Abstract
To increase the fluorescence intensity in the fluorescent antibody test(FAT), fluorescein labeled avidin-biotinylated antibody complex(ABC)was tested for the detection of Renibacterium salmoninarum antigen in the carrier fish of BKD. In comparison with indirect FAT, fluorescence intensity observed by ABC method was increased about 50% by multiscan fluorescent method using 96 well microtiter plate. The detection rate of R. salmoninarum antigens in kidney smears of masu salmon was also increased about 50% in comparison with indirect FAT.
The procedure of the ABC method is as follows:A tissue smear was incubated for 30 minutes with antibody against R. salmoninarum. After several washings with phosphate buffered saline, goat anti-rabbit IgG covalently linked to biotin was added and allowed to react for 30 minutes. The preparation was washed again and a solution containing fluorescein-labeled avidin was added andallowed to react for 30 minutes. After final rinsing, the smear is mounted in glycerol buffered to pH 9.0 and observed by fluorescence microscope.
