Characterization of the secreted MPT53 antigen of Mycobacterium tuberculosis

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.     

Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis.

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.     

结核分枝杆菌MPT64重组母牛分枝杆菌免疫学特性

目的:研究结核分枝杆菌MPT64重组母牛分枝杆菌疫苗免疫学特性.方法:用分泌表达MPT64的重组母牛分枝杆菌疫苗免疫BALB/c小鼠,ELISA法检测免疫小鼠的特异性抗体滴度和抗体亚类.分离免疫小鼠脾淋巴细胞,检测淋巴细胞增殖、IFN-γ和IL-12产生水平、CD4+细胞和CD8+细胞数、脾淋巴细胞特异性CTL杀伤效应.毒株攻击后对脾脏细菌负荷计数.结果:MPT64重组母牛分枝杆菌疫苗免疫可诱导小鼠高水平的体液免疫应答,免疫小鼠脾淋巴增殖明显,IFN-γ和IL-12含量增加,CD4+和CD8+细胞百分比明显增加,CTL杀伤效应明显,对MTB H37Rv攻击后有一定的保护作用.结论:MPT64重组母牛分枝杆菌疫苗可诱导小鼠有效的体液和细胞免疫应答,有可能作为新型TB疫苗候选.     

Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis.

Proteins that are actively secreted by Mycobacterium tuberculosis generate immune responses in the infected host. This has prompted the characterization of protein components of mycobacterial culture filtrates to develop subunit vaccines and immunodiagnostic reagents. Fractionation of filtrates of M. tuberculosis cultures has yielded an abundant protein called MPT63, which has an apparent molecular mass of 18 kDa. We report the molecular cloning and nucleotide sequence of the mpt63 gene, purification of recombinant MPT63 antigen from Escherichia coli cells, and serological characterization of MPT63. Nucleotide sequence analysis of mpt63 identified an open reading frame encoding a protein of 159 amino acids (aa) consisting of a 29-aa secretion signal peptide and a 130-aa mature MPT63 protein. Recombinant MPT63 protein, purified from E. coli cells, and native MPT63, purified from M. tuberculosis culture filtrates, were indistinguishable in serological assays. Thus, the recombinant protein constitutes a valuable reagent for immunological studies. MPT63 evoked humoral immune responses in guinea pigs infected with virulent M. tuberculosis by the aerosol route. The mpt63 gene is found only in species of the M. tuberculosis complex, as shown by DNA hybridization experiments. Moreover, polyclonal antibody against MPT63 does not cross-react with proteins of a common environmental mycobacterial species, Mycobacterium avium. The absence of cross-reactive epitopes makes MPT63 an attractive candidate as an M. tuberculosis complex-specific diagnostic reagent. In particular, evaluation of MPT63 as an M. tuberculosis complex-specific reagent for diagnostic skin testing is under way.     

Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis.

The T-cell response of human donors to secreted antigen fractions of Mycobacterium tuberculosis was investigated. The donors were divided into five groups: active pulmonary tuberculosis (TB) patients with minimal and with advanced disease, Mycobacterium bovis BCG-vaccinated donors with and without contact with TB patients, and nonvaccinated individuals. We found that patients with active minimal TB responded powerfully to secreted antigens contained in a short-term culture filtrate. The response to secreted antigens was mediated by CD4+ Th-1-like lymphocytes, and the gamma interferon release by these cells was markedly higher in patients with active minimal TB than in healthy BCG-vaccinated donors. Patients with active advanced disease exhibited depressed responses to all preparations tested. The specificity of the response to secreted antigens was investigated by stimulating lymphocytes with narrow-molecular-mass fractions of short-term culture filtrate obtained by the multielution technique. Considerable heterogeneity was found within the donor groups. Patients with active minimal TB recognized multiple secreted targets, but interestingly, six of eight patients demonstrated a predominant recognition of a low-mass (< 10-kDa) protein fraction which induced high levels of gamma interferon release in vitro. Only a few of 12 previously characterized secreted antigens were recognized by T cells isolated from TB patients, suggesting the existence of a number of as yet undefined antigenic targets among secreted antigens.     

Molecular Characterization of MPT83: a Seroreactive Antigen of Mycobacterium tuberculosis with Homology to MPT70

The Mycobacterium bovis antigens MPB70 and MPB83 are homologous cross-reactive proteins. It has been reported previously that MPB83 is glycosylated and exists in two forms with apparent molecular masses of 23 kDa and 25 kDa, whereas the apparent molecular mass of MPB70 is 22 kDa. Using a monoclonal antibody, SB10, which recognizes an epitope common to both MPB70 and MPB83, we compared the expression of these proteins in M. bovis BCG, virulent M. bovis and virulent Mycobacterium tuberculosis by Western blotting of bacterial lysates. The previously described pattern of high and low producing substrains of BCG for MPB70 was also applicable for MPB83. Virulent M. bovis was found to express high levels of MPB70 and MPB83. Immunoblotting experiments using sera from Balb/c mice infected with live M. tuberculosis H37Rv revealed that although the MPB83 homologue of M. tuberculosis, MPT83, is expressed at low levels in M. tuberculosis when grown in vitro, the protein is highly immunogenic during infection with live bacteria. A clone from a mycobacterial shuttle cosmid library of M. tuberculosis H37Rv was isolated which expressed both MPT70 and MPT83. Genetic analysis of this cosmid revealed that MPT70 and MPT83 were encoded by separate genes with the gene encoding MPT83 situated 2.4 kb upstream of mpt70. Both genes are transcribed in the same direction. The gene encoding MPT83 was cloned and DNA sequencing revealed an open reading frame of 660 bp encoding a protein with a predicted molecular mass of 22 kDa. Recombinant MPT83 was expressed in Escherichia coli from the native AUG initiation codon by translational coupling. In E. coli MPT83 was expressed as a 23 kDa antigen whereas in the rapid growing mycobacterium Mycobacterium smegmatis the protein was expressed as a 25 kDa protein indicating post-translational modification of the protein by M. smegmatis. In recombinant M. smegmatis MPT83 was predominantly cell associated whereas MPT70 was secreted into the culture medium. Amino acid sequence comparison between MPT83 and MPT70 revealed a 61% identity between the proteins, although little homology was apparent at the amino terminus. In MPT83 this region contained a typical lipoprotein signal peptide cleavage motif and a putative signal motif for O glycosylation. Both these motifs were absent from the amino acid sequence of MPT70.     

Monoclonal antibodies to a 28,000 mol. wt protein antigen of Mycobacterium leprae.

Monoclonal antibodies (MoAb) have been used to analyse a protein antigen from Mycobacterium leprae with a subunit mol. wt of 28,000 daltons. Three different patterns of species specificity were observed with two antibodies being specific for M. leprae, two partially specific, and one broadly cross-reactive amongst all mycobacteria. Competitive binding and sandwich assays demonstrated that the specific and partially specific antibodies recognized closely related regions of the molecule while the cross-reactive antibody recognized a spatially separate epitope on the same polypeptide chain. Identification of specific and cross-reactive epitopes on a single antigenic molecule may be of considerable importance for understanding the functioning of the cell-mediated immune system during leprosy infection and the use of MoAb for such analyses is discussed.     

Characterization of a novel Mycobacterium bovis secreted antigen containing PGLTS repeats.

Serum from naturally infected cattle was used to identify a novel Mycobacterium bovis antigen from an expression library. The first recombinant product identified was a fusion protein with lacZ (55 kDa). A clone containing the whole gene was also obtained. This clone expressed a 38-kDa protein. A rabbit serum against the recombinant antigen reacts in M. bovis supernatants with two proteins of 36 and 34 kDa. The new protein was called P36/P34. The gene cloned has a deduced amino acid sequence with a predicted molecular mass of 28 kDa, showing a characteristic signal sequence for exportation. The protein bears partial homology to a 28-kDa protein from M. leprae. An interesting feature of the P36/P34 sequence is that it contains several PGLTS repeats, which are not present in the M. leprae protein. Antigenic determinants seem also to be conserved between the two proteins because sera from leprosy patients recognized the recombinant M. bovis protein. The discrepancy among the molecular mass deduced from the sequence (28 kDa), that of the recombinant protein in Escherichia coli (38 kDa), and that of the native protein in M. bovis (36 and 34 kDa) could be attributed to posttranslational modifications or to the high proline content that may alter the migration properties of the protein. This antigen seems to be immunodominant during bovine tuberculosis, because 8 of 9 serum specimens from diseased cattle are reactive. The homology among the M. leprae 28-kDa protein, the protein described in this article, and a recently described M. tuberculosis protein suggests the existence of a new protein family in mycobacteria.     

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.     

Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-gamma) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-gamma by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-gamma and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.     

Recombinant early secreted antigen target 6 protein as a skin test antigen for the specific detection of Mycobacterium tuberculosis infection

Although the delayed-type hypersensitivity skin test reaction to tuberculin purified protein derivative (PPD) is used worldwide for tuberculosis (TB) detection, it is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacille Calmette-Guérin (BCG) vaccination or infection with non-tuberculous Mycobacteria. As a result, there is an urgent need for a more specific diagnostic tool for TB. This study reports the skin reactions of guinea pigs and human volunteers to recombinant early secreted antigen target 6 (rESAT6), a secretory protein found only in MTB, M. bovis and few other mycobacterial species. These volunteers had varying histories of BCG vaccination and exposure to MTB, allowing us to determine the specificity of their response to TB exposure. Our results show that 1.0 microg of the purified MTB rESAT6 antigen elicited a positive skin response in both animals and humans exposed to MTB, as well as in animals exposed to M. bovis and M. marinum, all species of Mycobacteria that contain the gene for early secreted antigen target 6 (ESAT6). ESAT6 appears to be more specific to MTB infection than PPD, as demonstrated by the fact that we saw no skin responses in the BCG-vaccinated volunteers, nor in the guinea pigs sensitized with BCG vaccine, or with Mycobacteria that do not contain the gene encoding ESAT6. We believe that this is the first report of the use of a rESAT6 protein in a skin test in human volunteers, and that these data support its use in the specific detection of MTB infection.     

Characterization of the Delayed Type Hypersensitivity-Inducing Epitope of MPT64 from Mycobacterium tuberculosis

Mycobacterium tuberculosis secretes several proteins into the extracellular environment, some of which are restricted to the M. tuberculosis complex. One of these antigens is MPT64. Recently, the authors showed that native as well as recombinant MPT64 is able to distinguish between an M. tuberculosis infection and a BCG Danish 1331 vaccination. Improved distinction between tuberculin purified protein derivative (PPD) sensitivity conferred by an M. tuberculosis infection and that induced by a BCG vaccination or infection with environmental mycobacteria would be useful in the control of tuberculosis. In this study, the authors report the mapping and characterization of a Dth-inducing epitope by the use of synthetic peptides in guinea-pigs vaccinated with BCG Danish 1331 or Tokyo. Studies with overlapping synthetic peptides have pinpointed the biological activity to a single Dth-inducing epitope at the carboxyterminal region of MPT64 consisting of 15 residues between amino acids Gly-173 and Ala-187, the core epitope (CE15). A fine mapping using truncated versions of CE15 indicates the epitope is restricted to 13 residues between amino acids Val-174 to Glu-186. However, the optimal Dth reactivity is obtained by CE15. Different modifications of CE15 revealed that a lysine tree construction improves the skin reactivity to a maximum level approaching that of the reactivity to tuberculin PPD.     

Structure of the Mycobacterium tuberculosis antigen 88, a protein related to the Escherichia coli PstA periplasmic phosphate permease subunit.

We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in lambda gt11. The gene encodes a 403-amino-acid-residue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane alpha-helical domains and presents a significant homology to the PstA protein of Escherichia coli. In its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS of E. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria.     

RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection

We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge with Mycobacterium tuberculosis. We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing the M. tuberculosis MPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection against M. tuberculosis challenge in mice. This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine. This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies.     

Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae.

Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both. Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria. Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M. leprae 65-kDa protein. Of the 14 distinct MAbs, 1 recognized an epitope found only on M. leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied. Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.     

The Mycobacterium leprae antigen 85 complex gene family: identification of the genes for the 85A, 85C, and related MPT51 proteins.

The genes for two novel members (designated 85A and 85C) of the Mycobacterium leprae antigen 85 complex family of proteins and the gene for the closely related M. leprae MPT51 protein were isolated. The complete DNA sequence of the M. leprae 85C gene and partial sequences of the 85A and MPT51 genes are presented. As in M. tuberculosis, the M. leprae 85A, 85C, and previously identified 85B component genes are not closely linked on the genome. However, the MPT51 genes of both species localize close to the respective 85A component genes. Like the 85B component, the M. leprae 85A-MPT51 and 85C antigens are recognized by T cells from healthy contacts and leprosy patients.     

Cloning, expression and significance of MPT53 for identification of secreted proteins of Mycobacterium tuberculosis

Based on our N -terminal amino acid sequence of MPT53 and a deduced DNA sequence, we searched for the corresponding gene in the Mycobacterium tuberculosis genomic sequence at the Sanger centre, localizing mpt53 close to mpt70 and mpt83. The gene was cloned and expressed, followed by purification of MPT53 to homogeneity from recombinant M. smegmatis culture fluid. In MPT53 there is 60 % identity with the active site of thioredoxin of M. tuberculosis (MPT46) with two cysteins in a CXXC motif, but MPT53 could not serve as an alternative substrate for thioredoxin reductase. Testing for IgM and IgG1 anti-MPT53 in cattle sera showed that MPT53 is immunogenic following natural and experimental infection with M. bovis. Cloning of mpt53 represents cloning of the last of the 10 proteins originally defined as "secreted proteins" of M. tuberculosis and M. bovis based on determination of their "Localization index" (LI) (J Gen Microbiol 1991;137 : 875-84). The need for a precise definition of the term "secreted protein" is discussed. So far we have observed full concordance between occurrence of an LI value indicating secretion of a protein and occurrence of a signal sequence in the corresponding gene. Signal sequence independent protein secretion in mycobacteria may occur for a limited number of proteins and remains to be established.     

An HLA-DRw53-restricted T-cell epitope from a novel Mycobacterium leprae protein antigen important to the human memory T-cell repertoire against M. leprae.

Cellular immunity mediated by T cells plays a major role in protection against intracellular infections, including leprosy, a chronic disease caused by Mycobacterium leprae. In this work, we describe CD4+ T-cell clones, isolated from healthy humans immunized with M. leprae, which recognize a novel M. leprae protein antigen previously isolated from a lambda gt11 DNA expression library. On the basis of the deduced primary structure of the carboxyl-terminal part of the antigen, we have used a synthetic-peptide approach to exactly define the T-cell epitope recognized. Importantly, major histocompatibility complex restriction studies showed that the epitope is presented by an HLA-DRw53 molecule which is frequently expressed in many populations. In addition, we have demonstrated that a long-term cell-mediated immunity response against the peptide epitope is present after immunization with M. leprae. In conclusion, the M. leprae T-cell epitope described here fulfills the primary criteria for subunit vaccine candidates against leprosy.     

MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex.

Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host. To identify and purify novel proteins in the filtrates of M. tuberculosis cultures, a bacteriophage lambda library of M. tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum. Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes. Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues. We called this gene mtc28. The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide. The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif. Thus, MTC28 is a new member of a group of proline-rich antigens found in M. tuberculosis and Mycobacterium leprae. As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M. tuberculosis complex. Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium. The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M. avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis.     

Dynamic evolution and immunoreactivity of aptamers binding to polyclonal antibodies against MPT64 antigen of Mycobacterium tuberculosis

Antibody responses can be useful markers of tuberculosis infection. However, the established immunoassay diagnostic method is limited by antigenic variability. Replacing the recombinant proteins with aptamers may overcome these antigenic challenges. In this study, we systematically monitored the selection process of aptamers against anti-MPT64 antibodies of Mycobacterium tuberculosis to obtain more aptamers for developing a multisite system to increase the sensitivity of TB serological diagnosis. Twelve high-affinity aptamers with distinctive secondary structures were obtained by analyzing the dynamic evolution of aptamers against anti-MPT64 antibodies in the process of system evolution of ligands by exponential enrichment (SELEX). Pocket and stem-loops were found to be the basis of these aptamers binding to antibodies. Point mutations of highly conserved nucleotides in the pocket and stem-loop structures resulted in decreased affinity of aptamers to targets. To test the potential of these aptamers for future use in a serological diagnostic tool, three high-affinity aptamers with different epitope specificities were applied as capture aptamer in an enzyme-linked immunosorbent assay (ELISA) with sera of TB patients. The results showed that three aptamers all effectively bound anti-MPT64 antibodies from TB patients and had high specificity and sensitivity. These aptamers with high immunoreactivity in human sera may represent an efficient and promising analogue of MPT64 and have potential to substitute MPT64 as a nucleic acid antigen in the serological diagnosis of TB. Moreover, these aptamers with different epitope specificities may facilitate the development of a sandwich assay platform or a multisite system to effectively capture more targets in sera.