Rapid Immunochromatographic Detection Of Edwardsiella Tarda

Edwardsiella tarda is an important fish pathogen, which is the agent of edwardsiellosis leading to mass mortality amongst commercially important freshwater and marine fishes. It is urgent to establish a rapid and convenient method for edwardsiellosis diagnosis.Various sizes of colloidal gold pellents were prepared by reduction of chloroauric acid with sodium citrate and monitored by the scanning of UV-visible (UV-vis) light at 400-600 nm and the observation with transmission electron microscopy. Rabbit polyclonal antibody (PAb) against E. tarda was labeled with gold particles of 30 nm and dispensed onto glass-fiber as conjugating pad. Rabbit polyclonal antibody was immobilized at the upper position of nitrocellulose (NC) membrane as test line (T). Goat anti-rabbit IgG was immobilized at the bottom of the nitrocellulose membrane as control line (C). The test strips were prepared by assembly of sample pad, conjugate pad, analytical membrane and absorbent pad. The detection limit of the test strip was 1×107 CFU/ml. The test strip showed high specificity to E. tarda and no cross-reactivity with other bacterial strains.Monoclonal antibody (MAb) specific to E.tarda was prepared by standard procedure. Simply, five female Balb/c mice were immunized intraperitoneally with purified OMPs for three times. The dispersed spleen lymphocytes of the mice with the highest ELISA titre were prepared and fused with myeloma cells to obtain hybridomas. Two positive hybridomas selected by indirect ELISA and cloned by limited dilution were cultured and injected intraperitoneally into the Balb/c mice to produce ascitic fluid. After 7-14 days, ascitics was collected and purified with Protein G agarose column. The isotype of two MAbs were identified to be IgG2a by using mouse monoclonal antibody isotyping kit.MAb and PAb were used to develop the test strips for rapid detection of E. tarda. MAb labeled with gold particles of 30 nm was laid on glass-fiber as conjugate pad. The rabbit polyclonal antibody was micro-sprayed onto a NC membrane at a position that would become the capture test line (T). Goat anti-mouse IgG antibody was micro-sprayed on the same NC membrane at a position that would become the control line (C). Then, the test strips were prepared with assembly of sample pad, conjugate pad, analytical membrane and absorbent pad. The detection limit of the test strip was 5×105 CFU/ml. The test strips recognized all 20 isolates of E. tarda, but no cross-reaction occurred with other bacterial strains.Strip test was compared with dot blotting and loop mediated isothermal amplification (LAMP) methods. The detection limit of the test strip (5×105 CFU/ml) was higher than that of dot blotting (1×105 CFU/ml) and LAMP method (1×103 CFU/ml). However, independent on other reagents and expensive equipments, the strip test could be used at pond site, and the results is obtained within 3-10 min.