Preparation and characterization of monoclonal antibodies against Vibrio harveyi

Abstract

The pathogenicities of four isolates of Vibrio harveyi, a bacterium that cause luminescent disease in Penaeus monodon were investigated by immersion method. P. monodon post larva were reared in sea water containing ~10[superscript 7] CFU ml[superscript -1] of bacteria. The 50% mortality rates of V. harveyi 639 and 1526 were shorter (39 and 43 h, respectively) than those of the other two isolates, VG and VG1 (49 and 50 h, respectively) due to the higher hemolytic and degradative enzymatic activities in V. harveyi 639 and 1526. Therefore, V. harveyi 639 and 1526 were used to produce monoclonal antibodies in order to provide an immunological tool for identification of the bacteria. Thirty-one monoclonal antibodies were obtained and can be divided into 9 groups according to their specificities to various proteins range from 8-95 kDa. Most of the antibodies were specific to V. harveyi without cross-reactivity to other Vibrio spp. and several closely related Gram-negative bacteria tested in this study. Some antibodies could be used to detect V. harveyi with high sensitivity up to 5 X 10[superscript 4] CFU ml[superscript 1] (50 cells/spot) by dot blot assay and some antibodies could be used to detect V. harveyi infection in tissues by immunohistochemistry. Three monoclonal antibodies that showed slight cross-reactivity to Aeromonas hydrophila, Salmonella Typhimurium and Escherichia coli were also isolated. Surprisingly, three specific monoclonal antibodies could be used to differentiate between V. harveyi 1526 and 639, VG, VG1 by Western blotting and could be used to differentiate V. harveyi between these four isolates and other 18 isolates by dot blot assay. Therefore, this study provided an immunological tool for specific identification not only to V. harveyi but also to differentiate some serotypes among this bacteria as well. Further development of highly sensitive assay and convenient kit using these monoclonal antibodies for detection of V. harveyi are inprogress