TY - JOUR
T1 - Optimization of recombinant vaccinia-based ELISPOT assay
AU - Shata, Mohamed Tarek
AU - Shan, Mei Mei
AU - Tricoche, Nancy
AU - Talal, Andrew
AU - Perkus, Marion
AU - Prince, Alfred
N1 - Funding Information:
All chimpanzee studies were conducted in accordance with Institutional Animal Care and Use Committee-approved protocol #458 PT-0 and the NIH Guide for the Care and Use of Laboratory Animals. This work was supported in part by NIH grant No RO1-AI47349. We acknowledge the dedicated assistance of the technical team at Southwest Foundation for Biomedical Research, Texas, in conducting this study. We also acknowledge Ms. Marie Fuderanan for her editorial assistance.
PY - 2003/12
Y1 - 2003/12
N2 - 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 μg/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.
AB - 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 μg/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.
KW - Cidofovir
KW - ELISPOT
KW - Recombinant vaccinia
UR - http://www.scopus.com/inward/record.url?scp=0345059075&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2003.10.002
DO - 10.1016/j.jim.2003.10.002
M3 - Article
C2 - 14659919
AN - SCOPUS:0345059075
SN - 0022-1759
VL - 283
SP - 281
EP - 289
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -