Sandwich enzyme-linked immunosorbent assay for Vibrio vulnificus hemolysin to detect V. vulnificus in environmental specimens

Abstract

Vibrio vulnificus hemolysin, purified by quantitative isoelectric focusing, was used to prepare rabbit and goat anti-hemolysin. The resulting antibodies were used as capture and detector antibody reagents in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect V. vulnificus in environmental samples. By this technique, 4 laboratory-maintained V. vulnificus strains and 33 environmental V. vulnificus isolates were detected. Also, the technique distinguished five other Vibrio species from V. vulnificus, and when it was used in combination with colistin-polymyxin-cellobiose agar, 31 non-V. vulnificus isolates were excluded. This sandwich ELISA compared favorably with the current Food and Drug Administration standard immunoassay in confirming presumptive V. vulnificus colonies from environmental specimens: oysters, sediment, and seawater. Among 340 presumptive V. vulnificus colonies, the sandwich ELISA detected 95% of the confirmed V. vulnificus colonies. Equally important, the technique correctly distinguished 99% of the non-V. vulnificus colonies. The sandwich ELISA offers time-saving and labor-saving advantages over the currently accepted immunoassay.

An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies

“…Thus, detection techniques have been focused on the recovery of biotype 1, usually by employing enrichment in alkaline peptone broth followed by isolation on selective media (21,27,33). Since these methods require checking of numerous colonies and are unable to recover nonculturable cells, recent molecular techniques, such as immunoassays and PCR, have been proposed to circumvent these problems (6,16,17,29,34,38). Because clinical and environmental strains of biotype 1 are phenotypically indistinguishable and possess a high degree of DNA similarity (36,39,41), all of these methods have been based on species-specific traits of V. vulnificus.…”

Growth of Aeromonas species on increasing concentrations of sodium chloride

“…1994 ). However, with the exception of V. vulnificus ( Parker & Lewis 1995) and V. parahaemolyticus ( Holt et al . 1994 ), contaminants of oysters, the other species of the genera are not considered to be important foodborne pathogens.…”

Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19

“…To reduce cross‐reactivity with Aeromonas strains belonging to other serogroups and with other genera, antiserum was absorbed with lyophilised cells of A. sobria CECT837 and Edwardsiella tarda CECT849 [18]. Adsorption was carried out by the Parker and Lewis [19] procedure, using a mixture of antisera from A. hydrophila EO63 and from A. hydrophila A19.…”