Isolation of environment-derived Mycobacterium leprae from soil in Bombay.
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Animals↗
Biomedical subjects
T M Buchanan
Publications and source records attributed to T M Buchanan.
Seroepidemiologic screening for antibodies to LAV/HTLV-III in Sri Lanka, 1980-1982.
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Acquired Immunodeficiency Syndrome↗
Use of a polysulfone membrane support for immunochemical analysis of a glycolipid from Mycobacterium leprae.
Polysulfone membranes have been used as a solid support for chromatography and immunoblotting of phenolic glycolipid I from Mycobacterium leprae. These membranes have an advantage over other supports such as nitrocellulose and silica gel in that very little non-specific background binding of antibodies occurs and assays can readily be carried out with IgM antibodies from human sera. An example of use of the polysulfone chromatography system for detection of phenolic glycolipid I in sera from leprosy patients is described.
Antigens, Bacterial↗
Detection of phenolic glycolipid I in sera from patients with lepromatous leprosy.
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Antibodies, Bacterial↗
Immunochemical characterization of a protein associated with Mycobacterium leprae cell wall.
A panel of nine monoclonal antibodies to Mycobacterium leprae were used to characterize a protein antigen of the bacillus. Two monoclonal antibodies (IVD8 and IIIE9) were specific for M. leprae and reacted with an epitope (CWPa) present on a protein molecule associated with the cell wall fraction of M. leprae. This protein, designated cell wall-associated protein (CWP), lost its immunoreactivity upon treatment with trypsin and had an apparent molecular weight of 65,000, though additional lower-molecular-weight forms of the protein were observed by immunoblotting. Four other cross-reactive epitopes (CWPb, CWPc, CWPd, and CWPe) were defined on the same molecule using seven independent monoclonal antibodies. Therefore, M. leprae possesses a trypsin-sensitive, heat-stable protein associated with the cell wall which contains at least one species-specific and four cross-reactive antigenic determinants.
Antibodies, Monoclonal↗
A spot test for detection of antibodies to phenolic glycolipid I.
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Antibodies, Bacterial↗
Monoclonal antibodies to a 28,000 mol. wt protein antigen of Mycobacterium leprae.
Monoclonal antibodies (MoAb) have been used to analyse a protein antigen from Mycobacterium leprae with a subunit mol. wt of 28,000 daltons. Three different patterns of species specificity were observed with two antibodies being specific for M. leprae, two partially specific, and one broadly cross-reactive amongst all mycobacteria. Competitive binding and sandwich assays demonstrated that the specific and partially specific antibodies recognized closely related regions of the molecule while the cross-reactive antibody recognized a spatially separate epitope on the same polypeptide chain. Identification of specific and cross-reactive epitopes on a single antigenic molecule may be of considerable importance for understanding the functioning of the cell-mediated immune system during leprosy infection and the use of MoAb for such analyses is discussed.
Antibodies, Monoclonal↗
Biochemical alterations in the serum of armadillos (Dasypus novemcinctus) infected with Mycobacterium leprae. A preliminary report.
Armadillos (Dasypus novemcinctus) were inoculated with Mycobacterium leprae isolated from lepromas taken from untreated lepromatous patients or from the spleen of an armadillo previously infected with human M. leprae. The effect of the infection on the serum levels of lactic dehydrogenase (LDH), alkaline phosphatase (AlkP), glutamate-oxalacetate (GOT) and glutamate-pyruvate (GPT) transaminases was investigated. In general, there was a good correlation between positive evidences of infection and alterations in the levels of LDH, GOT, and GPT. Although elevations in LDH levels were more striking, elevations in GOT and GPT levels were more consistent with the disease. When an absolute increase in the total LDH activity was not observed in a M. leprae-infected animal, an increase in the level of LDH isozyme V was still clearly evident. Serum levels of alkaline phosphatase were not affected by the disease. The ratio GOT/GPT (greater than 1.0) in the infected animals reflected and supported the chronic nature of the disease and the liver involvement. The enzymatic alterations are not, however, specific for leprosy.
Alanine Transaminase↗
Monoclonal antibody activity against native and denatured forms of gonococcal outer membrane proteins as detected within ultrathin, longitudinal slices of polyacrylamide gels.
Monoclonal antibodies were used to analyze the antigenic properties of denatured and native forms of gonococcal outer membrane proteins. The protein samples were only partially dissociated by treatment for 30 min at 40 degrees C with 0.1% (w/v) SDS, 0.5% (v/v) Triton X-100, and then processed by polyacrylamide gel electrophoresis without boiling. The resulting pattern included the native aggregated and trimeric forms of protein I and III as they exist in the gonococcal outer membrane, as well as the denatured monomeric forms. Two methods were compared to analyze these gels: gel immunoradioassay (GIRA), and Western blotting. With GIRA longitudinal 50 micron thin slices, up to 40 identical copies per gel, were produced with a microtome cryostat. These slices were exposed to the monoclonal antibody and antibody binding was detected by 125I-protein A and autoradiography. Serotype-specific, monoclonal antibodies reacted most commonly with the native polymeric form of gonococcal protein I and less frequently recognized the denatured, monomeric form. Monoclonal antibodies that recognized the polymeric form of protein I frequently produced antibody-mediated, complement-dependent, bactericidal activity for gonococci bearing the same protein I serotype. The antigen specificity of these functionally relevant antibodies could not be characterized by the Western blotting procedure, which produced incomplete transfer to nitrocellulose paper of the polymeric, high molecular weight protein aggregates. A third technique, radioimmunoprecipitation using partial dissociating conditions, did not permit differentiation between proteins I- and III-specific monoclonals after analysis of the precipitated material by denaturing SDS electrophoresis.
Animals↗
Humans respond predominantly with IgM immunoglobulin to the species-specific glycolipid of Mycobacterium leprae.
The immunoglobulin classes of the antibody response to the species-specific phenolic glycolipid antigen of Mycobacterium leprae have been characterized for serum specimens from 78 patients with leprosy. These patients included the entire clinical spectrum from paucibacillary to multibacillary disease, including polar tuberculoid (TT; 11 patients), borderline tuberculoid (BT; 15), borderline (BB; 17), borderline lepromatous (BL; 13), and lepromatous (LL; 22)--clinical classifications according to Ridley-Jopling criteria. In each patient group, the levels of IgM antibody to phenolic glycolipid were significantly higher than levels of IgG or IgA. Inhibition experiments with purified antigen showed that antibodies to the phenolic glycolipid dominated the human IgM antibody response to the surface of M. leprae.
Antibodies, Bacterial↗
Generation and characterization of monoclonal antibodies to the phenolic glycolipid of Mycobacterium leprae.
Nine cloned cell lines producing antibodies to the unique phenolic glycolipid of Mycobacterium leprae have been established as a result of fusions with spleens from mice immunized with the glycolipid complexed with methylated bovine serum albumin. One of the antibodies was relatively nonspecific, binding to a related glycolipid from Mycobacterium kansasii, but the remaining antibodies were specific for the M. leprae lipid. Some of the antibodies required the intact (trisaccharide) carbohydrate portion for recognition of the glycolipid antigen, whereas others recognized partially hydrolyzed forms lacking one or two sugar residues. Monoclonal antibodies directed at the terminal saccharide of the glycolipid showed the greatest specificity for M. leprae in enzyme-linked immunoassays. These antibodies brightly labeled whole mycobacteria in indirect immunofluorescence experiments, demonstrating the surface location of M. leprae-specific determinants of the glycolipid antigen. In addition to their use in providing information about the antigenic properties of the phenolic glycolipid, these antibodies have potential applications for elucidating the roles of glycolipid in the pathogenesis of leprosy.
Animals↗
Analysis of the antigen specificity of the human serum immunoglobulin G immune response to complicated gonococcal infection.
The antigen-specific basis of human serum immunoglobulin G antibody response to complicated gonococcal infection was studied in 13 patients by using the Western blot technique for transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose paper. Of 13 patients (8 with disseminated gonococcal infection, 4 with pelvic inflammatory disease, 1 with gonococcal epididymitis), 12 reacted with protein I antigens and 9 with lipopolysaccharide (LPS). Sera from eight patients reacted with both protein I and LPS, whereas sera from four reacted only with protein I, and one sera reacted with LPS alone. One serum with antibody to both protein I and LPS by Western blot analysis was tested for bactericidal activity before and after adsorption of antibody to LPS. Removal of antibody to LPS reduced the bactericidal titer of this serum from 1:100 to 1:50, indicating that antibody to both antigens may be bactericidal for Neisseria gonorrhoeae.
Adsorption↗
Ultrastructural localization of specific gonococcal macromolecules with antibody-gold sphere immunological probes.
In an effort to determine the ultrastructural location of specific macromolecules on the surface of intact microorganisms and in experimentally infected tissues, a new method of rapidly conjugating antibodies to gold spheres via a staphylococcal protein A intermediary has been developed. This new technique provides the excellent density of marking and versatility of sphere size provided by existing gold methods, but decreases preparation time, decreases the chance of bacterial contamination of antibody reagents, and increases specificity of marking. Staphylococcal protein A-coated gold spheres were conjugated with antibodies from rabbits immunized with purified gonococcal pili. The resulting gold-antibody conjugates allowed demonstration of antibody binding to pilus structures of the same gonococcal strain whose pili were used to raise the antibody and demonstration of the lack of antibody recognition of pilus structures on two other gonococcal strains. The failure of gold spheres to attach to isogenic nonpiliated clones of the homologous gonococcus indicated the absence of pilus antigens on the surface of these organisms. The use of a double label--small gold spheres conjugated to anti-pilus antibody and larger gold spheres conjugated to anti-protein I antibody--allowed the simultaneous localization of two gonococcal antigens.
Antibodies, Bacterial↗
Antibodies to mycobacterial arabinomannan in leprosy: correlation with reactional states and variation during treatment.
An enzyme-linked immunosorbent assay was used to measure antibody to mycobacterial arabinomannan in serial serum specimens obtained over the initial 12-31 months of therapy from nine patients with leprosy. The antibody level in pretreatment sera was directly proportional to the quantity of Mycobacterium leprae present in each patient as assessed by six-site scrapings (r = 0.75). The three patients with the lowest antibody levels (OD 0.1-0.3) had uncomplicated courses and their levels declined slowly with treatment. Three patients with intermediate antibody levels (OD 0.7-1.1) each experienced a reversal reaction during therapy; serial antibody titers in all three followed a triphasic pattern over the course of the reaction. The two patients who developed erythema nodosum leprosum during therapy had extremely high levels of antibody initially (OD greater than 1.5), which fell slowly with time and which were unaffected by the reactional state. The pretreatment antibody level to arabinomannan reflects the amount of M. leprae present and may have predictive value for the development of reactional states.
Adrenal Cortex Hormones↗
Production and characterization of a murine monoclonal antibody recognizing a shared mycobacterial polysaccharide.
An IgM monoclonal antibody specific for mycobacterial arabinomannan was produced by the fusion of splenocytes from BALB/c mice immunized with purified arabinomannan with NSI/1 myeloma cells. Specificity was demonstrated by gel-radioimmunoassay, and by inhibition of binding using the purified polysaccharide. The monoclonal antibody recognized the arabinomannans from all 18 species of mycobacteria tested, including Mycobacterium leprae. This antibody expands the number of defined mycobacterial antigens against which monoclonal antibodies have been produced, and has potential application in studies concerning the pathogenesis of mycobacterial disease.
Animals↗
A serological test for leprosy with a glycolipid specific for Mycobacterium leprae.
A phenolic glycolipid from Mycobacterium leprae was purified and used as antigen in an enzyme-linked immunosorbent assay. Antibodies directed against the lipid were seen in serums from leprosy patients but not in serums from uninfected controls or patients infected with other mycobacteria, including Mycobacterium tuberculosis. The antibody response distinguished between the Mycobacterium leprae lipid and the structurally related phenolic glycolipid from Mycobacterium kansasii. This assay has considerable potential as a specific serodiagnostic test for leprosy infection.
Enzyme-Linked Immunosorbent Assay↗
Confirmation of association of protein I serotype of Neisseria gonorrhoeae with ability to cause disseminated infection.
Previous work indicates that strains of Neisseria gonorrhoeae isolated in Seattle, Wash., and Atlanta, Ga., show an association between serotypes 1 and 2 of protein I of the outer membrane and the ability to cause disseminated infection (T.M. Buchanan and J.F. Hildebrandt, Infect. Immun. 32:985-994, 1981). By using the same serotyping system, we confirmed the association between those serotypes and both disseminated infection and serum resistance in strains from North Carolina. Some strains of the same serotype had protein I species with different apparent molecular weights.
Bacterial Proteins↗
Development of an enzyme-linked immunosorbent assay using arabinomannan from Mycobacterium smegmatis: a potentially useful screening test for the diagnosis of incubating leprosy.
A carbohydrate antigen composed predominantly of arabinomannan has been purified from Mycobacterium smegmatis and used in an enzyme-linked immunosorbent assay to detect anti-mycobacterial antibodies in human sera. Sera from 117 controls, 25 tuberculosis patients, 124 leprosy patients and 256 household contacts of leprosy patients were tested. When compared with the control group, 56% of tuberculosis patients, 27% of patients with tuberculoid leprosy, 77% of borderline leprosy cases, and 95% of patients with lepromatous leprosy had elevated titers. Nine percent of the household contact group had abnormally high levels of antibody. The relevance of these findings to the serodiagnosis of incubating leprosy and the management of household contacts of leprosy patients is discussed.
Adolescent↗