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 protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease 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. Protease could 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 ofprotease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5～6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 104 cell/mL in the indirect ELISA, while 105 cell/mL in the dot-ELISA.
作 者： ZHANG Zhendong ZHANG Peijun MO Zhaolan WANG Chunling YU Yang 作者单位： ZHANG Zhendong,WANG Chunling(Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;Graduate School of the Chinese Academy of Sciences, Beijing 100039, China)ZHANG Peijun,MO Zhaolan,YU Yang(Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China)
刊 名： 海洋学报（英文版） SCI 英文刊名： ACTA OCEANOLOGICA SINICA 年，卷(期)： 2005 24(4) 分类号： P7 关键词： Vibrio anguillarum extracellular products protease dot-ELISA indirect ELISA Western blot