Method for detecting Anti-colibactin-producing bacteria antibody, and reagent or kit for detecting Anti-colibactin-producing bacteria antibody

Abstract

The present invention provides: a method for detecting an antibody against colibactin-producing bacteria in a subject; and a reagent or kit for detecting an antibody against colibactin-producing bacteria in a subject.　Provided is a method for detecting an anti-colibactin-producing bacteria antibody in a sample, said method including a step for bringing the sample into contact with at least one of the lipopolysaccharides from among O2, O4, O6, O18, and O50 serotypes of Escherichia coli. Provided is a reagent or kit for detecting an anti-colibactin-producing bacteria antibody, said reagent or kit containing a sample and at least one of the lipopolysaccharides from among O2, O4, O6, O18, and O50 serotypes of Escherichia coli.

Description

Method for Detecting Anti-Colibactin-Producing Bacteria Antibody and Detection Reagent or Kit 【0001】 The present invention relates to a method for detecting an anti-colibactin-producing bacteria antibody in a biological sample derived from a mammal, and a detection reagent or kit therefor. 【0002】 Colibactin induces gene mutations in the host by inducing cleavage of the host DNA double strand and interstrand crosslinking (Non-Patent Document 1). The formation of colorectal cancer is known to involve the presence of Escherichia coli strains carrying gene clusters involved in the biosynthesis of colibactin, called the clb cluster or pks cluster (Non-Patent Document 2). A subgroup of colibactin-producing bacteria (clb+) of Escherichia coli is classified into the symbiotic B2 phylogenetic group, inhabits the human large intestine, and is presumed to be involved in the development of colorectal cancer as a result of symbiosis. Contact between the clb+ strain and eukaryotic cells is thought to cause cell cycle arrest in the G2 / M phase of the cell and / or the formation of megakaryocytes by colibactin. The detection rate of the clb cluster gene from biopsy samples of colorectal cancer patients is 67%, which has been reported to be higher than that of healthy subjects (21-27%). 【0003】 Colibactin is a genotoxin secreted by bacteria belonging to the family Enterobacteriaceae, and this secretory bacterium is known to have a 54-kilobase genomic island encoding polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and PKS-NRPS hybrid megasynthetase, which are necessary for colibactin biosynthesis (Non-Patent Document 3). The gene cluster involved in colibactin production contains genes for various enzymes, including peptidase ClbP as one of them. Based on its crystal structure and other findings, ClbP is suggested to activate precolibactin, a colibactin precursor, into a genotoxic final product (Non-Patent Documents 4 and 5). Patent Document 1 discloses a method for high-throughput measurement of colibactin-producing bacteria in clinical specimens using a fluorescent probe specifically activated by ClbP. The fluorescent probe is also useful for identifying high colibactin-producing strains from Escherichia coli strains. 【0004】Non-patent documents 6 and 7 disclose the structure of N-myristoyl-D-asparagine (N-myr-Asn) as a prodrug motif of colibactin, and a method for detecting it by LC-MS. To date, research on precolibactin (colibactin precursor) has been conducted using clb+ strains formed by gene mutations, and a variety of chemical structures have been identified as candidate precolibactin substances. However, these substances have not been isolated from wild-type clb+ E. coli strains, and it remains unclear whether they actually exist as precolibactin in colibactin-producing bacteria present in the human large intestine. 【0005】 Patent Document 2 discloses a method for detecting colibactin-producing E. coli in a sample using an antibody that specifically binds to colibactin-producing E. coli, and a method for specifically controlling colibactin-producing bacteria. 【0006】 International Publication No. 2019 / 044736, International Publication No. 2023 / 223796 【0007】 Nougayrede, JP et al., Science Vol. 313 (5788), pp. 848-851 (2006)Putze, J. et al., Infect. Immun. Vol. 77 (11), pp. 4696-4703 (2009)Homburg, S. et al., FEMS Microbiol. Lett. Vol. 275 (2), pp. 255-262 (2007)Dubois, D. et al, J. Biol. Chem. Vol. 286 (41), pp. 35562-35570 (2011)Brotherton, CA and Balskus, EP, J. Am. Chem. Soc. Vol. 135 (9), pp. 3359-3362 (2013)Bian, X. et al., Chembiochem. Vol. 14 (10), 1194-1197 (2013)Brotherton CA et al., Org. Lett., Vol.17, pp. 1545-1548 (2015) 【0008】As described above, Patent Documents 1 and 2 disclose methods for identifying / detecting colibactin-producing bacteria, but currently, no method has been reported for detecting antibodies against colibactin-producing bacteria in subjects infected with these bacteria. 【0009】 The present invention aims to provide a method for detecting antibodies against colibactin-producing bacteria in a target, as well as a detection reagent or kit. 【0010】 As a result of diligent research, the present inventors have discovered that antibodies specific to colibactin-producing bacteria recognize the lipopolysaccharide of O6 serotype Escherichia coli as an antigen, and have completed the present invention. Accordingly, the present invention provides the following: (1) A method for detecting anti-colibactin-producing bacterial antibodies in a sample, comprising the step of contacting the sample with at least one of the lipopolysaccharides of O2, O4, O6, O18, and O50 serotype Escherichia coli. (2) The method according to (1), comprising the step of contacting the sample with the lipopolysaccharide of O6 serotype Escherichia coli. (3) The method according to (1) or (2), wherein the sample is a sample taken from a subject. (4) The method according to (3), wherein the subject does not have diarrheal symptoms. (5) The method according to (3) or (4), wherein the subject is a human. (6) The method according to any one of (1) to (5), wherein the sample is serum, urine, or feces. (7) A reagent or kit for detecting an anti-colibactin-producing bacterial antibody, comprising at least one of the lipopolysaccharides of serotypes O2, O4, O6, O18, and O50 of Escherichia coli. (8) The reagent or kit according to (7), comprising the lipopolysaccharide of serotype O6 of Escherichia coli. This specification includes the disclosures of Japanese Patent Application No. 2024-213338, which forms the basis of the priority of this application. 【0011】 According to the present invention, antibodies against colibactin-producing bacteria in a target can be detected. 【0012】This is a photograph showing the results of the Western blot in Test Example 2 (1). It shows the results of detecting cell lysates of colibactin-producing Escherichia coli E. coli-50 and colibactin-non-producing strain #A using antibody δ. The numbers in the figure indicate the amount of protein loaded into each well. This is a graph showing the results of competitive ELISA in Test Example 2 (3). It shows the results of detecting suspensions of antibody δ and PS at various concentrations using an LPS-immobilized plate and labeled antibody. This is a graph showing the number of clinical strains listed in the NCBI database for each O antigen serotype and the prevalence rate of clb clusters in Test Example 5. 【0013】 1. Definitions In this specification, “subject” refers to any of the following: humans, primates including chimpanzees, pet animals such as dogs and cats, domestic animals such as cattle, pigs, horses, sheep and goats, rodents such as mice and rats, or mammals kept in zoos. In this specification, the subject is preferably a human. In this specification, the subject is preferably free from diarrheal symptoms, and even if diarrheal symptoms occur, it is preferable that at least 3 days, 7 days, 10 days, 14 days, 21 days, or 30 days have elapsed since the diarrheal symptoms disappeared. In this specification, “sample” refers to a sample taken from the subject, for example, blood (e.g., whole blood, serum, plasma), urine, feces, milk, tissue or cell extract, nasal secretions, saliva, or mixtures thereof. Preferably, it may be serum, urine, or feces. 【0014】 In this specification, the term "colibactin-producing bacteria" is not limited to any type of bacteria that has the ability to produce colibactin. Examples include Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, and Klebsiella aerogenes. The presence or absence of colibactin-producing ability in bacteria can be confirmed, for example, using the method described in Patent Document 1. 【0015】In this specification, "anti-colibactin-producing bacterial antibody" refers to an antibody that specifically binds to colibactin-producing bacteria, and more specifically, an antibody that binds to colibactin-producing bacteria but does not bind to non-colibactin-producing bacteria. The antibody isotype referred to herein may be IgG, IgM, IgA, IgD, IgE, or a mixture thereof. 【0016】 The antigens on the cell surface of Escherichia coli are broadly classified into four types: O antigen (cell), H antigen (flagellar), K antigen (capsule), and ciliary antigen. The "serotype" of Escherichia coli is a typing method that primarily classifies cells by combining the O antigen (O serotype) and H antigen (H serotype). In this specification, "O2 serotype," "O4 serotype," "O6 serotype," "O18 serotype," and "O50 serotype" Escherichia coli refer to Escherichia coli whose O antigen is the O2 glycan antigen, O4 glycan antigen, O6 glycan antigen, O18 glycan antigen, and O50 glycan antigen, respectively. The O serotype of Escherichia coli can be identified, for example, using the previously reported method of multiplex PCR (Jun Iguchi, Japanese Journal of Bacteriology, 71(4): 209-215 (2016)). While the genotypes in E. coli O2 and O50 serotypes are the same as OgGp7, their serotypes are known to be different. As described later, a high colibactin positivity rate has been confirmed overall in E. coli groups possessing the OgGp7 genotype. Consequently, it has also been confirmed that the detection rate of anti-colibactin-producing antibodies is high for antigens derived from E. coli of both O2 and O50 serotypes possessing this genotype. 【0017】 In this specification, "reagent" refers to a single component, and "kit" refers to a kit consisting of multiple components. Here, a container containing a reagent is considered to constitute "one component" together with the reagent. 【0018】In this specification, "antibody" refers to a protein that is the target of detection in the present invention and specifically binds to a particular substance (antigen) contained in the sample. The antibody refers to an antibody that binds to the antigen defined above on colibactin-producing bacteria and does not bind to bacteria that do not produce colibactin. The isotype of the detected antibody is not particularly limited and may be any of IgG, IgA, IgM, IgE, etc., and can be appropriately selected depending on the type of sample. 【0019】 2. Method for detecting antibodies against anti-colibactin-producing bacteria The first embodiment of the present invention is a method for detecting antibodies against anti-colibactin-producing bacteria. The method of this embodiment is characterized by including the step of contacting a sample with at least one of the lipopolysaccharides (LPS) of O2, O4, O6, O18, and O50 serotypes of Escherichia coli. In the method of this embodiment, the sample is preferably a biological sample taken from a subject. According to the method of this embodiment, it is possible to determine whether or not antibodies against anti-colibactin-producing bacteria are present in the sample of the subject, thereby making it possible to determine whether or not there is a current or past infection with colibactin-producing bacteria. 【0020】 The LPS to be brought into contact with the sample preferably contains at least the LPS of the O6 serotype of Escherichia coli, out of the five types of lipopolysaccharides (LPS) from the O2, O4, O6, O18, and O50 serotypes of Escherichia coli. The LPS to be brought into contact with the sample may be only one type, or it may be a combination of two or more LPS. The combination of two or more LPS is not particularly limited, but may include O6 and O2, O6 and O4, O6 and O18, O6 and O50, O6 and O2 and O4, O6 and O2 and O18, O6 and O4 and O50, O6 and O18 and O50, O6 and O4 and O18 and O50, O6 and O2 and O4 and The combinations can be O50, O6 and O2 and O4 and O18, O6 and O2 and O4 and O18 and O50, O2 and O4, O2 and O18, O2 and O50, O4 and O18, O4 and O50, O18 and O50, O2 and O4 and O18, O2 and O4 and O50, O2 and O18 and O50, O4 and O18 and O50, or O2 and O4 and O18 and O50. 【0021】The method of this embodiment specifically comprises reacting an antibody in a sample extracted from a target with at least one of the lipopolysaccharides (LPS) of O2, O4, O6, O18, and O50 serotypes of Escherichia coli. Examples of such immunoassays include direct competitive assays, indirect competitive assays, and sandwich assays. Examples of such immunoassays include chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), immunoturbidimetry (TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioactive immunoassay (RIA), latex agglutination assay, fluorescence immunoassay (FIA), and immunochromatography. The principles and specific methods of the above immunoassays are all well known to those skilled in the art. 【0022】 In the method of this embodiment, the detection of anti-colibactin-producing bacterial antibodies may be qualitative or quantitative. A qualitative method may involve comparing the signal obtained from the sample with a preset cutoff to determine the presence or absence of anti-colibactin-producing bacterial antibodies in the sample. A quantitative method may involve performing an immunoassay on a standard solution containing a known amount of antibody, similar to the sample, and calculating the amount of antibody in the sample by comparing the signal intensities obtained from the sample and the standard solution. Furthermore, the method of this embodiment may include steps of detecting antibodies in a standard solution and calculating the amount of antibody-colibactin-producing bacterial antibodies in the sample from the detection results of the standard solution. 【0023】 In the method of this embodiment, if the antibody against colibactin-producing bacteria is qualitatively determined to be "positive," or if the quantitative value exceeds the judgment criterion, it can support the determination that the subject from which the sample was taken is infected with colibactin-producing bacteria, or has been infected with colibactin-producing bacteria in the past. 【0024】 Hereinafter, a sandwich ELISA will be described as an exemplary aspect of this embodiment, but the scope of the present invention is not intended to be limited to the sandwich ELISA. 【0025】(1) Preparation of the antigen plate The ELISA in this embodiment can use a plate on which the antigen is immobilized, preferably a 96-well microwell plate (immunoplate). The LPS to be used as the antigen is at least one of the lipopolysaccharides (LPS) of O2, O4, O6, O18 and O50 serotypes of Escherichia coli, or a combination thereof (O6 and O2, O6 and O4, O6 and O18, O6 and O50, O6 and O2 and O4, O6 and O2 and O18, O6 and O2 and O50, O6 and O4 and O18, O6 and O4 and O50, O6 and O18 and O50, O6 and O4 and O18 and O50, The possible combinations are O6, O2, O18, and O50; O6, O2, O4, and O50; O6, O2, O4, and O18; O6, O2, O4, O18, and O50; O2, O4; O2, O18; O2, O50; O4, O18, O4, O50; O18, O50; O2, O4, O18, O2, O4, and O50; O2, O18, and O50; O4, O18, and O50; or O2, O4, O18, and O50. In particular, it is preferable to use LPS from at least O6 serotype Escherichia coli alone or in combination with other antigens. The means for preparing LPS are not particularly limited, but for example, it can be prepared by culturing Escherichia coli in which the O antigen serotype has been confirmed and extracting it from the bacterial cells. Alternatively, it can be chemically synthesized. When extracting from cultured bacterial cells, the method for extracting and purifying LPS from the cells is not particularly limited, and any known method can be used. For example, the hot phenol method can be used. By combining the reaction of RNase and DNase with the normal hot phenol method, polysaccharides excluding nucleic acids can be extracted from cells. For example, after homogenizing or sonicating the bacterial cells, proteins can be degraded with proteinase, and then nucleic acids can be degraded using RNase and DNase. After that, phenol can be added and heated, the residue removed, and then ethanol can be added and cooled to obtain polysaccharides containing LPS as a precipitate. 【0026】Alternatively, instead of purified LPS, unpurified bacterial cells of known serotypes may be used as the antigen. When using bacterial cells, for example, freeze-dried bacterial cells may be used. The bacterial cells may be at least one of the O2, O4, O6, O18, and O50 serotypes of Escherichia coli, or a combination thereof (O6 and O2, O6 and O4, O6 and O18, O6 and O50, O6 and O2 and O4, O6 and O2 and O18, O6 and O2 and O50, O6 and O4 and O18, O6 and O4 and O50, O6 and O18 and O50, O6 and O4 and O18 and O50, O6 and O2 and O1 This can be 8 and O50, O6 and O2 and O4 and O50, O6 and O2 and O4 and O18, O6 and O2 and O4 and O18 and O50, O2 and O4, O2 and O18, O2 and O50, O4 and O18, O4 and O50, O18 and O50, O2 and O4 and O18, O2 and O4 and O50, O2 and O18 and O50, O4 and O18 and O50, or O2 and O4 and O18 and O50). When using freeze-dried products of multiple serotypes of E. coli in combination, the freeze-dried products may be mixed together, or the bacterial cell mixture may be freeze-dried. 【0027】 The amount of LPS solidified on the plate is not particularly limited, but can be, for example, 0.1 to 1000 μg / well, particularly 1 to 100 μg / well, and even more preferably 5 to 50 μg / well. LPS can be solidified on the plate by, for example, suspending LPS in PBS, PBS containing 0.05% Tween (T-PBS), carbonate buffer, etc., preparing a suspension, dispensing this into a plate, and letting it stand under refrigerated conditions (e.g., 0 to 10°C, particularly 4 to 5°C) for 12 hours or more, particularly 18 hours or more, and even more preferably 24 hours or more. After washing the plate, it is preferable to further block it using a BSA / PBS solution or the like. 【0028】(2) Reaction between the sample and the antigen plate. Liquid samples (e.g., serum or urine) can be diluted with PBS or the like before use. Solid samples can be homogenized to liquefy them, for example, and then diluted with PBS or the like before use. Diluted samples can be dispensed onto an antigen-immobilized plate and reacted with the antigen by shaking or standing at 20-40°C, for example, 37°C, for 5 minutes to 3 hours, especially for about 30 minutes to 2 hours. If the sample contains antibodies against anti-colibactin-producing bacteria, the antibodies will bind to the antigen immobilized on the plate. 【0029】 (3) Antibodies bound to the labeled antibody-immobilized antigen can be detected using the labeled antibody. The antibody used as the labeled antibody may be any of the following: anti-IgG antibody, anti-IgM antibody, anti-IgA antibody, anti-IgE antibody, and anti-IgD antibody, or a combination thereof. In particular, a combination of anti-IgG antibody, anti-IgE antibody, and anti-IgA antibody is preferred. If the target is a human, a combination of anti-human IgG antibody, anti-human IgM antibody, and anti-human IgA antibody is preferred. 【0030】 Any known label may be used, such as enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, or biotin (streptavidin). The use of enzymes is particularly preferred. Examples of enzymes that can be used include horseradish peroxidase (HRP) and alkaline phosphatase (ALP). 【0031】 The labeled antibody can be diluted with PBS or similar and dispensed into wells at concentrations of, for example, 0.01 ng to 0.1 μg / well, particularly 0.01 to 20 ng / well, and even further 0.02 to 5 ng / well. The antibody can then be reacted with the antigen-bound antibody (anti-colibactin-producing bacterial antibody) by shaking or standing at 20 to 40°C, for example 37°C, for 5 minutes to 3 hours, particularly 30 minutes to 2 hours. 【0032】(4) Anti-colibactin-producing bacterial antibodies contained in the sample can be detected by measuring the label of the labeled antibody bound to the detection antibody. If the label is an enzyme, a suitable substrate solution can be selected and used depending on the enzyme used. In this case, the substrate solution can be dispensed into each well, colored, and then the absorbance at a predetermined wavelength can be measured. As substrates, if the enzyme is HRP, tetramethylbenzidine (TMB) (measurement wavelength 450 nm), o-phenylenediamine (measurement wavelength 490 nm), etc. can be used. If the enzyme is ALP, p-nitrophenyl phosphate (pNPP) (measurement wavelength 405 nm), etc. can be used. 【0033】 3. Reagent or Kit for Detection of Anti-Colibactin-Producing Antibodies The second embodiment of the present invention is a reagent or kit for detection of antibodies against anti-colibactin-producing bacteria. The reagent or kit of this embodiment is characterized by containing at least one of the lipopolysaccharides of O2, O4, O6, O18, and O50 serotypes of Escherichia coli. More specifically, the reagent or kit of this embodiment is a reagent or kit for carrying out the method described in section "2. Method for Detection of Anti-Colibactin-Producing Antibodies". 【0034】 The reagent or kit of this embodiment is a reagent or kit for immunoassay, comprising reacting an anti-colibactin-producing bacterial antibody with at least one of the lipopolysaccharides of O2, O4, O6, O18, and O50 serotypes of Escherichia coli. Examples of such immunoassays include direct competitive assays, indirect competitive assays, and sandwich assays. Other examples of such immunoassays include CLEIA, CLIA, TIA, EIA (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), RIA, latex agglutination assay, FIA, and immunochromatography. The principles and specific methods of the above immunoassays are all well known to those skilled in the art. 【0035】 The following describes a sandwich ELISA kit as an example of one embodiment of this invention, but the scope of the present invention is not intended to be limited to sandwich ELISA systems. The kit of this embodiment may include, for example, an antigen-immobilized plate, a diluent, a labeled antibody reagent, a substrate, a stop solution, and a washing solution. 【0036】 (1) Antigen-immobilized plate The kit of this embodiment may include an antigen-immobilized plate. The antigen-immobilized plate may be, for example, a 96-well microwell plate on which at least one of the lipopolysaccharides (LPS) of O2, O4, O6, O18, and O50 serotypes of Escherichia coli that serve as antigens are placed, or a combination thereof (O6 and O2, O6 and O4, O6 and O18, O6 and O50, O6 and O2 and O4, O6 and O2 and O18, O6 and O2 and O50, O6 and O4 and O18, O6 and O4 and O50, O6 and O18 and O50, O6 and It can be prepared by solidifying O4, O18, and O50, O6, O2, O18, and O50, O6, O2, O4, and O50, O6, O2, O4, and O18, O6, O2, O4, O18, and O50, O2 and O4, O2 and O18, O2 and O50, O4 and O18, O4 and O50, O18 and O50, O2, O4, O18, and O50, O2, O4, O18, and O50, or O2, O4, O18, and O50). The means for preparing LPS are not particularly limited, but at least one of the lipopolysaccharides (LPS) of O2, O4, O6, O18, and O50 serotypes of Escherichia coli, or a combination thereof (O6 and O2, O6 and O4, O6 and O18, O6 and O50, O6 and O2 and O4, O6 and O2 and O18, O6 and O2 and O50, O6 and O4 and O18, O6 and O4 and O50, O6 and O18 and O50, O6 and O4 and O18 It can be O50, O6 and O2 and O18 and O50, O6 and O2 and O4 and O50, O6 and O2 and O4 and O18, O6 and O2 and O4 and O18 and O50, O2 and O4, O2 and O18, O2 and O50, O4 and O18, O4 and O50, O18 and O50, O2 and O4 and O18, O2 and O4 and O50, O2 and O18 and O50, O4 and O18 and O50, or O2 and O4 and O18 and O50). In particular, it is preferable to use LPS of at least O6 serotype Escherichia coli alone or in combination with other antigens. The means for preparing LPS are not particularly limited, but for example, it can be prepared by culturing Escherichia coli in which the O antigen serotype has been confirmed and extracting it from the bacterial cells. The amount of LPS solid phase in the plate is not particularly limited, but can be, for example, 0.1 to 1000 μg / well, particularly 1 to 100 μg / well, and even more specifically 5 to 50 μg / well. 【0037】(2) Diluent The kit of this embodiment may include a diluent. The diluent can be used to dilute the sample and / or other reagents. Examples of diluents include PBS, T-PBS, etc. The pH can be 5 to 9, particularly 6 to 8, and even more specifically 6.5 to 7.5. 【0038】 (3) Labeled antibody reagent The kit of this embodiment may include a labeled antibody reagent. The antibody used as the labeled antibody can be any of the following: anti-IgG antibody, anti-IgM antibody, anti-IgA antibody, anti-IgE antibody, and anti-IgD antibody, or a combination thereof. In particular, a combination of anti-IgG antibody, anti-IgE antibody, and anti-IgA antibody is preferred. If the target is human, a combination of anti-human IgG antibody, anti-human IgM antibody, and anti-human IgA antibody is preferred. As the label, any known label such as enzyme, radioisotope, fluorescent substance, luminescent substance, colored particle, colloidal particle, or biotin (streptavidin) may be used. In particular, the use of an enzyme is preferred. As an enzyme, for example, HRP, alkaline phosphatase, etc. can be used. The concentration of the labeled antibody should be higher than the concentration used when actually used for detection, and it is preferable to dilute it immediately before use. For example, it can be configured to be diluted 10 to 1000 times, and particularly 20 to 200 times in volume. 【0039】 (4) Substrates The kit of this embodiment may include a substrate corresponding to the enzyme used when the label is an enzyme. If the enzyme is HRP, TMB, o-phenylenediamine, etc. can be used as substrates. If the enzyme is ALP, pNPP, etc. can be used. The substrate may be provided in solution or in solid form. If it is a solution, it can be diluted immediately before use. If it is a solid, it can be dissolved immediately before use. 【0040】 (5) Stop solution The kit of this embodiment may include a stop solution if the label is an enzyme. For example, 0.1 to 2 N sulfuric acid can be used as the stop solution. 【0041】(6) Washing Solution The kit of this embodiment may include a washing solution. The washing solution is used to wash and remove unreacted samples and components in the test solution in the microplate. As the washing solution, for example, PBS, T-PBS, etc. can be used. Also, the pH can be set to 5-9, particularly 6-8, and more preferably 6.5-7.5. The washing solution may be prepared, for example, at a 10-fold concentration and diluted with water or the like immediately before use. 【0042】 (7) Others In the kit of this embodiment, each test solution may contain, as necessary, pH buffers such as Tris, MES, HEPES, PIPES, chelating agents such as EDTA, preservatives, etc. Also, the kit of this embodiment may include an instruction manual, a storage seal bag, etc. 【0043】 [Test Example 1] Preparation of Colibactin-producing Bacteria-binding Antibody (1) Immunization The colibactin-producing strain 50 (a high colibactin-producing bacterium (Hirayama, Y. et al., Org. Lett. Vol. 21, No. 12, pp. 4490-4494 (2019)), hereinafter also referred to as "E. coli-50") was cultured in an LB liquid medium at 37 °C for 20 hours. Bacterial cells were collected from the culture solution, washed twice with PBS(-), and then suspended in PBS(-) to a concentration of 1×10 １０ cells / mL, and heat-treated at 60 °C for 60 minutes. 100 μL of the heated E. coli suspension was mixed with 100 μL of Sigma Adjuvant System (Catalog No. S6322, Sigma) to prepare a mixed solution, and this was subcutaneously injected into a mouse (BALB / c, female, 8 weeks old) at 50 μL per leg. Two weeks later, the mouse was sacrificed, and the spleen and inguinal lymph nodes were collected. 【0044】(2) The spleen and below-knee lymph nodes from which cells were collected were each cut in half, and the cells were pushed into a petri dish with deck tweezers and suspended in 5 mL of FBS(+). After passing the cells through a 100 μm cell strainer, they were transferred to a 50 mL centrifuge tube. The petri dish and cell strainer were washed with 10 mL of FBS(+), and the cells were centrifuged (1000 rpm, 5 minutes, room temperature). The supernatant was discarded, the pellet was tapped, and the cells were suspended in FBS(+) and counted. In addition, mouse myeloma P3U1 cells were counted, and 10 ８ The cells were suspended in FBS(+) to a concentration of cells / mL. Splenocytes / lymph node cells:P3U1 were added to a 50 mL centrifuge tube and suspended in a ratio of 3-4:1. After centrifugation (1000 rpm, 5 minutes, room temperature), the cells were resuspended in 30 mL of FBS(-) and centrifugated again (1000 rpm, 5 minutes, room temperature). After removing the supernatant, the pellet was tapped. 1 mL of PEG solution was added at a rate of 1 mL / min and allowed to stand for 1 minute. Then, 30 mL of FBS(-) was added at a rate of 1 mL / min while shaking, and the remaining 20 mL was added slowly. After centrifugation (1000 rpm, 5 minutes, room temperature), the supernatant was discarded and the pellet was tapped. The cells were suspended in HAT medium and dispensed into 96-well microwell plates, 200 μL per well, and stored at 37°C in 5% CO2. ２ The cells were cultured under the same conditions (all subsequent cell cultures were performed under the same conditions). The cell condition was checked, and the cell culture supernatant was collected. 【0045】(3) Measurement of antibody titer by ELISA E. coli - 50 and colibactin - non - producing E. coli #A (hereinafter also referred to as "#A") were each cultured in LB liquid medium at 37°C for 20 hours. Here, #A is a strain confirmed to be a colibactin - non - producing bacterium by the method described in Patent Document 1. After culturing, the cells were washed three times with pure water, rapidly frozen using liquid nitrogen, and then freeze - dried. They were suspended in PBS(-) with 0.05% Tween (hereinafter T - PBS) so that each strain had a concentration of 0.5 mg / mL. 100 μL of the suspension of E. coli - 50 or #A was dispensed into each well of a 96 - well microplate (Nunc Immunoplate, catalog No. 439454) and left standing at 4°C for one day or more for solid - phase immobilization. After washing this plate three times with T - PBS, 1% BSA (dissolved in T - PBS) was added, and it was left standing at room temperature for 2 hours for blocking. This was washed three times with T - PBS to obtain antigen - immobilized wells. A large number of antibody - producing cells (hybridomas) obtained by the cell fusion in (2) above were each cultured for one week. The culture supernatant was added to each well at 100 μL or 50 μL each and left standing at 4°C overnight. After washing four times with T - PBS, 100 μL of 0.5 ng / mL HRP - labeled goat anti - mouse IgG + IgM + IgA antibody was added and left standing at 37°C for 1 hour. After washing four times with T - PBS, 100 μL of substrate solution (0.04% o - phenylenediamine) was added, and it was left standing at room temperature for 30 minutes in the dark. 450 μL of 2N sulfuric acid was added to stop the reaction, and the absorbance at 490 nm of each well was measured with a microplate reader. 【0046】 Supernatants with an absorbance at 490 nm of 0.09 or more were obtained from 6 samples from the inguinal lymph nodes and 96 samples from the spleen. These supernatants were serially diluted to monoclonize and 753 cell lines were obtained. For these cells, culture and ELISA were performed in the same manner as above, and 641 positive cell lines were obtained. After culturing them by serial dilution, there were 15 cell lines with a difference in absorbance (ΔABS) exceeding 0.5 between the plate of E. coli - 50 and the plate of #A. 【0047】(4) Selection of antibodies that specifically bind to colibactin-producing bacteria The binding specificity to colibactin-producing Escherichia coli was examined for monoclonal antibodies of 15 hybridoma strains. For a total of 43 strains of Escherichia coli, including E. coli-50, Nissle 1917 strain (colibactin-producing strain), strain #A (non-colibactin-producing strain), 38 strains obtained from clinical samples (22 colibactin-producing strains, 16 non-colibactin-producing strains), and clbP gene-deficient strains of E. coli-50 and Nissle 1917, solid-phase wells were prepared in the same manner as described in (3). All 38 strains obtained from clinical samples were strains in which colibactin production / non-production was confirmed by the method described in Patent Document 1. 【0048】 Fifteen hybridoma strains obtained in (3) and one hybridoma strain (non-specific reaction strain) that showed positive results for both E. coli-50 and #A were cultured for one week, and 100 μL of the culture supernatant was dispensed into the antigen-immobilized wells of the 43 E. coli strains. ELISA was then performed in the same manner as described in (3). Table 1 shows the ELISA results for monoclonal antibodies (antibody α, antibody β, antibody γ, antibody δ) and antibodies (antibody ε) from four hybridoma strains (α, β, γ, δ) of 24 colibactin-producing strains, and Table 2 shows the ELISA results for antibodies from non-specific reaction strains (antibody ε) derived from four hybridoma strains (α, β, γ, δ) of 19 colibactin-non-producing strains. In the table, "Judgment" indicates the result of ELISA performed using antibody δ, with an absorbance of 0.09 as the cutoff, where 0.09 or higher was considered positive and less than 0.09 was considered negative. The results above confirmed that nonspecific reaction strains were positive in all E. coli strains. On the other hand, 15 hybridoma strains containing antibodies α to δ were found to be negative in all non-colibactin-producing strains except for the clbP gene-deficient strains of E. coli-50 and Nissle 1917. 【0049】[Test Example 2] Epitope Determination of Monoclonal Antibody δ (1) Western Blotting Cells of colibactin-producing Escherichia coli E. coli-50 and colibactin-non-producing Escherichia coli #A were suspended in PBS and sonicated. The cells were then loaded onto acrylamide gel so that the protein content was 0.3 to 5.0 μg, and SDS-PAGE was performed. After transferring the gel-like protein to a membrane, Western blotting was performed according to a standard procedure using 10 ng / mL antibody δ (cell culture supernatant diluted 150,000 times in PBS) as the primary antibody and HRP-labeled sheep anti-mouse IgG+IgM+IgA antibody as the secondary antibody. The substrate used was Immunostar® LD (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Figure 1 shows the results of the Western blotting. The ladder-like band of the E. coli-50 strain suggested that the antibody reacts with proteins having a repeating structure. Furthermore, these band patterns closely resemble those of lipopolysaccharide (LPS) in previously reported Gram-negative bacteria (see, for example, Pourcel et al., Antibiotics, 9, 339(2020) Figure 1), suggesting that the antibody δ epitope is located on LPS. 【0050】 (2) Extraction of lipopolysaccharide (LPS) and acquisition of polysaccharide (PS) from colibactin-producing bacteria E. coli-50 was inoculated into 3 L of culture medium and cultured at 37°C for 24 hours, after which the cells were collected. After washing twice with 10 mL of PBS(+), the cells were resuspended in 10 mL of PBS(+) and sonicated on ice for 10 minutes. Proteinase K was added at 100 μg / mL and treated at 65°C for 1.5 hours. RNase (40 μg / mL), DNase (20 μg / mL), 20% MgSO ４ (1 μL / mL) and chloroform (4 μL / mL) were added and incubated overnight at 37°C. Equivolute 90% phenol was added and heated at 70°C for 15 minutes. Centrifugation was performed at 4°C and 8500 × g for 15 minutes, and the supernatant was collected. 10 times the volume of ethanol was added to the supernatant and allowed to stand overnight at -20°C. Centrifugation was performed at 4°C and 2000 × g for 10 minutes, and the resulting precipitate was washed with 70% ethanol. Approximately 50 mg of LPS was obtained using the above procedure. 【0051】 1 mg of LPS was dissolved in 1 mL of 1% aqueous acetic acid solution and heated at 100°C for 2 hours. After heating, the solution was concentrated to dryness and then separated with water / chloroform. The aqueous layer was separated and concentrated again to dryness to obtain 0.5 mg of PS. 【0052】 (3) Epitope determination by competitive ELISA method An LPS suspension was prepared by adding T-PBS to LPS to a concentration of 0.1 mg / mL. 100 μL / well of this LPS suspension was dispensed into a Nunc immunoplate (#439454) and allowed to stand at 4°C for at least one day to solidify. After washing the plate three times with T-PBS, a 1% BSA / T-PBS solution was dispensed and allowed to stand at room temperature for 2 hours to block. After washing the plate three times with T-PBS, 100 μL / well of a mixed suspension of antibody δ and PS was added and incubated at 37°C for 1.5 hours. The mixed suspension was prepared containing 0.1 μg / mL of antibody δ and PS serially diluted to 10 μg to 1 mg / mL. After incubation, the plates were washed five times with T-PBS, and 100 μL / well of 0.5 ng / mL HRP-labeled sheep anti-mouse IgM antibody solution was added. The plates were incubated at 37°C for 1 hour. The plates were washed five times with T-PBS, 100 μL / well of chromogenic reagent (0.04% o-phenylenediamine) was dispensed, and the plates were left to stand at room temperature for 30 minutes in the dark. The reaction was stopped by dispensing 50 μL / well of stop solution (2N sulfuric acid solution), and the absorbance at 490 nm of each well was measured using a multiplate reader. 【0053】 Figure 2 shows the results of the competitive ELISA method. The absorbance decreased as the concentration of added PS increased, indicating a concentration-dependent effect. This suggests that the antigen of antibody δ is PS, and that the O antigen of E. coli-50 is the epitope of antibody δ. 【0054】[Test Example 3] Identification of O antigen in colibactin-producing bacteria. 43 strains of Escherichia coli used in Test Example 2 (4) were cultured overnight at 37°C in LB medium. After cell collection, DNA was extracted using a bacterial genome extraction kit (MonoFas® (Animos Co., Ltd.)). The amount of dsDNA was measured using a Qubit Fluorometer (ThermoFisher), and a DNA solution was prepared to 2 ng / μL and used as a template. 【0055】 The genotype of the O antigen in each strain was determined using the previously reported method of multiplex PCR (see Jun Iguchi, Japanese Journal of Bacteriology, 71(4): 209-215 (2016)). Multiplex PCR determines the O antigen genotype by targeting the O antigen synthesis gene (mainly wzx or wzy) of Escherichia coli with PCR primers. For multiplex PCR, the primer mixes provided by the Iguchi Laboratory, Faculty of Agriculture, Miyazaki University, PrimeSTAR® GXL Premix Fast and Dye Plus (both from Takara Bio Inc.) were used. After mixing the template and the above reagents, PCR (1) was performed under the conditions in Table 3. For strains from which PCR products could not be obtained in PCR (1), PCR (2) was performed under the conditions in Table 4. The obtained PCR products were subjected to 2% agarose gel electrophoresis, and the genotype of the O antigen was determined from the position of the obtained bands. Table 5 shows the relationship between the O antigen genotype and the O antigen serotype. Strains for which PCR products could not be confirmed under the above PCR conditions were classified as "OgUT (unclassifiable)". 【0056】 Tables 1 and 2 show the O antigen genotypes of each strain. All strains that were colibactin-producing and showed a "+" result on ELISA were confirmed to be O6 serotype. Furthermore, no O6 serotype was found among the non-colibactin-producing strains tested. In addition, the results in Tables 1 and 2 suggest that not only antibody δ, but also antibodies α, β, and γ bind to O6 serotype Escherichia coli LPS (O6 antigen). 【0057】 【0058】 【0059】 【0060】 【0061】 【0062】 [Test Example 4] Detection of Anti-Colibactin-Producing Antibodies (1) Antigen Preparation Escherichia coli (E. coli-50 strain) with serotype O6 was cultured in LB medium at 37°C for 20 hours. After washing the collected cells, they were rapidly frozen using liquid nitrogen and then freeze-dried. As a control, the same procedure was performed on Escherichia coli strain #A, which does not produce colibactin. 【0063】 (2) Detection of anti-colibactin-producing bacterial antibodies by ELISA A bacterial suspension was prepared by adding T-PBS to lyophilized bacterial cells to a concentration of 0.5 mg / mL. 100 μL / well of this bacterial suspension was dispensed into a 96-well microplate and allowed to stand at 4°C for at least one day to solidify. After washing the plate three times with T-PBS, a 1% BSA / T-PBS solution was dispensed and allowed to stand at room temperature for 2 hours to block. The plate was washed three times with T-PBS to obtain a bacterial solidification plate. 【0064】 Serum samples were obtained from two human subjects (positive) in whom infection with colibactin-producing bacteria was detected using the method described in International Publication No. 2019 / 044736, and from two human subjects (negative) in whom infection was not detected, by collecting blood multiple times on different days. Each serum sample was diluted 100-fold with T-PBS, and 100 μL / well was dispensed into each bacterial cell-immobilized plate and incubated at 37°C for 1.5 hours. After incubation, the plates were washed five times with T-PBS, and 100 μL / well of a 0.5 ng / mL HRP-labeled anti-human IgG+IgM+IgA antibody solution was added, followed by incubation at 37°C for 1.5 hours. The plates were washed five times with T-PBS, and 100 μL / well of SuperSignaltm ELISA Femto Substrate (Thermo, #37075) was added, and the luminescence was measured. For each sample, the value obtained by dividing the measurement value on the E. coli-50 solid-phase plate by the measurement value on the control #A solid-phase plate was calculated. The results are shown in Table 6. 【0065】 【0066】 As shown in Table 6, a significantly higher concentration of antibodies binding to LPS from E. coli-50 strain was confirmed in the blood of human subjects who tested positive for colibactin. 【0067】 [Test Example 5] Prevalence of various O antigens in Escherichia coli possessing known colibactin-producing gene clusters For 1853 types of Escherichia coli found in human samples registered with NCBI, the prevalence of the clb cluster, a colibactin-producing gene cluster, was calculated for each O antigen serotype. Figure 3 summarizes the relationship between each serotype and the prevalence of the clb cluster. Escherichia coli possessing the O6 antigen was found in greater numbers than Escherichia coli with other serotypes, and its clb cluster prevalence was also very high at 90.5%. Although the number of Escherichia coli possessing the O4 and O18 antigens was small, their clb cluster prevalence was also high at approximately 90%. Furthermore, about half of the Escherichia coli possessing the O20 / O50 antigen possessed the clb cluster, but the number of Escherichia coli possessing it was large. This suggests that screening Escherichia coli possessing the O2, O6, O4, O18, and O50 antigens can be used to assess the risk of colorectal cancer due to colibactin. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A method for detecting anti-colibactin-producing bacterial antibodies in a sample, comprising the step of contacting the sample with at least one of the lipopolysaccharides of serotypes O2, O4, O6, O18, and O50 of Escherichia coli.

2. The method according to claim 1, comprising the step of contacting a sample with lipopolysaccharide of O6 serotype Escherichia coli.

3. The method according to claim 1, wherein the sample is a sample taken from the subject.

4. The supplement to the law according to claim 3, wherein the subject does not have diarrheal symptoms.

5. The method according to claim 3, wherein the subject is a human.

6. The method according to claim 1, wherein the sample is serum, urine, or feces.

7. A reagent or kit for detecting anti-colibactin-producing bacterial antibodies, comprising at least one lipopolysaccharide from serotypes O2, O4, O6, O18, and O50 of Escherichia coli.

8. The reagent or kit according to claim 7, comprising lipopolysaccharide of O6 serotype Escherichia coli.

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