KR102016385B1 - Method for Providing Information for Diagnosing Tuberculosis Using GABA as a Biomarker - Google Patents

Abstract

The present invention relates to a tuberculosis diagnostic kit and an information providing method for diagnosing tuberculosis using a new biomarker that is easy to quantify as a small molecule compound and that can be usefully used for early diagnosis of tuberculosis without the psychological and physical burden on patients. More specifically, the present invention relates to a method for providing information necessary for diagnosing tuberculosis, wherein the expression level of GABA is quantitatively analyzed from a sample collected from a subject, and a diagnostic kit for tuberculosis comprising an agent for measuring the expression level of GABA.

Description

Method for Providing Information for Diagnosing Tuberculosis Using GABA as a Biomarker}

The present invention provides a method for providing information for diagnosing tuberculosis using a new biomarker, which can be easily quantitated as a small molecule compound, and can be usefully used for early diagnosis of tuberculosis without psychological and physical burden on a patient, and a tuberculosis diagnostic kit. will be.

Tuberculosis is an infectious disease caused by infection with mycobacterium, in particular Mycobacterium tuberculosis. It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis and approximately 800 million new cases occur each year. Most infected people are asymptomatic and about one-tenth of them develop, and more than half of them die if they are not treated properly. Typical symptoms are cough with bloody phlegm, chills, cold sweats, and weight loss, which can affect any organ in the body, causing a variety of symptoms depending on the affected organ.

Early diagnosis of tuberculosis is essential for preventing the spread of tuberculosis and appropriate treatment, but tuberculosis is one of the difficult diseases to diagnose. The gold standard for tuberculosis diagnosis is to identify the growth of Mycobacterium tuberculosis in selected media. However, because mycobacterium grows slowly, these cultures take three to 12 weeks. Sputum smears are widely used in clinical laboratories for their fast ending, but they are less sensitive. PCR-based nucleic acid amplification and immunological methods have made great progress in the early and early diagnosis of tuberculosis. However, endogenous amplification inhibition factors or poor quality control of tuberculosis bacteria that result in false-positive or false-negative results can interfere with clinical use of PCR methods. Therefore, new biomarkers or new diagnostic methods for the diagnosis of tuberculosis are required. The present inventors have disclosed that in Korean Patent Nos. 10-1643748 and 10-1476781, microRNAs can be used as biomarkers for the diagnosis of tuberculosis. Small molecule compounds are faster, simpler and more accurate to analyze than genes or proteins, making them more useful as biomarkers. However, no small molecule compounds have been reported as biomarkers for tuberculosis.

Gamma-aminobutyric acid (GABA) is a non-proteinaceous amino acid that is distributed in nature and is an essential component of the central nervous system such as the brain and spinal cord. It is known as a major inhibitory neurotransmitter. . GABA is involved in many physiological controls. Animals activate the blood flow in the brain and increase the oxygen supply to the brain to promote the metabolism of brain cells, reduce triglycerides in the blood and improve liver function. It is effective in preventing adult diseases, and has a neurostable function for pain relief, depression and anxiety. In the case of stressed modern people, especially test takers and alcohol-ingestors, low levels of brain and blood GABA have been reported. Extremely low levels of GABA in the body cause seizures, convulsions and epilepsy. Due to this physiological action, GABA is used as an intravenous injection to improve cerebral blood flow and cerebral metabolism in stroke, head trauma, and cerebral artery sequelae, and is also sold as an oral prescription drug.

In recent years, the interest as a functional food material as well as the use as a pharmaceutical is also increasing. GABA is widely distributed in various vegetables, fruits, rice, soybeans, fermented foods, intestinal microorganisms, and if it is supplemented with food, the required amount is 500-3000mg per day, and it is known that GABA can be lowered to 80mg per day. In addition, the development of green tea, rice germ, germinated brown rice and the like with increased GABA content is increasing interest of the food industry and consumers (registered patent No. 10-0367055, etc.).

Recently, GABAergic components such as GABA-A ion channels, GABA-B receptors, GABA transporters, and enzymes involved in the synthesis or degradation of GABA have been reported in peripheral immune cells. However, the correlation between TB infection and GABA is not known.

Patent Registration No. 10-1643748 Patent Registration No. 10-1476781 Patent Registration No. 10-0367055

It is an object of the present invention to provide a biomarker for diagnosing tuberculosis useful for early and early diagnosis of tuberculosis and a method for providing information for diagnosing tuberculosis using the same.

Another object of the present invention is to provide a kit for diagnosing tuberculosis, which can be used to measure the expression level of the biomarker.

The present invention for achieving the above object relates to a method of providing information necessary for the diagnosis of tuberculosis, characterized in that the expression level of GABA from the sample collected from the subject quantitative analysis.

The method for providing information necessary for diagnosing tuberculosis of the present invention is characterized by using a low molecular compound GABA as a biomarker. A "biomarker" refers to a marker that can distinguish between normal and pathological conditions, predict a therapeutic response, and be objectively measurable. Genes such as RNA and DNA, which are commonly used as biomarkers, require pre-treatment required for analysis because total RNA is isolated from the cell to measure expression level, cDNA is synthesized from the cell, and amplified by PCR. The process is complex and time consuming. On the contrary, GABA, which is a low molecular compound, does not require the pretreatment as described above, and thus can be analyzed by a simpler process.

 Infection of tuberculosis bacteria or BCG into peripheral blood-derived mononuclear cells resulted in a rapid change in expression of GABA and significantly decreased expression compared to normal cells without tuberculosis bacteria. In animal experiments with mice, the mice infected with tuberculosis also showed a significant decrease in the expression of GABA in the serum and tissues of normal groups not infected with tuberculosis.

From this, it was confirmed that the infection of Mycobacterium tuberculosis can be significantly diagnosed by measuring the expression level in the biological sample of the individual to be diagnosed. The sample may be blood of suspected tuberculosis infection or tissue suspected of infection with Mycobacterium tuberculosis, but blood is more preferably used as a sample in consideration of ease of collection and psychological and physical burden on potential patients. If the measured GABA expression level is lower than the GABA expression level determined in the normal control group, it can be diagnosed as tuberculosis.

The expression level of GABA may be used by any method known in the conventional GABA assay. For example, an immunoassay using an antigen-antibody reaction may be performed as in the method described in the following Examples, or may be measured by chromatography after converting GABA in a sample to a derivative capable of UV detection. It is not limited by the method.

The present invention also relates to a diagnostic kit for diagnosing tuberculosis, comprising an agent for measuring the expression level of GABA. For example, the agent may be an antibody labeled to be readable as an antibody specific for GABA. The diagnostic kits of the present invention may further include one or more other configurations required for analysis, such as instructions for use, test tubes, buffers.

As described above, according to the present invention, since GABA, which is a low molecular compound, is used as a biomarker, tuberculosis can be diagnosed quickly and safely without burdening the patient with physical, physical, and time burden compared to conventional tuberculosis infection test, and is useful for early diagnosis of tuberculosis. .

1 is a confocal fluorescence microscopy image showing the change in expression level of GABA caused by Mycobacterium tuberculosis or BCG infection in macrophages.
Figure 2 is a graph showing the change in the expression level of GABA caused by Mycobacterium tuberculosis or BCG infection in macrophages.
Figure 3 is a confocal fluorescence microscope image showing the change in expression level of GABA due to Mycobacterium tuberculosis infection in the lung tissue of the mouse.
Figure 4 is a graph showing the change in the expression level of GABA caused by Mycobacterium tuberculosis infection in mouse lung tissue.
5 is a graph showing changes in the expression level of GABA due to Mycobacterium tuberculosis infection in the serum of mice.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, such an embodiment is only an example for easily describing the content and scope of the technical idea of the present invention, whereby the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the present invention based on these examples.

EXAMPLE

Example 1 Analysis of GABA Expression by Mycobacterium Tuberculosis Infection in Peripheral Blood-derived Monocytes

1) Preparation and culture of bone marrow-derived macrophages

Primary bone marrow-derived macrophages (BMDM) isolated from C57BL / 6 mice (Samtako Bio Korea) were supplemented with DMEM (Lonza) supplemented with 10% FBS (Lonza), penicillin G (100 IU / mL) and streptomycin (100 μg / mL). ) Was cultured in the medium. BMDM was suspended at a concentration of 1 × 10 ⁵ / mL and attached to a 100 pie petri dish at 37 ° C. and 5% CO ₂ . After 2 hours the cells were collected and centrifuged. Macrophage colony-stimulating factor (M-CSF) was added to the cell medium at a concentration of 25 ng / ml, and the cells were differentiated into macrophages for 3 to 5 days and used for experiments. Lymphocytes, not differentiated macrophages, were removed by mechanical destruction.

2) Analysis of GABA Expression Following Mycobacterium Tuberculosis Infection

J. Immunol on bone marrow-derived monocytes prepared in 1). ERFP-labeled Mycobacterium tuberculosis ( M. tuberculosis ) or BCG prepared according to the method described in 198, 3283-3295, 2017 is added so that the multiplicity of infection (MOI) is 1: 5 per culture cell. Were infected at 37 ° C. for the time described in the figure (during 4, 6, 18 hours). To remove extracellular mycobacterium tuberculosis, the mononuclear cells infected with Mycobacterium tuberculosis were washed three times with PBS buffer on a coverslip, and then treated with 4% paraformaldehyde for 15 minutes and Triton X-100 (Sigma) for 10 minutes. After treatment with anti-GABA (A2052, Sigma-Aldrich), the primary antibody at room temperature for 2 hours, excess primary antibody was removed by washing with PBS. Subsequently, Alexa Fluor 488-conjugated anti-rabbit IgG (A17041) was incubated at room temperature for an additional 1 hour by treating with a secondary antibody. Nuclei were treated for 2 minutes using DAPI. As a control, the same sample that did not infect Mtb-ERFP was used.

Cultured mononuclear cells were observed for fluorescence images using confocal laser-scanning microscope (TCS SP8; Leica, Wetzlar, Germany). FIG. 1 is an observed fluorescence image with scale bars showing 15 μm, and FIG. 2 is a graph showing average fluorescence intensities (MFIs) of GABA measured from FIG. 1. 1 and 2 it can be seen that the expression of GABA is significantly reduced in the macrophages infected with Mycobacterium tuberculosis or BCG.

Example 2 Analysis of Expression of GABA in Blood and Tissues of M. tuberculosis Infected Mice

As in-vitro experiments confirmed that GABA expression was reduced by tuberculosis infection in macrophages, in-vivo experiments in mice were performed (n = 4 / group). C57BL / 6 mice were infected by intravenous injection of Mtb at a concentration of 1 × 10 ⁶ CFU. The control group was injected with PBS instead of Mtb. After 14 days of infection, mice were sacrificed and lung tissue was harvested and fixed in 10% formalin for at least one day. After making a tissue block with paraffin wax, sections were made of 4 μm thick, and after deparaffinization and antigen regeneration, the primary antibody, anti-GABA (A2052, Sigma-Aldrich), was reacted overnight at 4 ° C. with PBS buffer. After washing three times, Alexa Fluor 594-conjugated anti-rabbit IgG (A21207) was reacted with a secondary antibody at room temperature for 2 hours. Then, washed three times with PBS buffer again, using a gold antifade reagent with DAPI (P36935, invitrogen) to prepare a sample by staining and fixing the nucleus. Figure 3 is a confocal fluorescence image of the stained tissue, Figure 4 is a quantitative expression of GABA expression from the image of the control group. 3 and 4 show that the expression of GABA was significantly decreased by the infection of Mycobacterium tuberculosis in the in-vivo experiment as in the in-vitro experiment.

Separately, sera from mice infected with Mycobacterium tuberculosis were collected and ELISA was performed using a Mouse ELISA Kit (Novatein Biosciences; NB-E20434) for detecting GABA according to the user's manual. 5 is a graph of the measured serum GABA concentration, it can be seen that the concentration of GABA in serum also significantly reduced during Mycobacterium tuberculosis infection.

Claims (4)

Quantitative analysis of the expression level of GABA from the sample collected from the test subject, providing information necessary for the diagnosis of tuberculosis, characterized in that the diagnosis of tuberculosis when the GABA expression level is lower than the GABA expression level determined in the normal control group Way.
The method of claim 1,
The sample is a method for providing information, characterized in that the blood of suspected tuberculosis infection or tissue suspected of TB infection.
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