Related Experiment Videos
Use of immunogold preembedding technique to detect hepatitis A viral antigen in infected cells
T Cromeans1, C Humphrey, M Sobsey
1School of Public Health, University of North Carolina, Chapel Hill 27599.
Abstract:
Localization of virus and viral antigen in cell cultures infected with a rapidly replicating isolate of strain HM-175 of hepatitis A virus (HAV; pHM-175) was accomplished by using immunogold probes. Cells infected under one-step growth curve conditions were prefixed with 2% paraformaldehyde and 0.1-0.001% saponin at appropriate times postinfection for detection of maximum virus and viral antigen. An indirect labeling technique was employed using monoclonal antibody to HAV followed by 5 nm gold-antimouse IgG conjugate. Cells were then fixed by standard electron microscopy techniques and thin sectioned. This prefixation technique allowed penetration of the immunogold probes and moderate preservation of ultrastructure. Within infected cell cytoplasm, numerous antigenic sites were labeled with six to 200 gold particles. Two types of cells were infected with HAV and somewhat different results were obtained with the two cell types. In BS-C-1 cells, where a cytopathic effect (CPE) was not observed, myelin figures were immunogold labeled or frequently were located near immunogold-labeled sites. Vesicles containing viruslike particles (14-22 nm) were also observed. A significant observation in infected FRhK-4 cells was the presence of multivesicular bodies labeled with immunogold. Microfilaments were commonly seen near the multivesicular bodies. Our results demonstrate that the choice of prefixation method for immunogold labeling should be empirically determined for the cell type and condition.
Insights
Hepatitis A virus (HAV) was localized in infected cells using immunogold probes. Different cell types showed distinct viral antigen distribution and ultrastructural changes, highlighting the importance of prefixation methods.
Area of Science:
- Virology
- Cell Biology
- Microscopy
Background:
- Hepatitis A virus (HAV) is a significant human pathogen.
- Understanding HAV replication and antigen localization is crucial for developing antiviral strategies.
- Electron microscopy with immunogold labeling offers high-resolution visualization of viral components within cells.
Purpose of the Study:
- To localize hepatitis A virus (HAV) and its viral antigen in infected cell cultures.
- To investigate the ultrastructural changes associated with HAV infection.
- To evaluate the effectiveness of immunogold labeling techniques for HAV detection.
Main Methods:
- Infection of cell cultures (BS-C-1 and FRhK-4) with HAV (strain HM-175).
- Prefixation with paraformaldehyde and saponin for immunogold probe penetration.
- Indirect immunogold labeling using anti-HAV monoclonal antibody and gold-conjugated secondary antibodies.
- Standard electron microscopy for thin sectioning and imaging.
Main Results:
- Immunogold labeling detected numerous viral antigenic sites within the cytoplasm of infected cells.
- BS-C-1 cells showed labeled myelin figures and vesicles with virus-like particles, without observable cytopathic effect.
- FRhK-4 cells exhibited immunogold-labeled multivesicular bodies, often associated with microfilaments.
Conclusions:
- The study successfully localized HAV and its antigen in cell cultures using immunogold labeling.
- Distinct cellular responses and viral antigen localization patterns were observed in different cell types.
- Empirical determination of prefixation methods is essential for successful immunogold labeling in specific cell types and experimental conditions.