DEVELOPMENT OF A RAPID DETECTION METHOD ON MONOCLONAL ANTIBODIES CONJUGATING TO COLLOIDAL GOLD AGAINST PATHOGENIC AEROMONAS CAVIAE IN FISH

The purpose of this study was to prepare Aeromonas caviae hybridoma cell lines, and to establish the rapid, super sensitive detection method of ‘double-antibody-sandwich’ membrane colloidal gold basing on monoclonal antibodies that was used for the rapid diagnosis of Aeromonas caviae. In this study, BALB/c mice were immunized with Aeromonas caviae killed by formalin as the dosage of 2.5×107cell per mouse and 5.0×107cell per mouse respectively. The hybridoma cell lines which consistently secreted monoclonal antibody (McAb) against Aeromonas caviae were obtained through cell fusion. The specificity of the McAb was analysed by indirect ELISA. Then the subtypes were identified, the titer and affinity constant were measured, and its specificity was analyzed. Tests were made to identify the antigen epitope of the two McAbs by combined experimental site and indirect ELISA with LPS of Aeromonas caviae. The McAb conjugating to colloidal gold against Aeromonas caviae was established, and its specificity, sensitivity, repeatability was estimated respectively. From over hundreds of positive hybridomas which secreted anti-Aeromonas caviae McAbs, two strains of hybridomas were screened out, and designated with 3F3 and 2C9C3. The subtypes of the McAb were IgG1 and IgM. The titer of 3F3 and 2C9C3 McAb produced by ascites fluid were 1︰106 and 1︰105. The McAbs had high relative affinity. The two strains of monoclonal antibodies were targeted to different antigen epitope: 3F3 was targeted to the LPS of Aeromonas caviae, while 2C9C3 was targeted to the non-LPS sites of Aeromonas caviae. Basing on the McAbs, the rapid detection McAbs conjugating to colloidal gold against Aeromonas caviae was developed to detect the thalli antigen of Aeromonas caviae specifically. Therefore, the results demonstrated that this method had high specificity, sensitivity and repeatability. It could serve as an effective detection measure for clinic, and as the method of the rapid identification for Aeromonas caviae in aquaculture and monitoring the epidemic of Aeromonas caviae.