KR20170002347A - Method and kit for diagnosis of tuberculosis based on Mycobacterium tuberculosis antigen-specific antibody reaction - Google Patents

Abstract

Disclosed are a method and a kit for distinguishing active, dormant, or recurrent tuberculosis by using a specimens reaction specific to the combination of antigens selected from 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens. Specifically, disclosed are a method and a kit for diagnosing or detecting whether a specimen negatively identified from a sputum smear test has active tuberculosis. The method or the kit according to the present invention can diagnose whether a specimen identified to be sputum smear negative, which is hard to diagnose with a conventional method, has active tuberculosis, dormant tuberculosis, or recurrent tuberculosis with high selectivity and specificity. Thus, the method or the kit can rapidly differentiate tuberculosis independently or with a conventional smear test.

Description

[0001] The present invention relates to a method for diagnosing tuberculosis using tuberculosis-specific antigen,

The present invention relates to a method for differentiating an active tuberculosis, a latent tuberculosis, or recurrent tuberculosis using an antibody reaction test against a tuberculosis-specific antigen, and in particular, an early diagnosis of tuberculosis which is sputum negative.

Tuberculosis (TB) is caused by mycobacteria It is an infectious disease caused by tuberculosis (M. tb) , which is the leading cause of death worldwide. Approximately one-third of the world's population is potentially infected with tuberculosis, and in 2012 about 8.6 million new cases were reported to WHO.

Currently, a widely used method for diagnosing tuberculosis is to identify tuberculosis by clinically isolating the tuberculosis (acid-fast bacillus (AFB)) stain or culture. Although the stain dyeing is advantageous in that it rapidly detects a patient with a high infectivity by rapidly detecting the Mycobacterium tuberculosis, the sensitivity is low to 20-80% as compared with the culture test, and the immune system such as a child, an elderly person or acquired immunodeficiency Failure rates are high in injured patients. The incidence of non-tuberculous mycobacterial isolates is increasing in Korea, so smear testing alone is not enough to diagnose active tuberculosis. In addition, although the culture method is more sensitive than the smearing method, it takes about 2-6 weeks for the result to come out, and it is necessary to use an experimental facility, which can not be applied in developing countries.

Another method is to detect latent TB infection (LTBI) by immunological methods such as tuberculin test (TST) and IGRA (IFN-γ release assay). Although this method has high specificity, both TST and IGRA can not distinguish between active tuberculosis and latent tuberculosis in areas where tuberculosis frequency is high as in Korea. This means that there is currently no diagnostic criteria to distinguish between active tuberculosis and latent tuberculosis.

Another method, the serological diagnostic method, is simple and inexpensive, but it has shown inconsistent results depending on country, ethnicity and used antigen (Steingart KR, 2011. PLoS Med. 8: e1001062).

However, the incidence of tuberculosis in the world is increasing, so accurate and universal diagnosis of tuberculosis, which is high in sensitivity and specificity and easy to use in the field, is necessary. Especially, if the sputum is negative, the accuracy is lowered. Therefore, if there is a supplementary measure, accurate and fast results will be obtained.

Korean Patent No. 0361345 describes the antigenic composition of Mycobacterium tuberculosis but is not described when the sputum is negative.

U.S. Patent No. 8,906,388 describes a method for detecting M. tuberculosis antigens, but this is also not described when the sputum is negative.

Therefore, even if the sputum is spoken negative, it is necessary to make a diagnosis of tuberculosis which can be easily used with high sensitivity and specificity.

The present invention distinguishes active tuberculosis from latent tuberculosis or recurrent tuberculosis using a selective antigen-specific antibody test, and specifically diagnoses tuberculosis infection in patients who have been negative in sputum smears.

In one embodiment, the subject is provided with the steps of providing 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens in order to provide information to diagnose whether a specimen is active tuberculosis, latent tuberculosis or recurrent tuberculosis ; Contacting each of the antigens with a sample to form an antigen-antibody complex for each antigen by the contact; Detecting the antigen-antibody complex; And determining the result of the detection. In the step of determining or analyzing the detection result, it is preferable that the detection result of i) the normal control group for the 38 kDa, 16 kDa, ESAT-6, CFP-10 and LAM antigens The results are shown in Table 1. The results were as follows: (1) active tuberculosis when the antigen-antibody complex formation was high compared with the results; (ii) the same or higher than the results of the normal control for the 38 kDa, 16 kDa, ESAT-6, CFP- Of patients with latent tuberculosis, iii) the 38-kDa, CFP-10, and LAM antigens are judged to be recurrent tuberculosis when the antigen-antibody complex formation is higher compared to the normal control group and / or the new patient control group A method for determining whether or not a tuberculosis infection is caused by using a tuberculosis antigen-specific antibody reaction.

In one embodiment, the sample used in the method according to the present invention is a sample judged negative in the sputum pheromone test, wherein in the determining step, antibody formation to the 38 kDa and 16 kDa antigens is compared with a normal control And if it is higher, it is judged that the specimen is an active tuberculosis.

In one embodiment, the sample that may be used in the method according to the present invention includes, but is not limited to whole blood, serum, plasma, sputum, urine, pleural fluid, ascites, cerebrospinal fluid or bronchoalveolar lavage.

Detection of an antigen-antibody complex in the methods according to the present invention may be carried out using known methods, including, but not limited to, immunization including radial immunodiffusion, immunoelectrophoresis or reverse-flow electrophoresis Precipitation assay, RIA (Radioimmunoassay) or ELISA (Enzyme Linked Immunosorbent Assay).

Detection of an antigen-antibody complex in the method according to the present invention may further comprise the use of a detection antibody, wherein the detection antibody specifically binds to human IgG and comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or color particles comprising colloidal gold particles or color latex particles.

The method according to the present invention can be carried out in the form of a dip stick, an ELISA or a lateral flow analysis method.

The method according to the present invention can also be used in combination with the sputum smear test method, but is not particularly limited in order, and can be used before, after, or together with the smear test.

In another aspect, the disclosure also includes a support and a detection antibody, wherein the 38 kDa, 16 kDa, ESAT-6, CFP-10 and LAM antigens are adsorbed and used in the methods disclosed herein, wherein the detection antibody is specific for human IgG A chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or colored particles comprising colloidal gold particles or color latex particles, wherein the kit comprises active tuberculosis, latent tuberculosis, or a recurrent tuberculosis detection or diagnostic kit in Invitro.

In one embodiment, the support includes, but is not limited to, a solid support such as a microplate, microarray, chip, glass, bead or particle, or membrane.

The kit according to the present invention may be used for, but is not limited to, dip stick, ELISA, or lateral flow analysis.

We use a speci fi c response of a combination of antigens selected from 38 kDa, 16 kDa, ESAT-6, CFP-10 and LAM antigens to distinguish active, latent or recurrent tuberculosis, A method and a kit for diagnosing or detecting whether a specimen judged to be negative in an active tuberculosis is an active tuberculosis. The method or kit according to the present invention diagnoses whether the sputum-negative specimen which is difficult to diagnose by the conventional method is active tuberculosis or latent tuberculosis and whether the tuberculosis is the first onset tuberculosis or recurrent tuberculosis with high sensitivity It is possible to diagnose tuberculosis accurately and quickly, alone or in combination with existing smear methods.

Figure 1 shows the personnel involved in the experiments conducted herein. A total of 159 active TB patients, 51 TB contact groups, and 133 healthy controls were recruited from National Mokpo Hospital and Severance Hospital. Of these, genotypes of M. tuberculosis were confirmed in 94 patients with tuberculosis. Based on the QFT-IT test and TST, 26 contact groups were identified as LTBI and 54 as healthy controls for normal. Among 91 patients with tuberculosis in total 94 patients, 55 patients were negative in sputum smear test and 36 patients were positive.
Figure 2 shows the tuberculous antigen-specific IgG response according to infection status and TB recurrence. (a) Tuberculosis patients (n = 94) had significantly higher IgG responses to all tested antigens compared to LTBI (n = 26) and control (n = 54), while LTBI and control There was no significant difference in IgG response. (b) The results of the comparison of the TB antigen-specific IgG responses between recurrent tuberculosis (n = 36) and newly developed cases (n = 58). In the case of recurrence, IgG responses to 38 kDa, CFP-10, and LAM were significantly higher than those in the newly emerged cases. IgG responses to 38 kDa and LAM were also different between the newly emerged and control groups, while ESAT-6 and CFP-10 were not different between the groups. Intermediate levels of IgG response were indicated by red horizontal bars (* P <0.05, ** P <0.01, *** P <0.001). (c) Intermediate IgG responses to LAM were significantly higher in AFB-smear positive patients (AFB + ve, n = 36) than in patients negative (AFB-ve, n = 55) However, all AFB-smear negative patients had significantly higher IgG responses than the control in response to all tested antigens.
Figure 3 shows the effect of sex on antigen-specific IgG responses in patients with tuberculosis. Mycobacterial antigen - specific IgG responses were compared between male tuberculosis patients (n = 64) and female tuberculosis patients (n = 30). In response to all antigens except the ESAT-6 antigen, the IgG response was significantly higher in male patients. Intermediate levels of response were indicated by red horizontal bars (* P <0.05, ** P <0.01, *** P <0.001).

This article identifies active, latent, or recurrent tuberculosis using specimen-specific responses to combinations of antigens selected from 38 kDa, 16 kDa, ESAT-6, CFP-10 and LAM antigens, It is based on the discovery that tuberculosis infection can be diagnosed in a patient who has a negative test.

In one aspect, we used an antigen-specific serum antibody (IgG) to elucidate the diagnosis of active tuberculosis, latent tuberculosis, or recurrent tuberculosis in sputum negative tuberculosis (38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens.

In one embodiment, the method according to the present invention comprises administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens in order to provide information for diagnosing whether the sample is active, latent, ; Contacting said antigen with a sample to form an antigen-antibody complex by said contacting; And detecting the antigen-antibody complex, wherein the detection results include: i) comparing the results of the 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens against normal control, Of the active tuberculosis is higher than that of the normal control group for the above 38 kDa, 16 kDa, ESAT-6, CFP-10 and LAM antigens, Tuberculosis, and iii) the 38 kDa, CFP-10, and LAM antigens were identified as recurrent tuberculosis when the antigen-antibody complex formation was higher than in the normal control group and / or the new patient group And a method for determining whether or not a tuberculosis infection is caused.

According to the method according to the present invention, the specimen can distinguish between active tuberculosis, latent tuberculosis and recurrent tuberculosis by selective antigen-specific antibody reaction test regardless of sputum donor test (see Table 1, FIGS. 2a and 2b) .

The term 'sputum mark test' is used to detect tuberculosis bacillus. It refers to a method of selectively staining only tubercle bacillus with a microscope by sputum of sputum to be examined. Mycobacterium tuberculosis is called acid-fast bacilli (AFB) because it does not decolorize by strong acid once it has been stained with red fuchsin. The results are confirmed by the Ziehl-Neelsen method, fluorescence staining method, and the like. If the diagnosis of tuberculosis is confirmed by sputum smears, tuberculosis is diagnosed. If it is not confirmed, it is diagnosed as negative. In the case of the above-mentioned voice, it includes not only the case where there is no tubercle bacillus, but also the case where the sputum can not be spoken like a child.

The 'active tuberculosis' is a condition in which tuberculosis that can induce tuberculosis causes active proliferation, not latency, and may be positive in sputum test (smear, culture, nucleic acid amplification (PCR)), (Chest x-ray, CT, etc.), and active tuberculosis.

The term 'latent TB infection' refers to a condition that is infected with tuberculosis, but is not clinically diagnosed as tuberculosis, and is negative in tuberculosis tests such as tuberculosis and radiation, and can not be transmitted to others.

The term "recurrent tuberculosis" refers to a case in which a person who had previously suffered from tuberculosis is treated again for tuberculosis after treatment, and includes both a case of recurrence due to drug resistance and a case where the patient stops medication and recurs. It is necessary to judge the recurrence of tuberculosis because the duration of the use of the drug varies depending on the recurrence of tuberculosis.

The term 'new patient' or 'new patient control' refers to a patient who has been diagnosed with active tuberculosis for the first time in his life and who has no previous history of tuberculosis.

In addition, according to the method of the present invention, it is possible to diagnose tuberculosis early even when it is judged as negative in sputum smears, and it is possible to diagnose whether such tuberculosis is active, latent or recurrent (see FIG. 2C) .

Accordingly, in another embodiment, the present invention provides a method for detecting 38-kDa and 16-kDa antigens in the step of detecting an antigen-antibody complex when the sample is judged to be sputum smear negative, wherein the antigen-antibody complex formation is higher The present invention relates to a method for judging whether or not a tuberculosis infection is caused by using a tuberculosis antigen-specific antibody reaction, comprising the step of early judging the sample as active tuberculosis.

The 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens herein are proteins that are the major antigen of Mycobacterium tuberculosis . The '38 kDa 'antigen is a lipoprotein-binding protein with strong immunogenicity of Mycobacterium tuberculosis. The '16 kDa' antigen is a protein involved in the alpha -crystalline family of low molecular weight heat shock proteins. The 'ESAT-6' (early secreted antigen target 6 kDa) antigen is a secreted protein that is a powerful T cell antigen produced by Mycobacterium tuberculosis. The 'CFP-10 (culture filtrate protein 10 kDa)' antigen is also a protein secreted by Mycobacterium tuberculosis. The 'LAM (lipoarabinomannan)' antigen is a major cell wall element that inhibits host cell maturation and inhibits T cell proliferation, thereby allowing M. tuberculosis bacteria to become pathogenic. Such protein antigens are known and can be synthesized and purified through recombinant methods, for example, see the methods disclosed herein.

The sample according to the present invention is a sample derived from a person suspected of having tuberculosis and includes, but is not limited to, whole blood, serum, plasma, sputum, urine, pleural fluid, multiple fluid, cerebrospinal fluid or bronchoalveolar lavage It is not. In one embodiment according to the present invention, blood, particularly serum, is used.

In the present study, the control group was a healthy control group with no clinical tuberculosis symptoms and no active lesions on the chest X-ray, ie, a healthy person who was both negative in TST and IGRA (QuantiFERON-TB Gold In-Tube; QFT-IT) It is a person or a sample derived therefrom.

In addition, the latent tuberculosis group is a healthy person who does not have clinical tuberculosis symptoms as in the control group. It is a healthy person or a resultant sample that does not have an active lesion on chest X-ray but has positive test on IGRA test.

Also, the 'new patient control group' is the first person who has been diagnosed with active tuberculosis in Korea and has no previous history of tuberculosis.

In the methods according to the present invention, antigen-antibody complexes can be detected using a variety of known methods. For example, the antigen-antibody complex can be detected by immunoprecipitation assay, radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA), including radial immunodiffusion, immunoelectrophoresis or reverse-flow electrophoresis. In one embodiment according to the present invention, an ELISA method, in particular a sandwich ELISA, is used, in which case the detection antibody described below is also used.

The intensity of the final signal of the sample is analyzed through the antigen-antibody complex detection process and compared with the signal of the sample of the normal control group. If it is higher than this, diagnosis of active, recurrent or latent tuberculosis can be made as described above.

In one embodiment according to the present application, for the detection of the antigen-antibody complex, the antigen is labeled with a labeling substance described later, or a detection antibody or a secondary antibody is used.

The detection antibody that can be used in the method according to the present invention is specifically bound to a human IgG, and the detection antibody can be labeled with a substance that can be detected using visual or various image detection equipment.

In one embodiment according to the present invention, the antigen or detection antibody according to the present invention is a labeling substance which is selected from the group consisting of peroxidase such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, Beta-galactosidase, urease, catalase, asparginase, ribonuclease, malate dehydrogenase, starch, and the like. A staphylococcal nuclease, a triosephosphate isomerase, a glucose-6-phosphate dehydrogenase, a glucoamylase, and an acetylcholine Catalyzes a chemical reaction in the presence of a specific substrate such as acetylcholine esterase to form a detectable chromogenic reaction or light But is not limited to, an enzyme capable of releasing the enzyme.

In another embodiment, the antigen or detection antibody according to the present invention is a bioluminescence, chemiluminescence, electroluminescence, electrochemiluminescence and / or radioimmunoassay that emits light of a wavelength different from that of the light irradiated by light. Chromophores used in photoluminescence, for example, green fluorescent proteins as proteins; Examples of the organic compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorecamine. But are not limited to,

In another embodiment, the antigen or detection antibody according to the present invention can be labeled with various radioisotope materials.

Detection of a labeling substance in the present invention can be performed, for example, by a scintillation counter in the case of a radioactive isotope. For example, in the case where the labeling substance is a fluorescent substance, detection with a spectroscope, a phosphoimaging device, Can be performed by the same method. When labeled with an enzyme, it can be carried out by measuring the chromogenic product which is indicated by the conversion of the chromogenic substrate by the enzyme in the presence of a suitable substrate. It can also be detected as a color comparison of the chromogenic products produced by enzymatic reactions through comparison with appropriate standards or controls.

In one embodiment, the antigen or detection antibody according to the present invention is, for example, a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or nanoparticles such as colloidal gold particles or colored latex particles, and the like.

In another aspect, the disclosure also includes a solid support and a detection antibody, wherein the 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens are adsorbed, and the detection antibody used in the method according to the present invention, IgG, wherein the antigen or detection antibody comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or nanoparticles comprising colloidal gold particles or colored latex particles, wherein the active tuberculosis negative tuberculosis or the recurrent tuberculosis tuberculosis is diagnosed or diagnosed.

It is possible to refer to the above description of the configurations included in the kit according to the present application that overlap with those described above.

In the kit according to the present invention, the antigen according to the present invention may be a microwell plate such as a 96 well microwell plate, a bead or particle comprising colloidal gold particles or colored latex particles or a cellulose or nitrocellulose, polyethersulfone, polyvinylidene, Fluorine, fluoride, nylon, charged nylon and polytetrafluoroethylene, and the like. As a method for adhering or coating, known methods can be used, for example, those described in the Examples herein can be referred to.

In one embodiment, the method or kit according to the present invention can be used in a sandwich immunoassay such as an ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay), or the like. Such methods include adding a sample to an antigen coupled to a bead, membrane, slide, or microwell plate made of a solid substrate such as glass, plastic (e.g., polystyrene), polysaccharide, nylon or nitrocellulose , A labeling substance capable of direct or indirect detection such as a radioactive substance such as ³ H or ¹²⁵ I as described above, a fluorescent substance, a chemiluminescent substance, hapten, biotin, digoxigenin, etc., Can be detected qualitatively or quantitatively through binding of an enzyme conjugated with an enzyme such as horseradish peroxidase, alkaline phosphatase, or malate dehydrogenase capable of coloring or emitting light. Immunoassay methods are also described in Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984. An ELISA kit can be prepared by mixing a secondary detection antibody labeled with the above substance, such as a reagent capable of detecting the bound antibody, such as chromophores, an enzyme (e. G., Conjugated with an antibody) And the like.

In other embodiments, the methods or kits according to the invention may be used in the form of an array or chip comprising a microarray. Antigens can be attached to the surface of a support such as glass or nitrocellulose, and array fabrication techniques are described, for example, in Schena et al., 1996, Proc Natl Acad Sci USA. 93 (20): 10614-9; Schena et al., 1995, Science 270 (5235): 467-70; And U.S. Pat. Pat. Nos. 5,599,695, 5,556,752, or 5,631,734. Fluorescence intensity can be measured using a scanning confocal microscope, for example Affymetrix, Inc. Or from Agilent Technologies, Inc., and the like.

The method or kit according to the invention is also used in the form of a dip stick rapid kit according to the analytical mode. In the case of dip stick, a technique widely used in the field of POCT (Point of Care Treatment), in which an antigen according to the present invention is bound to a substrate such as nitrocellulose and is contacted with a sample such as serum, When the one end is immersed in a serum sample, the sample is detected in such a manner that the sample migrates the substrate by the capillary phenomenon and develops color when bound to the antibody in the substrate.

The kits herein can also be used for lateral flow analysis according to the analysis mode. A lateral flow assay is a method for quantitatively or qualitatively measuring a specific substance contained in a specimen, for example, a specific nucleic acid or protein, for example, a nitrocellulose membrane (for example, And a specific nucleic acid or protein in the sample is detected through an antigen-antibody reaction by moving the analyte by a chromatographic method using a developing medium.

The kits herein may also include instructions on how to use the biomarkers in accordance with the present disclosure. In addition, a control-specific antibody or a reagent capable of detecting bound antibody may be further included.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Materials and Methods

Subjects: Two hospitals in Korea recruited 159 patients with pulmonary tuberculosis, 51 patients with recent tuberculosis infection, and 133 healthy persons. During the period from February 2011 to December 2012, 73 TB patients (mean age: 46) were recruited and enrolled at National Mokpo Hospital, and during the period from November 2010 to December 2013, 86 TB (Mean age: 32 years), 51 non-symptomatic tuberculosis patients (mean age: 44 years), and 133 healthy subjects (mean age: 31 years) were enrolled and registered at Severance Hospital (Fig. 1) . All participants received written consent and informed consent for diagnosis and immunization experiments. Active pulmonary tuberculosis was diagnosed by diagnosis of tubercle bacilli in sputum smear / culture and chest X-ray examination (American Thoracic Society, 2000. Am J Respir Crit Care Med 1981: 1376-1395), 94 patients The infectious M. tuberculosis major genotype was confirmed by molecular genotyping (Warren RM et al., 2004. Am. J. Respir. Crit. Care Med. 169 (5): 610-614). LTBI and the control group were tested using the TST and QFT-IT test (American Thoracic Society. 2000. Am. J. Respir. Crit. Care Med. 161: 1376-1395; Pai M et al., 2004. Lancet Infect. 761-776). Of the 51 tuberculosis contact groups, 26 had a positive IFN-γ response in the QFT-IT test; Twenty-nine of 26 patients had a double-positive response in the TST (≥10 mm) and QFT-IT tests, whereas seven of the 26 patients had negative TST results (<10 mm). Of the 133 healthy individuals who had never been in contact with tuberculosis patients, 54 subjects with dual negative responses in the TST (<10 mm) and QFT-IT tests were used as controls. Subjects who received immunosuppressive agents or those with cancer, diabetes, or renal disease were excluded. In the present study, the test group consisted of 94 patients with tuberculosis, 26 patients with latent tuberculosis infection (LTBI), and 54 healthy control subjects (Fig. 1). Ethical approval was obtained from the National Mokpo Hospital Ethics Review Board (Approval No. 2010-001) and Severance Hospital Ethics Review Board (Approval No. 4-2010-0213).

TST : Tuberculin PPD solution (0.1 ml of RT-23, Statens Serum Institute, Copenhagen, Denmark) was injected intradermally into all participants. Induration size was measured at 48 and 72 hours, and those measured at 10 mm or more were classified as positive.

Preparation of blood samples: 4 ml of blood (VACUETTE ^® serum tube, Greiner Bio-One GmbH, Frickenhausen, Germany) was centrifuged to separate serum samples. In the QFT-IT test, a total of 3 ml of blood was transferred to three QFT-IT tubes (Nil, M. tb Ag tube (ESAT-6, CFP-10 and TB7.7 peptide antigens) and mitogens (PHA) Cellestis, CA, USA]. Plasma was collected after incubation at 37 [deg.] C for 24 hours and IFN- [gamma] ELISA was performed according to the manual (QuantiFERON-TB Gold, Cellestis).

Molecular genetic analysis of Mycobacterium tuberculosis: LSP analysis (large-sequence polymorphism analysis) with ^{MTB / Beijing genotyping ® (Optipham-} M & D, Wonju, South Korea) to determined the M. tuberculosis genotype (Warren RM et al based on a., 2004. Am. J. Respiration Crit Care Mod 169 (5): 610-614). Specifically, the mycobacterial suspension was boiled in 400 μl buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) in a Robenstein-Jensen medium slant (Becton Dickinson and Company, . Veriti ^® 96- well Fast Thermal Cycler was performed using the DNA as a template in a gene amplification using the ^{(® Applied Biosystems, Life technologies,} CA, USA), the product was analyzed by agarose gel electrophoresis.

Preparation of Recombinant Protein Antigens: The 38 kDa and 16 kDa antigens were obtained from Standardia Diagnostics, Inc. (Suwon, South Korea) and LAM was obtained from Mycobacteria Research Laboratory, Colorado State University (Fort Collins, Colorado, USA). Vector construction for expression of recombinant ESAT-6 and CFP-10 was performed using NdeI and BamHI (New England Biolabs, USA) of pET11a_KB and the gene was amplified using M. tb H37Rv genomic DNA (Colorado State University, USA) Lt; / RTI &gt; Plasmids encoding ESAT-6 and CFP-10 were transformed into E. coli for overexpression of the protein and recombinant proteins were purified using Ni-NTA resin (Qiagen, CA, USA) and MonoQ anion exchange column (GE Healthcare Life Sciences) (AKTA FPLC, GE Healthcare Life Sciences, PA, USA) according to the manufacturer's instructions.

Antibody ( IgG ) Reaction Assay for Antigen : 2 μg / mL (38 kDa, 16 kDa, 1 mM) was added to 96-well microplates (Corning Inc., NY, USA) containing carbonate- bicarbonate buffer ESAT-6, CFP-10) or a 0.1 μg / mL (LAM) concentration of Mycobacterium tuberculosis antigen was coated at 4 ° C. for 18 hours. The plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked with PBST (PBST-NGS) containing 5% NGS (normal goat serum) for 1 hour at 37 ° C Respectively. The blocked plates were incubated with human serum samples (1: 300 dilution in PBST-NGS) for 2 hours at 37 ° C, then washed again with TBST and incubated with peroxidase conjugated anti-human IgG antibody (1: 10,000 dilution in PBST-NGS, Calbiochem, CA, USA) at 37 ° C for 1 hour. The plate was again washed with TBST and tetramethylbenzidine (KPL, MD, USA) was added. The reaction was stopped with 2.5 NH ₂ SO ₄ and the absorbance (OD) was measured at 450 nm using an ELISA microplate reader (Molecular Devices, CA, USA).

Statistical analysis: Kruskal-Wallis and Dunn's multiple comparison of TB-specific IgG antibody responses measured in active TB, contact with TB patients (LTBI group), and healthy control (control) Test. The IgG antibody response, drug resistance, AFB staining, tuberculosis genotype, and BMI and sex of the patients based on recurrence of tuberculosis were analyzed by Mann-Whitney test.

Example  1. Active tuberculosis, Latent tuberculosis  And receiver operating characteristic (ROC) curve analysis for anti-TB-specific antibody (IgG) responses in the control group

The ROC curve for the tuberculous antigen - specific IgG response was analyzed in the patient group. AUC, sensitivity, specificity and cut-off value were calculated by analyzing the ROC curves of the different test groups tested. The results are shown in Tables 1 and 2.

[Table 1] Analysis of the ROC curve for the tuberculous antigen-specific IgG response in different patient groups

IgG responses to 38 kDa, 16 kDa, and LAM serum were highly sensitive and specific enough to diagnose active or latent tuberculosis. In addition, AUC, sensitivity and specificity were higher in recurrent group than in new-onset group.

[Table 2] Characteristics in male and female patients

Male patients were more likely to have tuberculosis recurrence, drug resistance, and AFB-smear + ve (% AFB-smear + ve) than female patients. However, there was no significant difference in age and BMI between the two groups. * The fold difference was calculated by dividing the male patient number by the female patient number.

Example  2. Active TB, LTBI  And anti-tuberculous antibody-specific serum (IgG) responses in the control group

IgG responses were evaluated in a healthy control group that was negative in both active TB, LTBI, and TST and QFT-IT tests to determine the response to infection-specific antigen-specific serum antibodies (IgG). As a result, we found that significantly higher IgG responses were seen in all the antigens tested by active TB patients than in the control (P <0.001 or P <0.01, Fig. 2a). The area under the ROC curve for the 38 kDa-specific IgG response was highest (AUC = 0.7695, P <0.001), followed by LAM and 16 kDa (Table 1). Although the ESAT-6 and CFP-10-specific IgG responses were different between active TB and controls (Fig. 2a), the response was lower in AUC (<0.7) and sensitivity (55.3% and 51.1% There was no diagnostic value (Table 1). IgG responses to all the tested antigens identified active TB and LTBI (P <0.01, Figure 2a), with the highest AUC (0.7756, P <0.001), sensitivity (67.0%), and specificity (80.8%) (Table 1). This means that LAM-specific IgG responses can be biomarkers that can diagnose LTBI and active tuberculosis to a reliable extent.

Example  3. Based on the history of patients with tuberculosis Tuberculosis antigen -Specific antibody in serum ( IgG ) Confirmation of reaction

Of 94 active TB patients, 58 were newly diagnosed and 36 had previously had tuberculosis. These 36 patients were defined as recurrent patients, including those who had recurred tuberculosis after successful treatment, and those who voluntarily stopped taking medication or had drug resistance and failed initial treatment. IgG responses to all tested antigens were significantly higher in the relapsed group than in the control group, and AUC, resistance, and specificity were higher in relapse than in the newborn than in the control (Table 1). In particular, the sensitivity of the CFP-10-specific IgG response was almost doubled between the recurrence group (72.2%) and the newly developed group (36.2%) (Table 1). In addition, the sensitivity and specificity (81% and 78%, respectively) of the 38 kDa diagnostic value (AUC = 0.8593, P <0.001) IgG responses to 16 kDa and LAM were not significantly different or significantly different in the recurrent and newly-emerging patients, but there was a difference between the newly-emerging group and the control group (FIG. 2b).

Example 4. Confirmation of effect of drug resistance on antibody (IgG) in serum

Of the 88 patients tested for drug susceptibility, a total of 23 patients were classified as drug-resistant tuberculosis, of which two were extensive drug-resistant (XDR) and three were multiple drug resistance (MDR) patients. Serum antibody (IgG) responses to all tested antigens were not significantly different in these patients compared to drug-sensitive Mycobacterium tuberculosis (P> 0.05, data not shown). TB antigen-specific IgG responses were also unchanged in 24 patients with a BMI <18.5 and in 64 patients with a normal range of BMI (18.5 <BMI <25).

Example  5. Tuberculosis antigen -Specific antibody in serum ( IgG ) Analysis of Correlation between Reaction and Isolation of Mycobacterium tuberculosis

The anti-TB-specific antibody (IgG) response to AFB-staining was analyzed (FIG. 2C). Analysis of AFB-smear positive patients (AUC = 0.8909, P <0.001) and negative patients' curves (AUC = 0.6857, P <0.001) IgG response was significantly higher (P < 0.01, Figure 2c). These differences in LAM-specific IgG responses between AFB-smear positive and negative TB patients (P <0.01) indicate that they are more associated with bacillary load compared to other antigens tested.

However, in 8 kDa- and 16 kDa-specific IgG responses, there was no difference in IgG response among smear-positive tuberculosis patients according to smear test results, but IgG levels in smear negative patients were significantly higher (AUC> 0.7, P < 0.001). The 38 kDa antigen has sensitivity and specificity of 40-89% and 44-100% in the previous TB serologic diagnosis (Abebe F et al., 2007 Scand. J. Immunol. 66 (2-3): 176-191) , And the results are different depending on the experimental subject and the laboratory that has been tested. However, in the present invention it was observed that AFB-smear negative patients had a high diagnostic value of approximately 70% sensitivity and specificity for the 38 kDa antigen (AUC> 0.7, P <0.001). These data suggest that measuring IgG responses to 38 kDa may be an early diagnosis of tuberculosis, especially in patients with sputum-smear negative or those who can not sput with sputum.

In addition, antigen-specific IgG responses did not depend on the mycobacterial strain (data not shown). The proportion of patients infected with Beijing strains was about 4 times higher (81% vs. 19.1%) than patients infected with non-Beijing strains, but this was not correlated with the tuberculous antigen-specific IgG response, And the limited number of patients with XDR / MDR in the cohort.

Example 6. Analysis of sex effect on IgG response

The present inventors have shown that in response to all antigens except ESAT-6, significantly higher antigen-specific IgG responses were seen in male patients (n = 64) than in female patients (n = 30) . These differences were partly due to 38 kDa- and LAM-specific IgG responses (P <0.001). In the case of recurrent tuberculosis, the rates of median age, BMI, and mycobacterial genotype were similar between the two groups, while drug resistance and AFB-smear positive were more than two times higher in males than females, respectively (Table 2) .

It is estimated that gender affects antibody-specific IgG responses in part because of the frequency and severity of tuberculosis in male and female patients. Tuberculosis prevalence is about two times higher for males than females, which means that men are more susceptible to tuberculosis. More frequent exposure of men to tuberculosis and other sex-specific factors may affect TB antigen-specific antibody responses.

Thus, serological diagnostic testing of tuberculosis by detection of a combination of antigens consisting of 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM is a fast and economical point- of-care test. It is useful to distinguish between active tuberculosis and latent tuberculosis and to diagnose recurrent patients. Especially, in sputum-smear negative cases, it will contribute to early diagnosis of recurrent tuberculosis.

Claims (8)

In order to provide information for the diagnosis of active tuberculosis, latent tuberculosis or recurrent tuberculosis,
38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigen;
Contacting each of the antigens with a sample to form an antigen-antibody complex for each of the antigens by the contact;
Detecting the antigen-antibody complex; And
If the detection result of i) the formation of antigen-antibody complexes against the 38 kDa, 16 kDa, ESAT-6, CFP-10 and LAM antigens is higher than that of the normal control group,
Ii) If the antigen-antibody complex formation for the 38 kDa, 16 kDa, ESAT-6, CFP-10, and LAM antigens is lower than that of the active tuberculosis control but is judged positive in the IGRA (IFN-γ release assay) The specimen is referred to as latent tuberculosis infection, and
Iii) determining that the specimen is recurrent tuberculosis when the antigen-antibody complex formation for the 38 kDa, CFP-10, and LAM antigens is higher compared to the normal control and the new patient control. Determination of the incidence of tuberculosis infection using antibody response.
The method according to claim 1, wherein the specimen is whole blood, serum, plasma, sputum, urine, pleural fluid, multiple fluid, cerebrospinal fluid or bronchoalveolar lavage.
The method according to claim 1, wherein the detection of the antigen-antibody complex is performed by immunoassay analysis, RIA (Radioimmunoassay) or ELISA (Enzyme Linked Immunosorbent Assay) including radial immunodiffusion, immunoelectrophoresis or reverse current electrophoresis Lt; / RTI &gt;
4. The method according to any one of claims 1 to 3,
Wherein the detection of the antigen-antibody complex comprises the use of a detection antibody, wherein the detection antibody specifically binds to human IgG, wherein the detection antibody comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or color particles comprising colloidal gold particles or color latex particles.
5. The method of claim 4, wherein the method is performed with a dip stick, ELISA or lateral flow analysis method. A kit for use in the method according to any one of claims 1 to 3,
38 kDa, 16 kDa, ESAT-6, CFP-10 and a support on which the LAM antigen is adsorbed and a detection antibody,
Wherein said detection antibody specifically binds to human IgG and comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or colored particles comprising colloidal gold particles or color latex particles, wherein the kit is for detecting or diagnosing active tuberculosis, latent tuberculosis, or recurrent tuberculosis infection in Invitro.
7. The kit of claim 6, wherein the support is a microplate, microarray, chip, glass, bead or particle, or membrane.
7. The kit of claim 6, wherein the kit is for dip stick, ELISA, or lateral flow analysis.

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