栉孔扇贝急性病毒性坏死症病毒单克隆抗体的制备及ELISA检测

利用蔗糖密度梯度离心技术从自然发病的栉孔扇贝 (Chlamysfarrreri)组织分离纯化急性病毒性坏死症(Acutevirusnecrobioticdisease,AVND)病毒 ,并以此为抗原免疫Balb c小鼠。将免疫小鼠的脾细胞与NS_1骨髓瘤细胞进行融合 ,最终筛选出 4株能稳定传代并分泌该病毒特异性单克隆抗体 (MonoclonalAntibody ,MAb)的杂交瘤细胞系。应用胶体金标记免疫电镜技术在超微水平上对这 4株MAbs进行测定表明 ,它们对AVND病毒均具有高度的特异性 ,并且所识别的特异性位点均位于病毒粒子囊膜的纤突上。应用该单克隆抗体对不同养殖季节的一龄贝进行间接ELISA检测发现 ,7月中旬至 7月底病毒感染率与感染强度均处于当年的最高峰 ,与这一时期栉孔扇贝所表现出的最高死亡率完全吻合

Monoclonal antibodies prepared and used for detection of Acute virus necrobiotic disease virus in scallop Chlamys farreri by indirect ELISA

For developing monoclonal antibodies against acute virus necrobiotic disease (AVND) virus, mice of Balb/c strain were immunized with AVND virions which were isolated from the infected scallop Chlamys farreri. The spleen cells from immunized mice were then fused with NS-1 myeloma cells and the hybradomas were screened by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Finally, 4 stable MAbs of IgG isotype were obtained. Moreover, the combined position of these 4 MAbs to this virus was examined by immunogold electron microscopy (IEM). The results demonstrate that all 4 MAbs recognized epitopes on the envelope of the virions. Subsequently, a MAb-based ELISA was developed and used for detection of the infection rate and densities of the scallops which were sampled during different seasons from mid-April to mid-October, 2003. The result exhibited that both of the infection rate and infection densities sharply rose in mid-July and reached to the spikes, which right corresponds with their mortality during this period.