PMID: 18720846Aug 30, 2008Paper

A flow cytometric assay for the expression of interferon gamma in T lymphocytes and its application in the study of EIAV-induced immune response

Wei sheng wu xue bao = Acta microbiologica Sinica
Yuezhi LinJian-Hua Zhou

Abstract

The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-gamma expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-gamma expression in different subtypes of T lymphocytes in EIAV-infected horses. Peripheral blood mononuclear cells (PBMC) were prepared from horses inoculated with EIAV vaccine stain FDDV (fetal donkey dermal-attenuated virus vaccine), virulent strain LV or saline (health control), were stimulated in vitro with FDDV or PMA + Inomycin. Brefeldin A was added into the cell culture to block protein secretions. Stimulated cells were then labeled with monoclonal antibodies specific for equine CD4 and CD8 surface markers, as well as an IFN-gammaspecific monoclonal antibody. Flow cytometry was applied to detect CD4+ and CD8+ lymphocytes expressing IFN-gamma. IFN-gamma positive cell population isolated from FD...Continue Reading

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