WO2020027617A1

WO2020027617A1 - Method for diagnosing scrub typhus using o. tsutsugamushi-derived outer membrane vesicle

Info

Publication number: WO2020027617A1
Application number: PCT/KR2019/009654
Authority: WO
Inventors: 이진수
Original assignee: 이진수
Priority date: 2018-08-03
Filing date: 2019-08-02
Publication date: 2020-02-06
Also published as: KR102132964B1; KR20200015276A

Abstract

The present invention relates to a kit for diagnosing scrub typhus, the kit comprising antibodies binding to outer membrane vesicle antigens derived from O. tsutsugamushi, and a method for diagnosing scrub typhus using the kit. In the method for diagnosing scrub typhus according to the present invention, outer membrane vesicle antigens can be directly measured in the blood to diagnose the disease, without the use of antibodies or genes. Accordingly, the method is very simple and has excellent sensitivity and specificity, and thus, scrub typhus can be effectively diagnosed.

Description

Diagnosis of Tsutsugamus Disease Using Tsutsugamus-derived Outer Vesicles

The present invention relates to a method for diagnosing Scrub typhus using O. tsutsugamushi- derived outer membrane vesicles, and more particularly, to a diagnostic kit for Tstsugamushi disease comprising an antibody that binds to Tsutsugamushi-derived outer membrane vesicle antigens. The present invention relates to a method for diagnosing Tsutsugamushi disease using the kit.

Scrub typhus is an important febrile disease not only in Korea but also in Asia. It is a fever caused by O. tsutsugamushi infection. The incidence of patients is reported throughout the year, but in Korea, patients occur mainly in autumn, and acute and chronic infections cause pneumonia, encephalitis, rash, fever and headache. Regionally, Tsutsugamushi's disease is a large area from Japan to the north, Australia to the south, and the Arabian peninsula to the west. Recently, cases have been reported in India, China, and Russia. Recently, the incidence of Tsutsugamushi disease has increased, and the importance of public health is increasing. In Korea, the incidence of patients has increased sharply since 2004, with 6,480 cases reported in 2006, 5,671 cases in 2010, 8,604 cases in 2012, and 10,365 cases reported in 2013, with a high incidence rate of 10.4 to 20.0 per 100,000 population. Headquarters Infectious Diseases Portal http://www.cdc.go.kr/npt/). The incidence of fall patients is related to the temperature and humidity of the summer, and it is expected that the warming will increase the number and spread of Tsutsugamushi disease epidemic areas. In addition, the burden of disease caused by Tsutsuga-Mushi disease is expected to increase due to the increase in the exposure chance of infection due to the change in the density of carrier hair mites, the use of environmentally friendly farming methods or low pesticides, and the change in lifestyle.

Tsutsugamushi disease has a mortality rate of up to 60% without antimicrobial treatment, and mortality has improved with effective antimicrobial treatment, but it may be late in the treatment or severe complications such as shock, respiratory failure, and encephalitis in the elderly. After treatment, chronic infections, systemic malaise, and muscle pain may persist for months (Rajapakse S et al., Trans R Soc Trop Med Hyg. 1; 111 (2): 43-54, 2017).

Therefore, it is important to accurately diagnose Tsutsugamushi disease early. An important finding of the clinical diagnosis of Tsutsuga's disease is the formation of 5-10 mm in diameter on the tick bite. However, the reporting rate of skins varies greatly from region to region, with 46% in Gangwon-do and 80% in south Chungcheong Province. In addition, the skin is not a disease-specific finding because it also occurs in other Rickettsia diseases other than Tsutsugashi disease, and 50% of cases without characteristic skins are present in patients with Tsutsugashi disease, so the rash does not accompany or early diagnosis. This is necessary.

Conventional diagnostic methods include the method of measuring the antibody produced by the immune response in the blood after infection with Tsutsugamu bacteria (IFA, ELISA), the method of amplifying and confirming the bacterial genes in blood or other tissues (PCR), or directly culturing the bacteria. And the like are used.

The antibody test requires follow-up antibody test for diagnosis, the hemagglutination test (HA) has low sensitivity, and the immunofluorescent antibody test requires skilled test techniques and expensive fluorescence microscopy. In addition, a standardized test raises the issue of sensitivity, with Taiwanese studies showing negative IFA in 15% of patients with characteristic skin and O. tsutsugamushi PCR positive (Saisongkorn W et al. Trans R Soc Trop Med Hyg 98: 360-366). Even up to four-fold increase in antibody titer was possible to confirm only up to 80%.

PCR testing is expensive and has the problem that the target sample and the appropriate PCR target gene region are not clear. PCR with blood can only be diagnosed with rickettsiaemia. The sensitivity and specificity of the test method are different according to the primers used for PCR, and unlike the antibody method, sensitivity to antibiotic administration is affected. It is known that the sensitivity of blood PCR within 3 days of antibiotic administration is reduced by 60% and decreases by 10% after 4 days (Kim DM et al. Clin Microbiol Infect . 16: 447-51, 2010).

Culture tests can be used to diagnose infections, but for the cultivation of blood or tissues, a laboratory cell culture system with a safety facility of BSL-3 is required, an experienced experimenter is required, and more than one month to identify bacteria. It is difficult to actually help the patient's care.

Recently, a new etiology has been identified that can lead to chronic infections caused by infection with Tsutsugamus (Chung MH et al., J Korean Med Sci . 27 (3): 231-5, 2012). Is very limited and difficult by culture test.

Thus, the existing diagnosis method of Tsutsugamus disease does not provide sufficient diagnosis and various limitations require a new diagnosis method.

Accordingly, the present inventors have made intensive efforts to develop a simple and excellent method for diagnosing Tsutsugamushi disease, which can overcome the limitations of existing diagnostic methods. As a result, extracellular vesicles, ie outer membrane vesicles secreted by O. tsutsugamushi bacteria, The antigen of the outer membrane vesicle) was measured in a mammal-derived sample, confirming that Tsutsugamus disease can be diagnosed, and the present invention was completed.

발명의 요약Summary of the Invention

An object of the present invention is Scrub typhus comprising an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi , which can more easily and accurately diagnose Tsutsugamushi disease using blood. The present invention provides a diagnostic composition, a diagnostic kit, and a method for providing information necessary for diagnosing Tsutsugamushi disease using the kit.

In order to achieve the above object, the present invention provides a composition for diagnosing Scrub typhus comprising an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi .

The present invention also provides a capture antibody (c-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen; And a detection antibody (d-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen and is conjugated with a label.

The present invention also comprises the steps of: (a) contacting a biological sample of a subject with a capture antibody that specifically binds to the Tsutsuga mucosa-derived outer membrane vesicle antigen to bind the antigen and the capture antibody in the sample; (b) binding the antigen to the detection antibody by contacting the antigen to which the capture antibody binds to a detection antibody conjugated specifically to the Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugated with a label; And (c) provides a method for providing information necessary for diagnosing Tsutsugamushi disease by measuring the level of the labeling substance conjugated to the detection antibody and comparing with the level of the control.

The present invention also provides a method for diagnosing Scrub typhus using an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi .

The present invention also provides a capture antibody (c-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen; And a detection method (Scrub typhus) using a detection antibody (d-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugates a label.

The present invention also comprises the steps of: (a) contacting a biological sample of a subject with a capture antibody that specifically binds to the Tsutsuga mucosa-derived outer membrane vesicle antigen to bind the antigen and the capture antibody in the sample; (b) binding the antigen to the detection antibody by contacting the antigen to which the capture antibody binds to a detection antibody conjugated specifically to the Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugated with a label; And (c) measuring the level of the labeled substance conjugated to the detection antibody and comparing the level with the level of the control group.

The present invention also provides the use of an antibody specifically binding to Orientia Tsutsugamushi- derived outer membrane endoplasmic reticulum antigen for the diagnosis of Scrub typhus.

The present invention also provides a capture antibody (c-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen; And a detection antibody (d-Ab) that specifically binds to the outer membrane vesicle antigen derived from Tsutsuga mucobacteria and conjugates a label, for use in diagnosing Scrub typhus.

The present invention also provides the use of an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi for the production of a composition for diagnosing Scrub typhus.

The present invention also relates to a capture antibody (c-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen for the production of a kit for diagnosing Scrub typhus; And it provides a use of a detection antibody (d-Ab) that specifically binds to the Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugated with a label.

Figure 1 shows the OD value of ELISA (OT-OMV ELISA; O. tsutsugamushi derived outer membrane vesicle ELISA) according to the outer membrane vesicle concentration.

2 shows that ELISA OD values correlate with a significant linear increase with increasing envelope vesicle concentration.

Figure 3 shows the ROC analysis graph of the IFA and OT-OMV ELISA assay.

Figure 4 shows a schematic diagram for the sandwich ELISA.

발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

As described above, in the absence of the characteristic findings of Tsutsuga's disease, differential diagnosis with acute febrile diseases such as leptospirosis, rash fever and nephrotic bleeding fever is difficult, and when appropriate treatment is delayed or untreated at first, the patient has pneumonia, Meningitis, renal failure, and multi-organ failures and deaths, and some of the infected people are chronically infected, causing complications. Therefore, there is a need for development of a method for effectively diagnosing Tsutsugamushi disease.

In the present invention, Tsutsugamushi disease was diagnosed by collecting a sample of a patient infected with Orientia Tsutsugamushi , preferably blood, and measuring the antigen of the outer membrane vesicles secreted by Tsutsugamushi bacteria. ELISA was performed using an antibody that specifically binds to the outer membrane vesicle antigen of Tsutsuga mush to measure the antigen of the outer membrane vesicle secreted by the Tsutsuga mucin. This significantly improves the accuracy of diagnosis compared to conventional indirect immunofluorescence (IFA) for diagnosing Tsutsuga's disease and PCR for genotyping, thereby completely replacing the conventional diagnostic method and providing confidence in the diagnosis result. Significantly improves.

Accordingly, the present invention relates to a composition for diagnosing Scrub typhus comprising an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia tsutsugamushi .

In the present invention, the outer membrane endoplasmic reticulum antigen is preferably TSA56 (56-kDa type-specific antigen), 47 kDa antigen, ScaA or chaperonin (chaperonin (60 kDa antigen)), and more preferably TSA56 (56). -kDa type-specific antigen, wherein the TSA56 antigen is one of various antigens included in an outer membrane vesicle (OMV), and most preferably, the TSA56 may be represented by the amino acid sequence of SEQ ID NO. have.

47-kDa protein is an important surface antigen located in the outer membrane of O. tsutsugamushi . It has group-specific characteristics of bacteria and activates epitopes of B lymphocyte cells (Choi et al., Am J Trop Med Hyg , 7 (1): 30-37, 2017).

Sca proteins are autotransporter proteins that are involved in bacterial attachment to cells and induce immunity to the host (Ha et al., Clin Vaccine Immunol . 19 (9): 1442-51, 2012).

The 60 kDa chaperonin protein is expressed in the groEL gene and is secreted under bacterial stress, leading to a well maintained bacterial morphology (DH Paris et al., Clin Microbiol Infect . 15 (5): 488-495, 2009 ).

The term outer membrane vesicle of the present invention can be understood as the same as the outer membrane vesicle (OMV), also called exosomes.

The outer membrane vesicles may have an average diameter of 20 to 200nm. In addition, the Tsutsugamu endoplasmic reticulum contains the main antigen of Tsutsugamu bacteria.

As the bacteria grow, they secrete vesicles, such as droplets of 20-250 nm in diameter, from the outer membrane. The vesicles secreted by bacteria are called outer membrane vesicles (OMV), meaning they are derived from the outer membrane. Outer endoplasmic reticulum has been identified in several strains, including Escherichia coli, Neisseira meningitides, and Staphylococcus aureus . The outer membrane vesicles produced by bacteria are involved in various roles such as material transport, interactions between bacteria, survival and pathogenesis of bacteria. The outer membrane endoplasmic reticulum is formed by protruding bacterial envelopes like envelopes and thus includes outer membrane proteins, lipopolysaccharides (LPS), phospholipids, and periplasma contents. That is, since the outer membrane vesicles are derived from bacterial cell membranes, the membranes of the outer membrane vesicles contain various important antigenic substances and the like.

Recently, O. tsutsugamushi has also been found to produce and secrete outer membrane vesicles, and the outer membrane vesicles of O. tsutusgamushi also contain various antigens unique to bacteria. Lee et al., J Korean Med Sci . 30 (7): 866-70, 2015.

In the present invention, the antibody may be a whole form of an antibody (hereinafter referred to as "antibody") or a functional fragment thereof. The whole antibody may be in the form of a monomer or a multimer having two or more whole antibodies bound thereto. The functional fragment of the antibody is an antibody having the heavy and light chain variable regions of the whole antibody, which means to recognize substantially the same epitope that the whole antibody recognizes. Functional fragments of the antibody include, but are not limited to, single chain variable region fragments (scFv), (scFv) 2, Fab, Fab 'and F (ab') 2. The single chain variable region (scFv) refers to an antibody fragment in which a heavy chain variable region and a light chain variable region are linked through a linker peptide to take the form of a single chain polypeptide.

The antibody can be modified by binding to various molecules such as enzymes, fluorescent materials, radioactive materials and proteins. Modified antibodies can be obtained by chemically modifying the antibody. Such modification methods are commonly used in the art. In addition, the antibody is obtained as a chimeric antibody in which a variable region derived from a non-human antibody and a constant region derived from a human antibody are combined, or complementarity derived from a non-human antibody. It may be obtained as a humanized antibody in which a constant region is combined with a frame work region (FR) derived from a human antibody including a crystal site. Such antibodies can be prepared using methods known in the art.

The Tsutsugamushi bacteria of the present invention may be uniform, which is described by the scientific name O. tsutsugamushi , and preferably, the Tsutsugamushi Boryeong antigen type (NCCP NO. 14794).

In another aspect, the present invention provides a capture antibody (c-Ab) that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi ; And it relates to a kit for diagnosing Scrub typhus (Scrub typhus) comprising a detection antibody (d-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugated with a label.

As used herein, the term "diagnosis" refers to determining whether an object for a particular disease or condition, that is, the subject being tested, currently has a particular disease or condition.

The capture antibody and the detection antibody may be a monoclonal antibody or a polyclonal antibody designed to specifically bind to the antigen to be detected. The capture antibody and the detection antibody may bind to the same antigen but bind to different sites of the antigen so that the capture antibody and the detection antibody bind to the antigen to form a capture antibody-antigen-detection antibody conjugate.

The capture antibody of the present invention is a polyclonal antibody capable of binding to several epitopes of the Tsutsuga mucosa outer membrane vesicles, and the detection antibody binds to TSA56 (56-kDa type-specific antigen) of the Tsutsuga mucosa outer membrane vesicles as a monoclonal antibody. It may be characterized by.

In the present invention, the capture antibody is preferably a polyclonal antibody capable of binding to the epitope of the outer membrane vesicles, and more preferably an anti-Tsutsugamushi borne serotype polyclonal antibody ( O. tustusgamushi serotype Boryong polyclonal antibody), but is not limited thereto.

In the present invention, the detection antibody is preferably a monoclonal antibody that binds to TSA56 (56-kDa type-specific antigen) of the Tsutsugamu outer membrane vesicles, and in the embodiment of the present invention, an FS-15 monoclonal antibody is used. Although used, it is not limited thereto.

The term "detection" of the present invention encompasses quantitative and / or qualitative analysis, including the detection of presence, absence, and expression level. Such methods are known in the art, and those skilled in the art will appreciate You will be able to choose the appropriate method.

In the present invention, the capture antibody may be attached to a substrate.

The substrate may be any solid substrate conventionally used for solid-based immunoassay methods, and may use wells of microtiter plates widely used in existing ELISA methods. Can be, for example, a flat bottom plate, U-shaped bottom plate depending on the shape; Avidin-coated plate, streptavidin-coated plate, glutathione coated plate, anti-GST coated plate (anti-GST coated plate) depending on ligand or protein coating ); Depending on the substrate material, a polystyrene plate, a polypropylene plate, or the like may be used, but is not limited thereto. The method of immobilizing the capture antibody on the substrate is not particularly limited and may be any method commonly used in immunoassay methods. For example, in one embodiment of the present invention, the capture antibody may be immobilized by coating the capture antibody on the substrate.

As such, the method of immobilizing one of the antigen-antibody binding pairs on a solid phase may be particularly effective when applied to a diagnosis using a liquid sample such as a body fluid. When the antibody is attached to a solid substrate and the antigen in the body fluid is detected, the signal is obtained after allowing the antibody to bind to the antigen and then removing all remaining non-body fluid and substances that interfere with the analysis by the body by washing. There is an advantage that the risk of a positive signal is significantly reduced.

As used herein, 'antigen detection' is used to mean not only determination of antigen presence but also quantitative analysis of antigen.

In the present invention, the kit can analyze the outer membrane vesicles derived from Tsutsugamu bacteria in the sample using various detection methods. Qualitative and quantitative assays that can be used with the kits herein include, for example, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich immunoassay (based on antigen-antibody reactions) sandwich ELISA), immunofluorescence assay (IFA), immunoluminescence assay (Immunochemiluminescence Assay) or immunohistochemical staining (Immnohistochemical staining) can be used.

Such methods are known and are described, for example, in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., HumanaPress, NJ, 1984; mass spectroscopy: Petricoin et al. 2002. Lancet 359: 572-77; eTag systems: Chan-Hui et al. 2004. Clinical Immunology 111: 162-174; microparticle-enhanced nephelometric immunoassay: Montagne et al. 1992. EurJ Clin Chem Clin Biochem. 30: 217-22, and the like.

In the present invention, short of using a sandwich immunoassay (sandwich ELISA) is preferable, and more preferably O. OT-OMV ELISA (O. tsutsugamushi derived outer membrane vesicle ELISA) in ELISA for the exciter of somjom tsutsugamushi derived using the It is not limited to this.

The kit may further include reagents known in the art for use in enzyme-immunoassays in addition to antibodies that specifically bind to Tsutsugamusi-derived outer membrane vesicle antigens. For example, such reagents may include negative control serum, positive control serum, wash solution, substrate solution capable of measuring enzymatic activity, and reaction stop solution. In particular, it may include a chromogenic substrate solution, a washing solution and an enzyme stopping solution to react with the labeling substance conjugated to the antibody.

The labeling material of the antibody conjugate is preferably a conventional coloring agent that performs a color reaction, horseradish peroxidase (HRP), basic dephosphatase (alkaline phosphatase), colloid (coloid), fluorescent material (fluorescein), radioisotope and pigment ( dye).

The chromogenic substrate solution is preferably used according to a label, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid )], OPD (o-phenylenediamine) and the like can be used. At this time, the color development substrate is more preferably provided in a dissolved state in a buffer solution (0.1M NaOAc, pH 5.5).

In the present invention, as a result of confirming the concentration of the outer membrane vesicles by taking a sample, preferably blood, from a Tsutsugamosis patient and a normal person, it is confirmed for the first time that the concentration of the outer membrane vesicles in the blood of Tsutsugamosis patients is significantly increased. The present invention provides a kind of in vitro analysis method for diagnosing Tsutsugamus disease by measuring Tsutsugamu-derived outer membrane vesicles (OMV) in the blood.

Accordingly, in another aspect, the present invention provides a method for preparing a biological sample of a subject in (a) a capture antibody (c-Ab) that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi . Contacting to bind the antigen and the capture antibody in the sample; (b) contacting the antigen with the detection antibody by contacting a detection antibody (d-Ab) that specifically binds to the Tsutsuga mucosa-derived outer membrane vesicle antigen and the labeling substance is conjugated to the antigen bound to the capture antibody; And (c) measuring the level of the labeled substance conjugated to the detection antibody and comparing the level with the level of the control group.

In the present invention, when the level of the labeling material of step (c) is higher than that of the control group, it may be characterized as being determined to be Tsutsugamushi disease.

In the present invention, the capture antibody is preferably attached to a substrate, but is not limited thereto.

In one embodiment of the present invention, Indirect Immunofluorescence Assay (IFA) and PCR assays, which are generally known as gold standard tools for diagnosing Tsutsugamushi disease, are compared to diagnostic methods using Tsutsugamushi envelope membrane vesicle measurement. The analysis was performed on patients and non-infected individuals with typical clinical features of Tsutsugamu infection.

The OT-OMV ELISA was 100% positive in 9 patients diagnosed with IFA when analyzed by Tsutsugamu disease patients. In addition, 14 positive and IFA negative patients were OT-OMV ELISA 57.1. % Were positive, and 19 OT-OMV ELISA positive patients were PCR positive 42.1% and IFA positive 47.3%. The existing methods were both negative and clinically diagnosed. Two patients were positively diagnosed by OT-OMV ELISA, which means that the OT-OMV ELISA method was the best in comparison to the test method for 25 patients with Tsutsuma's disease. The results were much better than the IFA test, which is the gold standard for diagnosis, and the diagnostic performance was better than the recently introduced PCR test. The sensitivity of the existing IFA test was 36.0%, the specificity was 85.7%, the positive predictive value was 81.8%, and the negative predictive value was 42.9%, but the sensitivity of the OT-OMV ELISA test was 76.0%, the specificity was 100%, Positive predictive value was 100.0% and negative predictive value was 70.0%. The OT-OMV ELISA test showed better sensitivity and specificity than the conventional IFA test.

As used herein, "biological sample" refers to a substance or mixture of substances that includes one or more components capable of detecting a biomarker and refers to cells, tissues or fluids derived from organisms, in particular humans, such as whole blood, urine, plasma, serum, saliva. This includes, but is not limited to, sputum and urine. It also includes cells or tissues derived directly from an organism as well as cultured in vitro. Various samples may be used for the detection of Tsutsugashimu-derived outer membrane vesicles according to the present application, but are not limited thereto. The sample may be pretreated by filtration, distillation, extraction, concentration, inactivation of interference components, addition of reagents, etc. before use.

In one embodiment according to the present invention a blood sample is used. Blood sample, as used herein, refers to a collection of similar cells obtained from the blood of a subject, including a patient and a normal person. The source of blood sample may be fresh, frozen and / or preserved blood or any blood component and includes blood, plasma or serum.

As used herein, the term "subject" refers to any single individual in need of treatment, including mammals, such as humans, cattle, dogs, guinea pigs, rabbits, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings, or subjects who participated in epidemiologic studies or used as controls.

The term "uninfected" or "normal person" refers to a normal healthy adult who has no history of being diagnosed with Tsutsumi's disease in a medical institution and has no history of fever disease with a crust or rash suspected of Tsutsugamus disease. .

Such a method according to the invention can be used alone or in combination with known methods. For example, in addition to the blood sample analysis result, clinical information other than the blood sample of the patient may be additionally used to provide the information on the diagnosis, monitoring or prognosis as described above.

In the present invention, the measurement can be used without limitation cytokine concentration measurement methods known in the art, preferably enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich immunoassay method (sandwich ELISA), immunofluorescence assay (Immuno-fluorence Assay, IFA), immunoluminescence assay (Immunochemiluminescence Assay) or immunohistochemical staining (Immnohistochemical staining) can be measured.

The ELISA generally refers to a method for measuring antigen or antibody amount using an antigen antibody reaction using an enzyme as a marker, generally referred to as enzyme immunoassay. Generally, enzymes (peroxidases, peroxidases, etc.) are bound to antibodies and quantitatively performed using the reactions, and microanalysis is possible because the reactions are usually completed in the wells of microplates.

In the present invention, as the labeling material capable of generating a detectable signal, enzymes, fluorescent materials, luminescent materials, radioactive materials and the like can be used. In particular, it is possible to use those selected from the group consisting of horseradish peroxidase (HRP), basic alkaline phosphatase (colloid), colloid (colloid), fluorescent (fluorescein), radioisotope and dye (dye). However, any of those that can be used for immunological assays other than those exemplified above may be used.

In yet another aspect, the present invention relates to a method for diagnosing Scrub typhus using an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi .

In still another aspect, the present invention provides a capture antibody (c-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen; And a method for diagnosing Scrub typhus using a detection antibody (d-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugates a label.

In another aspect, the present invention provides a method for preparing an antibody comprising: (a) contacting a biological sample of a subject with a capture antibody that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen to bind the antigen and the capture antibody to the sample; (b) binding the antigen to the detection antibody by contacting the antigen to which the capture antibody binds to a detection antibody conjugated specifically to the Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugated with a label; And (c) measuring the level of the labeling substance conjugated to the detection antibody and comparing the level with the level of the control group.

In another aspect, the present invention relates to the use of an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi , for the diagnosis of Scrub typhus.

In still another aspect, the present invention provides a capture antibody (c-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen; And a detection antibody (d-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane endoplasmic reticulum antigen and is conjugated with a label, for use in diagnosing Scrub typhus.

In another aspect, the present invention relates to the use of an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi for the manufacture of a diagnostic composition or kit for Scrub typhus.

In still another aspect, the present invention provides a capture antibody (c-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen for the production of a kit for diagnosing Scrub typhus; And it relates to the use of a detection antibody (d-Ab) that specifically binds to the Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugated with a label.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Target Patient

For the diagnosis of Tsutsugamosis disease, patients suspected of Tsutsugamosis infection were screened and blood was collected. All protocols were reviewed and approved by Inha University Hospital's Institutional Review Board (IRB), with informed consent from all patients included in this study.

The typical clinical characteristics of Tsutsuga's disease were fever, rash, and eschar, and were based on the diagnosis of an infectious physician. Data from patients diagnosed as Boryong serotype by IFA or PCR were analyzed. A total of 25 patients and 14 control blood without Tsutsugamus disease were analyzed.

Antibodies

Anti-O. Tustusgamushi serotype Boryong polyclonal antibody, a polyclonal antibody against Boryeong serotype Tsutsugamu bacteria, was prepared (Apclone, South Korea) and used as a capture antibody.

Monoclonal antibodies for use as detection antibodies are known by infecting the mouse abdominal cavity with the serotype Tsutsugamushi bacteria (Kang et al., Microbiol Immunol 43: 229-234, 1999; Song et al., Vaccine 19: 2-9, 2001) to prepare a monoclonal antibody against Tsutsugamu TSA56 (56-kDa type-specific antigen). In the present invention, FS-15 monoclonal antibody (Kim et al., Journal of Bacteriology and Virology 38 (1): 11-17, 2008) manufactured by the Department of Microbiology, College of Medicine, Inha University, was used.

Example 1 Confirmation of Tsutsugamus Infection Using Indirect Immunofluorescence Assay (IFA)

IFA analysis was performed on Boryong, Kawasaki, Gilliam, and Karp serotypes in the patients.

First, the patient's serum was obtained by treating EDTA with whole blood. Each serum was prepared by diluting twice with sequential dilution with PBS (phosphate buffered saline, pH = 7.4) in 1:40 to 1: 1280 microplates (U-bottomed microplates).

Dilution of the serum sample Boryeong (Boryong), Kawasaki (Kawasaki), Gilliam (Gilliam) and CAP (Karp) serotype of orientation thiazol tsutsugamushi (O. tsutsugamushi) are each ring-antigen of the cultured cells attached slide spots (each prototype antigen spots) and slides were reacted for 30 minutes at 37 ° C. in a moist chamber. The reaction was washed three times using PBS (PBST) containing 0.05% tween 20, and then treated with fluorescent conjugated goat anti-human IgG (Jackson laborotory, USA). Reacted for minutes. After washing three times as described above, it was dried and fixed with anti-fainting mounting media (Vectashild, Vector, CA). It was then observed at X200 magnification under a fluorescence microscope (Axioskop 2, CarlZeiss, Germany).

Tsutsugamushi disease diagnosed with Boryong serotype IFA was determined to be positive when IgG antisera titer was greater than 1: 160 dilution of serum based on long-term data collected from Inha University Hospital and experimental data. It was.

The results are shown in Table 4 below.

Example 2: Infected Tsutsugamus genotype analysis using PCR analysis

A buffy coat was obtained from the whole blood of each patient suspected of Tsutsugashi infection. DNA extraction was performed using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manual included in the kit. DNA was diluted in 50 μl of sterile distilled water, and nested PCR (nPCR) was performed to detect 56-kDa specific antigens of Orientia Tsutsugamus.

Two primer sets were prepared for nPCR performance (Gennotek). Primer sets for the first PCR were performed in the Bryong strain (Bryong strain, Genbank accession number: L04956), the karp strain (Karp strain, Genbank accession number: M33004), Gilliam (Gilliam), which encode a 56-kDa specific antigen gene. Strain, Genbank accession number: M33267) and Kawasaki strain (Kawasaki strain, Genbank accession number: M63383) were prepared based on the conserved DNA sequence, primer sequence information is as follows. The size of the amplified product amplified by the primary primer set is 1900 bp.

Primary primer set

[SEQ ID NO 1] 1F: 5'-ATAATTAATGTATTTTCGAACG-3 '

[SEQ ID NO 2] 2R: 5'-CCTKCAAAGGACTTTTAGCT-3 '

The primer set for performing the secondary PCR is as follows, and the size of the amplified product amplified by the secondary primer set is 1600 bp.

2nd primer set

[SEQ ID NO 3] 1Fn: 5'-AACACAGTGTTTTATAGATTGTTTA-3 '

[SEQ ID NO 4] 2Rn: 5'-RCATTAATTGCTACACCAAGT- 3 '

The first PCR amplification of the 56-kDa antigen gene used 1F and 2R primer sets and the second PCR amplification used 1Fn and 2Rn primer sets. The amplification products generated by the second PCR amplification were sequenced and identified through the National Center for Biotechnology Information (NCBI) blast program.

The results are shown in Table 4 below.

Example 3: Preparation of Tsutsugashimu envelope membrane vesicles

Outer membrane endoplasmic reticulum (OMV) was isolated and purified from Tsutsugamu bacteria for control experiments.

Cell culture, purification, and isolation of Tsutsugamu bacteria-infected outer membrane vesicles, ie, outer membrane vesicles (OMV), were performed according to the method of Korean Patent 10-1742236.

Example 4: Determination of Tsutsugamu-derived Outer Vesicle (OT-OMV) Concentration Using ELISA Method

To perform an enzyme-linked immunosorbent assay (ELISA), first, 0.1 M sodium phosphate buffer at a concentration of 3 μg / ml in a polystyrene 96-well plate (NUNC, Thermo Scientific, USA) Anti-O. tustusgamushi serotype Boryong polyclonal antibody capture antibody dissolved in (pH = 9.0) was diluted 1:40 and coated.

Then, after reacting overnight at 4 ° C., the solution remaining on the plate was removed. Each well was filled with 200 μl of PBS solution containing 1% BSA (GA biochem, Georgia, USA) and allowed to stand at room temperature for 2 hours, followed by 0.05% tween 20 (USB, ohio, USA). Wash three times with PBS buffer. 100 μl of the target sample was added to each well, followed by reaction at room temperature for 2 hours, and then washed three times in the same manner as above. The detection antibody was treated with FS-15 monoclonal Ab at a concentration of 3 ug / mL for 1 hour. Reacted.

Then, washed again three times with PBS buffer containing 0.05% Tween20, and then reacted with avidin conjugated horseradish peroxidase (BD Biosciences, USA) for 1 hour, containing 0.05% Tween20. Washed three times with PBS buffer and treated with 100 μl substrate (TMB, KPL, MD, USA). When sufficient color development appeared, the reaction was stopped by adding 100 µl of 1M sulfuric acid (H 2 SO 4) as a stop solution.

Next, the optical densities (OD) were measured at 450 nm using an ELISA reader (BioTek Instruments, USA).

The target samples were compared using the Tsutsugashimu-derived outer membrane vesicles purified in Example 3 and blood of normal persons. The OD value of the average blood test value of the normal person was found to be 0.1999 (Table 1), and the standard TEL was obtained using the Tsutsugashimu-derived outer membrane vesicles purified in Example 3 as a control for each concentration (Fig. 1).

Was measured by diluting an OMV derived from tsutsugamushi strains in stages in PBS, were the OD value of the ELISA increases with the OMV concentration (Table 2), OD value of R ² = 0.995, significant probability coefficient correlated with the increase OMV concentration Showed a significant linear increase correlation of 0.000 (FIG. 2). In addition, when the OMV concentration was 0, 0.5, 1, 2, and 4 ug / mL, the bivariate correlation coefficient value was R ² = 0.965 and the significance probability was 0.008, indicating that the OMV concentration was highly correlated with the OD value.

Example 4 Detection of Tsutsugashimu-derived Outer Vesicles in Tsutsugamu Patients Using ELISA Method

In the same manner as in Example 3, ELISA OD values were measured using blood of a Tsutsugashi patient.

As a result, the measured OD values in the patients exceeded all of the normal blood test mean values of 0.1999 (Table 3).

In addition, the PCR method of IFA and 2 of Example 1 and OT-OMV ELISA method was compared (Table 4).

(1 ~ 25: Tsutsugamushi patient, 26 ~ 39: Patients other than Tsutsugamushi disease, Clinical diagnosis-When diagnosed by clinical findings of infectious physician)

As a result, OT-OMV ELISA was 100% positive in nine patients diagnosed by Tsutsugamu disease. In addition, 14 positive and IFA negative patients were positive in OT-OMV ELISA 57.1%, and 19 positive OT-OMV ELISA patients were 42.1% positive in PCR and 47.3% in IFA. The existing methods were both negative and clinically diagnosed. Two patients could be positively diagnosed with OT-OMV ELISA.

In other words, the OT-OMV ELISA method was the best in comparison of the test methods for 25 patients with Tsutsugamushi disease, and was particularly superior to the IFA test, the current gold standard for diagnosis, and the diagnostic performance was better than that of the recently introduced PCR test. Excellent. The sensitivity of the existing IFA test was 36.0%, the specificity was 85.7%, the positive predictive value was 81.8%, and the negative predictive value was 42.9%, but the sensitivity of the OT-OMV ELISA test was 76.0%, the specificity was 100%, The positive predictive value was 100.0% and negative predictive value was 70.0%. The OT-OMV ELISA test showed better sensitivity and specificity than the conventional IFA test.

ROC analysis was performed. The AUC value was 0.609 for IFA and 0.880 for OT-OMV ELISA, and the p-value was 0.0007. That is, it was found that OT-OMV ELISA shows a superior diagnostic ability than the conventional IFA test (FIG. 3).

Tsutsugamus disease according to the present invention can be diagnosed by measuring the outer membrane vesicle antigen directly in the blood without using antibodies or genes, very easy and excellent sensitivity and specificity can be effectively diagnosed Tsutsugamus disease.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims

A composition for diagnosing Scrub typhus, comprising an antibody that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi .
The composition of claim 1, wherein the outer membrane vesicle antigen is TSA56 (56-kDa type-specific antigen), 47 kDa antigen, ScaA, or chaperonin (chaperonin (60 kDa antigen)) of Tsutsuga mucosa.
According to claim 2, wherein TSA56 is Tsutsugamushi disease diagnostic composition, characterized in that represented by the amino acid sequence of SEQ ID NO: 5.
A capture antibody (c-Ab) that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi ; And

A kit for diagnosing Scrub typhus, comprising a detection antibody (d-Ab) that specifically binds to a Tsutsuga mucosa-derived outer membrane vesicle antigen and conjugates a label.
The kit for diagnosing Tsutsugamushi disease according to claim 4, wherein the capture antibody is attached to a substrate.
[Claim 5] The kit for diagnosing Tsutsugamushi disease according to claim 4, wherein the capture antibody is a polyclonal antibody capable of binding to an epitope of the outer membrane vesicle.
Claim 6, wherein the capture antibody is a - tsutsugamushi bacteria polyclonal antibody (O. tustusgamushi polyclonal antibody) of tsutsugamushi disease diagnostic kit, characterized in that.
The kit for diagnosing Tsutsugamus disease according to claim 4, wherein the detection antibody is a monoclonal antibody that binds to TSA56 (56-kDa type-specific antigen) of the Tsutsuga mucosal outer endoplasmic reticulum.
The kit for diagnosing Tsutsugamushi disease according to claim 4, wherein the outer membrane vesicle antigen is TSA56 (56-kDa type-specific antigen), 47 kDa antigen, ScaA, or chaperonin (chaperonin; 60 kDa antigen) of Tsutsuga mucosa.
The kit according to claim 9, wherein the TSA56 is represented by the amino acid sequence of SEQ ID NO.
The method of claim 4, wherein the labeling substance is selected from the group consisting of horseradish peroxidase (HRP), basic alkaline phosphatase, colloid, colloid, fluorescein, radioisotope and dye Tsutsugamus disease diagnostic kit, characterized in that.
The method according to claim 4, wherein the enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich immunoassay (Immuno-fluorence Assay, IFA), immunoluminescence assay (IFA) Tsutsugamushi disease diagnostic kit, characterized in that carried out by (Immunochemiluminescence Assay) or immunohistochemical staining (Immnohistochemical staining).
Information needed to diagnose Scrub typhus, including the following steps:

(a) binding an antigen and a capture antibody to a sample by contacting a biological sample of the subject with a capture antibody (c-Ab) that specifically binds to an outer membrane vesicle antigen derived from Orientia Tsutsugamushi Making a step;

(b) contacting the antigen with the detection antibody by contacting a detection antibody (d-Ab) that specifically binds to the Tsutsuga mucosa-derived outer membrane vesicle antigen and the labeling substance is conjugated to the antigen bound to the capture antibody; And

(c) measuring the level of the labeled substance conjugated to the detection antibody and comparing it with the level of the control group.
15. The method of claim 13, wherein if the level of the labeling substance of step (c) is higher than that of the control group, it is determined that the disease is Tsutsugamushi disease.
The method of claim 13, wherein the label is selected from the group consisting of horseradish peroxidase (HRP), basic alkaline phosphatase (colloid), colloid (colloid), fluorescent material (fluorescein), radioisotope and dye (dye) Information providing method necessary for diagnosing Tsutsugamus disease.
The method of claim 13, wherein step (c) comprises: enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich immunoassay (sandwich ELISA), immunofluorescence assay (Immuno-fluorence Assay, IFA), immunoluminescence assay (Immunochemiluminescence Assay) or immunohistochemical staining method (Immnohistochemical staining) (Immnohistochemical) characterized in that the method for providing information necessary for diagnosing Tsutsugamushi disease.
The method of claim 13, wherein the capture antibody is attached to a substrate.
The method of claim 13, wherein the capture antibody is a polyclonal antibody capable of binding to an epitope of the outer membrane vesicles.
19. The method of claim 18, wherein the capture antibody is an anti-fungal tsutsugamushi polyclonal antibody (O. tustusgamushi polyclonal antibody) method provides information necessary for diagnosis tsutsugamushi disease, characterized in that.
The method of claim 13, wherein the detection antibody is a monoclonal antibody that binds to TSA56 (56-kDa type-specific antigen) of the Tsutsuga mucosal outer endoplasmic reticulum.
The method of claim 13, wherein the outer membrane vesicle antigen is TSA56 (56-kDa type-specific antigen), 47 kDa antigen, ScaA, or chaperonin (chaperonin (60 kDa antigen)) of Tsutsuga mucosa. How to Provide.
22. The method of claim 21, wherein the TSA56 is represented by the amino acid sequence of SEQ ID NO: 5.
The method of claim 13, wherein the biological sample is whole blood, serum, plasma, saliva, sputum or urine obtained from a mammal.