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.     

DOT-ELISA for detection of phenolic glycolipid PGL-Tb1 and diacyl-trehalose antigens of Mycobacterium tuberculosis.

A DOT-ELISA method for detection of 2,3-diacyl-trehalose (DAT, previously referred to as SL-IV antigen) and triglycosyl phenol phthiocerol di-mycocerosate (PGL-Tb1) antigens from Mycobacterium tuberculosis is described. The method enabled the detection of both antigens in 14 clinical isolates of M. tuberculosis from different geographic origins; the presence of the glycolipids was confirmed by chemical analysis. It was therefore concluded that the synthesis of both of these compounds is characteristic of the species.     

Production and characterization of a mouse monoclonal antibody to the glycolipid asialo-GM1

The glycosphingolipid asialo-GM1 (aGM1) is a true differentiation antigen of murine lymphoid cells. This glycolipid is highly immunogenic in the rabbit, but the antisera produced shows some cross reactivity with GM1, the naturally occurring sialylated derivative of aGM1. In the present study we examined the ability to raise anti-aGM1 antisera in the mouse. We compared the efficiency of several immunization methods in various strains of mice. The most effective procedure involved repeated intraperitoneal injections of aGM1-cholesterol rich particles in the NZB mouse. Hybrid B cell lines were generated by fusion of mouse myeloma cells with the splenocytes of an NZB mouse immunized with aGM1. The specificity of the antisera produced and of the monoclonal antibody secreted by one of these hybridomas (103HT30) was defined by ELISA and by immunostaining on thin layer chromatograms. The monoclonal antibody 103HT30 is an IgM. It reacted with aGM1 but not with any of the structurally-related ganglioside or neutral glycolipids tested. In particular, 103HT30 monoclonal antibody did not present any detectable cross-reactivity with GM1.     

Production of a monoclonal antibody specific for Mycobacterium avium and immunological activity of the affinity-purified antigen.

Three hybridomas which secrete antibodies to Mycobacterium avium were obtained by the fusion of p3u1 myeloma cells with spleen cells of mice immunized with M. avium culture sonicate. The reactivity of these monoclonal antibodies was determined in 16 species of mycobacteria by an enzyme-linked immunosorbent assay. An antibody, designated Avi-3, reacted only with M. avium and not with the reference strains of the other 15 species of mycobacteria tested, including M. intracellulare, M. paratuberculosis, and M. lepraemurium. Specificity of the antibody was confirmed by assay, using a specific DNA probe of M. avium complex in 29 M. avium complex isolates. An antigen was purified from M. avium culture sonicate on a monoclonal antibody Avi-3-coupled affinity column. The purified antigen gave a single band (molecular size, about 27 kilodaltons) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen Avi-3 showed strong skin test activity in guinea pig preimmunized with heat-killed M. avium but not in those sensitized with heat-killed M. intracellulare or M. bovis BCG. Purified protein derivative elicited positive skin reactions in all the immunized guinea pigs. When heat-killed M. avium was used as the immunogen, strong lymphoproliferative responses were observed in cultures stimulated with the antigen Avi-3. These results suggest that M. avium-specific antigen Avi-3 may facilitate the diagnosis of mycobacterial infections.     

Strain variations in the murine cellular immune response to the phenolic glycolipid I antigen of Mycobacterium leprae.

The cellular immune response to the Mycobacterium leprae-specific phenolic glycolipid I was examined in inbred mice immunized with M. leprae by in vivo delayed cutaneous hypersensitivity and in vitro lymphocyte proliferation. Whereas all mouse strains responded to M.leprae-induced delayed-type hypersensitivity and lymphocyte proliferation, only BALB.K was responsive in both assays to the glycolipid. Responsiveness was determined in part by non-H-2 genes, while the influence of H-2 genes was not apparent. Among congenic BALB/c mice differing only at Igh-C allotype loci, variations in responsiveness were found in both delayed-type hypersensitivity and lymphocytes proliferation assays, indicating a possible role for Igh-C loci-linked genes. Unresponsiveness in the lymphocyte proliferation assay to the glycolipid was inherited as a dominant trait in one set of responder X nonresponder F1 progeny. We conclude that after immunization with M. leprae organisms, the cell-mediated responses to the glycolipid, endowed with a single carbohydrate epitope, are under polygenic control, predominantly non-H-2-linked genes.     

Production of monoclonal antibody to characterize the antigen ofParagonimus westermani

We produced monoclonal antibodies against the adult antigen ofParagonimus westermani to investigate the expression of the stage-specific antigen of adult flukes. Two hybridoma cell lines, A1-2 and A4-1, were established. A1-2 reacted specifically only with the adult antigen in an enzyme-linked immunosorbent assay (ELISA), whereas A4-1 reacted with both adult and larval antigens. By immunoblotting analysis, A1-2 was found to react with four bands with molecular weights of 35000, 17000, 15500, and 12500 in the adult antigen but with none in the larval antigen. A4-1 was found to bind to the band of 27000 daltons in the adult antigen as well as those of 28000 and 26000 daltons in the larval antigen. By immunohistochemical methods, a positive reaction was observed only in the parenchymal tissues of the adult flukes with A1-2. A 4-1 reacted with the substance that located on the gut epithelium and in the luminal contents of both the adult and larval flukes. These results indicate that the adultP. westermani possesses an antigen or antigenic determinant specific to the adult stage, as well as that common to both the larval and adult stages.     

Production of monoclonal antibody to a late intracellular Epstein-Barr virus-induced antigen

A monoclonal antibody designated L2 was produced against a late intracellular protein induced by Epstein-Barr virus (EBV). This protein was expressed in cells producing virus but not in EBV genome-positive nonproducer cell lines, EBV genome-negative cell lines, or producer cultures cultivated in the presence of phosphonoacetic acid as determined by immunofluorescence. In addition, the antibody did not react with the membranes of infected cells indicating that it was not directed against an EBV-induced membrane antigen component. The monoclonal antibody was shown to recognize a glycoprotein with a molecular weight of approximately 125K by SDS-polyacrylamide gel electrophoresis. This glycoprotein was consistently found to be slightly larger when isolated from the P₃HR-1 cell line as opposed to the B-95-8 cell line. A similar difference was also noted by comparison of peptide maps of this protein isolated by immunoaffinity chromatography from the two cell lines. Serological studies indicated that this 125K glycoprotein was a major component of the viral capsid-antigen (VCA) complex as defined by immunofluorescence.     

Production and characterization of a monoclonal antibody to the complement fixation antigen of Coccidioides immitis.

Detection of complement-fixing antibody to coccidioidin by using the complement fixation test or an immunodiffusion assay for complement-fixing antibody (IDCF) is widely viewed as the most useful immunodiagnostic test for coccidioidomycosis. In this investigation, we report the production of an immunoglobulin G subclass 1 (IgG1) monoclonal antibody (MAb) to the IDCF antigen for use as a biospecific ligand for purifying the IDCF antigen on solid-phase immunosorbents and for use as a reagent for screening genomic or cDNA expression libraries from Coccidioides immitis. BALB/c mice were immunized by intramuscular injections of coccidioidin in adjuvant, followed by an intrasplenic booster injection of coccidioidin in saline. The spleen cells were fused with SP2/0 Ag14 myeloma cells, and the fusion products were screened for IgG antibody to coccidiodin by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned and evaluated for reactivity to the IDCF antigen by two-dimensional immunoelectrophoresis and by immunoblotting. An IgG1 Mab was produced that was specific for the IDCF antigen when evaluated by two-dimensional immunoelectrophoresis and immunoblotting. The epitope recognized by the MAb was heat labile (60 degrees C, 30 min) and susceptible to enzymatic digestion with pronase but was resistant to treatment with lipase, alpha-mannosidase, glucose oxidase, and endoglycosidase H. This heat-labile peptide epitope appears to be specific to C. immitis, as judged by the fact that the MAb was not reactive in immunoblots or enzyme-linked immunosorbent assays of histoplasmin or blastomycin.     

Isolation of the Mycobacterium leprae-specific glycolipid antigen, phenolic glycolipid-I, from formalin-fixed human lepromatous liver.

A Mycobacterium leprae-specific phenolic glycolipid antigen was purified from Formalin-fixed liver preserved from an advanced lepromatous leprosy patient. Its chemical and immunological properties were compared with those of phenolic glycolipid-I obtained from M. leprae-infected armadillo liver. Based on the findings that the glycolipids from the two sources have the same thin-layer chromatographic properties, infrared absorption spectrum, sugar composition, and seroreactivity, we conclude that large quantities of the phenolic glycolipid-I antigen are produced in human lepromatous leprosy lesions and that Formalin-fixed lepromatous livers and spleens from the prechemotherapeutic era are suitable sources of the glycolipid.     

Serological activity of a characteristic phenolic glycolipid from Mycobacterium leprae in sera from patients with leprosy and tuberculosis

Serological activity against a purified phenolic glycolipid from Mycobacterium leprae, which may be obtained in large amounts from M. leprae infected armadillo liver, was investigated using immunodiffusion and an enzyme linked immunosorbent assay (ELISA). Generally a good correlation was obtained between these techniques, but the ELISA was more sensitive and convenient. Relatively high IgG and IgM anti-glycolipid antibody levels were found in lepromatous leprosy patients. The antibody titres to the glycolipid were, however, low when compared with antibody titres to crude sonicates. Since the glycolipid is present in large quantities, this suggests that it is not very immunogenic. Antibody against the glycolipid especially of the IgM class, was demonstrable in some tuberculoid leprosy patients, although at much lower titres than in the lepromatous leprosy sera. In lepromatous leprosy patients that were skin smear negative after more than 5 years of treatment the IgG anti-M. leprae derived glycolipid activity had decreased markedly. The anti-IgG and IgM glycolipid antibody levels in tuberculosis patients did not differ significantly from the levels in appropriate normal healthy subjects. The glycolipid antibody levels in patients infected with M. kansasii, M. avium or M. intracellulare also fell within the range of normal healthy individuals.     

Evaluation of a commercial monoclonal antibody for detection of adenovirus antigen.

The Adenoclone monoclonal antibody for detection of adenovirus antigen was evaluated by enzyme immunoassay and a 72-h indirect fluorescent-antibody test in shell vials and compared with standard culture. The sensitivity and specificity of the Adenoclone test were both 100% with 114 cell culture isolates. With 310 direct clinical specimens, the Adenoclone enzyme immunoassay sensitivity varied from 14.3 to 66.6%, but the Adenoclone indirect fluorescent-antibody test sensitivity was 86.7%, with 100% specificity.     

Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a monoclonal antibody immunoblot assay

Throat swabs from patients with pharyngitis and sputum specimens from patients with atypical pneumonia were tested for the presence of a Mycoplasma pneumoniae polypeptide with a molecular weight of 43,000 with the use of an M. pneumoniae species-specific monoclonal antibody in an immunoblot assay. This 43,000-dalton polypeptide was detectable in 33 of 33 throat swabs from patients with pharyngitis that were positive for M. pneumoniae by conventional culture as well as a culture-amplified enzyme immunoassay. The 43,000-dalton polypeptide was also detected in three of three M. pneumoniae culture-positive sputum specimens. It was not detected in 3 sputum specimens culture-confirmed for Legionella pneumophila, 10 sputum specimens from normal persons, or 25 throat swabs also from normal persons. This immunoblot assay could be completed within five hours and may be an alternative method for detecting M. pneumoniae antigen directly in sputum or throat swab specimens.     

Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.     

Specificity of a Mycobacterium kansasii phenolic glycolipid (mycoside A) immunoserum.

The specificity of Mycobacterium kansasii anti-mycoside A antiserum prepared in rabbits injected with purified samples of the phenolic glycolipid was evaluated by an enzyme-linked immunosorbent assay. Chloroform-methanol extracts from representative strains of 23 mycobacterial species and 50 strains of M. kansasii showed that all strains of M. kansasii and the representative strain of M. gastri formed the antigen, whereas none of the remaining species (including M. leprae) formed it. Consequently, it was found that the antiserum was highly specific and useful for diagnostic purposes.     

Antibody response to phenolic glycolipid I in inbred mice immunized with Mycobacterium leprae.

The level of circulating antibody to phenolic glycolipid I of Mycobacterium leprae was determined in 18 inbred strains of mice after immunization with M. leprae organisms. By using a solid-phase radioimmunoassay with phenolic glycolipid I as test antigen, a continuous distribution of antibody levels ranging from high to low was observed. The level was found to be controlled by multiple genes, including both H-2 complex- and Igh allotype complex-linked genes. Low antibody response to phenolic glycolipid I was shown to be inherited as a dominant trait in three combinations of high X low responder F1 progeny.     

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.     

Antigen capture assay for detection of a 43-kilodalton Mycobacterium tuberculosis antigen.

This study describes the development of an enzyme-linked immunosorbent assay (ELISA) to detect Mycobacterium tuberculosis antigens in body fluids. A double-antibody sandwich procedure that used human and rabbit anti-M. tuberculosis immunoglobulin G antibodies was followed. The ELISA was able to detect as little as 0.8 micrograms of protein of M. tuberculosis sonic extract. Of 253 cerebrospinal fluid specimens submitted for analysis, 11 (4.3%) false-positive results were recorded. Analysis of 317 pleural and ascitic fluid specimens resulted in 6 (1.9%) false-positive recordings. No false-negative results were recorded for any of the body fluids tested. This technique is rapid (5.5 h) and sensitive, may be developed and used in many laboratories with limited resources, and may prove useful in the diagnosis of extrapulmonary and pulmonary tuberculoses. Analysis of these body fluids by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting indicated that the antibody used in the ELISA detects a mycobacterial antigen of 43 kDa. Such antigens were not detected in body fluids of nontuberculous patients.     

A monoclonal antibody to a rat hepato-renal membrane antigen.

A monoclonal antibody against a membrane glycoprotein of rat hepatocytes has been produced. The nature of this antibody designated as HAM.4 was analysed by cellular radioimmunoassay, flow cytofluorography and indirect immunoperoxidase procedures. The following characteristics of HAM.4 were elucidated. First, an immunohistochemical study revealed that this antibody stained preferentially the bile canalicular face of hepatocyte membrane. Secondly, HAM.4 cross-reacted with kidney, spleen and thymus as well as liver. The kidney expressed much more the antigen molecules detected by this antibody than the liver did. The antigen was located predominantly on the brush border of proximal tubules in kidney. Thus, HAM.4 would be useful for analysing one of the brush border antigens of renal tubules which has been thought to be a pathogenic antigen for inducing experimental membranous glomerulonephritis. Finally, HAM.4 failed to label the cell membrane of rat hepatoma cell lines examined, indicating that the antigen detected by HAM.4 may disappear from cell surface during the course of hepatocarcinogenesis.     

Production and characterisation of a human monoclonal antibody to cytomegalovirus and its use in an early nuclear fluorescence assay

A human monoclonal antibody to cytomegalovirus (CMV) was produced by transforming peripheral blood mononuclear cells of a patient with recent CMV infection. It is directed against a late antigen located in the nucleus of CMV infected fibroblasts at 24-72 hours postinfection and immuneprecipitates 65K and 48K proteins from 35S-labelled CMV infected cells. Results of its use in an early nuclear fluorescence assay for rapid diagnosis are presented.     

Characterization of extensively drug-resistant Mycobacterium tuberculosis clinical isolates in China

Thirteen extensively drug-resistant tuberculosis isolates which were highly resistant to a broad spectrum of antituberculosis drugs were identified from 1,926 clinical isolates in China. They had highly diverse mycobacterial interspersed repetitive-unit-variable-number tandem-repeat patterns. Most, but not all, of the drug target genes had mutations contributing to resistance to the corresponding drug.