Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent proteasesecreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity resultsshowed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount ofprotease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Proteasecould be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period.From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis ofserum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenicbacteria could be detected in the blood of the infected flounders but no protease was found. It was 5~6 h after infection that theprotease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease eitherincubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls ofquantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understoodgenerally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that thesensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount isapproximately 104 cell/mL in the indirect ELISA, while 105 cell/mL in the dot-ELISA.