红笛鲷弧菌病血清流行病学研究方法的建立

Establishment of Research Method on Serological Epidemiology of Vibriosis of Lutjanus sanguineus

以红笛鲷Lutjanus sanguineus弧菌病的主要病原菌——溶藻弧菌Vibrio alginolyticus为抗原制备兔抗血清,以辣根过氧化酶标记的羊抗兔血清为酶标二抗,建立了酶联免疫吸附试验检测技术。通过棋盘滴定法测定,得出溶藻弧菌菌悬液的最佳工作浓度为5×108mL-1,兔抗溶藻弧菌血清的最佳稀释度为1∶16000,酶标羊抗兔抗体的最佳稀释度为1∶2000。应用本实验建立的间接双抗体夹心法对红笛鲷进行现场检测,患病红笛鲷特异性抗体的检出率为70%,外观健康红笛鲷的检出率为20%。结果表明,本实验所建立的酶联免疫吸附试验(ELISA)检测法可以用于红笛鲷弧菌病的血清流行病学研究,而且可以检测出隐性感染状态下的红笛鲷。

The method of indirect double antibody sandwich ELISA was established to detect the serum specific antibody of vibriosis in Lutjanus sanguineus in this paper. The antiserum against Vibrio alginolaticus was obtained by immunizing rabbit against V. alginolaticus and the anti-rabbit IgG goat was marked by horseradish peroxidase （HRP） used in the ELISA detection. According to the results in this paper, the optimum concentration of the live V. alginolaticus resuspended in PBS, the antiserum against V. alginolaticus and horseradish peroxidase-labelled goat anti-rabbit IgG were 5 × 10^8 mL^-1, 1 ： 16 000 and 1 ： 2 000 respectively. The results showed that the serum specific antibody detected by ELISA from samples of diseased and healthy L. sanguineus was 70% and 20%, respectively. The results also indicated that the method established in this paper could be used not only to study on the serological epidemiology of vibriosis ofL. sanguineus, but also to recognize inapparent infective L. sanguineus.