KR20190032776A - Composition for stabilizing antigen for diagnosis of tuberculosis and method for diagnosis of tuberculosis - Google Patents

Abstract

According to the present invention, it is possible to provide a mixed composition for stabilizing tuberculosis antigen proteins for use in developing a diagnostic kit, and a method for diagnosing tuberculosis and for developing a tuberculosis diagnostic kit including the composition. Specifically, a stabilizing composition of the present invention can be applied to a tuberculosis diagnostic kit by stably preserving tuberculosis antigen proteins which are likely to be degraded by an environment such as heat. In addition, when a variety of tuberculosis antigens are used in combination, a suitable stabilizing composition can be prepared and applied to optimize stabilization conditions of the antigen proteins and diagnosis conditions of tuberculosis, thereby increasing the sensitivity in diagnosis of tuberculosis in tuberculosis patients.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for stabilizing an antigen for diagnosis of tuberculosis and a method for diagnosing tuberculosis,

The present invention was made by the Ministry of Health and Welfare funded by the Korea Health Industry Development Institute (KHIDI) in support of the healthcare technology R & D project (Field: Development of intractable tuberculosis ultra-north technology, Project No .: HI17C1000, Research Project: Development of korean high- Agency: Body Texmed Co., Ltd., Research institute: 2017.04.10 ~ 2019.12.31).

The present invention relates to an antigen-stabilizing composition for tuberculosis diagnosis.

The present invention also relates to a method for diagnosing tuberculosis using an antigen-stabilizing composition for tuberculosis diagnosis.

Tuberculosis is a disease caused by infection with Mycobacterium tuberculosis. It is estimated that 2.2 billion people, 1/3 of the world's population, are infected with tuberculosis and it is the second most common cause of adult infectious diseases following HIV / AIDS have. Tuberculosis is mainly caused by pulmonary tuberculosis that is invented in the lungs, but it can occur in all organs of the human body. Pulmonary tuberculosis is called pulmonary tuberculosis. Pulmonary tuberculosis, spinal tuberculosis, tuberculosis and intestinal tuberculosis can be classified according to their location. In the case of pulmonary tuberculosis, it is highly contagious, and the droplets discharged into the air through the coughing or sneezing of active tuberculosis patients are spread by the people around them.

On the other hand, Escherichia coli can be easily cultivated with a dissociation time of about 20 minutes, and other infectious bacteria are more proliferative than Mycobacterium tuberculosis, so that it is easy to identify. However, the tubercle bacilli have a proliferation rate of about 18-24 hours Do. As such, Mycobacterium tuberculosis requires a long-term medication for 6 months or longer to be treated because of its slow growth rate and its high fat-rich cell wall, and it needs to take a combination drug rather than a single anti-tuberculosis drug. In particular, if the initial treatment fails and the patient becomes resistant to the drug, the drug should be treated with a secondary drug. However, the treatment success rate is reduced to 50 to 60% even though it has problems related to cost and side effects. In Korea, the multidrug resistance rate due to treatment failure is high. Pulmonary tuberculosis as well as pulmonary tuberculosis for rapid cure is an early and accurate diagnosis.

Rapid and accurate diagnosis of tuberculosis leads to appropriate treatment, so that infectivity can be prevented early. There are various diagnostic methods for tuberculosis currently in use, and specifically, there are chest X-ray examination, chest computed tomography, skin reaction test, interferon gamma test, microbiological test method, serological test method and immunochromatography method . Among these diagnostic methods, the microbiological test method is used to diagnose active tuberculosis, and tuberculosis can be definitively diagnosed when smear is detected by sputum smear smear or culture test. However, there is a disadvantage that the smear test shows various sensitivities and the culture test takes a long time. Especially, when the sputum can not be discharged, it is difficult to apply the test. A serologic method can be used when the tubercle bacillus is not detected in the specimen because the tubercle bacillus is small in the tuberculosis lesion such as the sputum or the pulmonary tuberculosis, or when it is difficult to obtain the lesion itself. Is a method for detecting an antibody against a tuberculosis-specific antigen as a base. The current serologic diagnosis is lacking in sensitivity and specificity, and the World Health Organization does not recommend the use of the serological diagnosis. In order to solve these problems, it is necessary to develop a serologic diagnosis product with high sensitivity and specificity. In addition, the development of antigens is important in serologic tuberculosis diagnosis or immunological diagnosis. Furthermore, in order to apply the developed tuberculosis-specific antigens to the diagnostic method and to examine the role of the antigen, it is necessary to develop a composition for stabilizing the antigen. Tuberculous antigens are easily degraded by heat treatment. In order to prevent such degradation and preserve antigens for a long time, development of a stabilizing composition suitable for the characteristics of the antigens is required.

It is an object of the present invention to provide an antigen-stabilizing composition for diagnosis of tuberculosis.

It is another object of the present invention to provide a method for diagnosing tuberculosis using an antigen-stabilizing composition for tuberculosis diagnosis.

In order to solve the above problems,

(L-arginine), trehalose, L-glutamine, glucose, sucrose, mannitol and polyethylene glycol (PEG) The present invention provides a composition for stabilizing a tuberculosis diagnostic antigen.

According to one embodiment, L-arginine, L-glutamine, trehalose, or sucrose may be included alone in the range of 1 to 500 mM.

According to one embodiment, L-arginine in the range of 0.1 to 200 mM, trehalose in the range of 0.1 to 200 mM, L-glutamine in the range of 0.1 to 200 mM, Sucrose, mannitol in the range of 0.1 to 200 mM, and polyethylene glycol (PEG) in the range of 0.1 to 20%.

According to one embodiment, with L-glutamine ranging from 1 to 100 mM,

L-arginine in the range of 0.1 to 200 mM, trehalose in the range of 0.1 to 200 mM, sucrose in the range of 0.1 to 200 mM, mannitol in the range of 0.1 to 200 mM and a range of 0.1 to 20% (Polyethylene glycol (PEG)). ≪ / RTI >

According to one embodiment, the tuberculosis diagnostic antigen is one or more selected from the group consisting of tuberculosis antigen proteins ESAT-6 (Rv3875), CFP-10 (Rv3874), Mtb48 (Rv3881c), MPT64 (Rv1980c) and 16kDa . ≪ / RTI >

According to another embodiment of the present invention, there is provided a kit for diagnosing tuberculosis comprising the composition as described above.

According to another embodiment of the present invention, there is provided a method for diagnosing tuberculosis, comprising the step of reacting such a composition with a sample to analyze the tuberculosis infection.

Other details of the embodiments of the present invention are included in the following detailed description.

According to the tuberculosis diagnostic antigen-stabilizing composition of the present invention, by inhibiting and stabilizing the degradation of the tubercle antigen protein, it can play an important role in research on the characteristics of the antigen and long-term preservation. In addition, by providing a kit for diagnosing tuberculosis including the composition according to the present invention, it is possible to diagnose tuberculosis quickly and accurately with a simple method.

1 is a photograph showing electrophoresis results of CFP-10 protein.
2 is a photograph showing electrophoresis results of CFP-10 protein stored at 37 ° C for 1 day.
3 is a photograph showing electrophoresis results of CFP-10 protein stored at 37 ° C for 3 days.
4 is a photograph showing electrophoresis results of CFP-10 protein stored at 37 ° C for 5 days.
5 is a photograph showing electrophoresis results of CFP-10 protein stored at 37 ° C for 7 days.
6 is a photograph showing Western blot results of CFP-10 protein and tuberculosis patient serum.
7 is a photograph showing Western blot results of serum of CFP-10 protein and tuberculosis patient stored at 37 ° C for 3 days.
8 is a photograph showing electrophoresis results of 16 kDa of TB antigen protein stored at 37 캜 for 1 day.
9 is a photograph showing electrophoresis results of 16 kDa of TB antigen protein stored at 37 ° C for 7 days.
10 is a photograph showing electrophoresis results of 16 kDa of TB antigen protein stored at 37 ° C for 10 days.
11 (2E) is a photograph showing electrophoresis results of 16 kDa of TB antigen protein stored at 37 ° C for 18 days.
Fig. 12 is a photograph showing Western blot results of 16 kDa of TB antigen protein and tuberculosis patient serum stored at 37 캜 for 7 days.
13 is a photograph showing electrophoresis results of MTB48 protein stored at 37 ° C for 1 day.
14 is a photograph showing the result of electrophoresis of MTB48 protein stored at 37 ° C for 2 days.
15 is a photograph showing Western blot results of MTB48 protein and tuberculosis patient serum stored at 37 캜 for 2 days.
16 is a photograph showing electrophoresis results of MPT64 protein stored at 37 ° C for 7 days.
17 is a photograph showing electrophoresis results of MPT64 protein stored at 37 캜 for 14 days.
18 is a photograph showing Western blot results of serum of MPT64 protein and tuberculosis patient stored at 37 占 폚 for 10 days.
19 is a photograph showing electrophoresis results of ESAT-6 protein stored at 37 캜 for 2 days.
20 is a photograph showing electrophoresis results of ESAT-6 protein stored at 37 캜 for 3 days.
21 is a photograph showing Western blot results of serum of ESAT-6 protein and tuberculosis patient stored at 37 ° C for 3 days.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Hereinafter, the composition for stabilizing an antigen for tuberculosis diagnosis according to an embodiment of the present invention will be described in more detail.

The antigen used for diagnosing tuberculosis is preferably one or more selected from the group consisting of tuberculosis antigen proteins ESAT-6 (Rv3875), CFP-10 (Rv3874), Mtb48 (Rv3881c), MPT64 (Rv1980c) and 16 kDa May be used.

The tuberculosis antigen protein ESAT-6 (Rv3875) is a 6 kDa secreted protein produced by Mycobacterium tuberculosis and is used for the diagnosis of tuberculosis as a T cell antigen. The TB antigen protein CFP-10 (Rv3874) is a 10 kDa secreted protein produced by Mycobacterium tuberculosis. It is an antigen that contributes to the pathogen toxicity of Mycobacterium tuberculosis. In addition, it forms a complex with ESAT-6 to transmit virulence factors to host macrophages and leukocytes during infection.

The composition according to the present invention comprises at least one selected from the group consisting of L-arginine and trehalose.

L-arginine can improve stability against the tubercle antigenic proteins ESAT-6 (Rv3875), CFP-10 (Rv3874), Mtb48 (Rv3881c), MPT64 (Rv1980c) and 16 kDa (Rv2031c). In addition, trehalose can improve stability against CFP-10. Trehalose is a non-reducing sugar that allows the antigen to withstand severe conditions such as freezing and thawing, and L-arginine is an amino acid that inhibits the sensitivity of antigens to harsh conditions such as freezing or drying can do.

According to one embodiment, the present invention provides a pharmaceutical composition comprising one selected from the group consisting of L-glutamine, glucose, sucrose, mannitol, and polyethylene glycol (PEG) Or more. Each of the above compositions can improve the stability against the TB antigen protein, and the stability of the TB antigen protein can be further enhanced by the combination of any combination of the compositions. Specifically, each of the above compositions can further enhance the stabilizing effect of the antigenic protein when used together with trehalose or arginine, as compared with the case of using each composition alone. In addition, by adding L-glutamine to two or more mixed compositions containing, for example, L-arginine, aggregation of the antigen protein can be suppressed and stabilization can be improved.

According to one more specific embodiment, the tuberculosis antigen protein CFP-10 can be improved in stability in trehalose and L-arginine, respectively, and in a mixed composition, the ratio of the protein in the L-arginine, trehalose, mannitol, sucrose and L- Decomposition can be inhibited and stability can be greatly improved and comparative stability can be improved in L-arginine, trehalose and sucrose compositions, L-arginine, mannitol and sucrose compositions or L-arginine, trehalose and mannitol compositions.

In addition, the TBK antigen protein 16 kDa can be improved in stability in a single composition, L-arginine, and the stability can be improved even in a sucrose composition, and stability can further be improved in a mixed composition.

In addition, the TB antigen protein MTB48 can be improved in stability in an L-arginine composition, and degradation can be inhibited in L-glutamine and mannitol, respectively.

In addition, the tuberculosis antigen protein MPT64 can be relatively improved in stability in each composition.

In addition, the tuberculosis antigen protein ESAT-6 can be improved in stability in the L-arginine composition, and the stability can be further improved in the mixed composition.

According to one embodiment, the present invention provides L-arginine or L-arginine in the range of from 1 to 500 mM, for example from 1 to 200 mM, for example from 5 to 50 mM, as a sole composition or from 1 to 500 mM, Such as L-glutamine in the range of 1 to 50 mM, or trehalose in the range of 1 to 500 mM, such as 2 to 500 mM, such as 10 to 100 mM, or 1 to 500 mM, such as 2 to 500 mM, For example, sucrose in the range of 10 to 100 mM alone.

Also, according to one embodiment, L-arginine in the range of 0.1 to 200 mM, trehalose in the range of 0.1 to 200 mM, L-glutamine in the range of 0.1 to 200 mM, L-arginine in the range of 0.1 to 200 mM Sucrose in the range of 0.1 to 200 mM, mannitol in the range of 0.1 to 200 mM and polyethylene glycol (PEG) in the range of 0.1 to 20%, such as 0.1 to 7% And at least one selected from the group consisting of

Also, according to one embodiment, L-arginine in the range of 1 to 50 mM, trehalose in the range of 1 to 50 mM, L-glutamine in the range of 1 to 50 mM, Glucose, sucrose in the range of 1 to 50 mM, mannitol in the range of 1 to 50 mM and polyethylene glycol (PEG) in the range of 0.1 to 20%, for example, 0.1 to 7% And at least one selected from the group consisting of

According to one embodiment, a kit for diagnosing tuberculosis including the composition as described above can be provided and applied to a method for analyzing tuberculosis infection, so that it is possible to perform an early and early diagnosis of tuberculosis, so that latent or active tuberculosis infection It can be used for diagnosis.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Preparation Example: Tuberculous antigen protein

In order to obtain a tuberculosis antigen protein as a target to be stabilized, a specific tuberculosis antigen gene was amplified from mycobacterial chromosomal DNA and ligated to pET21a and pGEX4T-1, which are vectors for expressing E. coli, Respectively. The transformed Escherichia coli cells were overexpressed with a TB antigen protein and purified with His or GST columns, followed by purification with a PBS buffer using a desalting column. The purified tuberculous antigen protein was applied to the protein stabilizing composition. For the present invention, ESAT-6 (Rv3875, pET21a-ESAT6, His tag), CFP-10 (Rv3874, pGEX4T-CFP10, GST tag), MTB48 (Rv3881c, pET21a-Mtb48, His tag), MPT64 (Rv1980c, pET21a- MPT64, His tag) and 16 kDa (Rv2031c, pET21a-16kDa, His tag) recombinant TB antigen proteins were used. The antigen protein was diluted with PBS to a concentration of 0.5 mg / ml.

Example: Antigen stabilizing composition for diagnosis of tuberculosis

Sigma, P2263), Mannitol (Sigma, M9546), Polyethylene glycol compound (PEG, Sigma, P2263), Lucose (Sigma, -arginine (Sigma, A5006) and L-glutamine (Sigma, G3126) were used.

1000 mM glucose, 1000 mM sucrose, 1000 mM mannitol, 40% polyethylene glycol (PEG), 200 mM L-arginine (L- arginine, 100 mM L-glutamine.

The final concentrations for application of the examples were as shown in Table 1 below. In Table 1, the composition concentration unit of each composition except PEG is mM.

division L-arginine
(L-arginine) Trehalose
(trehalose) Glucose
(glucose) Sucrose
(sucrose) Mannitol
(mannitol) Polyethylene glycol
(PEG) L-glutamine
(L-glutamine) Example 1 33.3 Example 2 16.7 Example 3 16.7 Example 4 33.3 Example 5 6.7% Example 6 33.3 Example 7 1.3 Example 8 20 10 10 10 One% Example 9 20 10 10 10 10 Example 10 20 10 10 10 Example 11 20 10 10 Example 12 20 10 10 Example 13 20 10 10 Example 14 20 10 10 10 Example 15 20 10 10 10 Example 16 20 10 10 10 Example 17 20 Example 18 10

Experimental Example

First, freezing and thawing were repeatedly carried out in order to create a condition in which the protein was damaged and decomposed, or the protein was stored at 37 ° C to confirm the degree of degradation, and then it was confirmed by the stabilizing composition according to the Example Respectively. Specifically, the antigen proteins were exposed to each of the stabilizing compositions at 37 DEG C for one to twelve weeks to develop a protein stabilizing composition against heat. Each antigen protein was degraded under various conditions depending on the species, and the kind of the composition which showed improvement in the stabilization efficiency showed slight difference according to the protein. Protein was electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then stained with Coomassie blue and decolorized to examine the extent of degradation of the protein and the degree of protein preservation by the stabilizing composition according to the examples. Respectively. In addition, in order to confirm the applicability of the diagnosis of tuberculosis, the antigen protein preserved by the stabilizing composition and the serum of active tuberculosis patients were reacted and examined by Western blot analysis. Stabilizing Composition In order to apply the treated recombinant antigen protein to the diagnostic kit, the antibody response to tuberculous antigens detected in serum of patients with active tuberculosis confirmed by tuberculosis through smearing or culture was examined. To confirm the degree of stabilization of the recombinant tuberculous antigen protein on the stabilizing composition, 3.3 ug of the antigen protein was added to each example composition and stored at 37 캜 for each day. Controls not exposed to temperature and proteins exposed to date at 37 ° C were loaded in 5X SDS-PAGE sample buffer and incubated for 3 minutes. Then, 2.85 μg of protein was loaded on each SDS-PAGE gel and electrophoresed. The electrophoretic gel was confirmed to be degraded by Coomassie staining. To determine whether the antigenicity was maintained for diagnosis, electrophoretic gel was transferred to the nitrocellulose membrane and confirmed by using various active tuberculosis sera.

Experimental Example 1: Tuberculosis antigen protein CFP-10

To identify suitable stabilizing compositions for the recombinant antigen protein CFP-10 (Rv3874, pGEX4T-CFP10, GST tag), 3.3 μg of CFP-10 recombinant protein was added to each example composition and stored at 37 ° C. for 1 to 7 days Respectively. The CFP-10 protein exposed to the temperature unexposed controls and 1 to 7 days at 37 ° C was electrophoresed at 2.85 ug per well in 12% SDS-PAGE gel. The electrophoresis gel was checked for its degradation by Coomassie staining, M as a marker and C as a control, the TB antigen protein CFP-10.

Figure 1 shows the result of electrophoresis of the CFP-10 protein in the composition according to the Example without any harsh conditions. Figure 2 shows the results of the CFP-10 protein FIG. 3 shows the result of electrophoresis of the CFP-10 protein at 37 ° C for 3 days in the composition according to each example. FIG. 4 shows the results of electrophoresis of the CFP- 10 protein was stored at 37 ° C for 5 days and then electrophoresed. FIG. 5 shows the result of electrophoresis after storing the CFP-10 protein at 37 ° C for 7 days in the composition according to each example. Protein aggregation inhibits antibody reaction and immune cell activity, and inhibition and stabilization of protein aggregation can be confirmed by treatment with L-glutamine. In particular, as shown in Example 18 of Fig. 4, when L-glutamine alone was used, it was confirmed that the stability of the TFP protein CFP-10 was greatly improved, so that 1 to 100 mM of L-glutamine alone ≪ / RTI > can be identified.

In addition, sera from patients with active tuberculosis were used to confirm the maintenance of protein antigenicity. FIG. 6 is a Western blot result using serum of CFP-10 protein and tuberculosis patients which are not subjected to harsh conditions as a control, and FIG. 7 is a graph showing the results of Western blot analysis using CFP- The results were obtained by Western blotting using serum from tuberculosis patients after storage.

As shown in FIGS. 1-7, the CFP-10 protein was stable in Example 1 in the single composition and remained relatively stable in Examples 5, 6, and 18. In addition, the single composition and mixed composition of Example 18 inhibited the degradation and aggregation of proteins in Examples 9, 14, 15, and 16, demonstrating optimal stability. In Examples 11 to 13, It can be confirmed that it is stable.

Experimental Example 2: Tuberculosis antigen protein 16 kDa

In order to identify a stabilizing composition suitable for the recombinant antigen protein 16 kDa (Rv2031c, pET21a-16 kDa, His tag), 3.3 ug of 16 kDa recombinant protein was added to each example composition and stored at 37 ° C for 1 to 18 days. Controls not exposed to temperature and 16 kDa protein exposed for 1 to 18 days at 37 ° C were electrophoresed on 2.85 ug per well in 12% SDS-PAGE gel. The electrophoresed gel was checked for its degradation by Coomassie staining, M as a marker and C as a control, a TB antigen protein 16 kDa.

FIG. 8 shows the result of electrophoresis of the 16 kDa protein at 37.degree. C. for 1 day in the composition according to the embodiment, and FIG. 9 shows the results of the 16 kDa protein at 37.degree. C. for 7 days FIG. 10 shows the result of electrophoresis of the 16 kDa protein in the composition according to each example at 37 ° C. for 10 days, and FIG. 11 shows the results of the electrophoresis of the 16 kDa protein Was stored at 37 ° C for 18 days and then electrophoresed.

In addition, sera from patients with active tuberculosis were used to confirm the maintenance of protein antigenicity. FIG. 12 shows the result of Western blotting using 16-kDa protein in each composition at 37 ° C for 7 days and using serum from a patient with tuberculosis.

As shown in Figs. 8 to 12, the 16 kDa protein was stable in Example 6 at a single composition, and remained relatively stable in Example 3, and was relatively stable at a mixed composition.

Experimental Example 3: Tumorigenic protein MTB48

To identify suitable stabilizing compositions for the recombinant antigen protein MTB48 (Rv3881c, pET21a-MTB48, His tag), 3.3 μg of MTB48 recombinant protein was added to each example composition and stored at 37 ° C for 1-2 days. MTB48 protein exposed to temperature-uninhibited controls and 1 to 2 days at 37 ° C was electrophoresed on 2.85 ug per well in 12% SDS-PAGE gel. The electrophoresed gel was checked for its degradation by Coomassie staining, M as a marker and C as a control, the TB antigen protein MTB48.

FIG. 13 shows the result of electrophoresis after MTB48 protein was stored at 37 ° C for one day in the composition according to each example. FIG. 14 shows MTB48 protein in each composition according to each example stored at 37 ° C for 2 days After electrophoresis.

In addition, sera from patients with active tuberculosis were used to confirm the maintenance of protein antigenicity. FIG. 15 shows results of Western blotting using MTB48 protein in each composition for 2 days at 37 ° C and using serum from a patient with tuberculosis.

As shown in Figures 13 to 15, the MTB48 protein was stable in Example 6 at a single composition, and it can be confirmed that the MTB48 protein remains relatively stable in Examples 4 and 7.

Experimental Example 4: Tuberculous antigen protein MPT64

To identify suitable stabilizing compositions for the recombinant antigen protein MPT64 (Rv1980c, pET21a-MPT64, His tag), 3.3 μg of MPT64 recombinant protein was added to each example composition and stored at 37 ° C for 7-14 days. The MPT64 protein exposed to the control at 37 ° C for 7-14 days at 37 ° C was electrophoresed at 2.85 ug per well on 12% SDS-PAGE gel. The electrophoresed gel was confirmed by coomassie staining, M was a marker, and C was a control antigen, the TB antigen protein MPT64.

FIG. 16 shows the result of electrophoresis of MPT64 protein at 37.degree. C. for 7 days in the composition according to each example. FIG. 17 shows that MPT64 protein was retained at 37.degree. C. for 14 days in the composition according to each example After electrophoresis.

In addition, sera from patients with active tuberculosis were used to confirm the maintenance of protein antigenicity. 18 shows the result of Western blotting using the serum of a tuberculosis patient after storing the MPT64 protein at 37 DEG C for 10 days in each composition.

As shown in Figs. 16 to 18, MPT64 protein is more thermally stable than other antigen proteins, and overall stable results can be obtained in each composition.

Experimental Example 5: Tuberculosis antigen protein ESAT-6

To identify suitable stabilizing compositions for the recombinant antigen protein ESAT-6 (Rv3875, pET21a-ESAT6, His tag), 3.3 ug of ESAT-6 recombinant protein was added to each example composition and stored at 37 ° C for 2-3 days Respectively. ESAT-6 protein exposed to temperature-free controls and 2 to 3 days at 37 ° C was electrophoresed on 2.85 ug per well in 12% SDS-PAGE gel. The electrophoresed gel was checked for its degradation by Coomassie staining, M as a marker, and C as a control, the tuberculosis antigen protein ESAT-6.

FIG. 19 shows the result of electrophoresis after ESAT-6 protein was stored at 37 ° C for two days in the composition according to each example. FIG. 20 shows the results of ESAT-6 protein in the composition according to each example at 37 ° C And stored for 3 days.

In addition, sera from patients with active tuberculosis were used to confirm the maintenance of protein antigenicity. FIG. 21 shows results of Weston blotting using ESAT-6 protein in each composition for 3 days at 37 ° C and using serum from a patient with tuberculosis.

As shown in FIGS. 19 to 21, it can be seen that the ESAT-6 protein was stable in Example 6 at a single composition, and remained more stable in the mixed composition, showing useful results for diagnosis.

As can be seen from the above results, according to the present invention, the tuberculosis antigen protein can be stabilized to provide information for diagnosis of tuberculosis kits and tuberculosis. In addition, it was confirmed that the sensitivity of diagnosis of tuberculosis to patients can be improved by optimizing the diagnosis condition of tuberculosis.

The above description is only illustrative of the technical idea of the present invention, and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. It should be noted that the embodiments disclosed in the present invention are not intended to limit the scope of the present invention and are not intended to limit the scope of the present invention. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.

Claims (9)

One selected from the group consisting of L-arginine, trehalose, L-glutamine, sucrose, mannitol and polyethylene glycol (PEG) Of the total amount of the composition. The method according to claim 1,
Wherein the composition comprises L-arginine, L-glutamine, treahalose or sucrose alone in the range of 1 to 500 mM. The method according to claim 1,
L-arginine in the range of 0.1 to 200 mM, trehalose in the range of 0.1 to 200 mM, L-glutamine in the range of 0.1 to 200 mM, sucrose in the range of 0.1 to 200 mM, 0.1 Wherein the composition comprises at least one selected from the group consisting of mannitol in the range of 1 mM to 200 mM and polyethylene glycol (PEG) in the range of 0.1 to 20%. The method according to claim 1,
Together with L-glutamine ranging from 1 to 100 mM,
L-arginine in the range of 0.1 to 200 mM, trehalose in the range of 0.1 to 200 mM, sucrose in the range of 0.1 to 200 mM, mannitol in the range of 0.1 to 200 mM and a range of 0.1 to 20% Of polyethylene glycol (PEG). ≪ / RTI > The method according to claim 1,
Wherein the tuberculosis diagnostic antigen comprises at least one selected from the group consisting of tuberculous antigen proteins ESAT-6 (Rv3875), CFP-10 (Rv3874), Mtb48 (Rv3881c), MPT64 (Rv1980c) and 16kDa Diagnostic antigen-stabilizing composition. The method according to claim 1,
Wherein the composition diagnoses latent tuberculosis infection. The method according to claim 1,
Wherein the composition is for diagnosing active tuberculosis infection. A kit for the diagnosis of tuberculosis comprising the composition according to any one of claims 1 to 7. A method for diagnosing tuberculosis, comprising the step of reacting a composition according to any one of claims 1 to 7 with a sample to analyze the tuberculosis infection.

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