title: Optimization of recombinant vaccinia-based ELISPOT assay.
abstract: The ELISPOT assay has been considered as one of the most sensitive assays to measure antigen-specific CD8 T cells in vitro. Recently, recombinant vaccinia was successfully used to express internally processed target antigens in host cells in direct ex-vivo ELISPOT assays. However, the background in these assays was relatively elevated, and the risk of killing effector T cells was high. Therefore, we examined in this study an alternative approach where the replication of recombinant vaccinia virus was inhibited by the usage of Cidofovir in vitro. Our data indicate that recombinant vaccinia-infected target cells treated with Cidofovir retained their functional activity and present internally processed antigens more efficiently to T cells than non-treated ones. We also identify the optimum doses of Cidofovir to be in the range of 0.75-0.075 microg/ml. Thus, Cidofovir treatment of the target cells prior to antigen stimulation could be a useful methodology to increase the sensitivity of the ELISPOT assay.
authors.affiliation: Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, NY 10021, USA. mshata@NYBloodCenter.org
authors.forename: Mohamed Tarek
authors.initials: MT
authors.lastname: Shata
authors.forename: Mei Mei
authors.initials: MM
authors.lastname: Shan
authors.forename: Nancy
authors.initials: N
authors.lastname: Tricoche
authors.forename: Andrew
authors.initials: A
authors.lastname: Talal
authors.forename: Marion
authors.initials: M
authors.lastname: Perkus
authors.forename: Alfred
authors.initials: A
authors.lastname: Prince
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002470:Cell Survival / Q000187:drug effects; D000077404:Cidofovir; D003596:Cytosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D007371:Interferon-gamma / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D063065:Organophosphonates; D009943:Organophosphorus Compounds / Q000494:pharmacology; D002679:Pan troglodytes; D011994:Recombinant Proteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D014616:Vaccinia virus / Q000235:genetics*; D014779:Virus Replication
chemical_list: D063065:Organophosphonates; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D007371:Interferon-gamma; D003596:Cytosine; D000077404:Cidofovir