Research On Rapid Detection Of Vibrio Alginolyticus By The Methods Of Immunomagnetic Bead

In recent years, infectious diseases caused by bacteria, mainly Vibrio are spreading with the rapid development of the aquaculture intensive culture industry, resulting in great economic losses. As one of the dominant bacteria in the marine, the diseases caused by Vibrio alginolyticus take up a large proportion. The diseases of the fish, shrimp, molluscs and other aquaculture caused by V. alginolyticus brought about great economic losses. The method of treating bacterial diseases, for example V. alginolyticus, mainly relies on antibiotics, which not only contribute to the emergence of antibiotic resistant bacteria strains but also threat to human health. For the reasons above, it is very important to establish a fast accurate and sensitive method to detect V. alginolyticus, which can detect the potential threat of V. alginolyticus and reduce the use of antibiotics and improve the survival rate of cultured species. The method can also test the safety of aquatic products to avoide the V. alginolyticus to threat the human health.Immunomagnetic separation (MS) technique is an immunoassay technology based on immunomagnetic beads, which antigen-antibody bead complex formed in the reaction system produce directional movement in the magnetic field and separate the antigen, using the immunomagnetic beads enveloped by antibody as carrier and antigen-antibody specific respond. Immunomagnetic separation technique not only has the advantages of both the solid phase reagents and immunological specific reaction but also has the characteristics of the short separation time, fast, high separation efficiency and does not affect the biological characteristics and biological materials features. Therefore, in recent years, this new immunological techniques has become popular and have broad application prospects in the separation of biological macromolecules and cells. This paper is based on immunomagetic separation techniques to build the fast efficiency detection of V. alginolytics. The results are followig.1) We make the V. algnolytics antigen by phenol inactivated and immunize the New Zealand rabbits to get 150ml antiserum through carotid artery and the titer is 1:5120. Rabbit anti V. algnolytics IgG was purified by the protein A affinity chromatography and its concentration is 2.8579mg/ml. the titer detected by the improved ELISA is 1:256000.2) Immunomagnetic beads enveloped by purified IgG were detected by ELISA. The best combination occured the following conditions: the max enveloped IgG concentration (0.5μg/ml), the best enveloped IgG time (5min). The iron concentrations and pH are no significant influence on the combination of IgG and Immunomagnetic beads.3) The V. algnolyticus immunomagnetic detection technology has been preliminarily established. V. algnolyticus was detected by immunomagetic beads enveloped by rabbit anti V. algnolytics IgG. The best working concentration of 1ml sample was 20μL, and the best reaction of bonding Immunomagetic beads with bacteria was 20min. Our results showed that the sensitivity of detecting V. algnolyticus was up to 2.8CFU/ml.