Related Experiment Videos
In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy
J R Graybill1, M Patiño, J Ahrens
1Audie Murphy Veterans Administration Hospital, San Antonio.
Abstract:
Histoplasma capsulatum contains multiple antigens, among them the H antigen and M antigen, which are useful in serologic testing for histoplasmosis. We prepared 7 mouse monoclonal antibodies (5 IgG, 2 IgM) to histoplasmin, and compared these with polyclonal histoplasmin antibodies raised in rabbits and mice. Both monoclonal and polyclonal antibodies were high titered by ELISA. Colloidal gold immune electron microscopy (CGIEM) showed that polyclonal antibodies to histoplasmin or H antigen bound at multiple sites in the cell wall, cytoplasm, and nucleus of Histoplasma yeast cells. In contrast, antibodies to M antigen selectively label the cell membrane and antibodies to alkali soluble cell wall antigen label only the cell wall. Polyclonal antibodies cross reacted extensively with other fungi, both by ELISA and CGIEM. Monoclonal antibodies stained only cytoplasmic epitopes, but also cross reacted with other fungi by electron microscopy. Only periodate treated H antigen elicited polyclonal antibodies which were more specific than those of untreated H antigen or histoplasmin.
Insights
Monoclonal antibodies targeting Histoplasma antigens offer improved specificity for diagnosing histoplasmosis. Periodate treatment of H antigen further enhanced antibody specificity, crucial for accurate serologic testing.
Area of Science:
- Mycology
- Immunology
- Medical Diagnostics
Background:
- Histoplasma capsulatum possesses multiple antigens, including H and M antigens, vital for histoplasmosis serologic testing.
- Existing polyclonal antibodies show extensive cross-reactivity with other fungi, limiting diagnostic precision.
Purpose of the Study:
- To develop and characterize mouse monoclonal antibodies against Histoplasma antigens.
- To compare the specificity and binding sites of monoclonal and polyclonal antibodies using immunoelectron microscopy.
Main Methods:
- Production of seven mouse monoclonal antibodies (5 IgG, 2 IgM) against histoplasmin.
- Enzyme-linked immunosorbent assay (ELISA) for antibody titration.
- Colloidal gold immune electron microscopy (CGIEM) to visualize antibody binding sites.
- Comparison with rabbit and mouse-raised polyclonal antibodies.
Main Results:
- Both monoclonal and polyclonal antibodies demonstrated high titers by ELISA.
- Polyclonal antibodies bound to cell wall, cytoplasm, and nucleus; M antigen antibodies targeted the cell membrane; alkali-soluble antigen antibodies targeted the cell wall.
- Monoclonal antibodies primarily stained cytoplasmic epitopes.
- Extensive cross-reactivity with other fungi was observed for polyclonal antibodies and, to a lesser extent, for monoclonal antibodies.
- Periodate-treated H antigen elicited more specific polyclonal antibodies than untreated H antigen or histoplasmin.
Conclusions:
- Monoclonal antibodies offer potential for more specific detection of Histoplasma antigens.
- Antibody specificity is antigen-dependent, with M antigen and alkali-soluble cell wall antigen showing more restricted binding.
- Further refinement, such as periodate treatment of H antigen, can enhance antibody specificity for improved diagnostic applications.