Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy.
To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electron-dense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-labelling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10-100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.
D000818:Animals; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D002478:Cells, Cultured; D005293:Ferritins; D006046:Gold; D007158:Immunologic Techniques; D008854:Microscopy, Electron / Q000379:methods*; D013205:Staphylococcal Protein A; D013552:Swine; D015004:Yellow Fever / Q000382:microbiology* / Q000473:pathology; D015005:Yellow fever virus / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D013205:Staphylococcal Protein A; D006046:Gold; D005293:Ferritins