A simple immunoperoxidase plaque assay to detect and quantitate Marek's disease virus plaques.
We report an immunoperoxidase-based staining technique that can be used to rapidly and accurately detect and quantitate Marek's disease virus (MDV) plaques. Monolayer cultures were fixed and incubated with a monoclonal antibody specific for MDV. After washing, a second antibody of horseradish peroxidase-conjugated goat anti-mouse IgG was applied, incubated for 1 hr, and washed with phosphate-buffered saline. After the cultures were incubated with diaminobenzidine, CoCl2, and H2O2, the plaques appeared as black spots and were easily seen and counted. Significantly more immunoperoxidase-stained serotype 1 MDV plaques could be counted at 4 days postinoculation than were seen in unstained cultures. With serotype 2 MDV-infected cells, the difference in plaque counts was less dramatic. Nevertheless, at 3 days postinoculation, significantly more stained serotype 2 plaques were seen than unstained plaques. Immunoperoxidase staining of turkey herpesvirus plaques did not increase the sensitivity of viewing plaques. Similar numbers of stained and unstained plaques were seen at 2 days postinoculation. We also demonstrated that we could count serotype-specific MDV plaques in a mixed infection that contained all three serotypes.
Silva
R F
RF
USDA, Agricultural Research Service, East Lansing, Michigan 48823, USA.
Calvert
J G
JG
Lee
L F
LF
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D005347:Fibroblasts; D008381:Herpesvirus 2, Gallid / Q000145:classification / Q000254:growth & development* / Q000302:isolation & purification; D007124:Immunoenzyme Techniques; D007202:Indicators and Reagents; D008380:Marek Disease / Q000175:diagnosis* / Q000821:virology; D012703:Serotyping; D013997:Time Factors; D010948:Viral Plaque Assay / Q000379:methods
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents