Defence Research and Development Canada-Suffield, PO Box 4000, Station Main, Medicine Hat, AB, Canada.
J Virol Methods. 2010 Nov;169(2):274-81. doi: 10.1016/j.jviromet.2010.07.022. Epub 2010 Aug 1.
An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and by different operators demonstrated that the assay was reproducible (coefficient of variation 4.7-18.5% month-to-month; 3.3-8.8% person-to-person). The VEEV ECL assay exhibited no cross-reactivity with two closely related alphaviruses or with 21 heterologous biological agents. A genetically biotinylated recombinant VEEV antibody, MA116SBP, was evaluated for utility for detection of rE2; although functional in the ECL assay, the LOD was two log units higher (100 ng/ml vs 1 ng/ml) using MA116SBP than when chemically biotinylated antibody was used. The ECL assay detected VEEV at the lowest LOD (highest sensitivity) hitherto reported in the published literature and ECL assay results were generated in ∼60 min compared to a 6-8h period required for ELISA. Results have demonstrated a sensitive, rapid, and fully automated ECL immunoassay for detection and identification of VEEV.
电化学发光（ECL）免疫分析，结合了从一组单克隆和多克隆试剂中筛选出的化学生物素化和钌化抗体，用于检测和鉴定委内瑞拉马脑炎病毒（VEEV）。优化后的 ECL 分析的检测限（LOD）为 10(3)pfu/ml VEEV TC-83 病毒和 1 ng/ml 重组（r）VEEV E2 蛋白。ECL 分析的 LOD 比使用相同免疫试剂的夹心酶联免疫吸附测定（ELISA）低约一个对数单位。随着时间的推移和不同操作人员的 ECL 分析重复表明该分析具有可重复性（每月变异系数为 4.7-18.5%；人与人之间的变异系数为 3.3-8.8%）。VEEV ECL 分析与两种密切相关的黄病毒或 21 种异源生物制剂没有交叉反应。一种遗传生物素化的重组 VEEV 抗体 MA116SBP 被评估用于检测 rE2 的效用；尽管在 ECL 分析中具有功能，但与使用化学生物素化抗体相比，LOD 高两个对数（100ng/ml 与 1ng/ml）。ECL 分析在已发表文献中报道的最低检测限（最高灵敏度）下检测到 VEEV，并且与 ELISA 所需的 6-8 小时相比，ECL 分析在大约 60 分钟内产生结果。结果表明，该方法具有敏感、快速和完全自动化的 ECL 免疫分析，用于检测和鉴定 VEEV。
