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Title: A SIMPLE BLACK PLAQUE ASSAY TO DETECT AND QUANTITATE MAREK'S DISEASE VIRUS PLAQUES

Author
item Silva, Robert
item CALVERT, J - PFIZER ANIMAL HEALTH NE
item Lee, Lucy

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/6/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Marek's disease virus (MDV) is the causative agent for Marek's disease in chickens. The virus can be grown in susceptible cells in the laboratory, producing small discrete lesions. These lesions or plaques can be viewed and counted with the aid of a microscope, thereby determining how many infectious viruses there may be in a sample or vaccine vial. The difficulty is that the plaques are very small and are often indistinguishable from normal cells. We solved this problem by utilizing some chemicals and an antibody specific for MDV to develop a staining technique that stains MDV plaques. The procedure is simple and fast, resulting in plaques appearing as black spots that can be easily seen and counted with the naked eye. In addition, counting black plaques appears to be more accurate than the counting of conventional unstained plaques.

Technical Abstract: All three serotypes of Marek's disease virus (MDV) produce discrete plaques on duck and chicken embryo fibroblasts in cell culture. The resulting plaques can be viewed and counted with a microscope by anyone who has had some experience in culturing MDV. We report an immunoperoxidase-based staining technique that can be used by unskilled personnel to rapidly and accurately detect and quantitate MDV plaques. The procedure is simple and fast. Monolayer cultures are fixed and incubated with a monoclonal antibody specific for MDV. After washing, a second antibody of horseradish peroxidase conjugated goat anti-mouse IgG is applied, incubated for one hour and washed with phosphate-buffered saline. After the cultures are incubated with diaminobenzidine, CoCl2 and H2O2, the plaques appear as black spots and can be easily seen and counted with the naked eye. Counting early black plaques appears to be more accurate than counting unstained plaques. In addition, by using serotype specific monoclonal antibodies, it would be possible to differentiate between MDV serotypes.