免疫细胞化学法测定鱼类淋巴囊肿病毒(LCDV)滴度

Development of a Virus Titration Assay for Lymphocystis Disease Virus by an Immunocytochemical Staining Method

利用免疫细胞化学法测定鱼类淋巴囊肿病毒(LCDV)滴度。以牙鲆鳃细胞系(FG)作为感染细胞,将生长旺盛的FG细胞接种于48孔培养板中培养至形成细胞单层,用2倍连续稀释的LCDV粗提液分别接种FG细胞。固定各稀释度LCDV感染后的FG细胞,孵育抗牙鲆LCDV单克隆抗体,其后再运用生物素-亲合素反应系统,以碱性磷酸酶底物AP-Red试剂盒发色。倒置显微镜观察,被病毒感染的FG细胞的细胞质呈现红色,未被感染细胞的细胞质呈无色。记录各稀释度病毒感染的阳性细胞孔数,按Reed-Muench法计算组织细胞培养半数感染量(TCID50)。结果显示,免疫细胞化学法测得LCDV在FG的滴度为1.77×210 TCID50/mL。该法可以用来有效测定LCDV滴度,且结果直观、准确性较好,灵敏度较高。

An immunocytochemical staining method was developed for virus titration assay of lymphocystis disease virus（LCDV） in cultures of a flounder gill（FG） cell line.FG cells were propagated into a 48 well tissue culture plate and post inoculated with two-fold serially diluted LCDV,purified from lymphocystis cells of Japanese flounder Paralichthys olivaceus.After infection,the cell cultures were fixed and incubated with LCDV-specific monoclonal antibody in combination with biotin-avidin system.Infected cells and uninfected cells,stained with alkaline phosphatase substrate,could be differentiated by presence and absence of red color in the cytoplasm.Each positive well was scored microscopically based on the appearance of immunostained cells and the titer of the LCDV infectivity in FG cells was measured to be 1.77×210 TCID50 per mL by the immunocytochemical staining method.The immunocytochemical staining method is proved to be accurate and sensitive for measuring the titer of LCDV.