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 of protease 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 similar to 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 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Effects of La3+ on the active oxygen-scavenging enzyme activities in cucumber seedling leaves

The effects of La3+ on the antioxidant enzyme activities and the relative indices of cellular damage in cucumber seedling leaves were studied. When cucumber seedlings were treated with low concentrations of LaCl3 (0.002 and 0.02 mM), peroxidase (PO) activity increased, and catalase (CAT) activity was similar to that of control leaves at 0.002 mM La3+ and increased at 0.02 mM La3+, whereas superoxide dismutase (SOD) activity did not change significantly. The increase in the contents of chlorophyll (including chlorophylls a and b), carotenoids in parallel with the decrease in the level of malondialdehyde (MDA) suggested that low concentration of La3+ promoted plant growth. However, except the increase in SOD activity at 2 mM La3+, CAT and PO activities and the contents of pigments decreased at high concentrations of La3+ (0.2 and 2 mM), leading to the increase of MDA content and the inhibition of plant growth. It is suggested that lanthanum ion is involved in the regulation of active oxygen-scavenging enzyme activities during plant growth.