A method for detecting Helicobacter Swiss antibodies using the extracellular membrane fraction.

Abstract

Provided are a measuring reagent and a measuring method for a Helicobacter suis antibody using an outer membrane fraction of H. suis.　The reagent contains at least an H. suis outer membrane fraction or a protein contained in the outer membrane fraction and an anti-Ig antibody, and detects an antibody that binds to the H. suis outer membrane fraction of a subject-derived sample or to the protein contained in the outer membrane fraction.

Description

Technical Field

[0001] The present invention relates to a method for measuring antibodies against Helicobacter suis using the outer membrane fraction 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, the isolation culture method, the urea breath test (UBT), the measurement of H. pylori antibody levels in serum and urine (ELISA and latex agglutination method), the measurement of H. pylori antigen in feces (immunochromatography), and the rapid urease test (RUT) of gastric biopsy specimens are mainly adopted.

[0003] However, in recent years, with the decline in the infection rate of H. pylori due to the spread of the diagnosis and eradication of H. pylori, 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 referred to as 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 eradication 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 the outer membrane fraction of H. swiss. [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. However, there was a need for a method with higher sensitivity and specificity than conventional methods.

[0011] In this study, the inventors have novelly discovered that using the H. Swiss outer membrane fraction, or proteins contained within the outer membrane fraction, enhances the sensitivity and specificity of measuring anti-H. Swiss antibodies in infected individuals, thereby developing a new measurement method.

[0012] More specifically, the present invention relates to the following inventions: [1] A reagent for detecting the H. Swiss outer membrane fraction of a subject-derived sample, or antibodies that bind to proteins contained in the outer membrane fraction, comprising at least the H. Swiss outer membrane fraction, or proteins and anti-Ig antibodies contained in the outer membrane fraction. [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 the anti-H. Swiss outer membrane fraction of a subject-derived sample or an antibody that binds to a protein contained in the outer membrane fraction: (a) A step of contacting a sample derived from the subject with the H. Swiss outer membrane fraction or proteins contained in the outer membrane fraction, and (b) A step of detecting antibodies in the sample that are bound to the H. Swiss outer membrane fraction or proteins contained in the outer membrane fraction. [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 methods [6] to [8] is characterized by also detecting antibodies that bind to H. pylori in a sample derived from the test 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 incorporates the disclosure of Japanese Patent Application No. 2022-009302, which is the basis of the priority of this application.

Effects of the Invention

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

Brief Description of the Drawings

[0014] [Figure 1] A diagram showing the results of ELISA using human serum (diluted 3600-fold) as a sample and whole cells of H. suis or the outer membrane fraction of H. suis as an antigen, presented as measured values. [Figure 2] A graph showing the results of ELISA using human serum (diluted 3600-fold) as a sample and whole cells of H. suis (Figure 2A) or the outer membrane fraction of H. suis (Figure 2B) as an antigen. [Figure 3] A diagram showing the results of ELISA using mouse serum (diluted 3600-fold) as a sample and whole cells of H. suis or the outer membrane fraction of H. suis as an antigen, presented as measured values. [Figure 4] A graph showing the results of ELISA using mouse serum (diluted 3600-fold) as a sample and whole cells of H. suis (Figure 4A) or the outer membrane fraction of H. suis (Figure 4B) as an antigen. [Figure 5] A diagram showing the results of ELISA using human serum (diluted 3600-fold) as a sample and the outer membrane fraction of H. pylori or the outer membrane fraction of H. suis as an antigen, presented as measured values. [Figure 6] A graph showing the results of ELISA using human serum (diluted 3600-fold) as a sample and the outer membrane fraction of H. pylori (Figure 6A) or the outer membrane fraction of H. suis (Figure 6B) as an antigen. [Figure 7-1]This figure shows the measured values ​​of an ELISA using human serum (3600-fold dilution) as the sample and H. swiss outer membrane fraction or HsVA partial peptides of H. swiss (SEQ ID NOs: 1-5) as the antigen. [Figure 7-2] This figure shows the measured values ​​of ELISA results using human serum (3600-fold dilution) as the sample and H. swiss outer membrane fraction or HsVA partial peptides of H. swiss (SEQ ID NOs: 6-11) as the antigen. [Modes for carrying out the invention]

[0015] The method of the present invention will be described below. The present invention relates to a method for detecting antibodies against the outer membrane fraction of H. swiss bacteria, or proteins contained in the outer membrane fraction, 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. swiss in a subject, or a method for detecting H. swiss infection in a subject. Alternatively, it relates to a method for obtaining auxiliary data for diagnosing H. swiss 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 helicobacters; 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 outer membrane fraction, or proteins contained in the outer membrane fraction) In this specification, "H. Swiss outer membrane fraction" refers to a fraction prepared by collecting H. Swiss cell outer membrane fragments after ultrasonic disruption of H. Swiss. Ultrasonographic disruption can be performed by suspending H. Swiss cells in a buffer solution and using an ultrasonic disruptor. Examples of ultrasonic disruptors include the Bioruptor (BM Instruments) sample-sealed ultrasonic disruptor. By ultrasonic disruption, H. Swiss cells are destroyed, leaving behind cell outer membrane fragments. These cell outer membrane fragments can be recovered by centrifugation or filtration. "Proteins contained in the H. Swiss outer membrane fraction" refers to proteins derived from H. Swiss present in the fraction prepared by collecting H. Swiss cell outer membrane fragments after ultrasonic disruption of H. Swiss.

[0018] (H. Swiss outer membrane fraction, or method and reagent for detecting antibodies against proteins contained in the outer membrane fraction) Antibodies against the H. suisse outer membrane fraction refer to antibodies against antigens such as proteins and sugars contained in the H. suisse outer membrane fraction, 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 the H. suisse outer membrane fraction are referred to as antibodies against the H. suisse outer membrane fraction. Furthermore, antibodies against proteins contained in the H. suisse outer membrane fraction refer to antibodies against proteins among the antigens contained in the H. suisse outer membrane fraction. The H. suisse outer membrane fraction contains multiple proteins, and antibodies against proteins contained in the H. suisse outer membrane fraction may include multiple antibodies against various proteins. In this invention, antibodies against at least one of the proteins contained in the H. suisse outer membrane fraction are referred to as antibodies against proteins contained in the H. suisse outer membrane fraction. In practice, in a subject infected with H. suisse, as a result of the decomposition of H. suisse bacteria in the body, antibodies against the H. suisse outer membrane fraction, or antibodies against proteins contained in the outer membrane fraction, are produced in the subject.

[0019] Furthermore, the binding of an antibody to an antigen contained in the H. Swiss outer membrane fraction is referred to as the antibody binding to the Swiss outer membrane fraction.

[0020] Reagents for detecting the H. swiss outer membrane fraction, or antibodies against proteins contained in the outer membrane fraction, 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 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 an antibody against the H. Swiss outer membrane fraction or the proteins contained in the outer membrane fraction, or a sample containing the same concentration of H. Swiss outer membrane fraction or the antibodies against the proteins contained in the outer membrane fraction as the sample, and measuring whether detection is possible.

[0021] Antibodies against the H. suisse outer membrane fraction, or proteins contained within the outer membrane fraction, can be used as markers for H. suisse 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 the H. suisse outer membrane fraction or the proteins contained in the outer membrane fraction, which are produced by the subject's own immune system. The presence of these antibodies can be detected and confirmed by an antigen-antibody reaction using the H. suisse outer membrane fraction or the proteins contained in the outer membrane fraction. The detection of antibodies against the H. swiss outer membrane fraction, or proteins contained in the outer membrane fraction, using these proteins as the detection reagents, 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; immunochromatographic methods such as gold colloid agglutination; chromatographic methods such as ion exchange chromatography and affinity chromatography; turbidimetric assay (TIA); necrotic 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) Contacting a sample derived from a subject with the H. Swiss outer membrane fraction of the present invention, or a protein contained in the outer membrane fraction, (b) A step of detecting antibodies in the sample that are bound to the Swiss outer membrane fraction or proteins contained in the outer membrane fraction, and (c) A step in which subjects in which the H. suisse outer membrane fraction or antibodies bound to proteins contained in the outer membrane fraction are detected are determined to be infected with H. suisse, while subjects in which the H. suisse outer membrane fraction or antibodies bound to proteins contained in the outer membrane fraction are not detected are determined to be not infected with H. suisse.

[0024] In another aspect, the present invention relates to a method for determining H. swiss infection, comprising the following steps: (a) A step of contacting a sample derived from the subject with the H. Swiss outer membrane fraction or proteins contained in the outer membrane fraction. (b) A step of measuring the level of antibodies in the sample bound to the H. Swiss outer membrane fraction or proteins contained in the outer membrane fraction, 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 H. Swiss outer membrane fraction, or the proteins contained in the outer membrane fraction, have bound to antibodies in the subject-derived sample can be confirmed by the following example. For example, the H. Swiss outer membrane fraction, or the proteins contained in the outer membrane fraction, are bound to a solid-phase support, and the subject-derived sample is brought into contact with the H. Swiss outer membrane fraction, or the proteins contained in the outer membrane fraction. After that, the reaction system is washed to remove unbound antibodies. As a result, the reaction system contains a complex in which the H. Swiss outer membrane fraction, or the proteins contained in the outer membrane fraction, and antibodies in the subject-derived sample have bound. Subsequently, the labeled anti-Ig antibody is brought into contact with the H. Swiss outer membrane fraction, or the antibody from the subject bound to the protein contained in the outer membrane fraction, to the labeled anti-Ig antibody. After this, the reaction system is washed to remove the unbound labeled anti-Ig antibody, 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 H. Swiss outer membrane fraction, or the protein contained in the outer membrane fraction, has bound to the antibody in the subject-derived sample. By measuring the level of the labeled anti-Ig antibody, the binding level between the H. Swiss outer membrane fraction, or the protein contained in the outer membrane fraction, and the antibody in the subject-derived sample can also be measured. The anti-Ig antibody may be an anti-IgG antibody, an anti-IgM antibody, etc., but an anti-IgG antibody is preferred. Furthermore, a monoclonal antibody is preferably used as the anti-IgG antibody. Furthermore, 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 amount of antibody can be calculated from the measured value by creating a standard curve using a standard solution whose abundance is known in advance. Alternatively, a surface plasmon resonance sensor can be used to detect or measure the binding of antibodies in the H. Swiss outer membrane fraction, or proteins contained in the outer membrane fraction, to the test sample.

[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 outer membrane fraction, or the protein (antigen) contained in the outer membrane fraction, is immobilized. A sample potentially containing antibodies against the H. Swiss outer membrane fraction or the protein contained in the outer membrane fraction is added to the immobilized support and brought into contact with it, forming an antigen-antibody complex on the immobilized support. Furthermore, an enzyme-labeled antibody (anti-Ig antibody) specific to human antibodies is added and brought into contact with it, binding the anti-Ig antibody to the antigen-antibody complex on the immobilized 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 H. Swiss outer membrane fraction, or the proteins (antigens) contained in the outer membrane fraction, can be bound to the carrier by known methods such as physical adsorption or covalent bonding using functional groups. The amount to be immobilized is not particularly limited, but if the carrier is a 96-well microtiter plate, several ng to several tens of μg per well is desirable. Immobilization can be performed by contacting the carrier with a solution of the H. Swiss outer membrane fraction, or the proteins (antigens) contained in the outer membrane fraction to be immobilized. For example, the H. Swiss outer membrane fraction, or the solution of the proteins (antigens) contained in the outer membrane fraction, can be immobilized by dispensing it into the wells of a microtiter plate and leaving it for a certain period of time. After immobilizing the H. Swiss outer membrane fraction, or the proteins (antigens) contained in the outer membrane fraction, it is preferable to block the assay 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 are preferably humans. More preferably, the subjects are 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. Furthermore, the method of the present invention can be performed 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 present invention comprises a kit for measuring antibodies against the H. Swiss outer membrane fraction or proteins contained in the outer membrane fraction in a sample, the kit comprising at least the H. Swiss outer membrane fraction or a carrier on which proteins contained in the outer membrane fraction are immobilized, and further comprising labeled anti-Ig antibodies.

[0033] The H. Swiss outer membrane fraction of the present invention, or the proteins contained in the outer membrane fraction, 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.

[0034] In one embodiment, the present invention relates to a method for determining the presence of H. suisse in a subject by collecting a sample, which includes detecting antibodies against the H. suisse outer membrane fraction or proteins contained in the outer membrane fraction in the sample. In particular, the present invention relates to a method for determining infection with H. suisse in a subject, which includes detecting antibodies against the H. suisse outer membrane fraction or proteins contained in the outer membrane fraction. In this method, subjects from whom a sample is collected in which antibodies against the H. suisse outer membrane fraction or proteins contained in the outer membrane fraction are determined to have H. suisse present or to be infected with H. suisse.

[0035] 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 a substance is "detected" does not need to be based on absolute detection, but may be determined by comparison with other samples. In other words, the determination of whether or not a substance is 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 a substance is "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 the measured value of the test subject is equivalent to the measured value of the negative target. On the other hand, if the measured value of the test subject is higher than the measured value of the negative target, the method of the present invention will determine that H. swisii is present or that the subject is infected with H. swisii, and the measured value is "detected".

[0036] 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.

[0037] 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.

[0038] (Detection of antibodies against H. pylori or H. pylori antigen) This invention also includes a method for detecting the H. pylori outer membrane fraction, or antibodies that bind to proteins contained in the outer membrane fraction, and antibodies against H. pylori in the same subject, as well as reagents used in this method. Here, antibodies against H. pylori refer to antibodies against any component of the entire 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 the H. pylori outer membrane fraction, or antibodies that bind to proteins contained in the outer membrane fraction, and antibodies against H. pylori in 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 samples from the same subject may be the same or different for H. pylori and H. pylori. In other words, the detection of antibodies that bind to the H. pylori outer membrane fraction, or proteins contained in the outer membrane fraction, and the detection of antibodies against H. pylori may be performed simultaneously using the same sample, or separately using different samples collected at different times. The detection of antibodies against H. pylori may be performed in the same manner as the detection of antibodies against the H. pylori outer membrane fraction, or proteins contained in the outer membrane fraction.

[0039] In determining whether H. pylori has been detected, the determination does not need to be based on an absolute presence or absence of detection; it may be determined by comparison with other samples. In other words, the determination of whether or not it 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 it has been "detected" may be determined by comparison 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. pylori or a sample from a subject that is clearly not infected with H. pylori, and 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. 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 target, 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 target is within the range that the present invention pre-accepts.

[0040] The present invention comprises a kit for measuring antibodies against the H. Swiss outer membrane fraction, or proteins contained in the outer membrane fraction, and antibodies against H. pylori in a sample, the kit comprising at least a carrier immobilized with the H. Swiss outer membrane fraction, or proteins contained in the outer membrane fraction, and a carrier immobilized with components of either the whole H. pylori cell, and further comprising labeled anti-human Ig antibody.

[0041] Furthermore, H. pylori antigen may be detected in the sample derived from the subject. Infection with H. pylori can also be detected by detecting H. pylori antigen. Feces are preferred as the sample for detecting H. pylori antigen. The present invention comprises a kit for measuring the H. Swiss outer membrane fraction, or antibodies against proteins contained in the outer membrane fraction, and H. pylori antigen in a sample, the kit comprising at least a carrier on which the H. Swiss outer membrane fraction, or proteins contained in the outer membrane fraction, is immobilized and a carrier on which antibodies against H. pylori are immobilized, and further comprising labeled anti-human Ig antibody. [Examples]

[0042] 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.

[0043] (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.

[0044] 75cm 2A 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 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 five times). The disruption solution was prepared as a "H. swiss whole cell suspension," and the precipitate was obtained by centrifugation (30,190 G x 10 minutes) as the "H. swiss outer membrane fraction," which was then suspended in distilled water. For protein quantification, the Bio-Rad Protein Assay Kit was used with BSA as the standard protein.

[0045] A suspension of whole H. swiss cells or the outer membrane fraction of H. swiss cells (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) Tween 20). 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 to 200 μL / well 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. SuperBlue TMB Microwell Peroxidase Substrate (1-Component), Kirkegaard & Perry Laboratories, Inc. (KPL) was added at 50 μL / well, and after blue coloration, 50 μL / well of 1N hydrochloric acid was added to change the color to yellow. The absorbance at 450 nm (reference wavelength: 620 nm ~ 630 nm) was measured using a plate reader.

[0046] Figures 1 and 2 show the results of ELISA using human serum (3600-fold dilution). In subjects positive for H. suisse, absorbance was obtained for both the whole H. suisse cell and the H. suisse solubilized fraction antigens, but higher absorbance was obtained for the H. suisse solubilized fraction. Furthermore, H. pylori-infected individuals who were not infected with H. suisse, and uninfected individuals, showed low absorbance for both antigens. This demonstrates for the first time that antibodies against the H. suisse solubilized fraction are produced in the blood of humans infected with H. suisse, and that antibodies can be detected with high sensitivity by using the H. suisse solubilized fraction as an antigen. This reveals that using the H. suisse solubilized fraction enables highly sensitive diagnosis of H. suisse infection in humans, distinguishing it from H. pylori infection.

[0047] (Example 2) Measurement of mammalian specimens The cultured H. swiss SNTW101c strain was collected by centrifugation. The cells were suspended in PBS and administered to 4-week-old female C57BL / 6 mice at a dose of 1 x 10⁶. 8 Colony forming units (CFUs) were administered orally once for infection. Meanwhile, one H. pylori SS strain, stored at -80°C, was inoculated onto Nissui plate Helicobacter agar and cultured for 3 days at 37°C, 100% humidity, and microaerophilic conditions (5% O2, 12% CO2, 83% N2). The resulting colonies were then cultured for 3 days with shaking in 100 mL of Brucella broth (containing 10 mL of inactivated FBS and 0.4 mL of SR0147 Helicobacter pylori Selective Supplement (Dent)) at 37°C, 100% humidity, and microaerophilic conditions (5% O2, 12% CO2, 83% N2), after which the cells were collected by centrifugation. The cells were suspended in PBS and fed to 4-week-old female C57BL / 6 mice in 2x10⁶ doses. 8CFU was administered orally three times every other day. DNA was extracted from the gastric mucosa of mice infected with H. swiss and infection was confirmed by PCR. In addition, gastric mucosa of mice infected with H. pylori was suspended in buffer solution, spread on Nissui Helicobacter agar (Nissui Pharmaceutical), and cultured to confirm infection. Serum was collected from H. pylori-infected mice and H. swiss-infected mice four weeks after the last infection date. Serum was also collected from healthy mice that were not infected with either as a control (uninfected).

[0048] 100 μL / well of a H. swiss whole cell suspension (4 μg / mL) or H. swiss solubilized fraction (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. 200 μL / well of a blocking solution (1% BSA, PBS system pH 7.4) prepared from Blocker BSA 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. 50 μL / well of mouse serum diluted with the blocking solution was added dropwise and left at 37°C for 1 hour. The serum solution was discarded and washed three times with 200 μL / well of PBS-T. A 100,000-fold dilution of horseradish peroxidase-labeled secondary antibody (Goat Anti-Mouse Ig, Human ads-HRP, SouternBiotech, Inc.) using a blocking solution was added at 50 μL / well and incubated at 37°C for 1 hour. The secondary antibody solution was discarded, and the plates were washed three times with 200 μL / well of PBS-T. 50 μL / well of SuperBlue TMB Microwell Peroxidase Substrate (1-Component) was added, and after blue coloration, 50 μL / well of 1N hydrochloric acid was added to change the color to yellow. The absorbance at 450 nm (reference wavelength: 620 nm~630 nm) was measured using a plate reader.

[0049] Figures 3 and 4 show the results of ELISA using mouse serum (3600-fold dilution). In H. suiss-positive mice, absorbance was obtained for both the whole H. suisse cell and the H. suisse outer membrane fraction antigens, but higher absorbance was obtained for the H. suisse outer membrane fraction. Furthermore, H. pylori-infected mice and uninfected mice showed low absorbance for both antigens. This demonstrates for the first time that antibodies against the H. suisse outer membrane fraction are produced in the blood of H. suisse-infected mice, and that antibodies can be detected with high sensitivity by using the H. suisse outer membrane fraction as an antigen. This reveals that using the H. suisse outer membrane fraction allows for highly sensitive diagnosis of H. suisse infection in mice, distinguishing it from H. pylori infection.

[0050] (Example 3) 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 for ELISA (H. pylori extracellular membrane fraction) 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 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 the "H. pylori whole cell suspension," and the precipitate was obtained by centrifugation (30,190 G x 10 minutes) as the "H. pylori outer membrane fraction," which was then suspended in distilled water. For protein quantification, the Bio-Rad Protein Assay Kit was used with BSA as the standard protein.

[0051] (2) Preparation of H. Swiss antigen for ELISA (H. Swiss extracellular membrane fraction) 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.

[0052] 75cm 2 A 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 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 prepared as a "H. swiss whole cell suspension," and the precipitate was obtained by centrifugation (30,190 G x 10 minutes) as the "H. swiss outer membrane fraction," which was then suspended in distilled water. For protein quantification, the Bio-Rad Protein Assay Kit was used with BSA as the standard protein.

[0053] 100 μL / well of H. pylori outer membrane fraction and H. swiss outer membrane fraction (4 μg / mL) dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6) was added dropwise to a 96-well NUNC ImmunoPlate #468667 and left overnight at 4°C. The next day, the cell solution was discarded 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.

[0054] Figures 5 and 6 show the results of ELISA using human serum (3,600-fold dilution). In the H. pylori extracorporeal membrane fraction, the absorbance of the H. pylori infection group (gastric disease - A, B, C, D) was significantly higher than that of the H. pylori Swiss 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. pylori Swiss infection group: P=0.0209, H. pylori infection group vs. non-infection group: P=0.0202). Furthermore, in the H. Swiss extracellular membrane fraction, the absorbance of the H. Swiss infection group (gastric disease - E, F, G, H) was significantly higher than that of the H. pylori infection group (gastric disease - A, B, C, D) or the non-infection group (healthy individuals - A, B, C, D) (H. Swiss infection group vs. H. pylori infection group or non-infection group: P=0.0209). This indicates that by testing for both anti-H. pylori antibodies and anti-H. Swiss antibodies in the same subject, it is possible to diagnose H. Swiss infection not only in cases where H. pylori infection is present, but also in cases that were previously overlooked due to being H. pylori negative.

[0055] (Example 4) 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.

[0056] 75cm2 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 5 times). The disruption solution was prepared as a "H. swiss whole cell suspension," and the precipitate was obtained by centrifugation (30,190 G x 10 minutes) as the "H. swiss outer membrane fraction," which was then suspended in distilled water. For protein quantification, the Bio-Rad Protein Assay Kit was used with BSA as the standard protein.

[0057] 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).

[0058] 100 μL / well of H. pylori extracellular membrane fraction and HsvA antigen peptide (4 μg / mL each), dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6), was added dropwise to a 96-well NUNC ImmunoPlate #468667 and left overnight at 4°C. The next day, the bacterial solution was discarded 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.

[0059] Figures 7-1 and 7-2 show the results of ELISA using human serum (3,600-fold dilution). In the H. suisse outer membrane fraction, even the sample with the lowest absorbance for H. suisse infected individuals showed a high absorbance of 0.658 (Abs. 450nm~630nm), while the sample with the highest absorbance for H. pylori infected individuals who were not infected with H. suisse, and for uninfected individuals, the absorbance was only 0.461 (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 who were not infected with H. suisse, or for uninfected individuals. Therefore, it is clear that using the H. suisse outer membrane fraction allows for a diagnosis of H. suisse with superior sensitivity and specificity compared to detection systems using HsvA antigen peptides. [Industrial applicability]

[0060] 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 are incorporated herein by direct reference. [Sequence Listing Free Text]

[0061] Sequence IDs 1-11: Synthesis

Claims

1. A reagent for detecting the H. Swiss outer membrane fraction of a subject-derived sample, or antibodies that bind to proteins contained in the outer membrane fraction, comprising at least the H. Swiss outer membrane fraction, or proteins and anti-Ig antibodies contained in the outer membrane fraction.

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 the anti-H. Swiss outer membrane fraction or antibodies that bind to proteins contained in the outer membrane fraction of a subject-derived sample, comprising the following steps: (a) A step of contacting a sample derived from the subject with the H. Swiss outer membrane fraction or proteins contained in the outer membrane fraction, and (b) H. A step of detecting the Swiss outer membrane fraction, or antibodies in the sample bound to proteins contained in the outer membrane fraction.

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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