KR101524109B1 - Nano-biosensor bound with antibody against mycobacterium tuberculosis specific antigen and method for detecting mycobacterium tuberculosis using the same - Google Patents

Abstract

The present invention relates to a tuberculosis diagnosing method using a nano-biosensor, and more specifically, to the nano-biosensor which comprises a gold nano-probe and a magnetic micro-probe combined with an antibody against a mycobacterium tuberculosis specific antigen, and a method for detecting mycobacterium tuberculosis using the same. According to the present invention, there is no concern for infection of the mycobacterium tuberculosis, and the mycobacterium tuberculosis can be simply and effectively detected without an expensive apparatus, thereby early diagnosing the tuberculosis.

Description

TECHNICAL FIELD The present invention relates to a nanobiosensor to which an antibody against a Mycobacterium tuberculosis-specific antigen is bound, and a method for detecting the Mycobacterium tuberculosis using the same.

The present invention relates to a method for diagnosing tuberculosis using a nano-biosensor, and more particularly, to a nano-biosensor comprising a gold nanoprobe and a magnetic microprobe to which an antibody against a tubercle bacilli antigen is bound, and a method for detecting mycobacteria using the same.

In general, tuberculosis is a disease caused by infection with Mycobacterium tuberculosis , which is the number one disease incidence in Korea. Therefore, early diagnosis and treatment are important to obtain reliable information in a short period of time from patients suspected of being infected with tuberculosis.

Tuberculin skin test (TST) is a common method for diagnosing tuberculosis. However, its specificity is low and its reliability is lower than the criteria for tuberculosis infection. However, it is still widely used because of its high sensitivity and low cost. The culture method is also widely used. It is the most reliable test among the various methods of diagnosing tuberculosis, and it is a test to confirm tuberculosis by proliferation of tuberculosis. However, since the bacteria are separated and cultured, the risk of infection by the experimenter can not be ruled out, and it takes a minimum of 46 weeks to cultivate the bacteria.

Recently, the BACTEC system or the Mycobacteria Growth Indicator Tube (MGIT) method using a liquid medium has been implemented. This method has the disadvantage of requiring additional confirmation test to distinguish it from atypical mycobacteria, although it can confirm the proliferation of mycobacteria in about 2 weeks by shortening the incubation period by using liquid medium. In addition, there are problems such as waste disposal problems caused by the use of radioactive isotopes, concerns about infection of Mycobacterium tuberculosis, and the need for expensive equipment.

Accordingly, the present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, they have found that a gold nanoprobe and a magnetic microprobe having an antibody against the Mycobacterium tuberculosis secretion antigen bound thereto from a culture filtrate containing an antigen secreted by the growth of Mycobacterium tuberculosis The present invention has been accomplished based on the findings that it is possible to efficiently and rapidly detect the presence of tubercle bacilli by detecting a Mycobacterium tuberculosis-specific antigen in a culture solution without fear of infection by using the constructed nano-biosensor.

An object of the present invention is to provide a method for detecting Mycobacterium tuberculosis through the detection of a Mycobacterium tuberculosis detection nano-biosensor and a Mycobacterium tuberculosis secretion antigen using the same.

In order to achieve the above object, the present invention provides a nano-biosensor for detecting Mycobacterium tuberculosis comprising a metal nanoprobe immobilized with an antibody against a Mycobacterium tuberculosis secretion antigen and a magnetic microprobe immobilized with the antibody.

The present invention also provides an antibody against CFP10 comprising a heavy chain having the amino acid sequence of SEQ ID NO: 3 or 5 and a light chain having the amino acid sequence of SEQ ID NO: 4 or 6.

(A) contacting the culture filtrate containing an antigen derived from Mycobacterium tuberculosis with the nano-biosensor to form a nanocomposite composed of an antibody of an anti-mycobacterial antigen gold nanoprobe of a magnetic microprobe; And (b) separating the formed nanocomposite with a magnet, and measuring absorbance. The present invention also provides a method for detecting Mycobacterium tuberculosis.

According to the present invention, it is possible to diagnose tuberculosis at an early stage by detecting the mycobacterium tuberculosis easily and efficiently without any fear of infection against the tubercle bacillus and without expensive equipment.

Fig. 1 shows an artificial filter cap tube equipped with a syringe filter for culturing mycobacterial bacteria and equipped with a double syringe filter of 50 ml (above) and 15 ml (below).
FIG. 2 is a block diagram showing the parts for a 50 ml tube equipped with a double-syringe filter for culturing a Mycobacterium tuberculosis culture (1: 50 ml culture tube; 2: 50 ml culture tube cap; 3: syringe filter;
3 is an enlarged view of an assembled state for a 50 ml tube equipped with a double-syringe filter for culturing the Mycobacterium tuberculosis and an enlarged view of the culture tube cap (A: rib; B: syringe filter assembly).
Fig. 4 shows the binding strength of the phage antibody to the CFP10 according to the panning process.
Figure 5 shows an ELISA assay using a CFP10 antigen or an antimyc antibody. {(a): CFP10 ELISA, (b): CMyC ELISA}
Figure 6 is an analysis of the sequence of the selected antibodies. {(a): CFP10 [G2] sequencing; (b): CFP10 [G3] sequence analysis}
Figure 7 shows the ability to detect an antigen through a sandwich ELISA.
FIG. 8 is a conceptual diagram for detecting target antigens in a culture solution using a magnetophoretic immunoassay.
FIG. 9 shows the results of AFT (Artificial Filter Cap Tube) culture performance test based on comparison test between the magnetophoretic immunoassay and the BACTEC MGIT culture system.

Unless otherwise defined, 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.

Further, various elements and regions in the drawings are schematically drawn. Accordingly, the technical spirit of the present invention is not limited by the relative size or spacing depicted in the accompanying drawings.

As used herein, the term artificial filter cap tube (AFT) refers to a tube for culture of M. tuberculosis equipped with a double syringe filter.

In one aspect, the present invention relates to a nanobiosensor for detecting Mycobacterium tuberculosis containing a metal nanoprobe immobilized with an antibody against a Mycobacterium tuberculosis secretion antigen and a magnetic microprobe immobilizing the antibody.

In one embodiment of the present invention, a gold binding protein-bound antibody is attached to gold nanoparticles synthesized by a known room temperature synthesis method, and a metal cluster is attached to the surface of the magnetic microprobe in order to attach the antibody. And then the antibody against the Mycobacterium tuberculosis secretagogue was attached.

In the present invention, the antibody may be an antibody specific for Mycobacterium tuberculosis, preferably an antibody against CFP10, wherein the antibody against CFP10 comprises the heavy chain of SEQ ID NO: 3 or 5 and the heavy chain of SEQ ID NO: 4 or SEQ ID NO: 6 < / RTI >

 In the present invention, the metal may be selected from the group consisting of gold, silver, platinum, copper and zinc, and gold is preferably used.

In another aspect, the present invention relates to an antibody against CFP10 comprising a heavy chain having the amino acid sequence of SEQ ID NO: 3 or 5 and a light chain having the amino acid sequence of SEQ ID NO: 4 or 6.

In another embodiment of the present invention, a phage antibody against the CFP10 antigen was screened using a human antibody phage library. The antibody having high binding ability was screened by panning three times to analyze the sequence of the antigen recognition site, And finally two kinds of antibodies were obtained.

According to another aspect of the present invention, there is provided a method for preparing a nanocomposite comprising the steps of: (a) contacting a culture filtrate containing a Mycobacterium tuberculosis secretion antigen with the nanobiosensor to form a nanocomposite comprising an antibody of an antibody mucopolysaccharide antigen gold nanoprobe of a magnetic microprobe; And (b) separating the formed nanocomposite with a magnet, and then measuring the absorbance.

In another embodiment of the present invention, a diagnostic test for tuberculosis was conducted using a sample of sputum specimens from a clinical patient received from Chungnam National University Hospital. Since the time point at which the sample shows positive is different, the MGIT that has been used in commercial use is co-cultured. That is, when the positive reaction was confirmed in MGIT, the culture was stopped and sampling was performed in a tuberculosis culture tube equipped with a double syringe filter, and the secreted TB specific antigen was analyzed by magnetophoretic immunoassay. At this time, the sampled culture filtrate contains no tubercle bacillus and includes tuberculous antigen.

First, the culture filtrate was diluted to react with a magnetic microprobe having an antibody binding to the Mycobacterium tuberculosis secretion antigen, and then reacted with the gold nanoprobe to which the antibody was bound to form a nanocomposite with a sandwich structure. The magnet was placed on the bottom of the reaction vessel to separate the nanocomposite and the absorbance was measured. Absorbance was determined by measuring gold nanoprobe without complex. Ten specimens were analyzed for sensitivity and specificity of 80% and 100%, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Example  1: Tubing culture tube equipped with double syringe filter

An artificial filter cap tube (AFT) equipped with a 50 ml and 15 ml disposable double syringe filter for culturing mycobacteria was prepared. The culture tubes were used as commercialized parts, and only the cap was newly designed and manufactured (FIG. 1).

In order to obtain the supernatant to obtain only the secreted antigen of the Mycobacterium tuberculosis at the time of sampling, the prepared culture tube was designed and manufactured so that it can be repeatedly sampled by exchanging only the syringe filter for removing the M. tuberculosis.

11: Production of 50 ml tube

As shown in Fig. 1, a sealing portion was added between the culture tube and the syringe filter to improve the leakage (Fig. 2), and the syringe filter was marked with "A" Three ribs were added. Since the outer diameter (30.3 mm) of the syringe filter is larger than the bore diameter (292.66 mm) of the culture tube cap screw, the screw portion is forced through the press-fitting and the syringe filter assembly (labeled "B" Was made to be equal to the outer diameter of the syringe filter, thereby maintaining the fit state (Fig. 3).

12: Production of 15 ml tube

A 15 ml tube was prepared in the same manner as in Example 11 except that a 15 ml culture tube was used and the bore diameter (17.3 mm) of the tube cap screw and the outer diameter of the syringe filter (17.1 mm) were different.

Example  2: Mycobacterium tuberculosis antigen CFP Ten Recombinant protein  Production and refining

In order to produce the recombinant antigen CFP10, the gene was amplified by performing PCR using CFP10F '(SEQ ID NO: 1) and CFP10R' (SEQ ID NO: 2) using the genomic DNA of M. tuberculosis as a template (Shin et al ., Clin . Vaccine Immunol ., 15: 1788, 2008):

[SEQ ID NO: 1] CFP10F5'GGCCGG GGATCC ATGGCAGAGATGAAGACCG3 'BamHI

[SEQ ID NO: 2] CFP10R'5'GGCCGG GAATTC GAAGCCCATTTGCGAGGAC3 'EcoRI

(The underlined part represents the restriction enzyme site).

The amplified PCR product was digested with restriction enzymes BamHI and EcoRI and inserted into the BamHI / EcoRI site of pET28a (Novagen, Gibbstown, USA). The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), and isopropyl beta Dthiogalactopyranoside (IPTG) was added at 37 ° C to a final concentration of 1 mM at an OD ₆₀₀ value of 0.4-0.5. Induced overexpression. The cultured Escherichia coli cells were collected by centrifugation and suspended in 20 mM TrisHCl buffer (pH 8.0, 0.5 M NaCl, 20 mM imidazole and 1 mM phenylmethylsulfonyl fluoride (PMSF) And the cells were disrupted and purified by nickel NTA (NiNTA) agarose chromatography (Invitrogen, Carlsbad, USA).

Example  3: CFP 10 Antigen Selection and Production

31: Antibody screening

The phage antibody against the CFP10 antigen was selected using a human antibody phage library (Hong et al ., Sens . Actuat . B Chem, 156:. 271, 2011). The total number of phages in the library administered in Biopanning, in which 2 ml of a CFP10 antigen at a concentration of 10 占 퐂 / ml was coated on an immunotube and the library was reacted with the coated antigen to select the initial binding phage, And about 2 x 10 < ¹³ > CFU. To remove nonspecific phage, the remaining phage were infected into XL1Blue Escherichia coli after 10 washing steps. The phage was amplified and the primary sub-library was obtained. .

To determine whether polyclones recognize the CFP10 antigen, the phage prepared on a plate coated with CFP10 antigen or antimyc antibody was reacted and ELISA analysis was performed (FIG. 5). The myc analysis value showing the number of applied phage antibodies was similar, and the binding force of these phage antibodies to CFP10 was found to increase sharply during the panning of 3 times. In the 3rd panning, (Fig. 4).

The 96 monoclonal clones were selected and analyzed to identify antibodies that bind to CFP10 at a high rate. Sixty monophage sequences with strong signal were analyzed, and the monophage antigen recognition site of each monophage The sequence was analyzed and the binding force with the antigen was analyzed. Finally, two kinds of antibodies were selected and sequenced.

As a result, the antibody against the selected CFP10 contained an antibody consisting of the heavy chain represented by the amino acid sequence of CFP10 [G2] _HC and (SEQ ID NO: 3) and the heavy chain represented by the amino acid sequence of CFP10 [G2] _LC An antibody consisting of the heavy chain represented by the amino acid sequence of CFP10 [G3] _HC (SEQ ID NO: 5) and the heavy chain represented by the amino acid sequence of CFP10 [G3] _LC (SEQ ID NO: 6) 6 (b)). The variable region of the heavy chain CFP10 [G2] _HC (SEQ ID NO: 3) comprises a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 7, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 8, a CDR3 region consisting of the amino acid sequence of SEQ ID NO: Wherein the variable region of the light chain CFP-10 [G2] _LC (SEQ ID NO: 4) comprises a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 10, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 11, The variable region of the heavy CFP-10 [G3] _HC (SEQ ID NO: 5) comprises a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 14, The variable region of the light chain CFP-10 [G3]) _ LC (SEQ ID NO: 6) comprises a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 16, a CDR1 region consisting of the amino acid sequence of SEQ ID NO: The CDR2 region of SEQ ID NO was analyzed as comprising a CDR3 region consisting of the amino acid sequence of the 18 (Figure 6).

32: Antigen antibody reaction test

Antigen detection ability was analyzed by sandwich ELISA. The antiCFP10 antibody was diluted 2,000 times in a coating buffer and attached to an ELISA plate. After blocking, the antigen was added to induce the reaction. The detection antibody was rabbit antiCFP10 antibody produced by Chungnam National University Medical School and it was confirmed that lower concentration could be detected when antiCFP10 antibody was used as a capture antibody (FIG. 7).

33: Antigen antibody reaction test using actual sample

In order to measure the specificity of the developed antibodies, the antigens present in the culture medium of Mycobacteria including BCG and M. avium complex were detected. As shown in Table 1, it was confirmed that CFP10 antigen was specifically detected only in the culture medium of Mycobacterium tuberculosis.

Example  4: Gold Nano probe  Manufacturing and Self Micro probe  Produce

41: Gold Nano probe  Produce

Gold nanoparticles having a diameter of 25 nm were prepared by room temperature synthesis using phytochemicals as a reducing agent (Patent Application No. 1020090104313, JW Lee et al. , J. Mater . Chem ., 21: 13316, 2011). A phyto-chemical mixed solution was prepared by dissolving chloroauric acid (HAuCl ₄ .3H ₂ O, 1 mM) in 20 ml of distilled water to prepare a chloroauric acid solution and dissolving 10 mg of isoflavone in a 0.01 M gallic acid solution Respectively. While stirring the chloroauric acid solution, 300 μl of a phytochemical mixed solution was added and stirred for 2 hours to prepare gold nanoparticles.

100 μl of the above CFP10 [G2] antibody (32 μg / ml), to which gold binding protein (GBP) was bound, was added to 7.5 ml of the gold nanoparticle solution, and the mixture was mixed at 37 ° C. and 100 rpm for 30 minutes Followed by stirring to prepare gold nanoparticles coated with the antibody. To prevent aggregation and nonspecific adsorption, 700 B of BSA solution (1 mg / ml) was mixed and then stirred at 37 캜 for 1 hour at 100 rpm.

42: Self Micro probe  Produce

Gold cluster particles were attached to the surface of the magnetic microprobe to coat the surface of the microbeads with the antibody bound to the gold binding protein. 300 μl (10 mg / ml) of microbeads (Invitrogen, Carlsbad, USA) was self-separated and collected on the wall of the reaction vessel and washed with the same volume of 0.01 M PBS buffer (pH 7.4) . 300 쨉 l of the phytochemical mixed solution prepared in Example 2 was added to the washed beads and ultrasonicated for 20 minutes. Then, 2 ml of 1.27 mM chloroauric acid solution was added, ultrasonicated again for 20 minutes, and washed three times Finally, magnetic microbeads with gold clusters were fabricated. The microbeads were resuspended in 1 ml of PBS and 100 쨉 l of the antiCFP10 [G3] antibody (0.255 mg / ml) bound with Gold Binding Protein (GBP) was added and mixed. Lt; / RTI > at 100 rpm. To remove antibodies that were not immobilized on gold clusters attached to the surface of microbeads, they were washed three times and 1 ml of BSA solution (1 mg / ml) was added to prevent nonspecific adsorption. BSA was again removed by washing three times, and then redispersed in 8 ml of PBS solution.

Example  5. Using nanobiosensors Magnetic migration  Immune response and its use CFP 10 Antigen detection

500 μl of the magnetic microprobe (0.375 mg / ml, 2.62 to 4.50 × 10 ⁸ beads / ml) prepared in Example 42 and 150 μl of the culture solution of Mycobacterium tuberculosis (diluted with PBS: 1: 2) , 500 쨉 l of gold nano-probe (~ 4.25 × 10 ¹¹ particles / ml) prepared in Example 4-1 was added, and the mixture was slowly stirred at 37 ° C for 1 hour.

In the reaction vessel, a nanocomposite is formed in which a plurality of CFP10 target antigens and gold nanoprobe are bonded in a sandwich structure to one magnetic microprobe by an immune reaction, and the number of nanocomposites is proportional to the quantification of the incorporated CFP10 antigen. The nanocomposite (gold nanoprobe CFP10 antigen magnetic microprobe) formed by putting a magnet on the bottom of the reaction vessel was separated, and the concentration of the remaining gold nanoprobe was measured by measuring the absorbance using an ultraviolet visible spectrophotometer (UVvis spectrometer) 8).

Example  6. Tuber culture tube with double syringe filter ( Artificial filter cap  tube) and Magnetic migration  Tuberculosis diagnostic test using immunoassay

Samples of sputum specimens from clinical patients were co-cultured in a Mycobacterium growth indicator tube (MGIT) and an artificial filter cap tube (AFT) equipped with a double syringe filter prepared in Example 1 for the diagnosis of tuberculosis. At this time, the result of the MGIT incubator is for specifying the timing of each sample because the time at which each sample is positive is different. In other words, when a positive reaction was confirmed in MGIT, samples of AFT incubator were stopped and sampled, and the mycobacterial antigen CFP10, which was well cultured and secreted in the culture medium, was analyzed by magnetophoretic immunoassay.

Pretreatment of sputum specimens was performed by vortexing 4% NaOH to 23 times the volume of sputum and then vortexing until the viscosity disappeared. After washing with sterilized PBS, centrifugation (4,000 rpm, 4 ° C, 20 minutes) pellet) was resuspended in PBS.

Liquid culture was performed using MGIT 960 system. 7 mL of 7H9 broth, MGIT supplement and 800 μL of PANTA mixture were added to MGIT, and 500 μl of sputum pretreated sample was inoculated. The AFT culture was carried out by transferring broth media from AIT to AFT, adding 800 μl of MGIT supplement and PANTA mixture, and then inoculating 500 μl of sputum pretreated sample and control (sterilized PBS 1X) Respectively. After that, samples were taken and filtered to obtain culture filtrate, which was then diagnosed by magnetophoretic immunoassay, and compared with MGIT positive readings.

Ten specimens were cultured. The specimens consisted of 5 Mycobacterium tuberculosis (Mtb) and 5 Non-Tuberculosis Mycobacteria (NTM) determination samples. As shown in Table 2 and FIG. 9, in the comparative analysis using 10 clinical samples, the diagnosis of the magnetic resonance immunoassay showed a sensitivity of 80% and a specificity of 100%.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> ChungNam University Industry academic cooperation foundation          Chung-Ang University          PuSan University Industry academic cooperation foundation <120> Nano biosensor bound with antibody against Mycobacterium          tuberculosis specific antigen and Method for Detecting          Mycobacterium tuberculosis Using the Same &Lt; 130 > P14-B056 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 31 <212> DNA &Lt; 213 > CFP-10-F ' <400> 1 ggccggggat ccatggcaga gatgaagacc g 31 <210> 2 <211> 31 <212> DNA <213> CFP-10-R ' <400> 2 ggccgggaat tcgaagccca tttgcgagga c 31 <210> 3 <211> 114 <212> PRT <213> CFP-10 [G2] _HC <400> 3 Gln Leu Val Gln Ser Gly Gly Asp Leu Ile Lys Pro Gly Ser Ser Leu   1 5 10 15 Lys Leu Ser Cys Val Thr Ser Gly Phe Pro Phe Asp Asp Phe Ala Met              20 25 30 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly          35 40 45 Ile Thr Cys Leu Ser Gly Thr Ile Ser Tyr Ala Asp Ser Ala Lys Gly      50 55 60 His Phe Pro Leu Ser Arg Asn Asn Pro Gln Asn Ser Leu Ser Leu Glu  65 70 75 80 Met Lys Thr Arg Lys Pro Lys Asn Thr Ala Val Asn Tyr Gly Ala Lys                  85 90 95 Ser Phe Tyr Gly Leu Asp Val Trp Gly Asp Gly Thr Thr Val Ser Val             100 105 110 Ser Ala         <210> 4 <211> 101 <212> PRT <213> CFP-10 [G2] _LC <400> 4 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Tyr Ser Asn Ile Gly Thr Asn              20 25 30 Tyr Val Tyr Cys Tyr His Gln Leu Pro Gly Thr Ala Pro Asn Leu Ser          35 40 45 Ser Lys Arg Ile Leu Ser Gly Pro Gln Cys His Asp Arg Phe Ser Gly      50 55 60 Ser Ser Leu His Ser Ala Ser Leu Pro Ser Val Thr Pro Val Glu Asp  65 70 75 80 Glu Val Ile Ile Phe Val Pro His Gly Met Lys Ser Leu Asn Ala Tyr                  85 90 95 Phe Arg Gly Gly Thr             100 <210> 5 <211> 117 <212> PRT <213> CFP-10 [G3] _HC <400> 5 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr              20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met          35 40 45 Ala Phe Ile Trp Phe Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Met Gly Ile Ala Asp Gly Met Asp Val Trp Gly Gln Gly Thr             100 105 110 Thr Val Thr Val Ser         115 <210> 6 <211> 97 <212> PRT <213> CFP-10 [G3] _LC <400> 6 Leu Thr His Leu Pro Ser Thr Ser Gly Thr Pro Glu Gln Arg Val Ile   1 5 10 15 Met Ser Cys Ser Gly Ser Ala Pro Asp Arg Ile Tyr Thr Val Asn Trp              20 25 30 Tyr Leu Arg Ser Ser Thr Gly Pro Leu Thr Pro His Leu Ser Asn Asn          35 40 45 Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly      50 55 60 Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala  65 70 75 80 Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Phe Gly Gln                  85 90 95 Gly    

Claims (10)

A nano-biosensor for detecting Mycobacterium tuberculosis containing the following.
(i) a metal nanoprobe immobilized with an antibody against a tubercle bacilli secreted antigen to which a metal binding protein is bound; And
(ii) a magnetic microbe probe having a metal binding protein bound to a magnetic microbead having a surface coated with a gold cluster particle, and an antibody against a Mycobacterium tuberculosis secretory antigen having the above epitope and another epitope bound thereto.
delete delete The nano-biosensor for detecting Mycobacterium tuberculosis according to claim 1, wherein the Mycobacterium tuberculosis-derived antigen is CFP10.
5. The nano-biosensor for detecting Mycobacterium tuberculosis according to claim 4, wherein the antibody of the CFP10 has the heavy chain of SEQ ID NO: 3 or 5 and the light chain of SEQ ID NO: 4 or 6. [
The nano-biosensor according to claim 1, wherein the metal is selected from the group consisting of gold, silver, platinum, copper and zinc.
delete A method for detecting mycobacteria comprising the steps of:
(a) contacting the culture filtrate containing the Mycobacterium tuberculosis-producing antigen with the nano-biosensor of any one of claims 1 and 4 to 6, Forming a complex; And
(b) separating the formed nanocomposite with a magnet, and measuring the absorbance.
The method for detecting Mycobacterium tuberculosis according to claim 8, wherein the cultured filtrate in step (a) does not contain Mycobacterium tuberculosis but contains only Mycobacterium tuberculosis secretion antigen.
[9] The method of claim 8, further comprising the step of analyzing the presence or absence of the tubercle bacilli secretory antigen or the concentration of the tubercle bacilli antigen after the step (b).

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