains that grew well were isolated from the enriched microbial population.

[0058] (result) The ability of each isolated strain to degrade Micrococcus bacteria was examined, and one strain was found to be capable of degrading Micrococcus bacteria. Hereinafter, the strain that was found to be capable of degrading Micrococcus bacteria will be referred to as "Strain A."

[0059] [Experiment 2. Identification of strain A] (material) Forward primer for cloning (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)

[0060] (method) Strain A was identified by 16S rRNA gene analysis, morphological observation, and physiological and biochemical property tests.

[0061] 16S rRNA gene analysis was performed as follows: Genomic DNA was extracted from strain A, and the resulting 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 a KOD FX (Toyobo Co., Ltd.), and the amplified product after PCR was purified.

[0062] The purified PCR amplification products were used for cycle sequencing. Cycle sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit. The resulting reaction mixture was purified, and the purified solution was subjected to DNA sequence analysis (3730xl DNA Analyzer) to determine the base sequence of the 16S rRNA gene of the template DNA extracted from strain A.

[0063] Morphological observation and physiological and biochemical property tests were performed using an optical microscope, the method of BARROW et al. (Cowan and Steel's Manual for the Identification of Medical Bacteria 3rd Edition 1993, Cambridge University Press.), and API50CHB (bioMerieux, Lyon, France).

[0064] (result) The obtained 16S rRNA gene sequence (SEQ ID NO: 1) was subjected to homology analysis with the international nucleotide sequence database (DDBJ / ENA (EMBL) / GenBank). Among the type strains, the 16S rRNA gene sequence of this strain showed 98.1% identity with that of Tumebacillus permanentifrigoris Eurl_9.5. However, no microorganisms possessed a 16S rRNA gene sequence that perfectly matched the obtained sequence. Furthermore, strain A did not grow at 10°C, a characteristic not observed in Tumebacillus permanentifrigoris Eurl_9.5, the strain with the highest 16S rRNA gene homology. This suggests that strain A represents a new species distinct from conventional Tumebacillus species. Strain A was deposited internationally as Tumebacillus sp. NITE BP-02779.

[0065] The results of morphological observation and physiological and biochemical property tests of NITE BP-02779 are shown in Tables 1 to 4.

[0066] [Table 1]

[0067] [Table 2]

[0068] [Table 3]

[0069] [Table 4]

[0070] [Experiment 3. Evaluation of decomposition ability of excess sludge (dead bacteria) according to NITE BP-02779 - 1] (material) R2A medium: 3.2g of R2A Broth, DAIGO (manufactured by Nippon Pharmaceutical Co., Ltd.) dissolved in 1000mL of ultrapure water, sterilized by high-pressure steam. Excess sludge (killed bacteria)-containing inorganic medium: A medium containing 986 mL of substrate solution, 3.0 mL of solution A, 3.0 mL of solution B, 3.0 mL of solution C, 3.0 mL of solution D, and 1.8 mL of 1% phosphoric acid. The substrate solution and solutions A to D used were as follows. Substrate solution: After washing the excess sludge, it is mixed with 986 mL of ultrapure water to a turbidity (OD660) of 0.2, and then sterilized by high-pressure steam. Solution A: 4.35 g dipotassium hydrogen phosphate, 1.70 g monopotassium dihydrogen phosphate, 8.92 g disodium hydrogen phosphate dodecahydrate, and 0.34 g ammonium chloride were dissolved in ultrapure water, and the solution was adjusted to 200 mL and sterilized by high-pressure steam. Solution B: 4.50 g of magnesium sulfate heptahydrate dissolved in ultrapure water, adjusted to 200 mL, and sterilized by high-pressure steam. Solution C: 5.50 g of calcium chloride anhydrous was dissolved in ultrapure water, adjusted to 200 mL, and sterilized by high-pressure steam. Solution D: 0.05 g of iron chloride hexahydrate was dissolved in ultrapure water, adjusted to 200 mL, and sterilized by filtration using a 0.2 μm syringe filter.

[0071] (method) NITE BP-02779 was inoculated into R2A medium and cultured at 25°C for 24 to 48 hours. After the culture was completed, 50 μL of NITE BP-02779 culture solution and 5.0 mL of inorganic medium containing excess sludge (killed bacteria) were added to a test tube and incubated at 25°C and 200 rpm. The turbidity (OD660) of the test tube was measured over time using a simple turbidity meter (Miniphoto 518R Simple OD Monitor, manufactured by TAITEC). The number of days until the turbidity (OD660) of the inorganic medium containing excess sludge (killed bacteria) reached 50% of the turbidity (OD660) of the negative control was calculated. The turbidity (OD660) of inorganic medium containing target bacteria (killed bacteria) without NITE BP-02779 was used as the negative control.

[0072] (result) Addition of NITE BP-02779 reduced the turbidity of the excess sludge (dead bacteria), reaching 50% of the turbidity of the negative control in 3.2 days. Therefore, NITE BP-02779 was able to decompose excess sludge (dead bacteria).

[0073] [Experiment 4. Evaluation of decomposition ability of excess sludge (dead bacteria) according to NITE BP-02779 - 2] (material) The same experiment as in Experiment 3 was used.

[0074] (method) The reaction was carried out in the same manner as in Experiment 3. The reaction was carried out in quintuple, and all excess sludge remaining in the test tube was collected on the fourth day after the start of the reaction. The collected excess sludge was dried and then weighed, and a significance test (one-tailed t-test) was performed between the negative control group and the NITE BP-02779-added group.

[0075] (result) The results are shown in Figure 1. A significant decrease (p<0.01) in the dry weight of excess sludge was observed in the NITE BP-02779-added group compared to the negative control group. Therefore, the addition of NITE BP-02779 was able to reduce excess sludge.

[0076] [Experiment 5. Establishment of hybridomas] (material) Antigen: NITE BP-02779 was heat-treated, and the treated cells were mixed with Freund's complete adjuvant (Fujifilm Wako Pure Chemical Industries, Ltd.) Immunized animals: 5 female mice (B6D2F1 / Slc), 8 weeks old Myeloma cells: SP2

[0077] (method) Hybridomas were produced according to the hybridoma production and evaluation procedures shown in Figure 2. First, the antigen (bacterial cell count: approximately 1.0 × 10 9 Mice were immunized in the tail base with 1000 cells / animal. 17 days later, they were boosted with the same amount of antigen, and 21 days later, lymphocytes were collected from the iliac lymph nodes of the immunized mice. Hybridomas were formed by cell fusion between lymphocytes and myeloma cells, and then seeded into approximately 800 wells for culture. Hybridoma culture supernatant was collected from each well.

[0078] [Experiment 6. Hybridoma Selection] (material) 802 medium: 10g polypeptone, 2g yeast extract, 1g magnesium sulfate heptahydrate were dissolved in ultrapure water, the pH was adjusted to 7.0, and the volume was adjusted to 1000mL. The medium was then sterilized by high-pressure steam. 1% BSA-PBS: 250 mg of BSA (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in 25 mL of 1x PBS. Primary antibody solution: Hybridoma culture supernatant diluted 1 / 10 Secondary antibody solution: A solution of the following antibodies diluted 1 / 20,000 and mixed: HRP-labeled Donkey anti-Mouse IgG (H+L) (manufactured by Jackson ImmunoResearch) HRP-labeled Goat anti-Mouse IgG (Light Chain Specific) (Jackson ImmunoResearch) Chromogenic substrate: TMBZ (ThermoScientific)

[0079] (method) Solid-phase ELISA was performed according to the following procedure to evaluate the antigen-recognition ability of the antibodies in the hybridoma culture supernatant. NITE BP-02779 was cultured in 802 medium, harvested, washed with PBS, and adjusted to the desired concentration. The bacterial suspension was seeded onto an immunoplate (Nunc-Immuno Plate I, 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 blocking, 50 μL / well of primary antibody solution was added and incubated at room temperature for 1 hour. After incubation, the plate was washed three times with PBS, and 50 μL / well of secondary antibody solution was added and incubated at room temperature for 30 minutes. After incubation, the plate was washed three times with PBS, and 100 μL / well of chromogenic substrate solution was added and incubated at room temperature for several minutes. The reaction was stopped by adding 100 μL / well of 1M sulfuric acid. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices). Based on the results of solid-phase ELISA, five hybridomas (3A5, 4G4, 7D4, 4D9 and 6D9) shown in Table 5 were selected.

[0080] [Table 5]

[0081] [Experiment 7. Hybridoma Cloning] (method) The five selected hybridomas limiting dilution The cells were diluted using the same method and seeded onto 200 wells per well. Single colonies were confirmed by microscopic observation. The clonal culture supernatant was obtained from each well.

[0082] [Experiment 8. Clones Selection] (method) The antigen-recognition ability of the antibodies in the clone culture supernatant was evaluated using the same method as in Experiment 6. As a result of solid-phase ELISA, four clones (3A5-1H1, 4G4-1A9, 6D9-2G6, and 7D4-1G9) shown in Table 6 were selected. The obtained clones were deposited internationally at the depository institutions listed in Table 7.

[0083] [Table 6]

[0084] [Table 7]

[0085] [Experiment 9. Evaluation of clone culture supernatants for planktonic cells] (material) The same materials as in Experiment 6 were used, except that the clone culture supernatant obtained in Experiment 8 (1 / 10 dilution) was used as the primary antibody solution.

[0086] (method) Competitive ELISA was performed according to the following procedure to evaluate the antigen-recognition ability of antibodies in the clone culture supernatants. A bacterial solution of NITE BP-02779 prepared to a predetermined concentration was mixed with a primary antibody solution and left to stand at room temperature for 1 hour. 50 μL of the above mixture was added to each well of an immunoplate that had been immobilized and blocked with NITE BP-02779, and the mixture was allowed to react at room temperature for 1 hour. After the reaction, the plate was washed three times with PBS, and 50 μL of secondary antibody solution was added to each well, followed by a 30-minute reaction at room temperature. After the reaction, the plate was washed three times with PBS, and 100 μL of colorimetric substrate solution was added to each well, followed by a 5-minute reaction at room temperature. The reaction was stopped by adding 100 μL of 1 M sulfuric acid to each well. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices).

[0087] The ABS450nm when the competing bacterial cell concentration was 0 was taken as 100%, and the competing bacterial cell concentration when the ABS450nm showed 50% was calculated, and the clones were evaluated according to the following criteria. A: 1.25 x 10 8 < cells / mL B: 1.25 x 10 8 cells / mL or more 2.5×10 8 < cells / mL C:2.5×10 8cells / mL or more

[0088] (result) The results are shown in Table 8. All of the clone culture supernatants reacted with NITE BP-02779 (planktonic cells).

[0089] [Table 8]

[0090] [Experiment 10. Evaluation of clone culture supernatants against immobilized cells] (material) The same materials as in Experiment 6 were used, except that the clone culture supernatant obtained in Experiment 8 (1 / 10 dilution) was used as the primary antibody solution.

[0091] (method) The antigen-recognition ability of the antibodies in the clone culture supernatant was evaluated in the same manner as in Experiment 6. The solidified bacterial cell concentration is 5.0 x 10 8 The ABS450nm value at cells / mL was taken as 100%, and the concentration of immobilized bacterial cells was calculated when the ABS450nm value was 50%, and the clones were evaluated according to the following criteria. A: 1.0 x 10 8 < cells / mL B: 1.0 x 10 8 cells / mL or more 1.5×10 8 < cells / mL C:1.5×10 8 cells / mL or more 2.0×10 8 < cells / mL D:2.0×10 8 cells / mL or more

[0092] (result) The results of the solid-phase ELISA are shown in Table 9. The culture supernatants of all clones reacted with NITE BP-02779 (solid-phase bacterial cells). [Table 9]

[0093] [Experiment 11. Evaluation of cross-reactivity of clone culture supernatants] (material) The same materials as in Experiment 6 were used, except that the clone culture supernatant (1 / 10 dilution) obtained in Experiment 8 was used as the primary antibody solution, and the bacteria listed in Table 10 were used as the immobilized bacterial cells.

[0094] (method) The antigen cross-reactivity of the antibodies in the clone culture supernatant was evaluated in the same manner as in Experiment 6. The ABS450nm when NITE BP-02779 was used as the immobilized bacterial cells was set to 100%, and the cross-reactivity (%) for the bacterial cells listed in Table 10 was calculated. The value for the bacterial cell with the highest cross-reactivity (%) was used to evaluate the clones according to the following criteria. A: 0% to less than 20% B: 20% or more but less than 30% C: 30% or more but less than 40% D: 40% or more

[0095] (result) The cross-reactivity (%) of the four clones with the bacterial cells listed in Table 10 is shown in Figures 3 to 6. The evaluation of cross-reactivity is shown in Table 11. The culture supernatants of all clones showed high reactivity with NITE BP-02779, but low reactivity with other antigens. All four clones were found to produce antibodies with high antigen specificity.

[0096] [Table 10]

[0097] [Table 11]

[0098] [Experiment 12. Purification of Monoclonal Antibodies] (material) Clones: 4G4-1A9, 6D9-2G6 or 7D4-1G9 Culture medium: Serum-free Hybridoma-SFM (GIBCO) medium supplemented with human IL-6 (R&D Systems) at 1 ng / mL. Cation exchange chromatography binding buffer: 15.7 mM sodium phosphate buffer (pH 6.3) Cation exchange chromatography elution buffer: 1x PBS

[0099] (method) The clone was cultured in 100 mL of medium, and the culture supernatant was concentrated by ammonium sulfate precipitation. The concentrate was fractionated by cation exchange chromatography (column: HiTrap SP HP) to purify the monoclonal antibody.

[0100] [Experiment 13. Antibody Sensitization to Latex Beads] (material) Monoclonal antibodies: Monoclonal antibodies purified from the culture supernatant of clones (4G4-1A9, 6D9-2G6, or 7D4-1G9) in Experiment 12 (hereinafter referred to as 4G4-1A9 mAb, 6D9-2G6 mAb, and 7D4-1G9 mAb, respectively). Latex beads: #15713 (Polysciences, particle size 1.0 μm) Borate buffer solution: 6.18 g of boric acid was dissolved in ultrapure water, the pH was adjusted to 8.5, and the volume was adjusted to 1000 mL. 1% BSA-borate buffer: 250 mg of BSA dissolved in 25 mL of borate buffer. Storage solution: 1 g of BSA, 0.83 g of sodium chloride, and 5 g of glycerol dissolved in 100 mL of 100 mM phosphate buffer.

[0101] (method) 0.5 mL of latex beads were washed with 1 mL of borate buffer three times. 0.3 mL of 1 mg / mL monoclonal antibody solution was added to 1 mL of washed latex beads and mixed by inversion overnight at room temperature. After inversion, the supernatant was removed by centrifugation, and the beads were blocked with 1% BSA-borate buffer for 30 minutes. After removing the blocking solution by centrifugation, the beads were blocked twice by adding 1% BSA-borate buffer. 1 mL of preservative solution was added, and the beads were stored refrigerated as antibody-sensitized latex beads. 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.

[0102] (result) The absorbance (A280 nm) of the antibody solutions before and after antibody sensitization is shown in Table 12. The absorbance of all antibody solutions decreased before and after sensitization, confirming antibody sensitization of the latex beads.

[0103] [Table 12]

[0104] [Experiment 14. Latex agglutination test] (material) Antibody-sensitized latex beads prepared in Experiment 13 Other materials used were the same as those used in Experiments 11 to 13.

[0105] (method) 0.1 mL of antibody-sensitized latex beads were washed twice with 0.5 mL of borate buffer, and finally suspended in 4 mL of borate buffer. Next, NITE BP-02779 cultured in 802 medium was serially diluted to the desired concentrations with borate buffer. The serially diluted bacterial cell solution and antibody-sensitized latex beads suspended in borate buffer were each added to a U-shaped plate at 50 μL / well (100 μL total / well) and left to stand overnight at room temperature.

[0106] The agglutination image obtained with only the buffer solution was considered negative, and the agglutination image of each well was judged, and the test wells that showed positive agglutination images were classified into the following categories A to D based on the bacterial concentration. A: 2.0 x 10 6 < cells / mL B:2.0×10 6 cells / mL or more 2.0×10 7 < cells / mL C:2.0×10 7 cells / mL or more 2.0×10 8 < cells / mL D:2.0×10 8 cells / mL or more

[0107] (result) The results are shown in Table 13. All antibody-sensitized latex beads reacted with NITE BP-02779 (suspended cells), and an agglutination image was confirmed.

[0108] [Table 13]

Claims

1. A monoclonal antibody produced from a hybridoma having accession number NITE BP-03165, NITE BP-03166, NITE BP-03167 or NITE BP-03168.

2. A method for detecting Tumebacillus sp. NITE BP-02779 using the monoclonal antibody according to claim 1.

3. A detection kit for Tumebacillus sp. NITE BP-02779, comprising the monoclonal antibody according to claim 1.

4. A hybridoma having accession number NITE BP-03165, NITE BP-03166, NITE BP-03167 or NITE BP-03168.

Images