Comparison of immunogold methodologies for the detection of low copy number viral antigens in bluetongue virus (BTV)-infected cells.
Cells infected with bluetongue virus (BTV) were prepared for immunocytochemistry by freeze substitution, the progressive lowering of temperature technique and the Tokuyasu method. Sections containing virus-infected cells were incubated with specific monoclonal antibodies and colloidal gold probes to detect virus antigens of varying copy number; these BTV proteins were structural proteins VP2 and VP7 and the non-structural protein NS2. Protocols compared in this study represented those used in laboratories which handle infectious agents and as such, all samples were pre-fixed with minimum concentrations of glutaraldehyde to inactivate the virus. No statistical difference was found between the gold-labelling of sections prepared by the progressive lowering of temperature technique and freeze substitution. The results showed that cryo-sections yielded the best signal-to-noise ratio for all proteins examined in this study and were therefore the most sensitive system for the detection of low copy number proteins. The data and associated inferences relate to the system described in this paper and possibly other analogous systems.
Hyatt
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Australian Animal Health Laboratory, CSIRO, Geelong, Victoria, Australia.
Wise
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D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000032:analysis*; D001820:Bluetongue virus / Q000276:immunology*; D002213:Capsid / Q000032:analysis; D036022:Capsid Proteins; D002460:Cell Line; D015925:Cryopreservation; D016616:Cryoultramicrotomy; D017110:Freeze Substitution; D007150:Immunohistochemistry; D007181:Inclusion Bodies, Viral / Q000648:ultrastructure; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D013552:Swine; D014758:Viral Core Proteins / Q000032:analysis; D017361:Viral Nonstructural Proteins / Q000032:analysis
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; C442520:VP2 protein, Bluetongue virus; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C066862:VP7 protein, orbivirus