A bacterium LB110808-2 was isolated from the spleen of a moribund tilapia in August 2011 in Guangxi Province, and identified as Streptococcus agalactiae by 16 S rRNA gene. The antisera against strain LB110808-2 were obtained from the New Zealand white rabbit. The titer of the polyclonal antibody was 1:2560. This polyclonal antibody was used as primary antibody in indirect enzyme-linked immunosorbent assay (ELISA). The goat anti-rabbit IgG-HRP was used as secondary antibody. The indirect ELISA method of rapid detection of S. agalactiae was developed. The basic procedure of the indirect ELISA was as follows:S. agalactiae antigen was diluted with 0.05 mol/L Carbonate buffer (pH 9.6) and 100 μL diluted antigen was added to each well of the 96 well plate, followed by drying out at 60℃. The plate was washed three times with 300 μL of PBST (PBS+0.2% Tween 20) buffer, and 300 μL of 1% BSA blocking buffer (1% Albumin from bovine serum in PBST) was added to each well and the plate was incubated at 37℃ for l h. Subsequently, the plate was washed three times with PBST buffer, and each well was added with diluted positive serum or negative serum before incubating the plate at 37℃ for l h. Each well of the plate was added with goat anti-rabbit IgG-HRP after the plate was washed with PBST buffer three times. The plate was then incubated at 37℃ for l h, and was washed three times with PBST buffer. Each well was added 100 μL single-component TMB soluble substrate solution. The plate was then incubated at 37℃ for 20 min in dark place. The reaction was terminated with 50 μL 2 mol/L H₂SO₄. The value of OD₄₅₀ was determined by the ELISA reader.
The optimum coated concentration of the antigen was determined to be 10⁶ CFU/mL using chessboard titration method. The optimum antiserum concentrations of the primary antibody and the enzyme linked secondary antibody were determined to be 1:10 000 and l:1000, respectively. The sensitivity of the serum was tested, and the sensitivity of S. agalactiae that can be detected was 10³ CFU. Cross reactions of the antiserum with the strains of other common aquatic pathogenic bacteria were detected, and all the results were negative. Inhibition rate was 72.02% in inhibition test. The cross assay and the inhibition assay indicated that this method had high specificity. The method was optimized and standardized to detect the infected S. agalactiae isolated from the diseased tilapia from 2007 to 2013. All the 44 bacterial strains tested were positive, with a 100% positive detection rate. Next, we conducted the test of artificial infection using the S. agalactiae. Seventy percent of the fish were dead. Among the dead fish, 30 samples were detected from the brain tissue, and the positive detection rate was 100%. Among the live fish, 20 samples were detected from the brain, liver and spleen. None was positive from the brain tissue, but the liver and spleen were detected as positive, accounting for 25% and 50%, respectively. These results indicated that this assay can be used to detect not only the dead fish, but also the carrier. Thus, the established indirect ELISA method is very important for rapid and accurate diagnosis of tilapia infected by S. agalactiae at the early stage.