Application Of Time-resolved Fluoroimmunoassay For Detecting Pathogens In Eel Culture

Recently the aquaculture is rapidly developing in China, and its scale gets larger and larger.While the frequent occurrence of diseases especially from bacteria results in great financial loss in the intensive cultivation process with high density.Therefore, the establishment of a sensitive, specific and rapid detection technology for pathogens is important in the disease prevention and control. Time-resolved fluoroimmunoassay(TRFIA) is a non-radioactive immunoassay with high sensitivity, good specificity and simple manipulation. The detection for pathogens by TRFIA has never been reported in aquaculture.The systematic research of detecting pathogens by TRFIA in eel culture is carried out for the first time. The contents are as follows:1. Klebsiella oxytoca B12 is detected by direct TRFIA. The effect of particulate antigen on long-life fluorescence is analysed. The measuring time and the kinds of microtiter plate are optimized. The sensitivity of the assay is 1.0×105 cfu/mL(close to that of ELISA), which shows that TRFIA can also be applied to particulate antigen detection, and the sensitivity has a large space of improvement for this method.2. The detection for Aeromonas hydrophila B18 by indirect TRFIA is established. The sensitivity(1.0×104cfu/mL) is improved 10 times compared to that of the direct TRFIA.3. A double antibody sandwich TRFIA for Klebsiella oxytoca B12 is developed by optimizing immune time and the concentrations of coating IgG and Eu3+-IgG. The precision,sensitivity and specificity are assessed. The limit of detection is 5.0×103cfu/mL and the linear range is 5.0×103-1.0×107 cfu/mL with the correlation coefficients more than 0.99. This method can be applied to the real sample detection with high specificity and precision.4. The sensitivity of the TRFIA for Aeromonas hydrophila B27 can be further improved in biotin-streptavidin system. The assessment to precision, sensitivity, specificity and stability is made. The linear range of determination is 1.0×102-1.0×106 cfu/mL with a sensitivity of 1.0×102cfu/mL. The sensivity is superior to that of double-antibody sandwich TRFIA. This method also can be applied to the real sample detection with high specificity and precision. The reagents are valid for six months or more.5. The preliminary TRFIA is developed using synthesized rare earth chelate in our lab. The chelate itself is fluorescent compared to commercial reagents. Furthermore, the enhancedissociation is not used, which reduces steps and reduce the cost. The method has a fine potential.