Development Of A Rapid Detection Method For Pathogenic Aeromonas Caviae In Fish, Using Monoclonal Antibodies On Test Strips

Objective: Aeromonas caviae (A. caviae) is one of the most prominent pathogenic bacteria which in China has been reported that is harmful to our fresh water aquaculture. As a major causative agent of infections in fish, A. caviae becomes highly pathogenic and infective when the resistance of fish decreases or the fish is injured. In recent years, the outbreak of epizootic ulcerative syndrome associated with A. caviae was reported among the southern catfish. The diseases caused by A. caviae are characterized by rapid invasion, wide prevalence and high mortality, which can cause serious economic loss in fish keeping. At present, the commonly used diagnostic methods for A. caviae depends on clinical observation, dissection and common bacteriology tests that are tedious, time-consuming and have poor accuracy. It is obvious that the establishment of a rapid method with higher specificity is one of the better approaches to prevent and control the infection caused by A. caviae.This research is to prepare A. caviae hybridoma cell lines, analyze the biological characteristics and develop colloidal gold test strips to establish a rapid and accurate diagnostic method for A. caviae in aquacultureMethods: Balb/c mice were immunized with A. caviae killed by formalin as the dosage of 25μg per mouse and 50μg per mouse respectively. The hybridoma cell lines which consistently secreted monoclonal antibody (McAb) against A. caviae were obtained through cell fusion. The specificity of the McAb was analyzed by indirect ELISA. Then the subtypes were identified, the titer and relative affinity constant were measured. Tests were made to identify the antigen epitope of the McAbs by combined experimental site and indirect ELISA with LPS of A. caviae. The McAb conjugating to colloidal gold against A. caviae was established, and its specificity, sensitivity, repeatability, stability and clinic sample test were estimated respectively.Results: From many 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 10-6 and10-5. The McAbs had high relative affinity. The two strains of McAbs targeted to different antigen epitope:3F3 targeted to the LPS of A. caviae, while 2C9C3 targeted to the non-LPS sites of A. caviae. Basing on the McAbs, the rapid detection McAbs conjugating to colloidal gold against A. caviae was developed to detect the thalli antigen of A. caviae specifically. The results demonstrated that this method had high specificity and good repeatability (100%). The minimum detection dose of strips was 1.71×104 cfu/ml. The test results by the strips could be observed within 5 minutes. The strips could be used easily and exactly in clinical test.Conclusion: The results demonstrated that this method had high specificity, sensitivity, stability and repeatability. It could serve as an effective detection measure for clinic, and as the method of the rapid identification for A. caviae in aquaculture and monitoring the epidemic of A. caviae.