Preparation And Characterization Of Monoclonal Anti-body Against Vibrio Vulnificus

Vibrio vulnificus is an opportunistic pathogen of humans that has the capability of causing rare, yet devastating disease,it is a Gram-negative bacterium that is found in estuarine waters and frequently contaminates oysters and other seafood. Consumption of raw contaminated seafood or contamination of wounds with Vibrio vulnificus can lead to septicemia and wound infection. Respectively; septicemia can also be a complication of wound infection. Additionally, Vibrio vulnificus can cause gastroenteritis. Almost all persons who develop vibrio wound infections do so following infection of a pre-existing wound or one obtained while engaged in working or fishing activities in coastal waters. Mortality in the wound infection group is high. Especially Patients with chronic liver disease and diabetes mellitus have a much higher risk of septicemia and death.Vibrio vulnificus is the leading cause of reported death from seafood primary septicemia caused by Vibrio vulnificus infection has a high mortality and mortal outcome. Quick detection is the requirement for timely treatment which is essential to avoid a fatal outcome. However, with the increasingly drug resistance of antibiotics,the other instead of antibiotics may be expected to solve this problem.Nowadays, monoclonal antibody has been successfully employed to detection and treatment, and the effect is striking. As one of the pathogenic bacteria, there is no monoclonal antibody against vibrio vulnificus.To solve the problem of detection of infection and treatment against vibrio vulnificus, we have do some study to established the McAbs which is highly specific for Vibrio vulnificus to not only settle a solid foundation for development of rapid method of detecting Vibrio vulnificus infection but also provide theoretical proof for further investigation to treatment. A series of experiments and the methods and results were as follows:1. Hybridoma technology was used to produce mAbs against Vibrio vulnificus, by fusing of mouse Sp2/0 myeloma cells and spleen cells of BALB/c mouse immunized by inactivated vibrio vulnificus.The positive clones which produced mAbs against vibrio vulnificus were screened by enzyme linked immunosorbent assay (ELISA).After cloning, two strains of hybridoma named respectively1A2,2D6, and 1A2 stably secreting specific mAbs against vibrio vulnificus were obtained.Hydroperitoneum and supernatant titers of the antibodies were respectiveively 1×10-4,1×10-5 and 1×10-2,1×10-3 mensurating by ELISA. Identification of IgG subgroup showed that 1A2 were the IgG1 and 2D6 was the IgG2a. Indirect ELISA showed that the affinity of 1A2 is higher than 2D6, which is implied the further research on the treatment towards vibrio vulnificus. Bacteria cross-reaction showed that 2D6 did not have cross-reaction with Aeromonas.hydrophil, Aeromonas.sobria, VibrioAnguillarum, Edwardsiella tarda, and Vibrio alginolyticus, showing that 2D6 have high specificity and could be used to detect Vibrio vulnificus .The mAbs against Vibrio vulnificus might also be useful tool for studying biological properties of Vibrio vulnificus.2. Using 10LD50 of vibrio vulnificus challenging suckling mice, the protective properties of mAbs against vibrio vulnificus were studied. The results showed that the protective effect of mAb 1A2 was significant, and the protective rate of suckling mice was 62.50%.These results indicated that mAb 1A2 might be directed against neutralized epitopes of Vibrio vulnificus and have ability to neutralize or reduce the toxicity of Vibrio vulnificus, so it could be used to cure the infection of vibrio vulnificus.In conclusion, in these experiments we produced mAbs against Vibrio vulnificus for the first time, selected out mAb 2D6 intend to detect Vibrio vulnificus and 1A2 maybe used in the treatment.