Development Of The Colloidal Gold Immunochromatographic Strip For The Detection Of Riemerella Anatipestiper

Riemerella anatipestifer (RA) is one of the most important bacterial pathogen of ducks and causes a contagious septicemia, which is characterized by fibrinous pericarditis, airsacculitis and perihepatitis. Currently, at least 21 serotypes of R. anatipestifer have been identified, but the cross-protection among different serotypes is quite poor. Since RA infection causes similar syndromes of serositis with other bacterial infections in ducks, including Escherichia coli, Salmonella enterica and Pasteurella multocida, it is usually difficult to clinically differentiate RA from these bacterial pathogen infections. For the detection of R. anatipestifer, a number of assays have been reported, including slide and tube agglutination tests, immunofluorecent assay (IFA) and polymerase chain reaction (PCR) testing. But their procedures are very cumbersome, and an efficient, rapid and specific method for detection of RA is critically needed. In this study, the monoclonal antibodies against RA GroEL and TonB dependent outer membrane receptor (TbdR) were prepared. Based on the monoclonal antibodies, colloidal gold immunochromatographic strip for detecting R. anatipestifer was established.1 Prokaryotic expression and purification of R. anatipestifer GroEL and TbdRThe specific primers were designed according to the groEL and tbdR sequences of R. anatipestifer reported in GenBank. Based on the sequence analysis of the tbdR gene, the ORF of the groEL and tbdR genes (groEL 1629bp and tbdR-A 2472bp) and several partial fragments (tbdR-B 1020bp, tbdR-C 1335bp, tbdR-D 678bp) of the tbdR gene were expressed respectively. Genomic DNA of R. anatipestifer strain CH3 was used as the template for polymerase chain amplification (PCR), and the PCR products were cloned into plasmid pET-28a(+) and pET-30a(+). The positive recombinants were identified by PCR, endonuclease digestion and DNA sequencing. The recombinant expression plasmid pET28a-groEL?pET30a-tbdR-A?pET30a-tbdR-B?pET30a-tbdR-C?pET30a-tbdR-D were successfully constructed and expressed in E. coli BL21 (DE3). After induced by IPTG, the pET28a-groEL and pET30a-tbdR-D were successfully expressed and the molecular weight of the recombinant protein was 60kDa and 25kDa respectively, while the expression of pET30a-tbdR-A, pET30a-tbdR-B and pET30a-tbdR-C was failed. After purification, the recombinant GroEL and TbdR were used as antigens for immunization. It is concluded that the recombinant proteins have been successfully expressed and purified for monoclonal antibody (McAb) production.2 Preparation of monoclonal antibody to R. anatipestifer GroEL and TbdRThe purified recombinant GroEL and TbdR were used as the antigens for mice immunization respectively. BALB/c mice were immunized with either recombinant protein for three times, and the hybridoma technique was performed for the McAb development. Hybridoma clones were screened using indirect enzyme-linked immunosorbent assay (ELISA) and positive clones were subjected to sub-clone for three times. As a result, two stable hybridoma cell clones (1G2B6,1G2F10) producing anti-GroEL monoclonal antibody and two stable hybridoma cell clones (1A12,3F1) producing anti-TbdR monoclonal antibody was established. The isotype of McAb 1G2B6,1G2F10,1A12 was identified as IgG1, and McAb 3F1 as IgG2a. The titers of all the McAbs in the ascite were assessed over 1:102,400 by ELISA. Western blot showed that all the McAbs reacted well with R. anatipestifer serotypes 1,2, and 10 strains, but did not react with Escherichia coli, Salmonella enterica and Pasteurella multocida strains. The 4 monoclonal antibodies were specific to and cross-reactive with R. anatipestifer serotype 1,2, and 10 strains, which could be used for developing diagnostic kits in the further study.3 Development of the colloidal gold immunochromatographic strip for the detection of R. anatipestiferUsing trisodium citrate with reducing reaction, colloidal gold of 20nm was prepared. Studies on the optimal conditions about preparing the colloidal gold conjugates with McAbs were carried out. It was figured out that the optimal pH was 8.0 and the optimal McAb amount is 3?g/mL. The anti-GroEL McAb 1G2F10 labeled with colloidal gold was used as the detection regent, it was blotted on the test line while a goat anti-mouse IgG antibody was blotted on the control line of the nitrocellulose membrane. The monoclonal antibody-based colloidal gold immunochromatography strip was successfully developed for the detection of R. anatipestifer. Then a series of experiments were progressed to evaluate the specificity and sensitivity. The results showed that the method was specific to the detection of R. anatipestifer but not to other bacteria tested, including E. coli, S. enterica and P. multocida. The sensitivity assay showed that the detection limit for R. anatipestifer was 1×106CFU. Therefore, the high specificity and sensitivity, together with its advantages of rapid detection and easy operation, makes this method a suitable tool for detection of R. anatipestifer.