Method for detecting Helicobacter Swiss antibodies using whole bacterial cells

Abstract

Provided are a measuring reagent and a measuring method for a Helicobacter suis antibody using a whole bacterial cell of H. suis.　The reagent contains at least an H. suis whole bacterial cell and an anti-Ig antibody, and detects an antibody that binds with H. suis whole bacterial cell of a subject-derived sample.

Description

Technical Field

[0001] The present invention relates to a method for measuring antibodies against Helicobacter suis using whole cells of H. suis.

Background Art

[0002] Currently, it is known that infections by Helicobacter pylori (a microaerophilic gram-negative spiral bacterium that parasitizes the human stomach, hereinafter referred to as "H. pylori") are involved in chronic gastritis, gastric ulcers, duodenal ulcers, gastric cancer, gastric Mucosa-Associated Lymphoid Tissue (MALT) lymphoma, diffuse large B-cell lymphoma, etc. As methods for detecting H. pylori infection, mainly the isolation culture method, urea breath test (UBT), measurement of H. pylori antibody levels in serum and urine (ELISA and latex agglutination method), measurement of H. pylori antigen in feces (immunochromatography method), and rapid urease test (RUT) of gastric biopsy specimens are adopted.

[0003] However, in recent years, with the decline in the infection rate of H. pylori due to the spread of H. pylori diagnosis and eradication, NHPH (Non-Helicobacter pylori hericobacters; helicobacters other than H. pylori) has become a problem as a helicobacter that induces severe gastric diseases in humans other than H. pylori. NHPH includes Helicobacter suis (hereinafter, H. suis), H. bizzozeronnii, H. felis, H. salmonis, H. ailurogastricus, H. cynogasticus, H. baculiformis, H. mustelae, H. acinonychls, H. cetorum, and H. heilmannii, and among these, most found in the human stomach are H. suis.

[0004] H. pylori infects only primates, and infection from close relatives is assumed only in infancy, but H. suis infects from animals such as pigs and monkeys regardless of age.

[0005] H. suisse parasitized the stomachs of monkeys approximately 100,000 years ago. Later, 15,000 years ago, it began infecting pigs. With the domestication of pigs, the infection spread explosively, eventually infecting humans (Non-Patent Literature 1). MLST (Multi-Locus Sequencing Typing) analysis of 181 H. suisse strains isolated from around the world revealed that they formed independent clusters depending on the infected host animal. However, strains isolated from humans did not form independent clusters and were included in the pig cluster, leading to the conclusion that pigs were the source of human infection (Non-Patent Literature 2). Therefore, infection diagnosis and disinfection agents are needed for both pigs and humans.

[0006] As a method for testing for H. swiss infection, measurement of H. swiss antibody titers in serum (ELISA and latex agglutination method) has been reported (Patent Documents 1 and 2). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2016-10331 [Patent Document 2] International Publication No. WO2019 / 225639 [Non-patent literature]

[0008] [Non-Patent Document 1] Flahou et al.,ISM J.,Jan;12(1):77-86.doi:10.1038 / ismej.2017.145 [Non-Patent Document 2] E.Rimbara et al.,Proc Natl Acad Sci USA 2021 Vol.118 Issue 13 e2026337118.doi:10.1073 / pnas.2026337118. [Non-Patent Document 3] Haesebrouck F. et al., Helicobacter,16(4),339-340,2011 doi:10.1111 / j.1523-5378.2011.00849.x [Non-Patent Document 4] ADAugustin,et al.,Front.Med.,2019,6,188,doi:10.3389 / fmed.2019.00188(https: / / www.ncbi.nlm.nih.gov / pubmed / 31555648) [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a method and reagent for measuring antibodies against Helicobacter swiss using whole H. swiss cells. [Means for solving the problem]

[0010] Patent Document 1 discloses measuring antibodies in serum by immobilizing the H. swiss F2R2 protein on a plate. Patent Document 2 discloses measuring antibodies in serum by immobilizing the HsvA protein, which is said to have a higher antibody titer than the F2R2 protein, on a plate.

[0011] However, there was a need for a method with higher sensitivity and specificity than conventional methods. In this study, the inventors have novelly discovered that using whole H. suisse cells enhances the sensitivity and specificity of measuring anti-H. suisse antibodies in infected individuals, thus establishing a new measurement method.

[0012] More specifically, the present invention relates to the following inventions: [1] A reagent for detecting antibodies that bind to whole H. suisse cells in a subject-derived sample, including at least whole H. suisse cells and anti-Ig antibodies. [2] The reagent of [1], characterized in that the test subject is a mammal. [3] The reagent according to [1] or [2], characterized in that the sample is blood-derived. [4] Any of the reagents [1] to [3] further characterized by detecting antibodies that bind to H. pylori or antigens of H. pylori cell components in a subject-derived sample, which also contain H. pylori cell components or antibodies against H. pylori cell components. [5] One of the reagents [1] to [4], which is an ELISA or immunochromatographic reagent. [6] A method comprising the following steps, characterized by detecting antibodies that bind to whole anti-H. Swiss bacteria in a sample derived from a subject: (a) A step of bringing a sample derived from the subject into contact with whole H. swiss bacteria, and (b) A step of detecting antibodies in the sample that are bound to whole H. swiss bacteria. [7] The method of [6], characterized in that the subject is a mammal. [8] The method of [6] or [7], characterized in that the sample is of blood origin. [9] Furthermore, any of the following methods [6] to [8] is characterized by detecting antibodies that bind to H. pylori in a sample derived from the subject, comprising the following steps: (a) A step of bringing a sample derived from the subject into contact with H. pylori cell components, and (b) A step of detecting antibodies in the sample that are bound to H. pylori cell components.

[10] Furthermore, any of the methods [6] to [8] is characterized by detecting antigens of H. pylori cell components in a sample derived from the subject, including the following steps: (a) A step of contacting a sample derived from the subject with an antibody against H. pylori cell components, and (b) A step of detecting the antigen of the H. pylori cell component in the sample, which is bound to an antibody against the H. pylori cell component.

[11] A method for detecting H. swiss infection, wherein a subject is determined to be infected with H. swiss if antibodies are detected by measurement using any of the reagents [1] to [5] and / or by any of the methods [6] to

[10] . This specification includes the disclosures of Japanese Patent Application No. 2022-009397, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0013] By using the whole cells of H. suis, the anti-H. suis antibodies in the body of an infected person can be measured with high sensitivity. The whole cells of H. suis can be used to measure the presence or antibody titer of anti-H. suis antibodies in blood derived from a subject to be diagnosed for H. suis infection.

Brief Description of the Drawings

[0014] [Figure 1] A diagram showing the results of ELISA using human serum as a sample and whole cells of H. suis and HsvA antigen peptide as antigens in measured values. [Figure 2] A diagram showing the results of ELISA using human serum as a sample and whole cells of H. suis and HsvA antigen peptide as antigens in a graph. [Figure 3] A diagram showing the results of ELISA using human serum (diluted 3600-fold) as a sample and whole cells of H. pylori or whole cells of H. suis as antigens in measured values. [Figure 4] A diagram showing the results of ELISA using human serum (diluted 3600-fold) as a sample and whole cells of H. pylori (Figure 4A) or whole cells of H. suis (Figure 4B) as antigens in a graph. [Figure 5-1] A diagram showing the results of ELISA using human serum (diluted 3600-fold) as a sample and whole cells of H. suis or partial peptides (SEQ ID NO: 1-5) of HsVA of H. suis as antigens in measured values. [Figure 5-2] A diagram showing the results of ELISA using human serum (diluted 3600-fold) as a sample and whole cells of H. suis or partial peptides (SEQ ID NO: 6-11) of HsVA of H. suis as antigens in measured values.

Modes for Carrying Out the Invention

[0015] Hereinafter, the method of the present invention will be described. The present invention relates to a method for detecting antibodies against all H. suissiae bacteria in a biological sample of a subject, and a reagent for detecting said antibodies. The present invention also relates to a method for determining the presence of H. suissiae in a subject, or a method for detecting H. suissiae infection in a subject. Alternatively, it relates to a method for obtaining auxiliary data for diagnosing H. suissiae infection in a subject.

[0016] (H. Switzerland: Helicobacter suis) In this specification, "H. suisse" refers to the H. suisse species included in NHPH (Non-Helicobacter pylori hericobacters; Helicobacter other than H. pylori) or Helicobacter heilmannii sensu lato (Helicobacter heilmannii in the broad sense) among the more than 50 reported species of Helicobacter bacteria (Non-Patent Literature 3). H. suisse is known to infect the stomachs of animals other than humans, such as pigs, monkeys, wild boars, cats, and dogs. The infected mammals from which H. suisse has been isolated in this specification are not particularly limited and may be, for example, humans, monkeys, wild boars, or pigs.

[0017] (H. Swiss whole cell) In this specification, "H. swiss whole cell" refers to the components of the whole cell of H. swiss, and can be obtained, for example, by ultrasonically disrupting H. swiss. It is also called the H. swiss whole cell fraction. Ultrasonic disruption can be performed by suspending H. swiss cells in a buffer solution and using an ultrasonic disruptor. An example of an ultrasonic disruptor is the Bioruptor (BM Instruments), a sample-sealed ultrasonic disruption device. By ultrasonic disruption, the H. swiss cells are destroyed, and a lysate containing the whole cell components is obtained.

[0018] (Method and reagents for detecting antibodies against whole H. swiss bacteria) Antibodies against H. swiss whole cells refer to antibodies against components of H. swiss whole cells, such as proteins and sugars, i.e., components of H. swiss whole cells, and may include multiple antibodies against various substances. In this invention, antibodies against at least one of the antigens, such as proteins and sugars, contained in H. swiss whole cells are referred to as antibodies against H. swiss whole cells. In practice, in a subject infected with H. swiss, antibodies against H. swiss whole cells are produced in the subject as a result of the degradation of H. swiss cells in the body.

[0019] Furthermore, the binding of an antibody to an antigen contained in the entire H. swiss bacterium is referred to as the antibody binding to the entire H. swiss bacterium.

[0020] Reagents for detecting antibodies against whole H. swiss bacteria can be prepared as reagents applicable to known methods. Known methods include, for example, labeled immunoassays such as enzyme immunoassay (EIA), simplified EIA, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence immunoassay (FIA); immunoblotting; immunochromatography; chromatography; turbidimetric assay (TIA); nephrite assay (NIA); colorimetric assay; latex agglutination assay (LIA); particle counting assay (CIA); chemiluminescence assay (CLIA, CLEIA); sedimentation assay; surface plasmon resonance assay (SPR); resonant mirror detector assay (RMD); and comparative interference assay. Whether or not the reagent of the present invention can be applied to a desired measurement method can be confirmed by performing each measurement method using a sample containing antibodies against whole H. swiss bacteria or a sample containing antibodies against whole H. swiss bacteria at the same concentration as said sample, and measuring whether or not detection is possible.

[0021] Antibodies against all H. swiss bacteria can be used as markers for H. swiss infection.

[0022] In other words, whether or not a subject is infected with H. suisse can be determined by confirming the presence of antibodies against all H. suisse bacteria produced by the subject's own immune system. The presence of these antibodies can be detected and confirmed by an antigen-antibody reaction using all H. suisse bacteria. Detection of H. swiss using whole H. swiss cells can be performed using various immunoassay methods, including: labeled immunoassays such as enzyme immunoassay (EIA), simplified EIA, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence immunoassay (FIA); immunoblotting methods such as Western blotting; immunochromatography methods such as gold colloid agglutination; chromatography methods such as ion exchange chromatography and affinity chromatography; turbidimetric assay (TIA); nebulizer assay (NIA); colorimetric assay; latex agglutination assay (LIA); particle counting assay (CIA); chemiluminescence assay (CLIA, CLEIA); sedimentation assay; surface plasmon resonance assay (SPR); resonant mirror detector assay (RMD); and comparative interference assay.

[0023] In one aspect, the present invention relates to a method for determining H. swiss infection, comprising the following steps: (a) A step of bringing a sample derived from the subject into contact with whole H. swiss bacteria, (b) A step of detecting antibodies in the sample that are bound to whole H. swiss bacteria, and (c) A process in which subjects in which antibodies bound to whole H. swiss bacteria are detected are determined to be infected with H. swiss, while subjects in which antibodies bound to whole H. swiss bacteria are not detected are determined to not be infected with H. swiss.

[0024] In another aspect, the present invention relates to a method for determining H. swiss infection, comprising the following steps: (a) A step of bringing a sample derived from the subject into contact with the whole H. swiss bacterial cells of the present invention, (b) A step of measuring the level of antibodies in the sample that are bound to whole H. swiss bacteria, and (c) If the measured antibody level is higher than the antibody level measured in a negative control using the same method, the subject is determined to be infected with H. swiss; on the other hand, if the measured antibody level is equal to or lower than the antibody level measured in a negative control using the same method, the subject is determined not to be infected with H. swiss. Here, a negative control refers to a sample derived from a subject not infected with H. swiss. The antibody level refers to the quantitative value of the antibody, i.e., the antibody titer, which is expressed, for example, as the concentration in the sample. Furthermore, the measurement of the antibody level is included in antibody detection.

[0025] Whether or not the whole H. swift cells have bound to the antibodies in the sample derived from the test subject can be confirmed by the following example. For example, after contacting the sample derived from the test subject with the whole H. swift cells of the present invention, the reaction system is washed to remove unbound antibodies. As a result, a complex containing the whole H. swift cells and the antibodies in the sample derived from the test subject is present in the reaction system. Subsequently, the sample is contacted with a labeled anti-Ig antibody to bind the antibodies from the test subject bound to the whole H. swift cells to the labeled anti-Ig antibody. After this, the reaction system is washed to remove unbound labeled anti-Ig antibodies, and the label of the remaining labeled anti-Ig antibody is detected. If the label of the labeled anti-Ig antibody is detected, it can be determined that the whole H. swift cells and the antibodies in the sample derived from the test subject have bound. The level of the antibodies in the whole H. swift cells and the sample derived from the test subject can also be measured by measuring the labeling level of the labeled anti-Ig antibody. The anti-Ig antibody may be an anti-IgG antibody, an anti-IgM antibody, etc., but an anti-IgG antibody is preferred. Furthermore, monoclonal antibodies are preferably used as anti-IgG antibodies. Additionally, fragments with specific antigen-binding properties (antigen-binding fragments) such as Fab, Fab', F(ab')2, single-chain antibodies (scFv), VHH single-domain antibodies (nanobodies), dsFv, diabody, and minibody can also be used. Representative examples of such methods include ELISA and immunochromatography. For labeling anti-Ig antibodies, enzymes such as alkaline phosphatase and horseradish peroxidase, metal colloids such as gold colloid, silica particles, cellulose particles, magnetic particles, fluorescent particles, colored polystyrene particles, and colored latex particles are often used. When using colored particles such as metal colloid particles, colored polystyrene particles, or colored latex particles, coloring occurs due to aggregation of these labeling reagents, and this coloration is measured. When using the binding level as an indicator, the antibody amount can be calculated from the measured value by creating a standard curve using standard solutions whose abundance is known in advance. Alternatively, a surface plasmon resonance sensor can be used to detect or measure the binding of H. swiss whole cells to antibodies in a sample derived from the test subject.

[0026] Among the immunoassay methods described above, the sandwich method is preferred. The sandwich method itself is well known in the field of immunoassay and can be performed, for example, by immunochromatography or ELISA, which perform immunoassay in a lateral flow manner. All of these sandwich methods are well known, and the method of the present invention can be performed by a well known sandwich method. The ELISA method will be described below.

[0027] In an ELISA plate, the H. swiss whole cell component (antigen) is immobilized, and a sample potentially containing antibodies against the H. swiss whole cell is added to the immobilized support and brought into contact with it to form an antigen-antibody complex on the support. Furthermore, an enzyme-labeled antibody (anti-Ig antibody) specific to human antibodies is added and brought into contact with the support, causing the anti-Ig antibody to bind to the antigen-antibody complex on the support. Next, a substrate for the enzyme is added, the enzymatic reaction is carried out, and the resulting color is measured by measuring the absorbance to detect the antigen-antibody sandwich complex on the plate.

[0028] The antigen-antibody reaction can be carried out at 4°C to 45°C, preferably 20°C to 40°C, and more preferably 25°C to 38°C. The reaction time for each binding reaction is approximately 10 minutes to 18 hours, more preferably 10 minutes to 3 hours, and even more preferably 30 minutes to 2 hours.

[0029] The binding of H. swiss whole cell components (antigens) to the carrier can be carried out by known methods such as physical adsorption and covalent bonding using functional groups. The amount to be immobilized is not particularly limited, but when the carrier is a 96-well microtiter plate, several ng to several tens of μg per well is desirable. Immobilization can be carried out by bringing a solution of the H. swiss whole cell components (antigens) to be immobilized into contact with the carrier. For example, immobilization can be achieved by dispensing a solution of H. swiss whole cell components (antigens) into the wells of a microtiter plate and leaving it for a certain period of time. After immobilizing the H. swiss whole cell components (antigens), it is preferable to block the binding using a blocking solution containing bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin, etc., to prevent nonspecific binding during the assay.

[0030] Since H. suisse infects a wide range of mammals, the subjects in the method for determining the presence of H. suisse and the method for determining infection with H. suisse according to the present invention can be mammals such as humans, monkeys, pigs, cats, wild boars, dogs, rabbits, mice, and sheep, and preferably humans. Furthermore, the subjects are preferably human patients suffering from gastritis, chronic gastritis, goosebump gastritis, type A gastritis, gastric MALT lymphoma, diffuse large B-cell lymphoma, gastric cancer, gastric or duodenal ulcer, idiopathic thrombocytopenic purpura, functional dyspepsia, or Parkinson's disease. In addition, the method of the present invention can be carried out qualitatively, quantitatively, or semi-quantitatively (Non-Patent Literature 4).

[0031] In the method for determining the presence of H. swiss of the present invention, the specimen used can be, for example, a tissue sample or a liquid sample taken from a subject as a biopsy. The specimen is not particularly limited as long as it can be used as the subject of the method of the present invention, and examples include tissue, blood, plasma, serum, lymph, urine, feces, serous fluid, cerebrospinal fluid, synovial fluid, aqueous humor, tears, saliva, or fractions or processed products thereof. When antibodies are to be detected in the method for determining the presence of H. swiss of the present invention, preferred specimens are blood, plasma, serum, lymph, and urine.

[0032] The whole H. Swiss bacterial cells of the present invention can be appropriately prepared by methods well known to those skilled in the art with reference to the disclosure herein. Furthermore, the reagents of the present invention can be appropriately manufactured by methods well known in the art.

[0033] In one aspect, the present invention relates to a method for determining the presence of H. swiss in a subject from which a sample has been collected, comprising a method for detecting antibodies against all H. swiss bacteria in a sample. In particular, the present invention relates to a method for determining infection with H. swiss in a subject, comprising detecting antibodies against all H. swiss bacteria. In this method, a subject from which a sample has been collected in which antibodies against all H. swiss bacteria have been detected is determined to have H. swiss present or to be infected with H. swiss.

[0034] When the method of the present invention is performed by measuring the binding of antibodies in the sample to the test reagent, the determination of whether or not something has been "detected" does not need to be an absolute detection or non-detection, but may be determined by comparison with other samples. In other words, the determination of whether or not something has been detected may be based on the measured value rather than on a ± detection result. That is, in the method of the present invention, the step of "detecting" can be replaced with "measuring" as needed, and whether or not something has been "detected" may be determined by comparing it with a negative target based on the measured value of the target substance. For example, if the target substance is detected in a negative target, i.e., a sample that does not contain H. swisii, or a sample from a subject that is clearly not infected with H. swisii, if the measured value of the test subject is equivalent to the measured value of the negative target, even if it is detected in a trace amount, the method of the present invention will determine that H. swisii is not present or that the subject is not infected with H. swisii, and it is "not detected". On the other hand, if the measured value of the subject is higher than the measured value of the negative target, it will be determined that H. swisii is present or that the subject is infected with H. swisii, and it is "detected". Therefore, in the method of the present invention, the presence of a small measured value in a negative target is within the range that the present invention pre-accepts.

[0035] In the method of the present invention, antibody titers in samples from individuals infected with H. swiss and those not infected with H. swiss can be measured, and a cutoff value can be set. If the titer is above the cutoff value, it can be determined that H. swiss is present or that the individual is infected with H. swiss.

[0036] The cutoff value can be determined, for example, by ROC (receiver operating characteristic curve) analysis. Furthermore, the diagnostic accuracy (sensitivity and specificity) of the method of the present invention can be determined by ROC analysis. In ROC analysis, anti-H. Swiss antibodies are measured in samples taken from individuals infected with H. Swiss and individuals not infected with H. Swiss, and the sensitivity and false positive rate (1-specificity) at each cutoff value are calculated and plotted on a coordinate system with (1-specificity) on the x-axis and sensitivity on the y-axis. When the diagnostic accuracy is analyzed by ROC analysis, the sensitivity should be 80% or higher, preferably 85% or higher, and more preferably 90% or higher, and the specificity should be 75% or higher, preferably 80% or higher.

[0037] (Detection of antibodies against H. pylori) The present invention also includes a method for detecting antibodies that bind to whole H. pylori cells or proteins contained in the whole cells, as well as antibodies against H. pylori, in the same subject, and reagents used in the method. Here, antibodies against H. pylori refer to antibodies against any component of the whole H. pylori cell, i.e., bacterial components. For example, if a patient tests negative for H. pylori but exhibits symptoms of gastric disease, H. pylori infection may be the cause. By detecting antibodies that bind to whole H. pylori cells and antibodies against H. pylori from the same subject, it is possible to confirm not only the presence or absence of H. pylori infection, but also H. pylori infection in subjects that were previously overlooked due to being H. pylori negative. Early confirmation of infection allows for the determination of an appropriate treatment plan. The specimens from the same subject may be the same for H. pylori and H. pylori, or they may be different. That is, the detection of antibodies that bind to whole H. pylori cells and the detection of antibodies against H. pylori may be performed simultaneously using the same specimen, or they may be performed separately using different specimens collected at different times. The detection of antibodies against H. pylori can be performed using the same method as for detecting antibodies against whole H. swiss bacteria.

[0038] In determining whether H. pylori has been detected, the determination does not need to be based on an absolute detection; it may be determined by comparison with other samples. In other words, the determination of detection may be based on the measured value rather than on a ± detection result. That is, in the method of the present invention, the step of "detecting" can be replaced with "measuring" as needed, and whether or not it has been "detected" may be determined by comparison with a negative sample based on the measured value of the target substance. For example, if the target substance is detected in a negative sample, i.e., a sample that does not contain H. pylori or a sample from a subject that is clearly not infected with H. pylori, and the measured value of the subject is equivalent to the measured value of the negative sample, even if it is detected in a trace amount, the method of the present invention will determine that H. pylori is not present or that the subject is not infected with H. pylori, as it will be considered "not detected." On the other hand, if the measured value of the subject is higher than the measured value of the negative sample, it will be determined that H. pylori is present or that the subject is infected with H. pylori, as it will be considered "detected." Therefore, in the method of the present invention, the presence of a small measured value in a negative sample is within the range that the present invention pre-accepts. [Examples]

[0039] The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. All references cited throughout this application are incorporated herein by reference in their entirety.

[0040] (Example 1) Measurement of human specimens Measurement of anti-H. Swiss antibody titers in infected individuals (humans) using ELISA (Enzyme-Linked ImmunoSorbent Assay). Composition of H. Swiss medium Brucella Broth (BD BBL) 2.8 g Agar powder (BD BBL) 1.5 g; Pyruvate (Sigma) 0.1 g (to be added when preparing agar plates) Skirrow Supplement(2 mL / vial, Oxoid) 0.4 mL Vitox Supplement(10 mL / vial, Oxoid) 2 mL Amphotericin B(0.25 mg / mL) 2 mL Adjust the pH to 5 with 0.135 mL of concentrated hydrochloric acid. Deactivated fetal bovine serum (FBS) 20 mL Distilled water was added to bring the total volume to 100 mL.

[0041] 75cm 2 A slant medium was prepared by adding 9 mL of agar medium to a flask. H. swiss SNTW101c strain (Non-Patent Literature 2), isolated from a patient with goosebump gastritis stored at -80°C, was inoculated, and 12 mL of liquid medium was added. Shaking culture was performed for one week at 37°C, 100% humidity, and microaerophilic conditions (5% O2, 12% CO2, 83% N2). Subsequently, the culture medium was collected by centrifugation (13,420 G x 10 minutes). The cells were washed twice with PBS (phosphate-buffered saline, pH 7.4), suspended in distilled water, and subjected to sonication for 5 minutes under ice-cold conditions (using a Bioruptor II ultrasonic cell disruption device (set to High), repeating 30 seconds of ultrasonic treatment followed by 30 seconds of rest five times). The disruption solution was designated as "H. swiss whole cell suspension". For protein quantification, the Bio-Rad Protein Assay kit was used with BSA as the standard protein.

[0042] HsvA antigen peptide No. 16 represents 14 amino acid residues (SEQ ID NO: 6), which are part of HsvA (an outer membrane protein specifically present only in H. swiss, composed of approximately 3000 amino acid residues) disclosed in Patent Document 2. Since antibody testing cross-referencing this No. 16 peptide has shown excellent sensitivity and specificity in H. swiss infection testing, it is thought that peptide No. 16 is involved in the production of H. swiss-specific antibodies.

[0043] 100 μL / well of a H. swiss whole cell suspension (4 μg / mL) dissolved in 0.1 M carbonate-bicarbonate buffer (pH 9.4) was added dropwise to a 96-well NUNC ImmunoPlate #439454 and left overnight at 4°C. The cell solution was discarded the next day and washed three times with 200 μL / well of PBS-T (PBS containing 0.05% (V / V) Tween20). 200 μL / well of a blocking solution (1% (V / V) BSA, PBS system pH 7.4) prepared from Blocker BSA (bovine serum albumin) 10X in PBS (Thermo Scientific) was added dropwise and left at 37°C for 1 hour. The blocking solution was discarded and washed three times with 200 μL / well of PBS-T. Serum samples were collected from individuals infected with H. pylori (determined by existing testing methods such as urea breath test, antibody test, or antigen test) and from individuals infected with H. swiss (DNA was prepared from the subject's gastric biopsy, and H. swiss infection was determined by the real-time PCR method described in Patent Document 2 or by culturing from the gastric biopsy). Serum samples were also collected from healthy individuals not infected with either bacterium as a control (non-infected). 50 μL / well of human serum diluted with blocking solution was added and left at 37°C for 1 hour. The serum solution was discarded and washed three times with 200 μL / well of PBS-T. 50 μL / well of horseradish peroxidase-labeled secondary antibody (Goat anti-Human IgA+IgG+IgM(H+L), Jackson ImmunoResearch, Inc.), diluted 100,000 times with blocking solution, was added and left at 37°C for 1 hour. The secondary antibody solution was discarded, and the plates were washed three times with PBS-T 200 μL / well. 50 μL / well of SuperBlue TMB Microwell Peroxidase Substrate (1-Component), Kirkegaard & Perry Laboratories, Inc. (KPL) was added, and after blue coloration, 50 μL / well of 1N hydrochloric acid was added, causing a yellow coloration. The absorbance at 450 nm (reference wavelength: 620 nm~630 nm) was measured using a plate reader.

[0044] The results of ELISA using human serum are shown in Figures 1 and 2. For whole H. swiss bacteria, 1800-fold and 3600-fold dilutions of human serum (3 H. swiss-positive samples (patients A, B, C); 3 H. pylori-positive samples (patients D, E, F); 3 non-infected samples (patients G, H, I)) were able to distinguish between positive and negative results based on absorbance values ​​of 1.0 or higher. On the other hand, HsvA antigen peptide No. 16 could not distinguish between positive and negative results based on 200-fold, 800-fold, and 3200-fold dilutions of human serum (3 H. swiss-positive samples (patients A, B, C); 3 H. pylori-positive samples (patients D, E, F); 3 non-infected samples (patients G, H, I)). This indicates that whole H. swiss bacteria are significantly more sensitive and specific to antibodies against H. swiss than HsvA antigen peptide No. 16. Therefore, whole H. swiss bacteria are useful for specific infection diagnosis using antibodies in serum.

[0045] (Example 2) Measurement of human specimens Measurement of anti-H. pylori antibody titers and anti-H. Swiss antibody titers in infected individuals (humans) using ELISA (Enzyme-Linked ImmunoSorbent Assay). (1) Preparation of H. pylori antigen (whole H. pylori bacteria) for ELISA H. pylori strain TN2GF4 (Non-Patent Literature 2) was cultured for 48 hours with shaking in Brucella broth containing 10% (V / V) fetal bovine serum (FCS) at 37°C, 100% humidity, and microaerophilic conditions (5% O2, 10% CO2, 85% N2). The culture medium was then centrifuged (13,420 G x 10 minutes) to collect the bacterial cells. The cells were washed twice with PBS phosphate-buffered saline (pH 7.4), suspended in distilled water, and subjected to sonication for 5 minutes under ice-cold conditions (using a Bioruptor II ultrasonic cell disruption device (set to High), repeating 30 seconds of ultrasonic treatment followed by 30 seconds of rest five times). The disruption solution was designated as the "H. pylori whole cell suspension." For protein quantification, the Bio-Rad Protein Assay kit was used with BSA as the standard protein.

[0046] (2) Preparation of H. Swiss antigen (whole H. Swiss bacteria) for ELISA Composition of H. Swiss medium Brucella Broth (BD BBL) 2.8 g Agar powder (BD BBL) 1.5 g; Pyruvate (Sigma) 0.1 g (to be added when preparing agar plates) Skirrow Supplement(2 mL / vial, Oxoid) 0.4 mL Vitox Supplement(10 mL / vial, Oxoid) 2 mL Amphotericin B(0.25 mg / mL) 2 mL Adjust the pH to 5 with 0.135 mL of concentrated hydrochloric acid. Deactivated fetal bovine serum (FBS) 20 mL Distilled water was added to bring the total volume to 100 mL.

[0047] 75cm 2 A slant medium was prepared by adding 9 mL of agar medium to a flask. H. swiss SNTW101c strain (Non-Patent Literature 2), isolated from a patient with goosebump gastritis stored at -80°C, was inoculated, and 12 mL of liquid medium was added. Shaking culture was performed for one week at 37°C, 100% humidity, and microaerophilic conditions (5% O2, 12% CO2, 83% N2). Subsequently, the culture medium was collected by centrifugation (13,420 G x 10 minutes). The cells were washed twice with PBS phosphate-buffered saline (pH 7.4), suspended in distilled water, and subjected to sonication for 5 minutes under ice-cold conditions (using a Bioruptor II ultrasonic cell disruption device (set to High), repeating 30 seconds of ultrasonic treatment followed by 30 seconds of rest five times). The disruption solution was designated as "H. swiss whole cell suspension". For protein quantification, the Bio-Rad Protein Assay kit was used with BSA as the standard protein.

[0048] 100 μL / well of H. pylori whole cell suspension and H. swiss whole cell suspension (4 μg / mL) dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6) were added dropwise to a 96-well NUNC ImmunoPlate #468667 and left overnight at 4°C. The cell solution was discarded the next day and washed three times with 250 μL / well of PBS-T (PBS containing 0.05% (V / V) Tween20). 200 μL / well of blocking solution (1% (V / V) BSA, PBS system pH 7.0) was added dropwise and left at 37°C for 1 hour. The blocking solution was discarded and washed three times with 250 μL / well of PBS-T. Serum samples were collected from H. pylori-infected individuals with symptoms of gastric disease (determined by existing testing methods such as urea breath test, antibody test, and antigen test), and from H. swiss-infected individuals with symptoms of gastric disease (DNA was prepared from the subject's gastric biopsy, and H. swiss-infected individuals were determined by the real-time PCR method described in Patent Document 2 or by culture from the gastric biopsy). Serum samples were also collected from healthy individuals not infected with either bacterium as a control (non-infected). Human serum diluted with sample diluent (0.5% (V / V) BSA, PBS-based pH 7.0) was added at 50 μL / well and left at 37°C for 1 hour. The serum solution was discarded, and the samples were washed three times with 250 μL / well of PBS-T. 50 μL / well of horseradish peroxidase-labeled secondary antibody (Goat anti-Human IgG(H+L), Jackson ImmunoResearch, Inc.), diluted 20,000-fold with secondary antibody diluent (1.0% (V / V) BSA, PBS system pH 7.0), was added and left at 37°C for 1 hour. The secondary antibody solution was discarded, and the plates were washed three times with 250 μL / well of PBS-T. 50 μL / well of TMB One Component HRP Microwell Substrate, (Surmodics, Inc.) was added, and after developing a blue color, 50 μL / well of 0.17 M sulfuric acid was added, causing a yellow color change. The absorbance at 450 nm (reference wavelength: 620 nm~630 nm) was measured using a plate reader.

[0049] The results of ELISA using human serum (3,600-fold dilution) are shown in Figures 3 and 4. For whole H. pylori cells, the absorbance of the H. pylori infection group (gastric disease - A, B, C, D) was significantly higher than that of the H. swift infection group (gastric disease - E, F, G, H) or the non-infection group (healthy individuals - A, B, C, D) (H. pylori infection group vs. H. swift infection group or non-infection group: P=0.0209). This study revealed that by testing for both anti-H. pylori antibodies and anti-H. Swiss antibodies in the same subject, it is possible to diagnose not only the presence or absence of H. pylori infection, but also H. Swiss infection in individuals who were previously overlooked due to being H. pylori negative.

[0050] (Example 3) Measurement of human specimens Measurement of anti-H. Swiss antibody titers in infected individuals (humans) using ELISA (Enzyme-Linked ImmunoSorbent Assay). Composition of H. Swiss medium Brucella Broth (BD BBL) 2.8 g Agar powder (BD BBL) 1.5 g; Pyruvate (Sigma) 0.1 g (to be added when preparing agar plates) Skirrow Supplement(2 mL / vial, Oxoid) 0.4 mL Vitox Supplement(10 mL / vial, Oxoid) 2 mL Amphotericin B(0.25 mg / mL) 2 mL Adjust the pH to 5 with 0.135 mL of concentrated hydrochloric acid. Deactivated fetal bovine serum (FBS) 20 mL Distilled water was added to bring the total volume to 100 mL.

[0051] 75cm 2A slant medium was prepared by adding 9 mL of agar medium to a flask. H. swiss SNTW101c strain (International Publication WO2019 / 225639), isolated from a patient with goosebump gastritis stored at -80°C, was inoculated, and 12 mL of liquid medium was added. Shaking culture was performed for one week at 37°C, 100% humidity, and microaerophilic conditions (5% O2, 12% CO2, 83% N2). Subsequently, the culture solution was centrifuged (13,420 G x 10 minutes) to collect the bacterial cells. The bacterial cells were washed twice with PBS phosphate-buffered saline (pH 7.4), suspended in distilled water, and subjected to sonication for 5 minutes under ice-cold conditions (using a Bioruptor II ultrasonic cell disruption device (set to High), repeating 30 seconds of ultrasonic treatment followed by 30 seconds of rest 5 times). The disruption solution was designated as "H. swiss whole cell suspension". For protein quantification, the Bio-Rad Protein Assay Kit was used with BSA as the standard protein.

[0052] The HsvA antigen peptides (11 types) represent 14 amino acid residues that are part of HsvA (an outer membrane protein specifically present only in H. swiss, composed of approximately 3000 amino acid residues) as disclosed in international publication WO2019 / 225639. EKKAVQQMENSNPD (Peptide No. 11: SEQ ID NO: 1) EKKAVEQMENSNPD (Peptide No. 11 (TKY): SEQ ID NO: 2) EKDAVTSLKNSNSG (Peptide No. 11 (SH8): SEQ ID NO: 3), EKDAVTSLENSNSG (Peptide No. 11 (SH10): SEQ ID NO: 4), NQGTLEFLSNDVST (Peptide No. 19 (TKY): SEQ ID NO: 5), TNGQEVSASIDYNK (Peptide No. 16: SEQ ID NO: 6), AKLSNFASNDALPD (Peptide No. 23: SEQ ID NO: 7), PTTSSGASPDSSNP (Peptide No. 10: SEQ ID NO: 8), NVDNILNMPSTTSG (Peptide No. 20: SEQ ID NO. 9), TLTLEGTETFAQNS (Peptide No. 81: SEQ ID NO: 10), and ADIQSSQTTFANSV (Peptide No. 61: SEQ ID NO: 11).

[0053] A suspension of H. pylori whole cells and HsvA antigen peptide (4 μg / mL each), dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6), were added dropwise to a 96-well NUNC ImmunoPlate #468667 and left overnight at 4°C. The cell solution was discarded the next day and washed three times with 250 μL / well of PBS-T (PBS containing 0.05% (V / V) Tween20). 200 μL / well of blocking solution (1% (V / V) BSA, PBS system pH 7.0) was added dropwise and left at 37°C for 1 hour. The blocking solution was discarded and washed three times with 250 μL / well of PBS-T. Serum samples were collected from individuals infected with H. pylori (determined by existing testing methods such as urea breath test, antibody test, or antigen test) and from individuals infected with H. swift (DNA was prepared from the subject's gastric biopsy, and H. swift infection was determined by the real-time PCR method described in Patent Document 2 or by culturing from the gastric biopsy). Serum samples were also collected from healthy individuals not infected with either bacterium as a control (non-infected). Human serum diluted with sample diluent (0.5% (V / V) BSA, PBS-based pH 7.0) was added at 50 μL / well and left at 37°C for 1 hour. The serum solution was discarded, and the samples were washed three times with 250 μL / well of PBS-T. 50 μL / well of horseradish peroxidase-labeled secondary antibody (Goat anti-Human IgG(H+L), Jackson ImmunoResearch, Inc.), diluted 20,000-fold with secondary antibody diluent (1.0% (V / V) BSA, PBS system pH 7.0), was added and left at 37°C for 1 hour. The secondary antibody solution was discarded, and the plates were washed three times with 250 μL / well of PBS-T. 50 μL / well of TMB One Component HRP Microwell Substrate, (Surmodics, Inc.) was added, and after developing a blue color, 50 μL / well of 0.17 M sulfuric acid was added, causing a yellow color change. The absorbance at 450 nm (reference wavelength: 620 nm~630 nm) was measured using a plate reader.

[0054] Figures 5-1 and 5-2 show the results of ELISA using human serum (3,600-fold dilution). For whole H. suisse cells, even the sample with the lowest absorbance for H. suisse-infected individuals showed a high absorbance of 1.391 (Abs. 450nm~630nm), while for H. pylori-infected individuals not infected with H. suisse, the sample with the highest absorbance was only 0.591 (Abs. 450nm~630nm), clearly distinguishing between H. suisse-infected and uninfected individuals. On the other hand, with HsvA antigen peptides, none of the sequence peptides showed high absorbance for H. suisse-infected individuals, and the absorbance could not be clearly distinguished from that of H. pylori-infected individuals not infected with H. suisse or uninfected individuals. Therefore, it is clear that using whole H. suisse cells allows for a diagnosis of H. suisse with superior sensitivity and specificity compared to detection systems using HsvA antigen peptides. [Industrial applicability]

[0055] The method of the present invention enables highly sensitive diagnosis of H. swiss infection, distinguishing it from H. pylori infection. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference. [Sequence Listing Free Text]

[0056] Sequence IDs 1-11: Synthesis

Claims

1. A reagent for detecting antibodies that bind to H. swiss whole cells in a subject-derived sample, including at least H. swiss whole cells and anti-Ig antibodies.

2. The reagent according to claim 1, characterized in that the test subject is a mammal.

3. The reagent according to claim 1, characterized in that the sample is derived from blood.

4. Furthermore, the reagent according to claim 1 is characterized by also detecting antibodies that bind to H. pylori or antigens of H. pylori cell components in a subject-derived sample, which include H. pylori cell components or antibodies against H. pylori cell components.

5. The reagent according to claim 1, which is an ELISA or immunochromatographic reagent.

6. A method characterized by detecting antibodies that bind to anti-H. Swiss whole cells in a sample derived from a subject, comprising the following steps: (a) A step of bringing a sample derived from the subject into contact with whole H. swiss bacteria, and (b) H. A step of detecting antibodies in the sample that are bound to whole Swiss bacteria.

7. The method according to claim 6, characterized in that the subject is a mammal.

8. The method according to claim 6, characterized in that the sample is derived from blood.

9. Furthermore, the method according to claim 6 is characterized by detecting antibodies that bind to H. pylori in a sample derived from the subject, comprising the following steps: (a) A step of bringing a sample derived from the subject into contact with H. pylori cell components, and (b) H. A step of detecting antibodies in the sample that are bound to Helicobacter pylori cell components.

10. Furthermore, the method according to claim 6 is characterized by also detecting antigens of H. pylori cell components in a sample derived from the subject, including the following steps: (a) A step of contacting a sample derived from the subject with an antibody against H. pylori cell components, and (b) A step of detecting the antigen of the H. pylori cell component in the sample, which is bound to an antibody against the H. pylori cell component.

11. A method for obtaining supplementary data for detecting H. swiss infection in a subject, comprising detecting antibodies by measurement using a reagent described in any one of claims 1 to 5 and / or by the method described in any one of claims 6 to 10.

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