Serodiagnosis of infection with Mycobacterium leprae.
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Antibodies, Bacterial↗
Biomedical subjects
T M Buchanan
Publications and source records attributed to T M Buchanan.
Gonococcal pili: safety and immunogenicity in humans and antibody function in vitro.
In a phase I clinical trial, 39 volunteers received an initial and booster subcutaneous injection of 100 or 112 microgram of a pili vaccine prepared from Neisseria gonorrhoeae strain F62 in Tris buffer with an aluminum phosphate adjuvant (alum) or 220 microgram of the F62 pili vaccine prepared in ethanolamine, with or without alum. Antibody responses were quantitated with an enzyme-linked immunosorbent assay. All four groups had significant (P less than 0.0001) responses to the vaccine preparations with peak mean antibody responses one to three weeks after the booster. Differences in antibody responses among the groups were due to the administration of alum and not to use of Tris buffer vs. ethanolamine. In vitro, postimmunization sera enhanced phagocytosis of piliated strain F62 organisms by human polymorphonuclear leukocytes; preabsorption of the sera with strain F62 pili blocked this activity. Thus, gonococcal pili given subcutaneously are immunogenic and lead to production of functional serum antibodies.
Adjuvants, Pharmaceutic↗
Serological classification of Neisseria gonorrhoeae with monoclonal antibodies.
Hybrid cells producing monoclonal antibodies against antigens of Neisseria gonorrhoeae were obtained by the polyethylene glycol-mediated fusion of mouse myeloma cells and lymphocytes from mice immunized with gonococcal protein I or outer membrane proteins. From four fusions, 16 phenotypically stable, independently cloned hybrid cell lines were selected for continued study. Each of the cell lines produced a characteristically different monoclonal antibody which reacted in immunoprecipitation assays with a unique antigenic determinant on protein I of the outer membrane complex of the bacteria. In antibody binding, immunofluorescence, and coagglutination assays these antibodies each reacted with a restricted group of N. gonorrhoeae strains. None of the monoclonal antibodies reacted with 17 other different species of Neisseria or with Branhamella catarrhalis. When tested on 34 N. gonorrhoeae reference serotyping strains, the monoclonal antibodies demonstrated serological relationships between the strains which paralleled those observed with conventional polyvalent antisera. These antibodies now provide standardized reagents for the rapid and precise serological characterization of many strains of N. gonorrhoeae.
Animals↗
Production and partial characterization of monoclonal antibodies to Mycobacterium leprae.
Monoclonal antibodies to Mycobacterium leprae were produced by the fusion of BALB/c splenocytes and lymph node cells to BALB/c myeloma (NSI/1) cells. Eleven monoclonal antibodies were characterized as to their reactivity with M. leprae and 18 other mycobacterial species by enzyme-linked immunosorbent assay and immunofluorescence. Two monoclonal antibodies reacted only with M. leprae, and the other nine showed unique patterns of reactivity by enzyme-linked immunosorbent assay. One monoclonal antibody (IIH9) reacted with a 68,000-dalton protein present in extracts from M. leprae, M. tuberculosis H37Rv, M. gastri, and M. smegmatis. Potential uses for these antibodies in serological tests and immunochemical analyses are discussed.
Antibodies, Bacterial↗
Attachment of gonococcal pili to lectin-resistant clones of Chinese hamster ovary cells.
Pili facilitate the attachment of virulent Neisseria gonorrhoeae to host cells. Isolated pili and peptides from pili obtained by cyanogen bromide cleavage were used in attachment assays to Chinese hamster ovary cells and their lectin-resistant clones. Pili and the largest cyanogen bromide fragment (CNBrI) from the amino-terminal portion of the pilin molecule attached to a greater degree to the parent cell and showed 40 to 75% reduced attachment to clones deficient in cell surface oligosaccharides. The CNBrI fragment, with a molecular weight of approximately 10,000, bound specifically to host proteins with subunit molecular weights of 14,000 to 16,000 that were electrophoretically transferred onto nitrocellulose sheets from polyacrylamide gel patterns of host cells. Periodate or galactosidase treatment of pili or the CNBrI fragment markedly reduced attachment, suggesting the importance of galactose residues on pili for their attachment function. Similarly, highly purified exoglycosidase or trypsin treatment of the parent cell reduced attachment, suggesting that oligosaccharide moieties of cell surface components (glycoproteins or glycolipids or both) were receptors for pili attachment. This study indicated that the portion of the pilin molecule involved in attachment resides on the CNBrI fragment and that sugar moieties, both on pili and on the host cell, were required for optimal attachment.
Animals↗
Serology of Neisseria gonorrhoeae: coagglutination serogroups WI and WII/III correspond to different outer membrane protein I molecules.
The 125I-labeled tryptic peptides of the outer membrane protein I of 33 previously characterized serological reference strains of Neisseria gonorrhoeae were investigated by peptide maps in relation to their coagglutination W serogroup. Serogroup WI strains tended to have lower-molecular-weight protein I molecules than did WII strains, and WIII strains had the highest-molecular-weight protein I molecules, although the serogroup could not be predicted from the molecular weights of the protein I molecules for a given strain. All 13 strains belonging to serogroup WI were found to have 11 peptides in common, as judged by their migration with respect to one another and to the internal marker valine in the peptide maps. Common peptides isolated from a given strain were found to comigrate with the corresponding common peptides from other strains in the same serogroup under various electrophoretic conditions. The 20 strains belonging to serogroups WII and WIII were all found to have 10 common peptides by the same criteria. When common peptides from serogroup WI were compared with the common peptides of serogroups WII and WIII, only three of these peptides appeared to be similar. Thus, two different outer membrane protein I molecules seem to exist which are mutually exclusive. Protein IA molecules contain the antigens recognized as serogroup WI, and protein IB molecules contain the antigens that characterize serogroups WII and WIII.
Agglutination Tests↗
Detection of a gonococcal endo-beta-N-acetyl-D-glucosaminidase and its peptidoglycan cleavage site.
Neisseria gonorrhoeae contains several hydrolases which may be responsible for gonococcal cell lysis. One of these enzymes, an endo-beta-N-acetyl-D-glucosaminidase, has been extracted from supernatants of sonicated gonococci and partially purified by ammonium sulfate precipitation and affinity and ion-exchange chromatography. This enzyme has a different specificity than egg white lysozyme and cleaves the beta 1 leads to 4 glycosidic linkage between N-acetylglucosamine and N-acetylmuramic acid in gonococcal peptidoglycan.
Acetylglucosaminidase↗
Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein.
Principal outer membrane protein (protein I) of Neisseria gonorrhoeae was prepared nearly free of lipopolysaccharide (LPS) and substantially purified from other membrane proteins by chromatography of partially purified gonococcal outer membranes over Sepharose 6B in the presence of deoxycholate at pH 9.0. This protein I of nine separate antigenic types was coated to polystyrene tubes and used in the enzyme-linked immunosorbent assay (ELISA) to measure antibody to protein I or in inhibition tests to quantitate protein I antigen. No significant inhibition of the ELISA test was produced by purified LPS from the strain used to prepare each of the protein I types or by whole gonococci bearing the same LPS but different protein I antigens as the strain used to produce a given protein I antigen. Of 125 strains of gonococci used as whole organisms to inhibit the protein I ELISA, 124 (99%) typed with one or more of the nine protein I types, and 35% of these typed with a single protein I serotype. Sixty-one of 65 (94%) strains from Seattle and Atlanta patients with disseminated gonococcal infection contained protein I serotype 1, and 16 of 24 (64%) strains from Seattle patients with salpingitis bore one or both of protein I serotypes 1 and 2.
Antigens, Bacterial↗
Comparison of 22 species of Mycobacteria by immunodiffusion against an absorbed reference leprosy serum.
Lithium acetate antigenic extracts of 22 species of acetone-treated mycobacteria were tested by immunodiffusion precipitation for reactivity with a pool of sera from treated lepromatous leprosy patients (ARLS). This ARLS had been adsorbed with M. bovis (BCG, M. vaccae, cardiolipin, and lecithin to make it specific for M. leprae when used in an indirect immunofluorescence test. The ARLS produced two precipitin lines with M. leprae extract, one of which formed a line of identity with extracts of M. lepraemurium and M. bovis (BCG). Aso, recognition without reactions of identity was produced between ARLS and M. flavecens, M. gastri, M. gordonae, and M. nonchromogenicum. The ARLS did not recognize the 15 other species including the human pathogens, M. tuberculosis, M. intracellulare, M. kansasii, M. scrofulaceum, and M. marinum. These data suggest that serologic tests for M. leprae infection might be affected by antibodies to antigens shared by M. leprae and other mycobacteria. The significance of these shared antigens will depend upon the prevalence of human immune responses to mycobacteria containing the shared antigens in any given community.
Antigen-Antibody Reactions↗
Gonococcal salpingitis is less likely to recur with Neisseria gonorrhoeae of the same principal outer membrane protein antigenic type.
If protective immunity were to develop following an episode of gonococcal pelvic inflammatory disease (salpingitis), PID should recur with organisms bearing antigens that do not react with these immune mechanisms. To test this hypothesis, gonococci from 15 women with gonococcal PID, who experienced 19 subsequent episodes of gonococcal infection, were serotyped for their principal outer membrane protein (Protein I) antigens. Of nine cases in which the initial and subsequent infections involved the same Protein I serotype, none was associated with recurrence of PID. Of 10 cases in which the initial and subsequent infections involved different Protein I types, five were associated with recurrent PID (p = 0.02). These data suggest that an episode of gonococcal PID produces some immunity to repeated episodes of salpingitis with the same Protein I serotype, while not preventing reinfection with the same Protein I serotype. The immune response to Protein I antigen may thus provide serotype-specific protection against gonococcal salpingitis.
Antigens, Bacterial↗
Hydrolases from Neisseria gonorrhoeae. The study of gonocosin, an aminopeptidase-P, a proline iminopeptidase, and an asparaginase.
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Amino Acid Sequence↗
Immune-enhanced phagocytosis of Neisseria gonorrhoeae by macrophages: characterization of the major antigens to which opsonins are directed.
antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab')2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.
Antigen-Antibody Reactions↗
2-mercaptoethanol Brucella agglutination test: usefulness for predicting recovery from brucellosis.
Ninety-two patients with brucellosis were studied for 18 months, and 15 to 29 sera from each patient were tested by the standard tube brucella agglutination test and by the 2-mercaptoethanol (2ME) brucella agglutination test. The standard tube test remained positive (greaterr than or equal to 160) for 1.5 years in 44 of the 92 patients (48%), despite adequate antibiotic treatment. The 2ME titers remained positive (greater than or equal to 160) in only 8 of 92 patients (9%) after 1 year, and in only 4 of 92 patients (4%) after 1.5 years. Positive 2ME titers 1 year after initiation of treatment were present in 3 of 10 patients (30%) with drug allergies that interfered with antibiotic treatment, in contrast to only 5 of 82 patients (6%) without any allergies (P = 0.039). None of 84 patients with negative 2ME titers after 12 months of treatment had significant signs or symptoms of brucellosis, and none developed chronic brucellosis. In contrast, four of eight patients with positive 2ME titers after 12 months of treatment still had signs and symptoms of brucellosis and required further treatment. The 2ME test is superior to the standard tube test in determining the adequacy of antibiotic therapy, and a negative 2ME test is strong evidence against a diagnosis of chronic brucellosis.
Agglutination Tests↗
Determination of biogenic amines using heterocyclic cation and aromatic anion elution.
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Anion Exchange Resins↗
Amine content of vaginal fluid from untreated and treated patients with nonspecific vaginitis.
We examined the vaginal washings from patients with nonspecific vaginitis (NSV) to seek biochemical markers and possible explanations for the signs and symptoms of this syndrome. Seven amines were identified including methylamine, isobutylamine, putrescine, cadaverine, histamine, tyramine, and phenethylamine. These amines may contribute to the symptoms of NSV and may contribute to the elevated pH of the vaginal discharge. They may also be partly responsible for the "fishy" odor that is characteristic of vaginal discharges from these patients. Among the seven amines, putrescine and cadaverine were the most abundant and were present in all vaginal discharges from each of ten patients before treatment. These amines are produced in vitro during growth of mixed vaginal bacteria in chemically defined medium, presumably by decarboxylation of the corresponding amino acids. We hypothesize the anaerobic vaginal organisms, previously shown to be quantitatively increased in NSV, are responsible for the amine production, because metronidazole inhibited the production of amines by vaginal bacteria in vitro, and Haemophilus vaginalis did not produce amines. H. vaginalis did release high concentrations of pyruvic acid and of amino acids during growth in peptone-starch-dextrose medium, whereas, other vaginal flora consumed both pyruvic acid and amino acids in the same medium during growth. These findings suggest that a symbiotic relationship may exist between H. vaginalis and other vaginal flora in patients with NSV.
Amines↗
Immunoglobulin class responsible for gonococcal bactericidal activity of normal human sera.
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Adolescent↗
Immunochemical and structural integrity of surface protein antigens of mycobacteria during separation from armadillo liver tissue.
Surface proteins of Mycobacterium smegmatis were iodinated using the lactoperoxidase method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated two major surface proteins in the radiolabelled M. smegmatis. Both surface proteins were released from M. smegmatis using the nonionic detergent Triton X-100. The major surface component was sensitive to pronase digestion and contained no detectable carbohydrate. The second radiolabelled component was found to be of low molecular weight, resistant to pronase digestion and stained positive for carbohydrate by the periodic acid/Schiff method. Triton X-100 solubilized radiolabelled surface proteins were antigenic as assessed by a radioimmune precipitation test. When surface labelled M. smegmatis was mixed with armadillo liver tissue and separated from tissue using a method formerly employed by the World Health Organization Immunology of Leprosy Program for the purification of M. leprae, as much as 50% of the surface proteins of M. smegmatis was either released or destroyed. In addition, another twenty distinct proteins were released from M. smegmatis after treatment with Triton X-100. Similar losses of proteins from M. leprae may also occur using this procedure for M. leprae purification. Separation techniques employing surfactants and enzymatic treatment should be carefully evaluated since proteins lost during these procedures may prove relevant to human immune responses to M. leprae.
Animals↗
Identification of a Mycobacterium leprae specific protein antigen(s) and its possible application for the serodiagnosis of leprosy.
Acetone-killed Mycobacterium leprae separated from infected armadillo liver tissue without the use of proteases were treated with 0.2 M lithium acetate, 20 mM EDTA, pH 8.8 solution, and the concentrated antigen extract was analyzed by Ouchterlony immunodiffusion. The antigen extract gave a single immunoprecipitate when reacted with pooled lepromatous leprosy (LL) patients sera made highly specific for M. leprae by adsorption. Apparently identical precipitates were produced by reacting the antigen extract with sera of each of 15 treated LL patients, 5 of 7 patients with tuberculoid leprosy, and 3 of 4 M. leprae infected armadillos. Serum from 1 of 16 persons immunized with BCG and from none of 15 patients with chlamydial urethritis or brucellosis reacted with the antigen. Identically prepared extracts of M. smegmatis, M. phlei, M. vaccae, M. duvali and M. diernhoferi gave no immunoprecipitates with sera from LL patients or infected armadillos. Preliminary characterization indicates the antigen is protein since antigenicity was destroyed by pronase and/or heat treatment. The relative specificity of the protein antigen for M. leprae and the presence of antibody to this antigen in patients with leprosy suggest a possible role for this antigen in the serodiagnosis of leprosy.
Animals↗