| Summary: | 碩士 === 國立成功大學 === 生物學系 === 90 === Cobia (Rachycentron canadum) has been completely farmed recently with great success in Taiwan. It is selected by the aquaculture industry as the promising fish for cage aquaculture and target to be promoted to international market. However, after couple years of farming the survival rate decreased drastically. It is to believe that the causes of mortality are infectious diseases of bacterial source. To improve the prophylaxis and treatment of Cobia disease, a simple diagnostic method such as ELISA which can test Cobia immunization response would be desirable.
To develop ELISA test, a Cobia Ig was isolated by 45-50% ammonium sulfate precipitation and by gel filtration chromatography. The molecular weight of the Ig was estimated by reduced SDS-PAGE and non-reduced SDS-PAGE. The heavy chain and light chain of the Cobia Ig are found to be 75 kDa and 30 kDa, respectively. Since the molecular weight of the Cobia Ig monomer is about 190 kDa and the polymer is 670 kDa, the structure of the Cobia Ig was proposed to be tetramer. The Cobia Ig was injected into rabbit several times to induce rabbit anti-Cobia IgG. The secondary antibody was purified by Protein A Sepharose. As testing the specificity by immunoblotting, the rabbit anti-Cobia antibody did not react with human IgM, and the antibody titer has reached 1:32,000 by ELISA test. With this secondary antibody, the microtiter plate ELISA kit was developed. The ELISA kit was examined repeatedly with positive and negative serum of Cobia.
The most appropriate condition for antibody assay was 20μg antigen in each well, 1:50 dilution of the serum, and 1:10,000 dilution of the secondary antibody. Using the same condition in artificial immunization and non-immunization models in Vibrio anguillarum, the difference of OD ratio was found at least four folds. This kit also provides a way that can be used for the detection of the existence of specific antibody in Cobia serum. We look forward to employ the kit for epidemic study of Cobia in the future.
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