A rapid,simple enzyme immunoassay for detection of antibody to individual epitopes in the serodiagnosis of tuberculosis

The antibody response to individual epitopes has previously been analysed by competition assay using¹²⁵I- or enzyme labelled monoclonal antibodies. A modification of the test is described in which competition of human sera with unlabelled mouse monoclonal antibodies at the limiting dilution is revealed by peroxidase labelled antimouse IgG conjugate. Analysis of 54 sera from patients with pulmonary (36) and extrapulmonary (18) tuberculosis and 31 controls indicated that the modified test compares favourably with the test based upon directly labelled antibodies. Diagnostic sensitivity for five monoclonal antibodies evaluated was 11.1 % (TB78), 35.2 % (TB23 and TB68), 37.0 % (TB71) and 61.1 % (TB72) at 97.5 % specificity. For TB72, sensitivity was highest for pulmonary disease (69.7 %). The modified assay is also easier to standardise for screening new monoclonal antibodies using a single enzyme-labelled conjugate.     

Immunological activity of a 38-kilodalton protein purified from Mycobacterium tuberculosis.

A 38-kilodalton (kDa) protein antigen from Mycobacterium tuberculosis was purified by monoclonal antibody TB71-based affinity chromatography. This molecule carries two nonoverlapping epitopes recognized by monoclonal antibodies TB71 and TB72, which are expressed substantially more strongly by M. tuberculosis than by Mycobacterium bovis. However, cross-reactive determinants between these two species were revealed on the 38-kDa protein by a rabbit anti-BCG serum. An immunoradiometric assay based on the TB71 and TB72 antibody pair specifically determined 38-kDa-antigen concentrations in mycobacterial extracts. Antibodies in sera from tuberculosis patients estimated by binding to 38-kDa-antigen-coated microtiter plates were positively correlated with TB72 competing titers. Unlike antibodies, T-cell proliferative responses to the 38-kDa protein were expressed equally by 60% of tuberculosis patients and healthy BCG-vaccinated subjects. Similarly, delayed-type hypersensitivity skin reactions were elicited in both M. tuberculosis- and M. bovis-sensitized guinea pigs. The results suggest the immunodominance of the species-specific B-cell and cross-reactive T-cell stimulatory epitopes.     

Time course of anti-SL-IV immunoglobulin G antibodies in patients with tuberculosis and tuberculosis-associated AIDS.

Immunoglobulin G (IgG) and IgM antibodies against the SL-IV antigen of Mycobacterium tuberculosis in the sera of patients with tuberculosis with negative serology for human immunodeficiency virus (HIV) infection (TB group; n = 97), patients with tuberculosis with positive serology for HIV infection (TB-HIV group; n = 59), and healthy controls (n = 289) were determined by enzyme-linked immunosorbent assay. All sera were obtained at the onset of tuberculosis, i.e., when clinical symptoms appeared. Clinical specimens were collected and cultured for the isolation of M. tuberculosis, and treatment with antituberculous drugs was started. Sera were also obtained from patients in the TB group at fixed intervals during treatment; sera were available from 13 patients in the TB-HIV group before the onset of tuberculosis. The best specificity and positive predictive values were obtained with the IgG assays. In the IgG assays at specificities above 96.0%, the sensitivities of the tests were 45.3 and 72.8% for the TB and TB-HIV groups, respectively, and the sensitivity was 51.9% when data from both groups were combined for analysis. For the TB group, results of this study indicated that the levels of IgG antibodies remain high during treatment. Thus, repetitive serological assays may not be useful for treatment follow-up. In the TB-HIV group, 12 of 13 patients had IgG-specific antibodies against the SL-IV antigen between 1 and 30 months before the onset of tuberculosis, so we suggest that the IgG antibody assay against SL-IV may be helpful for identifying tuberculosis in patients infected with HIV.     

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.     

Analysis of the immunological humoral response toMycobacterium tuberculosis glycolipid antigens (DAT,PGLTb1) for diagnosis of tuberculosis in HIV-seropositive and -seronegative patients

Using an enzyme immunoassay (EIA) test, the concentrations of IgG antibodies against 2,3 diacyl trehalose (DAT) and phenolic glycolipid Tb1 (PGLTb1) were measured in the sera of 153 patients with active tuberculosis, 50 of whom were coinfected with HIV, and in the sera of 152 healthy blood donors, 149 asymptomatic HIV-seropositive patients, 12 HIV-seronegative patients with conditions simulating tuberculosis, 23 HIV-seropositive patients with disseminated infection caused by mycobacteria other than tuberculosis and 24 HIV-seropositive patients with pulmonary disease from whom mycobacteria was not isolated in culture. A slightly lower percentage (74 %) of the HIV-seropositive than the HIV-seronegative (77 %) tuberculosis patients were positive for anti-DAT and antiPGLTb1 IgG antibodies, with a specificity ranging from 91 to 95 %. There was no significant difference between EIA sensitivity in smear-positive and smear-negative patients with pulmonary tuberculosis for all HIV immune statuses and sites of disease (pulmonary vs. extrapulmonary). In HIV-seropositive patients, however, sensitivity was always lower for disseminated tuberculosis than for localized tuberculosis. Combining data for both the smear test and the EIA maximized sensitivity. The main value of the EIA test could be to provide early complementary information by antibody detection in patients with tuberculosis, particularly those with a negative smear test.     

Significance of circulating immune complexes in pulmonary tuberculosis

In the present study we have tried to demonstrate circulating immune complexes (CIC) in sera from patients with pulmonary tuberculosis (TB) by three techniques; latex agglutination; 3.5% PEG precipitation and determination of optical density at 280 nm and RIA of CIC using bovine spermatozoa. About 40 normal control sera and 100 TB patients sera were investigated for the presence of CIC. Seventeen per cent cases of pulmonary TB were positive by latex agglutination while none of the control was positive. Levels of CIC as detected by PEG precipitation and RIA were significantly elevated in patients as compared to normal controls. While IgG, IgA and IgM were elevated in the CIC of patients, IgM immunoglobulins were detected only in patients and not in controls. Detection of CIC may at times be useful in diagnosis, prognosis and therapeutic monitoring of disease processes, but it is the characterization of immune complexes (IC) and identification of the specific components of these complexes which holds the greatest potential for better understanding of disease mechanisms. CIC were precipitated using 3.5% PEG from sera of patients suffering from TB. The specific anti-TB antibody component of complex was determined using S. aureus protein A as a solid phase, Anti-BCG antibody and 125I-labelled TB antigen. The specific TB antigen component of the IC was dissociated thermally from TB antibody and assayed by a radioimmunoassay technique developed in our laboratory. Patients were classified into two groups. Those those sputum was positive for Mycobacterium tuberculosis by smear and/or culture and those whose sputum was negative. The TB antigen concentrations of CIC was higher 19.1 +/- 2.3 ng/ml (mean +s.e.) in sputum positive cases, and 9.9 +/- 1.9 ng/ml in sputum negative cases as compared to 2.2 +/- 0.3 ng/ml in controls. Patient groups were significantly different from controls as well as from each other (P less than 0.001). Anti-TB antibody ratios were 11.7 +/- 1.48, 5.1 +/- 1.5 and 0.6 +/- 0.1 in sputum positive, sputum negative and controls. The significance of differences between the groups was P less than 0.001. The effect of treatment administered over a period of 12 weeks or more was evaluated. It was observed that in patients with persistent demonstration of M. tuberculosis in the sputum, the TB antigen and TB antibody levels of CIC were consistently high. In patients who responded to anti-tubercular drugs the TB antigen levels decreased progressively while TB antibody levels remained high.(ABSTRACT TRUNCATED AT 400 WORDS)     

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 α-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.     

Tuberculin skin testing compared with T-cell responses to Mycobacterium tuberculosis-specific and nonspecific antigens for detection of latent infection in persons with recent tuberculosis contact.

The tuberculin skin test (TST) is used for the identification of latent tuberculosis (TB) infection (LTBI) but lacks specificity in Mycobacterium bovis BCG-vaccinated individuals, who constitute an increasing proportion of TB patients and their contacts from regions where TB is endemic. In previous studies, T-cell responses to ESAT-6 and CFP-10, M. tuberculosis-specific antigens that are absent from BCG, were sensitive and specific for detection of active TB. We studied 44 close contacts of a patient with smear-positive pulmonary TB and compared the standard screening procedure for LTBI by TST or chest radiographs with T-cell responses to M. tuberculosis-specific and nonspecific antigens. Peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, TB10.4 (each as recombinant antigen and as a mixture of overlapping synthetic peptides), M. tuberculosis sonicate, purified protein derivative (PPD), and short-term culture filtrate, using gamma interferon production as the response measure. LTBI screening was by TST in 36 participants and by chest radiographs in 8 persons. Nineteen contacts were categorized as TST negative, 12 were categorized as TST positive, and 5 had indeterminate TST results. Recombinant antigens and peptide mixtures gave similar results. Responses to TB10.4 were neither sensitive nor specific for LTBI. T-cell responses to ESAT-6 and CFP-10 were less sensitive for detection of LTBI than those to PPD (67 versus 100%) but considerably more specific (100 versus 72%). The specificity of the TST or in vitro responses to PPD will be even less when the proportion of BCG-vaccinated persons among TB contacts evaluated for LTBI increases.     

Humoral response to defined epitopes of tubercle bacilli in adult pulmonary and child tuberculosis

In order to investigate the humoral response to tuberculosis in different categories of patients, serum antibody levels to six epitopes ofMycobacterium tuberculosis in adult pulmonary and child tuberculosis were determined. Serum antibody titres were determined by competitive inhibition with radio-labelled murine monoclonal antibodies in 67 adults and 85 children with tuberculosis and in 79 age-matched controls. BCG vaccination (n=39) and self-healed tuberculosis (n=11) in adults gave rise to higher antibody titres to TB68, TB23 and TB72 epitopes (all p<0.003) when compared to non-vaccinated controls (n=18). TB68 titres were higher (p=0.006) in self-healed than in vaccinated adults. Adult sputum-negative patients (n=15) had higher titres to TB71 (p=0.015) and ML34 (p=0.02) epitopes compared to BCG-vaccinated healthy controls, while sputum-positive patients (n=41) had higher titres to all epitopes tested (all p<10^–4). The diagnostic sensitivity, with a 95 % specificity, was best with the combination of probes TB23, TB68, TB72 for sputum-positive (85 %) and TB78, ML34 (53 %) for sputum-negative patients. Antibody titres in children with tuberculosis were lower than in adult patients; diagnostic sensitivity in histologically or microbiologically proven cases (n=18) was only 44 %, while that in mediastinal lymphadenitis (n=67) was 13.5 %. This study suggests that the magnitude and specificity of the humoral response to tubercle bacilli varies with site and severity of infection; the implications for pathogenesis or protective immunity are discussed.     

Absence of antibodies to muramyl dipeptide in patients with tuberculosis or leprosy

The enzyme-linked immunosorbent assay (ELisa) was used to detect the presence of antibodies to muramyl dipeptide (MDP) in serum of patients with leprosy or tuberculosis. Using a conjugate of MDP-lysine to horse radish peroxidase, no such antibodies could be detected in sera of either patients or controls. Antibodies to a sonicate antigen of Mycobacterium tuberculosis were found in sera of all individuals tested and the binding of these antibodies to the M. tuberculosis antigen could not be inhibited by MDP. On the other hand, binding of MDP to anti-MDP antibodies, raised in rabbits, was largely inhibited by free MDP, slightly inhibited by M. tuberculosis antigen and was not inhibited by the patients' sera.     

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.     

Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

Rapid serodiagnosis of active pulmonary Mycobacterium tuberculosis by analysis of results from multiple antigen-specific tests

We have prospectively analyzed three antigens for serodiagnosis of tuberculosis (TB). These antigens were tuberculous glycolipid antigen, lypoarabinomannan polysaccharide antigen, and antigen 60 (A60), which was derived from purified protein derivatives. Of the 131 patients with active pulmonary TB, 57 were both smear and culture negative and 14 had chronic active pulmonary TB that remained smear positive for >12 months of chemotherapy. One hundred twenty healthy adults were controls. The percentages of patients positive in all three tests were 58.8% for smear-positive active pulmonary TB and 71.4% for chronic active pulmonary TB. When the results of the three serodiagnostic tests were evaluated in combination, the sensitivity increased to 91.5% in patients with active pulmonary TB and to 86.0% in smear- and culture-negative patients. The false-positive rate of the three-test combination was 12.5% in the healthy control groups. In conclusion, it was not possible to detect all of the antibodies against antigenic substances in the cell walls of the tuberculous bacilli in the sera of all TB patients by using available serodiagnostic tests. However, the combined use of tests with three separate antigens maximizes the effectiveness of serodiagnosis.     

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.     

Humoral immune response in human tuberculosis: immunoglobulins G, A, and M directed against the purified P32 protein antigen of Mycobacterium bovis bacillus Calmette-Guérin

The P32 protein antigen of Mycobacterium bovis BCG, identified as antigen 85A in the BCG reference system, was used to investigate the humoral immune response in human tuberculosis (TB). Immunoglobulin G (IgG), IgA, and IgM directed against P32 were measured by an enzyme-linked immunosorbent assay. Mean IgG and IgA antibody levels differed significantly (P less than 0.001) between active-TB patients (50 untreated and 52 treated) and healthy control subjects (111 unvaccinated tuberculin negative, 38 unvaccinated tuberculin positive, and 72 recently BCG vaccinated). Mean IgG antibody levels, but not mean IgA antibody levels, were higher (P less than 0.05) in patients with positive microscopic examination for acid-fast bacilli than in patients with negative microscopic examination. A positive relation was found between mean levels and the extent of disease. There was no difference in mean IgM antibody levels between patients and controls. By setting the upper normal limit at the 95th percentile of the 221 healthy subjects, the sensitivities were 46% in untreated and 63% in treated patients for IgG and 30 and 50%, respectively, for IgA. Of the untreated patients, 56% were positive for either IgG or IgA antibodies. Among the untreated patients with negative direct smear, 35% were positive for IgG and 24% were positive for IgA. When both immunoglobulin classes were combined, the serological test was positive in 47% of those patients. Neither naturally acquired tuberculin hypersensitivity nor BCG vaccination affected positivity frequencies in healthy subjects. Only active TB seemed to induce significant anti-P32 antibody levels and to be associated with positivity. A serological test with P32 as the antigen might therefore be helpful for the rapid diagnosis of TB.     

Monocyte chemotactic protein-1 production in patients with active pulmonary tuberculosis and tuberculous pleurisy

OBJECTIVE: The role of monocyte chemotactic protein (MCP)-1 in human pulmonary and pleural tuberculosis (TB) was assessed by examining its production in clinical samples from patients with active pulmonary TB and tuberculous pleurisy (TBP). METHODS: Serum was obtained from 26 active pulmonary TB patients [14 early TB (E-TB), and 12 chronic refractory TB (CR-TB)] and 15 healthy tuberculin reactors (HTRs). The monocytes and peripheral blood mononuclear cells (PBMCs) were separated and stimulated with purified protein derivatives (PPD) or the 30-kDa antigen of Mycobacterium tuberculosis. Pleural exudates were isolated from 25 patients with TBP and 24 non-TBP patients [malignancy and congestive heart failure (CHF)]. The MCP-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In sera, the MCP-1 levels of TB patients were similar to those of HTRs. For monocytes, CR-TB patients spontaneously expressed more MCP-1, compared with HTRs and E-TB patients. In addition, MCP-1 production of PPD- or 30-kDa antigen-stimulated monocytes was significantly elevated in CR-TB patients than that from E-TB. Interestingly, the E-TB patients had significantly depressed MCP-1 production by PBMCs in response to PPD or 30-kDa, compared with HTRs and CR-TB patients. In pleural effusions, MCP-1 levels were significantly higher in patients with TBP than in patients with CHF, but lower than in malignant effusions. CONCLUSIONS: The data suggest that MCP-1 production is not uniquely elevated systemically in TB patients, although MCP-1 production might be elevated by monocytes in the chronic phase of TB or with a local pleural infection.     

A rapid latex agglutination test for detection of antibodies in tuberculosis and Hansen's disease.

Antigens of Mycobacterium w, a saprophytic fast growing organism having antigenic epitopes cross-reactive with Mycobacterium leprae and Mycobacterium tuberculosis, were coated on to latex beads (0.33 micron Zn size), and the reactivity tested with sera of tuberculosis and Hansen's disease (HD) patients. Seventy nine percent of lepromatous leprosy (LL) and eighty five percent of pulmonary tuberculosis (TB) patients sera showed an agglutination reaction easily read by naked eye. Specificity of the test was further checked by testing sera of non-mycobacterial infection cases and all of them were found negative. Among apparently healthy controls, 4.3% were found positive from non-endemic and 8.8% from endemic area. The sensitivity of the assay is further enhanced from 78.7% to 90.4% and 85.7% to 91.6% in both LL HD and pulmonary tuberculosis respectively, by using immune complexes extracted from the patients sera. Potential of these antigen coated beads to detect the two major human mycobacterial disease, LL HD and pulmonary TB was also put evidence in a double blind study on coded sera samples obtained from various hospitals in India. The antigen coated beads are stable for upto 6 months at 4 degrees C. The latex slide agglutination test reported here, is simple, rapid, easy to perform and can be used even in rural areas of developing countries.     

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.     

Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis.

A serological survey was performed in groups of patients with active sputum smear-positive or smear-negative pulmonary tuberculosis, healthy household contacts, and controls. Sera were tested for titers of antibodies which bound to each of five purified mycobacterial antigens by enzyme immunoassay and for competition of binding to single epitopes, using six radiolabeled monoclonal antibodies directed toward corresponding molecules. The evaluation of diagnostic specificity was based on a positive score represented by titers above the cutoff point of 2 standard deviations above the mean titer of a control group. For smear-positive samples, the best sensitivity (83%) was achieved by exclusive use of the 38-kilodalton (kDa) antigen or its corresponding monoclonal antibodies. For smear-negative samples, levels of antibodies binding to the 19-kDa antigen showed a lower sensitivity of 62% compared with the control group or 38% compared with the contact group. Titers of antibody binding to the 14-kDa antigen were raised in Mycobacterium bovis BCG-vaccinated contacts, indicating that the greatest potential of this antigen may be in the detection of infection in a population for which tuberculin testing is unreliable. The results demonstrated the differing antibody responses to each of the tested antigens and distinct associations with the stage of infection or disease.