KR101135668B1 - Composition for diagnosis of M. Tuberculosis and Method for diagnosis using the same - Google Patents

Abstract

The present invention relates to the diagnosis of Mycobacterium tuberculosis, and more specifically, to a thisulfate sulfurtransferase CysA2 (CysA2) antigen, or further to a heat shock protein hspX (HspX) or periplasmic phosphate-binding protein PstS1 (PstS1). A method for diagnosing M. tuberculosis by contacting an antigen with a serum sample to determine whether an antigen-antibody complex is formed.

The diagnostic compositions and methods of the present invention can be used to more accurately diagnose M. tuberculosis.

Description

M. Composition for diagnosis of M. Tuberculosis and Method for diagnosis using the same}

The present invention relates to M. tuberculosis diagnosis, and more particularly, to a composition for diagnosing M. tuberculosis infection comprising CysA2 antigen, or additionally HspX or PstS1 antigen and CysA2 antigen, or additionally HspX or PstS1 antigen The present invention relates to a method for diagnosing M. tuberculosis by measuring whether an antigen-antibody complex is formed in contact with a sample.

TB (tuberculosis) is a worldwide disease that affects 7 to 800 million new patients every year, and one-third of the world's population is infected with Mycobacterium tuberculosis (Corbett EL et al., Arch Intern Med. 2003 May 12; 163 (9): 1009-21). For effective control of TB, it is important to identify and treat active disease patients early so that transmission between humans is blocked.

Traditional bacteriological diagnostic methods have low sensitivity and require a long time to diagnose, thus limiting the exact initial diagnosis of TB, causing treatment delays. Thus, the focus has been on developing rapid and reliable diagnostics for active TB based on sputum, blood and other clinical specimen tests. Such methods include molecular level methods such as PCR and IFN- [gamma] release assays useful for the detection of latent TB infections, which are rapid and sensitive methods. However, the method is not suitable for use in the field. Serological tests based on antibody detection are simple, inexpensive, and readily applicable in conditions commonly encountered in developing countries. In addition, the test is useful for the diagnosis of patients who are unable to produce enough sputum, are sputum smear smear, or are suspected of having extrapulmonary TB (Wilkins EG et al., Lancet. 1990 Sep 15; 336 (8716): 641-4).

Many mycobacterial antigens have been applied to detect antimycobacterial antibodies specific to the clinic. However, serological diagnostic methods that can have satisfactory levels of sensitivity and specificity for the diagnosis of TB due to heterogeneous immune responses due to the diversity of antigen recognition depending on disease stage as well as the immunogenetic differences of patients. Lyashchenko K et al., Infect Immun. 1998 Aug; 66 (8): 3936-40; Steingart KR et al., Clin Vaccine Immunol. 2009 Feb; 16 (2): 260-76.Epub 2008 Dec 3).

The present inventors have made a more accurate diagnosis of TB by using CysA2 antigen, which has not been used as a serological target in the past, while making efforts to develop a serological diagnostic method that can have a satisfactory level of sensitivity and specificity for the diagnosis of TB. It was confirmed that this can be done, and the present invention has been completed.

One object of the present invention is to provide a composition for diagnosing M. tuberculosis infection comprising a CysA2 antigen, or additionally an HspX or PstS1 antigen.

Another object of the present invention is to provide a method for diagnosing M. tuberculosis infection by measuring the formation of an antigen-antibody complex by contacting a CysA2 antigen, or HspX or PstS1 antigen, with a serum sample.

The present invention relates to a composition for diagnosing M. tuberculosis (Mycobaterium tuberculosis) infection comprising a CysA2 antigen.

CysA2 (Rv0815c; thiosulfate sulfurtransferase CysA2) is a sulfurtransferase involved in the formation of thiosulfate and is increased in standing culture of M. tuberculosis. However, the protein has never been disclosed as a serological target.

The diagnostic composition of the present invention preferably comprises a CysA2 antigen and additionally an HspX or PstS1 antigen. In addition, the diagnostic composition of the present invention may comprise a CysA2 antigen and further HspX and PstS1 antigen.

HspX (16kDa; heat shock protein hspX) is a heat shock protein that is overexpressed in the resting phase and is essential for the growth of mycobacteria in hypoxic microenvironmental conditions in host macrophages. A positive patient showed a sensitivity of 62%. The antigen is used incorporating in a commercially available kit (Pathozyme TB complex). PstS1 (38 kDa; periplasmic phosphate-binding protein PstS1) is the most widely studied serological antigen and is used as a major component in some commercially available kits (ICT Tuberculosis AMRAD-ICT, RAPID test TB and PATHOZYME-MYCO). The antigen shows a variety of specificity depending on the condition of the patient, low specificity in TB patients negative sputum test.

The diagnostic composition of the present invention includes a CysA2 antigen together with an HspX or PstS1 antigen, and thus, TB can be diagnosed more accurately with higher sensitivity when diagnosing TB using only the HspX and / or PstS1 antigen (see Table 3 and Table 4). ).

When the diagnostic composition of the present invention comprises two or more antigens, it may be included in the form of a cocktail (cocktail) of the antigen so that the two or more antigens can contact the serum sample at the same time. In addition, each antigen may be contained in a separate container so that each antigen may be separately contacted with a serum sample.

According to the present invention, the diagnosis of M. tuberculosis is an antibody that specifically binds to the diagnostic antigen of the present invention in the serum sample by measuring whether the antigen-antibody complex is formed by contacting the diagnostic antigen of the present invention with a serum sample. It can be determined by the method of confirming the existence of. As used herein, the term 'antigen-antibody complex' refers to a combination of an antigen protein of the present invention and an antibody that specifically recognizes it in a serum sample.

The antigenic protein of the present invention is derived from M. tuberculosis and can be produced by recombinant methods based on DNA sequences. When using a recombinant technique, a nucleic acid encoding a protein is inserted into an appropriate expression vector, the host cell is cultured so that the target protein is expressed in the transformant transformed with the recombinant expression vector, and then It can be obtained by recovering the protein.

The diagnostic composition of the present invention may further include reagents known in the art used for immunological analysis in addition to the diagnostic antigen.

Immunological analysis in the above may include any method that can measure the binding of the antigen and the antibody. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunosorbent assay (ELISA), immunoblotting, and PAR assays. Farr assay), immunoprecipitation, latex aggregation, erythrocyte aggregation, non-turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence.

Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

The label capable of generating a detectable signal enables qualitatively or quantitatively measuring the formation of an antigen-antibody complex, examples of which include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and redox. Molecular and radioisotopes can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidase, hexokinase, malate dehydrogenase, and glucose-6 Phosphate dehydrogenase, invertase and the like can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, fluorine isothiocyanate, etc. may be used. Ligands include biotin derivatives, and luminescent materials include acridinium esters, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, and the redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone and hydroquinone. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. However, any of those that can be used in immunological assays other than those exemplified above may be used.

On the other hand, the diagnostic composition of the present invention can be provided in a fixed state using a variety of methods known on a suitable carrier or support to increase the speed and convenience of diagnosis ( Antibodies : A Labotory Manual, Harlow & Lane Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, poly acrylamide, polyester, pam, filter paper, ion exchange resins, plastic films, plastic tubes , Glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible form, for example spherical (bead), cylindrical (test tube or inner surface of the well), planar (sheet, test strip).

Preferably, M. tuberculosis diagnostic composition of the present invention may be provided in the form of a kit or microarray.

The diagnostic kit may be provided, for example, in the form of a lateral flow assay kit based on immunochromatography to detect an antibody against the antigen of the present invention in a sample. Lateral flow assay kits include a sample pad to which a sample is applied, a release pad coated with a detection antibody, a developing membrane (e.g., nitro) in which the sample is moved and separated and an antigen-antibody reaction occurs. Cellulose) or strip, and an absorption pad.

The microarray generally attaches an antigen on a slide glass surface treated with a specific reagent to detect an antibody that specifically attaches to the antigen by an antigen-antibody reaction.

The present invention relates to a method for diagnosing M. tuberculosis by contacting a CysA2 antigen with a serum sample to determine the formation of an antigen-antibody complex.

The diagnostic method of the present invention preferably diagnoses M. tuberculosis by measuring the formation of an antigen-antibody complex by contacting a CysA2 antigen with HspX or PstS1 antigen in addition to a serum sample. In addition, M. tuberculosis can also be diagnosed by contacting the CysA2 antigen with additional HspX and PstS1 antigens with serum samples to determine the formation of antigen-antibody complexes.

When two or more antigens are contacted with a serum sample in the diagnostic method of the present invention, a cocktail of two or more antigens may be contacted with the serum sample. In addition, M. tuberculosis can be diagnosed by separately contacting each antigen with a serum sample and measuring the formation of an antigen-antibody complex for each antigen, and then combining the results.

Specific analytical methods for measuring the formation of antigen-antibody complexes by the above method, Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunity Precipitation assays, complement fixation assays, FACS, protein chips, and the like, but are not limited thereto.

According to the present invention, M. tuberculosis can be diagnosed more accurately with high sensitivity by contacting the serum sample with the CysA2 antigen alone or in combination with other antigens to diagnose M. tuberculosis.

In particular, the diagnostic composition and method of the present invention can be easily applied in the medical field because it can diagnose M. tuberculosis from serum samples.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Materials and Methods

1-1. patient

A total of 265 serum samples were obtained from TB patients and healthy controls (Table 1). Serum samples from lung TB patients were collected from Kunyang University Hospital (Daejeon, Korea) and Korea University Ansan Hospital (Ansan, Korea) two weeks before or within antituberculosis chemotherapy. TB diagnosis was based on clinical evaluation, sputum smear and culture, and / or radiographic findings. TB patients diagnosed by clinical and radiological findings were divided into AFB positive group (n = 101) including smear positive and / or culture positive patients and AFB negative group (n = 49) including smear and culture negative patients. Classified. Healthy control serum was obtained from 115 medical students with no clinical history associated with TB; All control patients were negative on smear cultures for chest X-rays and acid-fast bacilli. None of the subjects had a history of diabetes or steroid therapy and all were negative for human immunodeficiency virus.

This example was approved by the Ethics Committee of Konyang University and Korea University Ansan Hospital, with prior consent from each participant.

1-2. Antigen manufacturing

Five antigens (two natural and three recombinant) were prepared. Two natural antigens, Ag85 (antigen 85 complex) and pstS1 antigen , were synthesized using the method described by Lee et al. (Lee JS et al., Respirology. 2008 May; 13 (3): 432-7). Purification from the filtrate of M. Tuberculosis culture. Recombinant CFP-10 antigen was prepared according to the procedure disclosed by Shin et al. (Shin AR et al., Clin Vaccine Immunol. 2008 Dec; 15 (12): 1788-95. Epub 2008 Oct 22). Recombinant plasmids encoding hspX were provided by the TB Vaccine Testing and Research Materials. CysA2 and Rv0652 were cloned using pET28a. To produce a recombinant plasmid, each gene was amplified by PCR using genomic DNA of M. tuberculosis and the following primers as templates.

For CysA2,

Forward: 5'-CTCGAGATGGCACGCTGC-3 '(SEQ ID NO: 1)

Reverse: 5'-GAATTCTCAGCTTCCCAA-3 '(SEQ ID NO: 2)

For Rv0652,

Forward: 5'-GGATCCATGGCAAAGCTC-3 '(SEQ ID NO: 3)

Reverse: 5'-GAATTCCTACTTGACGGT-3 '(SEQ ID NO: 4).

PCR products of CysA2 were digested with XhoI and EcoRI, and products of Rv0652 were digested with BamHI and EcoRI. Both products were inserted into pET28a and clones formed were sequenced. Recombinant plasmids were transformed into E. coli BL21 cells for protein overexpression. The culture was performed at 37 ° C. until the optical density (OD) at 600 nm became 0.4 to 0.5, and then induced with 1 mM IPTG (isopropyl-D-thiogalactopyranoside) (ELPIS-Biotech, Daejeon, South Korea). Cells were recovered by centrifugation and suspended in 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 20 mM imidazole and 1 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO); Lysed by ultrasound. Recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose chromatography according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Each purification step was analyzed by dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and immunoblot using anti-His antibody (Invitrogen) with Coomassie brilliant blue dye.

3.Enzyme-linkded immunosorbent assay (ELISA)

Antibody titers were determined by ELISA as known (Shin AR et al., Clin Vaccine Immunol. 2008 Dec; 15 (12): 1788-95. Epub 2008 Oct 22). Polystyrene 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with a single antibody at 100 ng / well and a cocktail of two or three antigens at 50 ng / well overnight in 0.05 M carbonate buffer (pH 9.6). Serum samples were diluted 1: 200 and 0.1 ml was added to each well. After washing, 0.1 ml of peroxidase-conjugated goot anti-human IgG (Sigma) diluted 1: 9,000 was added to each well. The reaction was visualized using tetramethylbenzidine (Sigma) and 0.5% (vol / vol) H ₂ O ₂ , allowed to stand for 5 minutes and stopped with 1N H ₂ SO ₄ . OD was measured at 450 nm using an ELISA microplate reader (Molecular Devices, Sunnyvale, Calif.).

Example 2: Results

2-1. Purification of Recombinant CysA2 and Rv0652

In preliminary experiments with immunoblot using serum from TB patients, two specific proteins recognized by pooled serum were identified. To investigate their serodiagnostic potential, proteins CysA2 (Rv0815c) and Rv0652 were expressed as 6x His tagged proteins in E. coli and purified from ultrasonic extracts by Ni-NTA affinity chromatography. Purified proteins were analyzed by SDS-PAGE (FIG. 1) and detected by immunoblotting using anti-His antibodies. Purified CysA2 and Rv0652 showed bands of about 39 kDa and 23 kDa, respectively, on the PAGE gel.

2-2. Serological response to antigens of mycobacteria

Since the two proteins CysA2 and Rv0652 have never been disclosed as serological targets, their serological activity was compared with four proteins known as serological antigens (PstS1, Ag85, CFP-10, HspX). Serum IgG antibodies to each antigen were measured in AFB positive and AFB negative groups and healthy controls of lung TB patients (Table 2 and FIG. 2). As expected, there was a heterogeneous response to each antigen. Antibody responses to all tested antigens in lung TB patients were significantly higher than controls (P <0.001). However, there was no significant difference in response to antigen between the AFB positive and negative groups.

ROC curves for each antigen were constructed by constructing true positive rates with each specific value of the indicator variable for healthy control and TB patients. Indicator variables with high discrimination power will show curves with regions close to one. The AUC values of HspX, CysA2, PstS1 and CFP-10 ranged from 0.87 to 0.82 (Figure 3 and Table 3). Using the cut off value when the accuracy reached the maximum on the ROC curve for each antigen provided the highest sensitivity as HspX and CysA2 were recognized by the sera of TB patients 106/150 (71%). In contrast, cocktail ELISA with HspX, PstS1 and CysA2 had higher diagnostic sensitivity and produced the highest AUC values (0.90 to 0.93) compared to single antigen use (Table 3). Specificity of all tested antigens ranged from 90% in Ag85 to 96% in PstS1 based on the highest specificity. However, Rv0652 was not suitable for diagnostic purposes in view of AUC values and sensitivity.

To investigate the possibility of CysA2 as one of the diagnostic antigens, the combinatorial effects of the results obtained from CysA2, HspX and PstS1 antigens were analyzed. The sensitivity was increased to 95% by combining two antigens (CysA2 + HspX) and three antigens (CysA2 + HspX + PstS1) (Table 3). In particular, 18 of these were positive only in CysA2, 6 were positive only in HspX, 1 positive in PstS1, and 46 positive in three antigens. Specificity in the healthy control group was slightly reduced than the single antigen, but was found to be high (87-83%). These results suggest that CysA2 is useful for increasing the sensitivity for detecting M. tuberculosis specific antibodies, especially when combined with HspX or PstS1.

With cutoff values of 100% specificity, PstS1 and CysA2 provided higher sensitivity than other antigens (57% and 41%, respectively), and also cocktails or data combinations of two or three antigens The highest sensitivity was provided (Table 3).

2-3. Serological performance of samples and antigens in AFB negative and AFB positive patients

According to current diagnostic methods, the diagnosis of sputum smear smear and / or culture negative lung patients is more difficult than positive TB patients. Thus, differences between the sensitivity of each antigen in the AFB positive and negative groups were analyzed. For all antigens tested, the sensitivity to the samples of AFB positive patients was somewhat higher than that of AFB negative patients, but this difference was not significant (Table 4). The exception was PstS1 and its sensitivity was significantly lower in the AFB negative group than in the AFB positive group. This shortcoming of PstS1 was complemented by the cocktail and combined results of CysA2 and HspX.

Figure 1 shows the results of SDS-PAGE analysis for CysA2 (A) and Rv0652 (B) prepared according to the present invention.

FIG. 2 shows antibody response to Ag85, PstS1, CFP-10, HspX, CysA2, Rv0652 and mixtures thereof in AFB positive and negative lung TB patients and healthy controls.

3 shows ROC curves for six antigens and their cocktails in healthy controls and lung TB patients.

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Claims (6)

M. tuberculosis diagnostic composition comprising a CysA2 (thiosulfate sulfurtransferase CysA2) antigen. The method of claim 1, M. tuberculosis diagnostic composition further comprises a heat shock protein hspX (HspX) antigen or a periplasmic phosphate-binding protein PstS1 (PstS1) antigen. The method of claim 1, M. tuberculosis diagnostic composition, characterized in that it further comprises a HspX antigen and a PstS1 antigen. A method of providing information for M. tuberculosis diagnosis by contacting a CysA2 antigen with a serum sample to determine whether an antigen-antibody complex is formed. The method of claim 4, wherein Further providing information for M. tuberculosis diagnosis by contacting a HspX antigen or PstS1 antigen with a serum sample to determine whether an antigen-antibody complex is formed. The method of claim 4, wherein Further providing information for diagnosing M. tuberculosis by contacting the HspX antigen and PstS1 antigen with serum samples to determine the formation of antigen-antibody complexes.

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