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In situ locations of Mycobacterium leprae-specific antigens. Immunoelectronoptical studies
1Dept. Dermatology, Medical Faculty, Erasmus University, Rotterdam, The Netherlands.
Abstract:
Lipid or protein antigen sites in Mycobacterium leprae proper and in M. leprae -infected human or armadillo tissues were investigated by immunogold-electron microscopy. Simultaneous preservation of immunogenicity of antigens and conservation of ultrastructural details of M. leprae and host cells was aimed at by subjecting organisms and tissues, prior to immunolabelling, to differing fixation, embedding and ultramicrotomy techniques. The M. leprae-specificity of monoclonal antibodies (MoAbs) utilized in the study was tested first. Hereto, ultracryosections of M. leprae, M. tuberculosis and M. nonchromogenicum suspended in gelatin were employed. MoAb anti-phenolic glycolipid I (PGL I) and MoAb anti-36 kD were found to be specific for M. leprae. MoAb anti-65 kD also labelled the cytoplasm of M. tuberculosis. After incubation with MoAb anti-lipid MAIS, employed as control MoAb, no gold labelling of leprosy bacilli or host cells was seen. PGL-I immunogenicity was still present after "hard" fixation of M. leprae and host cells in glutaraldehyde-OsO4 and after Araldite embedding. This enabled the qualitative demonstration of PGL-I inside the cell wall and capsular area of M. leprae and in vacuoles of bacillated phagolysosomes of macrophages in Araldite-embedded human skin biopsies and armadillo liver parenchymal cells. Sites of 65 kD and, to a lesser extent, of 36 kD protein antigens in M. leprae were demonstrable only in ultracryosections of non-fixed organisms and not in Araldite sections. Results are discussed and recommendations for future investigations on M. leprae antigen sites are presented.
Insights
Investigating Mycobacterium leprae antigen sites using immunogold-electron microscopy revealed that phenolic glycolipid I (PGL-I) is detectable in host tissues. Protein antigens required non-fixed samples for visualization.
Area of Science:
- Microbiology
- Immunology
- Electron Microscopy
Background:
- Mycobacterium leprae causes leprosy, a disease with complex antigenicity.
- Understanding antigen localization is crucial for diagnostics and therapeutics.
Purpose of the Study:
- To investigate the localization of lipid and protein antigens of Mycobacterium leprae.
- To optimize immunogold-electron microscopy techniques for preserving antigenicity and ultrastructure.
Main Methods:
- Immunogold-electron microscopy was used to examine M. leprae and infected tissues.
- Various fixation, embedding, and ultramicrotomy techniques were tested.
- Monoclonal antibodies (MoAbs) specific to M. leprae antigens were employed and their specificity confirmed.
Main Results:
- Phenolic glycolipid I (PGL-I) was localized within M. leprae cell walls and in host cell vacuoles, retaining immunogenicity after harsh fixation and Araldite embedding.
- 36 kD and 65 kD protein antigens were primarily visualized in non-fixed ultracryosections, indicating sensitivity to fixation and embedding processes.
- Monoclonal antibodies anti-PGL-I and anti-36 kD demonstrated M. leprae specificity, while anti-65 kD also labeled Mycobacterium tuberculosis.
Conclusions:
- Phenolic glycolipid I is a robust antigen suitable for immunolocalization in fixed and embedded tissues.
- Protein antigen localization in M. leprae requires less stringent sample preparation, such as non-fixed ultracryosections.
- Optimized electron microscopy techniques are essential for accurate antigen site mapping in Mycobacterium leprae.