Microbead electrochemiluminescence immunoassay for detection and identification of Venezuelan equine encephalitis...

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.