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.     

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.     

Mycobacterium tuberculosis HBHA protein reacts strongly with the serum immunoglobulin M of tuberculosis patients

Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.     

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.     

Evaluation of synthetic pseudo cord-factor-like glycolipids for the serodiagnosis of tuberculosis.

A number of glycolipids were evaluated in an ELISA test for their serodiagnostic usefulness in tuberculosis. One hundred and twelve (112) sera belonging to bacteriologically confirmed TB patients, patients with pathologies other than tuberculosis and healthy individuals were examined against several synthetic "mirror" pseudo cord factors (analogues of trehalose-6,6'-dimycolate or TDM) using natural cord factor and another recently described natural glycolipid (SL-IV) of Mycobacterium tuberculosis as control antigens. Analysis of the results shows that all synthetic "mirror" pseudo cord factors, except one with a short 8-carbon chain, were better recognized by the sera of tuberculosis patients than natural cord factor, with sensitivity and specificity values in the ELISA test similar to those reported for M. tuberculosis species-specific SL-IV. Of all antigens tested in this study, BDA. TDA, a bis(N,N-dioctadecylamide) of "trehalose dicarboxylic acid", [(alpha-D-glucopyranosyluronic acid) (alpha-D-glucopyranosiduranic acid)], showed the highest serodiagnostic discriminating power (93% sensitivity and specificity). We postulate that either these artificial molecules are cross-reactants of similarly structured native glycolipids of M. tuberculosis or that they bear closer resemblance to actual phagosome-lysosome-modified antigens than to native mycobacterial ones.     

Serodiagnosis of tuberculosis: comparison of immunoglobulin A (IgA) response to sulfolipid I with IgG and IgM responses to 2,3-diacyltrehalose, 2,3,6-triacyltrehalose,and cord factor antigens

Nonpeptidic antigens from the Mycobacterium tuberculosis cell wall are the focus of extensive studies to determine their potential role as protective antigens or serological markers of tuberculous disease. Regarding this latter role and using an enzyme-linked immunosorbent assay, we have made a comparative study of the immunoglobulin G (IgG), IgM, and IgA antibody responses to four trehalose-containing glycolipids purified from M. tuberculosis: diacyltrehaloses, triacyltrehaloses, cord factor, and sulfolipid I (SL-I). Sera from 92 tuberculosis patients (taken before starting antituberculosis treatment) and a wide group of control individuals (84 sera from healthy donors, including purified protein derivative-negative, -positive, healed, and vaccinated individuals, and 52 sera from nontuberculous pneumonia patients), all from Spain, were studied. The results indicated a significantly elevated IgG and IgA antibody response in tuberculosis patients, compared with controls, with all the antigens used. SL-I was the best antigen studied, showing test sensitivities and specificities for IgG of 81 and 77.6%, respectively, and of 66 and 87.5% for IgA. Using this antigen and combining IgA and IgG antibody detection, high test specificity was achieved (93.7%) with a sensitivity of 67.5%. Currently, it is widely accepted that it is not possible to achieve sensitivities above 80% in tuberculosis serodiagnosis when using one antigen alone. Thus, we conclude that SL-I, in combination with other antigenic molecules, could be a useful antigen for tuberculosis serodiagnosis.     

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.     

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.     

Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis 38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis.     

Evaluation of Antigen-Specific Immunoglobulin G Responses in Pulmonary Tuberculosis Patients and Contacts

This study aimed to evaluate the serodiagnostic potential of immunoglobulin G (IgG) responses to Mycobacterium tuberculosis antigens in pulmonary tuberculosis (TB) patients, recent TB contacts with latent TB infection (LTBI), and healthy subjects. Infections were assessed using tuberculin skin tests, QuantiFERON-TB Gold In-Tube tests, drug susceptibility testing, and molecular genotyping of clinical isolates. Serum IgG responses to selective M. tuberculosis antigens, including the 38-kDa and 16-kDa antigens, lipoarabinomannan (LAM), and recombinant early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10), were determined. We found that the serum IgG responses to all antigens might differentiate between active TB and LTBI, with LAM having the highest diagnostic value (area under the curve [AUC] of 0.7756, P < 0.001). Recurrent TB cases showed significantly higher IgG responses to 38 kDa, CFP-10 (P < 0.01), and LAM (P < 0.05) than new cases, and male patients had higher levels of antigen-specific IgG than females (P < 0.05). Conversely, drug resistance and patient body mass index did not affect IgG responses (P > 0.05). LAM-specific IgG responses differentiated between acid-fast bacillus (AFB) smear-positive and -negative patients (P < 0.01), whereas antigen-specific IgG responses did not vary with the M. tuberculosis genotype (P > 0.05). Significantly higher IgG responses to 38 kDa and 16 kDa were observed in AFB smear-negative patients than in controls. These results suggest that assessment of serum IgG responses to selective purified M. tuberculosis antigens may help improve the diagnosis of active TB, particularly for sputum smear-negative patients or recurrent cases, and these may also help to differentiate between active TB and LTBI.     

Antimycobacterial antibody level in pleural, pericardial and cerebrospinal fluid of patients with tuberculosis

The goal of the study was to evaluate IgG, IgA and IgM mediated humoral immune response against 38kDa and 16 kDa or 38kDa and LAM mycobacterial antigens in pleural, pericardial or cerebrospinal fluid from patients with tuberculosis (TB) and to compare to non-tuberculous controls (NTB). 30 cerebrospinal fluids (CSF) (16 TB pts and 14 NTB pts), 17 pericardial fluids (6 TB and 11 NTB) and 20 pleural fluids (7 TB and 13 NTB) were examined. Commercially available ELISA-based assays (Pathozyme Tb complex plus, Myco G, A and M--Omega Diagnostic) were used. Tests were performed and cut off established according to manufacturer instruction. Mean IgG level against 38 + 16kDa was significantly higher in neurotuberculosis group compared to control (p<0.05). Sensitivity of the test in detecting neurotuberculosis was of 42% and specificity of 96%. Mean IgG, IgA and IgM against 38kDa + LAM level was higher in TB group compared to NTB in CSF. No difference was observed between TB and NTB group in pleural effusion. Antimycobacterial antibody levels were non-significantly increased in pericardial fluid in TB. The findings of the study indicate that TB is associated with the presence of detectable levels of antibodies in the CSF and pericardial effusion. Anti 38kDa + 16kDa IgG test can be used in combination with other diagnostic methods to increase diagnostic accuracy of neurotuberculosis.     

Humoral Immune Responses against the Mycobacterium tuberculosis 38-Kilodalton,MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.The control of tuberculosis (TB) remains challenging in China (18). Currently, the diagnosis of active TB mainly relies on clinical symptoms, radiologic findings, and the detection of Mycobacterium tuberculosis in clinical samples using smear staining and mycobacterial culture. However, the diagnosis of TB in smear- and culture-negative TB patients is difficult. The detection of M. tuberculosis-specific antibodies in human sera has been an important aid in diagnosis of TB. Notably, several antigens have been demonstrated to have merit in TB diagnosis, including the 38-kDa protein, which is commonly used in serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies suggest that the antibody responses to M. tuberculosis antigens are heterogeneous among individuals (17) so that the detection of antibodies against a single antigen usually has a low sensitivity for diagnosis of TB, especially for bacterium-negative cases. Therefore, it may be valuable to evaluate antibodies against the 38-kDa antigen and other major antigens for the diagnosis of active TB (14, 15).Notably, the MTB48, CFP-10 (culture filtrate protein 10), and ESAT-6 (6-kDa early secreted antigen target of M. tuberculosis) genes are conserved in M. tuberculosis and Mycobacterium bovis isolates but partially deleted or absent in M. bovis BCG as well as in most nontuberculous mycobacteria (NTM) (1-3, 10, 16). Importantly, the proteins encoded by these genes are immunogenic (7, 9, 12, 16). In this study, we cloned the 38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion proteins in Escherichia coli. Subsequently, we developed an enzyme-linked immunosorbent assay (ELISA) for the characterization of serum antibodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens in a population of 250 active TB patients and 260 healthy subjects. We found that characterization of antibodies against multiple M. tuberculosis antigens were valuable for the diagnosis of active TB.     

Progress in serodiagnosis of Mycobacterium tuberculosis infection

One-third of the world population is estimated to have Mycobacterium tuberculosis infection. Accurate and timely identification of infected individuals is critical for treatment and control. The current diagnostic methods lack the desired sensitivity and specificity, require sophisticated equipment and skilled workforce or take weeks to yield results. Diagnosis of extrapulmonary TB, TB-HIV co-infection, childhood TB and sputum smear-negative pulmonary TB pose serious challenges. Interest in developing serodiagnostic methods is increasing because detection of antibody is rapid, simple and relatively inexpensive, and does not require a living cell for detection. Three types of tests, namely screening tests to overcome diagnostic delay, specific tests for diagnosis of extrapulmonary TB and other bacteriologically negative cases, and tests for vaccine-induced immunity need critical consideration. Several factors must be considered to develop serodiagnostic methods for TB. Antigen recognition by infected individuals is highly heterogeneous due to stage of disease, differences in HLA types, strain of the bacilli, health of the patient and bacillary load. With advances in molecular biological techniques, a number of novel antigens have been identified. Some of these antigens have proven valuable in detecting specific antibodies in some of the most challenging TB patients. The best example is a fusion protein containing several M. tuberculosis proteins (e.g. CFP-10, MTB8, MTB48, MTB81 and the 38-kDa protein) which showed encouraging results in detecting antibodies in sera of patients, including TB-HIV co-infection. This review presents progress made in the serodiagnosis of TB during the last decade.     

Comparison of IgM, IgG and IgA responses to M.leprae specific antigens in leprosy

Antibodies of IgM, IgG and IgA classes against M.leprae specific antigens (PGL-I, ND-O-BSA, and NT-O-BSA) were determined in the sera of 80 leprosy patients (28 untreated, 34 treated lepromatous and 18 tuberculoid), 25 tuberculosis patients and 33 normal individuals of Northern Thailand. No strong distinction in reactivity could be found between the three antigens. The IgM antibody assay yielded more positive results than assays for IgG and IgA. It was found that the positivity rates of IgM antibodies to all three antigens were highest in untreated lepromatous leprosy (82%). In tuberculoid leprosy, the positivity rates of IgM, IgG and IgA to the antigens were more variable, ranging from 22 to 50 percent. Patients with tuberculosis and normal individuals did not produce IgM antibodies against the antigens. The results suggested that the determination of IgM against the three antigens is a more sensitive and specific test for active leprosy than those of IgG and IgA. The relationship between the duration of treatment and IgM antibody levels in lepromatous leprosy (LL) was studied. Untreated LL patients had significantly higher IgM and IgA antibody levels than treated patients. There was no difference in IgG antibody levels between the two groups, and the levels of both groups were higher than normal controls. Serial determination of IgM antibodies in 7 LL patients revealed that treatment was strongly associated with progressive decrease in IgM antibody levels against all three antigens.     

Use of multiepitope polyproteins in serodiagnosis of active tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Diversity of Antigen Recognition by Serum Antibodies in Experimental Bovine Tuberculosis

Tuberculosis in cattle remains a major zoonotic and economic problem in many countries. The standard diagnostic assay for bovine tuberculosis, the intradermal tuberculin test, has low accuracy. Therefore, alternative immunodiagnostic methods, such as serological assays, are needed for detection of infected animals. Development of an accurate serodiagnostic test requires a detailed understanding of the humoral immune responses during bovine tuberculosis and, in particular, identification of the key antigens of Mycobacterium bovis involved in antibody production. In this study, we characterized antibody responses in cattle experimentally infected with M. bovis. Sequential serum samples were collected every 3 to 4 weeks for up to 27 months postinfection. Circulating immunoglobulin G antibody levels were measured by an enzyme-linked immunosorbent assay using 12 highly purified recombinant proteins of M. bovis. Six proteins, ESAT-6, 14-kDa protein, MPT63, MPT70, MPT51, and MPT32, were identified as major seroreactive antigens in bovine tuberculosis. A remarkable animal-to-animal variation of antigen recognition by serum antibodies was observed. Kinetic analyses of the antibody production to individual antigens during infection revealed that the heterogeneous antigen recognition profile changed markedly in a given infected animal as disease progressed.     

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 approximately 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 (approximately 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.     

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.     

Lack of IgA subclass restriction in antibody response to phosphorylcholine, beta lactoglobulin and tetanus toxoid

Although there is IgG subclass restriction in the antibody responses to most antigens, our data indicate that the human IgA subclasses, IgA, and IgA2, do not demonstrate a similar antigen specific restriction. We did not find evidence for IgA subclass restriction in the antibody responses to phosphorylcholine (PC), beta lactoglobulin or tetanus toxoid. These antigens were chosen to represent carbohydrate-like versus protein antigens and antigens presented through the mucosal route versus the humoral route. For each of these antigens the proportion of antigen specific IgA that was IgA1 and IgA2 was similar to that of total serum IgA. IgA anti-PC, which is thought to be directed against the phosphorylcholine moieties found on certain bacterial polysaccharides, could be found in the serum of all individuals tested and constituted 0.063-0.088% of the total serum IgA. IgA anti-beta lactoglobulin and anti-tetanus toxoid could be measured only in the serum of selected individuals, usually those with known milk protein sensitivity, or those recently immunized with tetanus toxoid. The lack of marked subclass restriction of IgA responses to these antigens stands in contrast to results obtained by others for IgG antibodies, in which carbohydrates and proteins preferentially stimulate antibodies in different IgG subclasses.     

Mycobacterium tuberculosis malate synthase- and MPT51-based serodiagnostic assay as an adjunct to rapid identification of pulmonary tuberculosis

The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from approximately 80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis.