Antigens of Mycobacterium tuberculosis Recognized by Antibodies during Incipient, Subclinical Tuberculosis

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of ~12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (~90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.     

Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis

Clinical tuberculosis (TB), whether noncavitary or cavitary, is the late stage of a chronic disease process, since Mycobacterium tuberculosis is a slowly growing organism. Our studies have shown that the profiles of antigenic proteins expressed by the in vivo bacteria that elicit antibodies differ in cavitary and noncavitary TB. To gain insight into antigenic proteins expressed during incipient, subclinical TB, an expression library of M. tuberculosis genomic DNA was screened with sera obtained during subclinical TB from guinea pigs infected with aerosols of M. tuberculosis H37Rv. One of the proteins recognized by antibodies elicited during subclinical TB infection of guinea pigs is the 309-kDa PPE55 (Rv3347c) protein. Genomic hybridization studies suggest that the PPE55 gene is specific to the M. tuberculosis complex and is present in a majority of clinical isolates tested. Antibodies to the C-terminal, approximately 100-kDa fragment of PPE55 (PPE-C) were detectable in sera from 29/30 (97%) human immunodeficiency virus-negative/TB-positive (HIV(-) TB(+)) patients and 17/24 (71%) HIV(+) TB(+) patients tested but not in sera from purified-protein derivative-positive healthy controls, suggesting that the in vivo expression of PPE55 protein correlates with active M. tuberculosis infection. Anti-PPE-C antibodies were also detected in retrospective sera obtained months prior to manifestation of clinical TB from 17/21 (81%) HIV(+) TB(+) individuals tested, providing evidence that the protein is expressed during incipient, subclinical TB in HIV-infected humans. Thus, PPE55 is a highly immunogenic protein that may be useful for differentiating between latent TB and incipient, subclinical TB.     

Antigens of Mycobacterium tuberculosis expressed during preclinical tuberculosis: serological immunodominance of proteins with repetitive amino acid sequences

Four antigens of Mycobacterium tuberculosis that are expressed in vivo after aerosol infection but prior to the development of clinical tuberculosis (TB) in rabbits were identified by immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained 5 weeks postinfection. Three of the proteins identified, PirG (Rv3810), polymorphic GC-repetitive sequence (PE-PGRS; Rv3367), and proline-threonine repetitive protein (PTRP) (Rv0538), have multiple tandem repeats of unique amino acid sequences and have characteristics of surface or secreted proteins. The fourth protein, MtrA (Rv3246c), is a response regulator of a putative two-component signal transduction system, mtrA-mtrB, of M. tuberculosis. All four antigens were recognized by pooled sera from TB patients and not from healthy controls, confirming their in vivo expression during active infection in humans. Three of the antigens (PE-PGRS, PTRP, and MtrA) were also recognized by retrospective preclinical TB sera obtained, prior to the clinical manifestation of TB, from human immunodeficiency virus-TB patients, suggesting that they are potential candidates for devising diagnostic tests for active, preclinical TB.     

Homogeneity of antibody responses in tuberculosis patients

The goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grown Mycobacterium tuberculosis that are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins of M. tuberculosis were fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens of M. tuberculosis which suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936-3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients.     

Enzyme-linked immunosorbent assays using immune complexes for the diagnosis of tuberculosis

The serodiagnosis of tuberculosis has long been the subject of investigation, but we still lack a test with widespread clinical utility. The poor sensitivity and specificity of commercial assays precludes their use as the sole means of diagnosis. All of these assays use mycobacterial antigens adsorbed onto a surface. Little attention has been paid to changes in antigen conformation that may occur as a result of passive coating of these antigens to solid supports like polystyrene. Such changes may cause technical artifacts resulting in false-positive (FP) and false-negative (FN) reactions. We have developed two different enzyme-linked immunosorbent assay (ELISA) systems, in which human serum antibodies and target antigens of Mycobacterium tuberculosis are able to associate and dissociate freely in solution to form immune complexes. In one ELISA, rabbit antibodies against M. tuberculosis, passively coated in the ELISA wells, capture the immune complexes (ICs). In the other ELISA, the ICs are detected by these same rabbit antibodies but are first captured by passively coated goat anti-rabbit IgG. We have compared these two ELISA systems with an ELISA using M. tuberculosis antigens passively adsorbed to the solid polystyrene surface of the plate. We studied sera from 81 patients with tuberculosis and 47 healthy subjects. The differences between tuberculosis (TB) patients and healthy subjects were statistically significant in all three of our ELISA systems. However, the ELISA systems using soluble M. tuberculosis antigens distinguished better between TB patients and healthy subjects than the ELISA using surface-adsorbed M. tuberculosis antigens. We suggest that in the latter ELISA, passive adsorption of the target antigens induces conformational change, generating altered epitopes that are recognized by antibodies present in the serum from even healthy people. These altered conformational epitopes are recognized by antibodies that were originally evoked by antigens other than M. tuberculosis, known as heterophile antigens.     

Immunochromatographic IgG/IgM test for rapid diagnosis of active tuberculosis

For rapid diagnosis and discrimination between active tuberculosis (TB) and other pulmonary diseases, we evaluated the clinical usefulness of detection of serum immunoglobulin IgG and IgM antibodies raised against mycobacterial 38-kDa, 16-kDa, and 6-kDa antigens by a commercial rapid immunochromatographic IgG/IgM test (Standard Diagnostics, South Korea) in 246 serum samples from three groups of patients: (i) 171 patients with active TB (128 with pulmonary TB [pTB] and 43 with extrapulmonary TB [epTB]), (ii) 73 patients with pulmonary non-TB diseases, and (iii) two leprosy patients. The sensitivities of IgG and IgM in patients with active TB (pTB and epTB) were 68.4% and 2.3%, respectively. IgG had the best performance characteristics, with sensitivities of 78.1% and 39.5% in sera from patients with active pTB and epTB, respectively, and a specificity of 100%. The sensitivities of IgM were poor and were similar for pTB and epTB (2.3%). In contrast, specificity was very elevated (100%). The combination of IgG with IgM did not improve its sensitivity. IgG-mediated responses against the mycobacterial 38-kDa, 16-kDa, and 6-kDa antigens might constitute a clinically useful tool for presumptive diagnosis and discrimination of active pTB from other pulmonary diseases. Moreover, based on its simplicity and rapidity of application, it could be a screening tool for active pTB in poorly equipped laboratories.     

Patients with pulmonary tuberculosis develop a strong humoral response against methylated heparin-binding hemagglutinin

Reactivities of human sera against selected recombinant Mycobacterium tuberculosis antigens were assessed by enzyme-linked immunosorbent assay. The results obtained indicate that patients with tuberculosis (TB) do not develop a strong humoral response against PE_PGRS and PPE proteins or against the Ag85B and heparin-binding hemagglutinin (HBHA) recombinant antigens. Conversely, purified methylated HBHA was strongly recognized by sera obtained from TB patients compared to controls.     

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.     

Differential immunogenicity of novel Mycobacterium tuberculosis antigens derived from live and dead bacilli.

Mouse serum raised against killed antigen preparations of Mycobacterium tuberculosis failed to recognize most of the recombinant antigens of M. tuberculosis that were originally identified by reactivity to tuberculosis (TB) patient sera. Similar results were obtained with serum from guinea pigs immunized with live and killed mycobacteria. Antibodies raised against seven random TB patient serum-reactive antigens detected each of these antigens in the sonicate preparation. The nucleotide sequences of the genes for these seven antigens revealed that all represented hitherto unreported genes of M. tuberculosis. Our results suggest differential presentation to the host immune system of the same antigens derived from live and killed mycobacteria.     

Comparison of antibody responses to a potential combination of specific glycolipids and proteins for test sensitivity improvement in tuberculosis serodiagnosis

The humoral response to different proteinaceous antigens of Mycobacterium tuberculosis is heterogeneous among patients with active disease, and this has originated in the proposal to use a combination of several specific antigens to find an efficient serodiagnostic test for tuberculosis (TB). However, to date, comparisons of antibody responses to several antigens in the same population have been carried out without consideration of antigenic cell wall glycolipids. In the present study the presence of immunoglobulin G (IgG), IgM, and IgA antibodies to M. tuberculosis glycolipids (sulfolipid I, diacyltrehaloses, triacyltrehaloses, and cord factor) was compared with the response to four commercially available tests based on the 38-kDa protein mixed with the 16-kDa protein or lipoarabinomannan. Fifty-two serum samples from TB patients and 83 serum samples from control individuals (48 healthy individuals and 35 non-TB pneumonia patients) were studied. Three relevant results were obtained. (i) Smear-negative TB patients presented low humoral responses, but the sera which did react principally showed IgA antibodies to some glycolipidic antigens. (ii) TB patients exhibit heterogeneous humoral responses against glycolipidic antigens. (iii) Finally, test sensitivity is improved (from 23 to 62%) when IgG and IgA antibodies are detected together in tests based on different antigens (proteins and glycolipids). We conclude that it is possible to include glycolipidic antigens in a cocktail of specific antigens from M. tuberculosis to develop a serodiagnostic test.     

Comparison of two 2,3-diacyl trehalose antigens from Mycobacterium tuberculosis and Mycobacterium fortuitum for serology in tuberculosis patients.

Immunoglobulin G antibodies against two 2,3-diacyl trehalose (DAT) antigens from Mycobacterium tuberculosis (DATT) and Mycobacterium fortuitum (DATF) were studied by enzyme-linked immunosorbent assay of 356 serum samples. The sera were obtained from non-tuberculosis-infected individuals (282 serum samples) and tuberculosis patients (74 serum samples). Non-tuberculosis-infected individuals were healthy people (120 serum samples; positive purified-protein-derivative skin test, 60 patients; negative purified-protein-derivative skin test, 60 patients) patients with nontuberculosis lung disease (59 serum samples), contacts of sputum-smear-positive tuberculosis patients (57 serum samples), and human immunodeficiency virus-infected patients with nontuberculosis lung disease (46 serum samples). Of the 74 patients with tuberculosis, 14 were human immunodeficiency virus infected. The sensitivity of the method using DATT was 44.5%, and that with DATF was 48.6%. The specificities with both antigens were 99.1%. There were no significant differences between the mean values for both antigens (P = 0.2815). We therefore concluded that both antigens were interchangeable. As M. fortuitum, a fast-growing mycobacterium, could be a good source of antigen DAT, these results deserve consideration in the serology of tuberculosis.     

Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment

Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively.     

Assessing the serodiagnostic potential of 35 Mycobacterium tuberculosis proteins and identification of four novel serological antigens

Improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement presently available diagnostic methods. The aim of the present study was to identify novel serological targets for use for the future serodiagnosis of tuberculosis (TB). We cloned and expressed 35 M. tuberculosis proteins as recombinant proteins in Escherichia coli and analyzed their serodiagnostic potentials. By a two-step selection process, four superior seroantigens, TB9.7, TB15.3, TB16.3, and TB51, were identified, none of which has been described before. The four novel antigens were tested with panels of sera from smear-positive and smear-negative TB patients from areas both where TB is endemic and where TB is not endemic, with recognition frequencies ranging from 31 to 93% and with a specificity of at least 97%. The single most potent antigen was TB16.3, which had a sensitivity of 48 to 55% with samples from Danish resident TB patients and a sensitivity of 88 to 98% with samples from African TB patients. Importantly, the TB16.3 and the TB9.7 antigens were recognized by more than 85% of the samples from TB patients coinfected with human immunodeficiency virus, a patient group for which it is in general difficult to detect M. tuberculosis-specific antibodies.     

IgG anti-multiantigen sonicate of Mycobacterium tuberculosis (Mts) measured by ELISA-Mts

Laboratory diagnosis of tuberculosis (TB) is often difficult. In recent years the detection of TB by serologic means has been the subject of a number of investigations. We studied the diagnostic utility of enzyme-linked immunosorbent assay in pulmonary tuberculosis. Two serological tests, commercial Pathozyme-Myco IgG (Omega Diagn.), and self-prepared ELISA-Mts using mycobacteria sonicate were evaluated simultaneously with 132 serum samples. Frequency and level of anti-mycobacterial IgG were compared in sera from 68 patients with TB and in sera from 64 healthy volunteers. The sensitivities of the tests with sera from TB patients were, respectively 55.9% and 70.6%. The specificities of these tests were respectively 98.5% and 84.4%. By combining two tests, a maximum sensitivity of 76.5% was obtained with reciprocal drops in specificity to 82.8%. Despite that, it has been suggested that combinations of different antigens were attempted in order to improve the diagnostic yield.     

Epitope-specific antibody levels demonstrate recognition of new epitopes and changes in titer but not affinity during treatment of tuberculosis

Antibody levels rise during treatment of tuberculosis. This study examined when this rise occurred, whether there was recognition of new antigen binding sites (epitopes) on the same or different antigens, and how long specific antibody persisted. Forty patients with smear-positive pulmonary tuberculosis provided serum before and during treatment. Antibody levels were measured using a monoclonal antibody competition assay to epitopes restricted to the Mycobacterium tuberculosis complex and an enzyme-linked immunosorbent assay for lipoarabinomannan. Significant increases in antibody levels were apparent after 7 days of treatment. Five samples (12.5%) had positive titers to all epitopes at the start of treatment, and this increased to 23 (58%) during treatment. Antibody to epitopes with the poorest sensitivity (the TB23 epitope of the 19-kDa antigen and the TB78 epitope of hsp65) showed the greatest increases after treatment. Antibody to these two epitopes was also absent in some patients with relapsed tuberculosis until after treatment. Antibody titers showed a biphasic response, with a fall at 2 to 3 months of treatment. Sera from two patients showed changes in the affinity of epitope-specific antibody during treatment, whereas the majority did not. Those infected with isoniazid-resistant strains of M. tuberculosis showed a late rise in antibody. Antibody to the TB68 epitope of the 16-kDa alpha-crystallin homolog was short-lived, but it recurred with bacteriological relapse during treatment. Positive antibody titers persisted for at least 3 to 18 months after treatment. Diagnostic tests for tuberculosis should be evaluated using only pretreatment sera. Delayed antigenic recognition could be due to active suppression and/or failure to engage internal antigens of M. tuberculosis.     

Current issues on molecular and immunological diagnosis of tuberculosis

Laboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.     

Serodiagnostic potential of culture filtrate antigens of Mycobacterium tuberculosis

Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534-1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49-56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies.     

Comparison of Antigen-Specific T-Cell Responses of Tuberculosis Patients using Complex or Single Antigens of Mycobacterium tuberculosis

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-γ (IFN-γ) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (rGroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis . The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M . tuberculosis , M . tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well as the vaccine strain, Mycobacterium bovis bacillus Calmette–Guérin (BCG). In addition, M . tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN-γ secretion showed that the most frequently recognized antigen was ESAT-6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS. The frequency of ESAT-6 responders, as measured both by proliferation (18/19) and secretion of IFN-γ (16/19) was comparable to the results obtained with whole-cell M . tuberculosis , MT-CF and M . bovis BCG. We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T-cell specificities induced by natural infection is directed towards several unrelated culture filtrate as well as somatic-derived protein antigens. In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M . tuberculosis and that some antigens, such as ESAT-6, are recognized by a high number of individuals. Such antigens represent candidates to be used for development of specific diagnostic reagents or in subunit vaccines.     

Effect of chemotherapy on whole-blood cytokine responses to Mycobacterium tuberculosis antigens in a small cohort of patients with pulmonary tuberculosis

The development of genomic and proteomic tools has enabled studies that begin to characterize the molecular targets of an effective host immune response to Mycobacterium tuberculosis, including understanding the specific immune responses associated with tuberculosis (TB) disease progression, disease resolution, and the development of latency. One application of such tools is the development of diagnostic reagents and assays useful as a test of cure. Such a test could be of considerable importance for the evaluation of new therapeutics. We and others have previously described immunodominant proteins of M. tuberculosis, including both vaccine and diagnostic candidates. In the present study, we describe the changes in immune responses to a panel of 71 M. tuberculosis antigens in six patients during the course of therapy. The levels of six cytokines were measured in 24-h whole-blood assays with these antigens, revealing that gamma interferon (IFN-γ), tumor necrosis factor (TNF), and interleukin-10 (IL-10) were differentially regulated in response to a subset of antigens. Therefore, measuring the production of these three cytokines in response to a panel of carefully selected M. tuberculosis proteins during the course of TB therapy might be a promising path toward the development of a test of cure and warrants further validation in larger cohorts of pulmonary TB patients.     

结核分枝杆菌抗原分析及免疫交叉反应研究

目的：筛选和鉴定结核分枝杆菌特异性和保护性抗原，研究结核杆菌的免疫反应特点，以探索结核病诊断和治疗的新途径。方法：采用超声破碎和滤膜抽滤的方法分别得到菌体蛋白和滤液蛋白，通过Western blot试验用结核杆菌的单克隆抗体及结核病人血清来检测蛋白样品，把发生阳性反应的蛋白在BECKMAN LF3200／多肽氨基酸序列测定仪上进行N末端序列分析，并用结核杆菌的单抗对自身抗原组蛋白进行了检测。结果：结核杆菌的31kD和30kD蛋白与结核杆菌的单抗及病人血清反应均呈阳性，但与正常小鼠血清和健康人血清反应呈阴性。31kD和30kD蛋白的N末端序列分别为：Ala Glu Val Asp Trp Leu Val Phe Ala Val和Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr。结核分枝杆菌的单抗与自身抗原组蛋白能发生免疫交叉反应。结论：结核杆菌的31kD和30kD蛋白是免疫保护性抗原，对免疫交叉反应分子基础的进一步研究必将增加对结核免疫机理的了解。