Sandwich enzyme-linked immunosorbent assay for detection of Vibrio vulnificus in oysters

Abstract

Vibrio vulnificus has emerged in recent years as an agent of major public health concern because of its widespread contamination of the American oyster (Crassostrea virginica). Rapid assay tests are needed to assist in the detection of V. vulnificus in environmental samples. This study focuses upon the use of a sandwich enzyme-linked immunosorbent assay (ELISA), oriented toward V. vulnificus hemolysin, for detecting the agent. Vibrio vulnificus hemolysin, purified by quantitative isoelectric focusing, was used to prepare rabbit and goat anti-hemolysin. The resulting antisera were used as capture and detector antibody reagents in the sandwich ELISA. Using this technique, four standard V. vulnificus strains and 33 environmental V. vulnificus isolates were detected. In contrast, the technique distinguished five other Vibrio species from standard V. vulnificus and, when used in combination with Colistin-Polymyxin-Cellobiose agar, 31 non-V. vulnificus isolates were excluded. The sandwich ELISA, developed in this study, compared favorably with the current FDA-standard immunoassay in confirming presumptive V. vulnificus isolates from environmental specimens: oysters, sediment, and seawater. Of 340 presumptive V. vulnificus isolates, the sandwich ELISA detected 95% of the confirmed V. vulnificus isolates. Equally important, the technique correctly distinguished 99% of the non-V. vulnificus isolates from those specimens. The sandwich ELISA offers considerable time-saving and labor-saving advantages over the currently accepted FDA-standard immunoassay.