New Time-resolved Fluoroimmunoassay For Pathogenic Bacteria In Aquaculture

The aquaculture is advanced in China. Its scale is among the highest in the world. The infection of pathogenic bacteria that causes massive death of aquatic animals is a pressing problem for aquaculture. The commonly used florescent antibody and enzyme-linked immunosorbent assay suffer from high background fluorescence and low sensitivity. Time-resolved fluoroimmunoassay(TRFIA) with a high sensitivity has attracted much attention in chemistry and food science. We have detected Klebsiella oxytoca in TRFIA for the first time. On the basis, new TRFIA for pathogenic bacteria in aquaculture is carried out. The contents are as follows: 1. Aeromonas hydrophila B27 and Klebsiella oxytoca B12 are detected by dual-label TRFIA. The pathogens are coated on the microtiter plate. Both the sensitivities of Aeromonas hydrophila B27 and Klebsiella oxytoca B12 are 1.0×106 cfu/m L. The sensitivity has a large space of improvenment for this method. 2. To improve the sensitivity, pathogens antibodys are coated on microtiter plate. A double antibody sandwich dual-label TRFIA for Aeromonas hydrophila B18 and Klebsiella oxytoca B12 is developed. By optimizing the concentrations of coating IgG, Eu3+-IgG and Sm3+-IgG, the detection limits are 1.0×104 cfu/mL for Klebsiella oxytoca B12 and 6.0×104cfu/m L for Aeromonas hydrophila B27, respectively. The liner ranges of two pathogens are 1.0×104-1.0×107cfu/m L with the correlation cofficients of 0.99. This method can be applied to the real sample detection with high specificity and precision. 3. Firstly Aeromonas hydrophila B18 antibody is labeled using biotin, then the labeled streptavidin by alkaline phosphatase specially reacts with biotinylated antibody. The reaction substrate diflunisal phosphate(DFP) is added. By catalysis of enzyme, the product diflunisal of DFP forms high fluorescent complexes with t Tb3+-EDTA that are quantified with time-resolved fluorometry. Enzyme-amplified time-resolved fluorescence detection for Aeromonas hydrophila B18 is founded. The liner range of the method is 1.0×102-1.0×106cfu/m L with a sensitivity of 1.0×102 cfu/mL. The sensitivity is superior to that of double antibody sandwich dual-label TRFIA. The method also can be applied to the real sample detection with high specificity and precision. 4. We have already synthesized new chelate DTPA-pAS. However, the sensitivity is low with DTPA-pAS as a label. Using polylysine to inprove the label ratio, we develop a detection for Aeromonas hydrophila B18 with detection limit of 1.0×105cfu/m L.