Validation of a cell-based ELISA as a screening tool identifying anti-alphavirus small-molecule inhibitors

详细信息    查看全文

• 作者：Kevin B. Spurgers ; Clarence R. Hurt ; Jeffrey W. Cohen ; Lori T. Eccelston ; Cathleen M. Lind ; Vishwanath R. Lingappa ; Pamela J. Glass
• 关键词：BSA ; bovine serum albumin ; CHIKV ; Chikungunya virus ; CPE ; cytopathic effect ; EEEV ; eastern equine encephalitis virus ; ELISA ; enzyme-linked immunosorbent assay ; HRP ; horseradish peroxidase ; IC50 ; 50 % inhibitory concentration ; MAb ; monoclonal antibody ; MOI
• 刊名：Journal of Virological Methods
• 出版年：2013
• 出版时间：October, 2013
• 年：2013
• 卷：193
• 期：1
• 页码：226-231
• 全文大小：541 K

文摘

Venezuelan (VEEV), eastern, and western equine encephalitis viruses, members of the genus Alphavirus, are causative agents of debilitative and sometimes fatal encephalitis. Although human cases are rare, these viruses pose a threat to military personnel, and to public health, due to their potential use as bioweapons. Currently, there are no licensed therapeutics for treating alphavirus infections. To address this need, small-molecules with potential anti-alphavirus activity, provided by collaborators, are tested routinely in live alphavirus assays utilizing time-consuming virus yield-reduction assays. To expedite the screening/hit-confirmation process, a cell-based enzyme-linked immunosorbent assay (ELISA) was developed and validated for the measurement of VEEV infection. A signal-to-background ratio of >900, and a z-factor of >0.8 indicated the robustness of this assay. For validation, the cell-based ELISA was compared directly to results from virus yield reduction assays in a single dose screen of 21 compounds. Using stringent criteria for anti-VEEV activity there was 90 % agreement between the two assays (compounds displaying either antiviral activity, or no effect, in both assays). A concurrent compound-induced cell toxicity assay effectively filtered out false-positive hits. The cell-based ELISA also reproduced successfully compound dose-response virus inhibition data observed using the virus yield reduction assay. With available antibodies, this assay can be adapted readily to other viruses of interest to the biodefense community. Additionally, it is cost-effective, rapid, and amenable to automation and scale-up. Therefore, this assay could expedite greatly screening efforts and the identification of effective anti-alphavirus inhibitors.