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

Vibrio is the causative agent of vibrosis, one of the major bacterial disease affecting marine fishes, shrimps and shellfishs in the world. The distribution of vibrosis is worldwide, which cause great economic loss to our country. Meanwhile, some strains of Vibrio are pathogens of human acute gastroenteritis, which induced people fulminating food poisoning. The effect people showed poisoning symptoms like sporadic diarrhea and dysentery. Therefore, the detection of marine pathogenic vibrio has great significance for protection of human health and promoting healthy development of marine aquaculture industry. However, the detection method of pathogenic vibrio exists some disadvantages such as tedious process time-consuming and low integration, which unable to meet the needs of marine monitoring. Therefore, it is nessecery to develop a quick, simple, sensitive and high integration detection technology.In recent years, Immunomagnetic beads (IMB) method has been more and more popular in the study of Immunological techniques?in the world. As a matter of fact, IMB has the same advantages with the solid-phase reagent, and it also has the advantages of high efficient immunological specificity, Etc. In this study, IMB was used as a rapid method to detect environmental pathogenic marine vibrio. We optimized the physical and chemical factors of this method to establish a practical, simple, accurate IMB detection system for the rapid detection of environmental pathogenic marine vibrio. The results are as follows:1. Anti-sera were prepared form rabbit by ear vein injection of Vibrio antigen. We got:210 ml of rabbit anti-Vibrio anguillarum immune serum with a agglutination titer of 1:5120;150 ml of rabbit anti-V. alginolyticus immune serum with a agglutination titer of 1:5120;230 ml of rabbit anti-V. fluvialis immune serum with a agglutination titer of 1:2560;190 ml of rabbit anti-V. alginolyticus immune serum with a agglutination titer of 1:2560.We have prepared a large number of high titer immune serum for the follow-up study.2. The methods of immunomagnetic bead and protein A affinity chromatography were introduced to the IgG purification from the anti-sera. The results showed that: The bonding of IgG by immunomagnetic beads reached equilibrium within 10 min, and the maxium bonding was 0.227mg IgG/ml.Purified IgG exhibited two bands in SDS-PAGE gel, the molecular weight of heavy chain and light chain of IgG were 55 kD and 25 kD respectively.The IgG purified by immunomagnetic beads exhibited higher titer(1:307200) than IgG purified by protein A affinity chromatography examined(1:256000) by indirect ELISA. The process of Immunomagnetic beads separation could be finished within 10min which was shorter than that of protein A Sepharose affinity chromatography (20min).The IgG recovery efficacy of Immunomagnetic beads method (11.5mg/ml) was higher than that of protein A Sepharose affinity chromatography (10.25mg/ml).3. Constructed of immunomagnetic beads technique for detect V. anguillarum.That is: the best Concentration of antibody bonding magnetic bead was 0.4mg IgG/ml; the best reaction time was10 min; and the best reaction time of bonding magnetic bead with bacteria was 20min. By our test, the sensitivity of V. anguillarum examination by IMB ups to 0.13 CFU / ml.