Use and standardization of the Mycobacterium avium sensitin skin-test reagent.
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Humans↗
Biomedical subjects
S D Chaparas
Publications and source records attributed to S D Chaparas.
Dual skin testing with Mycobacterium avium sensitin and purified protein derivative in patients with M. avium complex infection or tuberculosis.
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Antigens↗
Skin testing with recombinant Mycobacterium intracellulare antigens.
The immunoreactivity of four recombinant Mycobacterium intracellulare beta-galactosidase fusion proteins, which correspond to 22, 40, 43 and 85 kDa M. intracellulare antigens, was assessed. Lymphoproliferative assays demonstrated that Escherichia coli lysates containing each of the fusion proteins stimulated T cells in vitro. Purified preparations of three of these recombinant M. intracellulare antigens (22, 43 and 85 kDa) also induced delayed-type hypersensitivity (DTH) reactions in sensitized guinea pigs. However, the skin test responses evoked by each of these antigens was not species-specific. Given these results, the potential utility as skin test reagents of the purified antigens or peptides derived from these proteins is discussed.
Animals↗
Immunological characterization of recombinant antigens isolated from a Mycobacterium avium lambda gt11 expression library by using monoclonal antibody probes.
Nontuberculous mycobacteria, particularly Mycobacterium avium, have been isolated from a significant percentage of patients with AIDS. Early detection of M. avium infection is difficult, and treatment regimens are often ineffective. Much needs to be learned about antigens and factors responsible for immunity to and pathogenesis of the disease. Specific antigens and diagnostic procedures for infection need to be developed. To address some of these problems, we have generated 25 different monoclonal antibodies against a serovar 4 strain of M. avium isolated from a patient with AIDS. Protease sensitivity studies have demonstrated that each of these antibodies recognizes a protein-associated epitope. Immunoblot analyses suggest that seven of these monoclonal antibodies react specifically with M. avium and M. intracellular epitopes. Immunoreactive bacteriophages were identified from an M. avium lambda gt11 expression library with two of these monoclonal antibodies (3808 C3 and 3954 B12). Lambda lysogens, generated from the immunoreactive bacteriophages, overproduced beta-galactosidase fusion proteins which were reactive with the two monoclonal antibodies in immunoblot assays. The purified fusion proteins were shown to elicit skin test reactions in sensitized guinea pigs.
Acquired Immunodeficiency Syndrome↗
Mycobacterium avium complex disease in patients with AIDS: seroreactivity to native and recombinant mycobacterial antigens.
Antibodies to Mycobacterium avium complex (MAC) antigens were measured by enzyme-linked immunosorbent assays and immunoblot analyses in sera from 20 patients with AIDS and disseminated MAC disease, 5 human immunodeficiency virus-seronegative patients with pulmonary MAC infections, and 20 healthy controls. Whereas enzyme-linked immunosorbent assay titers for healthy controls and patients with AIDS and MAC disease were comparable, human immunodeficiency virus-seronegative patients with MAC disease had higher anti-MAC antibody titers (P less than 0.01). Immunoblot analysis with the same sonic extracts indicated that each of the three groups had a limited heterogeneous response to M. avium antigens. No significant differences in immunoblot reactivities were detected. However, immunoblot studies with recombinant nontuberculous mycobacterial antigens revealed that sera from over 90% of the patients with MAC disease and only 25% of controls recognized a recombinant protein derived from a 35-kDa mycobacterial antigen. Although sonic extracts did not permit adequate discrimination of antibody reactivity in patients with MAC disease, recombinant antigens may be useful as indicators of disease.
Acquired Immunodeficiency Syndrome↗
Characterisation of plasmids extracted from AIDS--associated Mycobacterium avium isolates.
The plasmid profiles of 12 Mycobacterium avium strains isolated from 12 different patients with acquired immunodeficiency syndrome were analysed. Plasmids were identified in 9 of these strains. Plasmids were isolated from all 7 serovars 4 and 8 strains, a serovar 20a strain and an untypeable strain, but were not detected in either of 2 serovar 3b strains or an untypeable isolate. Southern blot hybridisations revealed that extracts derived from all of the plasmid-containing strains hybridised to a DNA probe prepared from known mycobacterial plasmid sequences. However, restriction analyses suggest that native plasmids which hybridised to the DNA probe and were similar in mass were not identical.
Acquired Immunodeficiency Syndrome↗
Isolation and characterization of recombinant lambda gt11 bacteriophages expressing four different Mycobacterium intracellulare antigens.
Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a lambda gt11 library with monoclonal antibodies to M. intracellulare. These four antibodies reacted with native M. intracellulare proteins of 54, 43, 40/38, and 22 kilodaltons. Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M. intracellulare derivation of the inserts. The physical maps of the immunoreactive phages were deduced by restriction enzyme digestions. The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting.
Antibodies, Bacterial↗
Production, characterization, and species specificity of monoclonal antibodies to Mycobacterium avium complex protein antigens.
The incidence of Mycobacterium avium-Mycobacterium intracellulare complex infections has increased in recent years primarily because a significant proportion of acquired immunodeficiency syndrome patients develop disseminated M. avium complex disease. In an effort to develop new tools to study these infections, we have produced eight monoclonal antibodies directed against M. avium. Western blot (immunoblot) specificity analysis and protease sensitivity assays indicate that four of these antibodies recognize M. avium-specific protein epitopes and two react with M. avium complex-specific peptide determinants. These monoclonal antibodies may be useful clinically in the diagnosis of M. avium complex disease and in the laboratory for isolation and characterization of native and recombinant M. avium complex antigens.
Acquired Immunodeficiency Syndrome↗
The 45 kilodalton molecule of Mycobacterium tuberculosis identified by immunoblotting and monoclonal antibodies as antigenic in patients with tuberculosis.
The object of this study was to discover new M. tuberculosis antigens which are recognized by patients with tuberculosis, because effective serodiagnostic tests are likely to require combinations of different antigens. In our early experiments using immunoblotting, the findings suggested that human sera from smear-negative tuberculosis patients bound to an antigen in the 45 kDa region. Subsequently, estimates of molecular weight in the immunoblots confirmed that the murine monoclonal antibody (MAB) HGT-6 and sera from patients both recognized the same 45 kDa molecule. An antibody-antibody competition assay between MAB HGT-6 and sera from smear-positive tuberculosis patients yielded a positive result in 23 out of 43 sera from patients, but in only four out of 23 from controls. This is further evidence that the 45 kDa antigen is recognized by tuberculous patients. We analysed whether a combination of the 45 kDa antigen results and those of known antigens might better discriminate between minimal smear-negative disease and healthy controls than could test with single antigens. There is no clinically useful laboratory test for smear-negative tuberculosis. In immunoblotting, combining the results with the 65, 45, 38 and 10 kDa antigens gave the best discrimination. This suggests that future serodiagnostic tests for minimal disease, such as the antibody-antibody competition assay, should contain a MAB against the 45 kDa antigen and possibly also against the 10 kDa antigen.
Antibodies, Monoclonal↗
Isolation and characterization of a recombinant lambda gt11 bacteriophage which expresses an immunoreactive Mycobacterium intracellulare protein in Escherichia coli.
Disseminated Mycobacterium avium-Mycobacterium intracellulare (M. avium complex) disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome. Because of the increasing importance of this disease, an M. avium complex lambda gt11 expression library was prepared. We screened the library with an absorbed anti-M. intracellulare serum and identified a recombinant phage which expressed a 190-kilodalton beta-galactosidase-M. intracellulare fusion protein. Lysates containing the 190-kilodalton fusion protein evoked strong humoral and cell-mediated responses. The immunoreactivity of the M. intracellulare recombinant protein suggests that antigens isolated from the expression library may be useful as skin test, serodiagnostic, or immunoprophylactic reagents for M. avium complex disease.
Animals↗
Isolation and characterization of recombinant lambda gt11 bacteriophages expressing eight different mycobacterial antigens of potential immunological relevance.
A genomic lambda gt11 DNA library of Mycobacterium tuberculosis was screened for expression of mycobacterial protein antigens with murine monoclonal antibodies. The reactivity patterns of the monoclonal antibodies ranged from those showing a limited interspecies reactivity to antibodies widely cross-reactive among different mycobacterial species. Twelve recombinant bacteriophages were isolated, containing eight mycobacterial genes (paa, pab, pac, pad, paeA, paeB, pafA, and pafB) encoding protein antigens. Physical maps of the phages were generated and the products of the recombinant genes were analyzed by immunoblotting techniques. PaeA and PaeB are distinct proteins but were shown to share an epitope. A similar condition was observed between PafA and PafB. Among the phages isolated, two groups expressed epitopes specific for M. tuberculosis and Mycobacterium bovis BCG. One group of phages produced an antigenic determinant which is found in M. tuberculosis and Mycobacterium marinum but not in M. bovis BCG.
Antibodies, Monoclonal↗
T lymphocyte responses to mycobacterial antigen in AIDS patients with disseminated Mycobacterium avium-Mycobacterium intracellulare infection.
Patients with acquired immunodeficiency syndrome (AIDS) who have Mycobacterium avium-Mycobacterium intracellulare (MAI) infection typically have widely disseminated disease, often fail to respond to multi-drug chemotherapeutic regimens, and show little or no inflammatory tissue response. To determine if this clinicopathologic state correlates with in vitro lymphocyte responses to specific antigen, peripheral blood mononuclear cells from 18 patients with AIDS who had MAI bacillemia were stimulated with either particulate (heat-killed bacille Calmette Guérin [BCG]) or soluble (M intracellulare) mycobacterial antigens. In comparison to reactive cells from healthy control subjects testing positive with purified protein derivative of tuberculin (PPD) or from MAI-colonized (non-AIDS) control subjects, cells from 16 (89 percent) patients with AIDS essentially failed to show any antigen-induced proliferative activity or secretion of gamma-interferon; however, in two patients, antigen-stimulated proliferation of gamma-interferon production was modest but within the range of responses of normal healthy control subjects. Thus, although an occasional patient with AIDS can develop disseminated MAI infection despite the presence of antigen-reactive cells in vitro, most MAI-infected patients with AIDS display a striking defect in responsiveness to both particulate and soluble mycobacterial antigens. Since treatment with gamma-interferon activates the mononuclear phagocyte in vivo, these results suggest a rationale for a trial of gamma-interferon therapy in patients with AIDS who have disseminated MAI infection.
Acquired Immunodeficiency Syndrome↗
Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis.
An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.
Animals↗
Radioimmunoassay for detecting Mycobacterium tuberculosis antigen in cerebrospinal fluids of patients with tuberculous meningitis.
A biotin-avidin radioimmunoassay for detecting Mycobacterium tuberculosis antigen has been developed. The assay involves sandwiching mycobacterial antigens from sonicate preparations and cerebrospinal fluids between two antibodies produced in burros and rabbits. The reaction is amplified by using biotinylated antibody to rabbit IgG and 125I-labeled avidin. The assay has a sensitivity of 20 ng/ml and shows less than 5% cross-reactivity with six other mycobacteria. We studied patients with untreated tuberculous meningitis, patients with treated tuberculous meningitis, patients with nonbacterial meningitis, and patients with bacterial meningitis. Antigen was detectable in two of 56 control samples (40 ng/ml). Hence, samples with greater than or equal to 80 ng of antigen/ml were considered positive. In patients with untreated tuberculous meningitis, antigen levels ranged from 20 to 10,000 ng/ml, and 15 (79%) of 19 samples were positive. In the treated group, only two (10%) of 17 samples were positive. This test promises to be a rapid adjunct in the early diagnosis of tuberculous meningitis.
Antigens, Bacterial↗
Production, characterization, and species specificity of five monoclonal antibodies to Mycobacterium tuberculosis.
The production and characterization of five monoclonal antibodies to Mycobacterium tuberculosis are described. Specificity of the monoclonal antibodies was tested against other mycobacterial species by an enzyme-linked immunosorbent assay and immunoblots. HGT 3a, an immunoglobulin M (IgM) antibody, recognizes a molecule of 38,000 molecular weight present only in the tuberculosis complex of M. tuberculosis and Mycobacterium bovis BCG. HGT 6, an IgG1 antibody, recognized two molecules with molecular weights of 43,000 and 45,000 and showed limited cross-reactivity. Three other antibodies, HGT 1, HGT 2, and HGT 4, all belonging to the IgG1 type, recognized multiple bands and showed broad reactivity among all mycobacterial antigens tested, Escherichia coli and Nocardia asteroides.
Animals↗
Cellular interactions contributing to observed tuberculin reaction.
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Cell Communication↗
Inhibition of cellular immunity by products of Aspergillus fumigatus.
A fraction, FB, from extracts of Aspergillus fumigatus which previously was found to display a high degree of cellular hypersensitivity in skin tests in sensitized guinea-pigs but a low ability to affect transformation of lymphocytes in vitro, was studied. This fraction was found to be capable of inhibiting lymphocyte transformation induced in vitro by tuberculin and by T and B cell mitogens. The inhibitory properties of FB on lymphocyte activation were heat labile, destroyed by proteolysis, and could be reversed by washing 24 h after addition. Antibody to FB did not reduce inhibition of lymphocyte transformation. Fluorescence labeling studies showed a uniform and non-capping distribution of FB on lymphocyte surfaces which resulted in exclusion of attachment of fluorescein-labeled Concanavalin A to the cell surface.
Animals↗
Determination of mycobacterial antigens in sputum by enzyme immunoassay.
An enzyme-linked immunosorbent assay (ELISA) was examined for its usefulness in detecting mycobacterial antigens in sputum. A double-antibody sandwich procedure was set up by using a commercially available hyperimmune serum directed against Mycobacterium bovis, BCG. The ELISA was able to detect 10 ng of protein per ml of BCG sonic extract. The system also clearly distinguished Mycobacterium tuberculosis organisms from Mycobacterium avium and Mycobacterium kansasii organisms. A total of 68 unknown sputum specimens submitted to the clinical laboratories for examination for tuberculosis were tested by ELISA. Of the 20 specimens that were smear positive and culture positive, 12 (60%) were positive by ELISA; 6 of the 11 (55%) smear-positive culture-negative samples were positive by ELISA; 1 of 2 (50%) of the smear-negative culture-positive samples was positive by ELISA; and only 3 of 35 (9%) of the smear-negative culture-negative samples were positive by ELISA. This approach offers promise as an aid in the presumptive differentiation of nontuberculous mycobacteria from the M. tuberculosis complex.
Antibody Specificity↗