对虾白斑综合征病毒胶体金免疫层析试纸条的优化及应用

Optimization and Application of Colloidal Gold Immunochromatographic Test Strip for Detection of White Spot Syndrome Virus(WSSV)

在已研制的对虾白斑综合征病毒(WSSV)胶体金免疫层析试纸条的基础上,对影响试纸条检测灵敏度的金标抗体、检测线抗体等关键因素进行优化,制备并纯化了5种IgG型的抗WSSV单克隆抗体2E6、2A3、3B7、4G9、5D2;金标抗体以Au-2E6和Au-2A3按1∶1(V/V)混合,检测线抗体以2A3、3B7、4G9、5D2按2∶4∶2∶1(V/V)混合,优化后的试纸条灵敏度提高到117.2ng/mL。使用该试纸条对人工感染WSSV克氏原螯虾(Procambarus clarkii)的鳃、肠、心脏、性腺、肌肉、肝胰腺、血淋巴等组织样品进行跟踪检测。发现感染后12h时,肠最早检测为WSSV阳性;24h时,鳃、血淋巴、肠、心脏、性腺、肝胰腺的检测结果为阳性,肌肉为阴性;36h时,各组织均检测为阳性,其中肠、性腺、血淋巴、鳃的阳性信号明显比其他组织强。研究表明,优化后的WSSV胶体金免疫层析试纸条可用于虾类感染WSSV早期的病毒检测,在对虾养殖中推广应用该试纸条,对控制白斑病暴发、减少经济损失有重要意义。

A sensitive colloidal gold immunochromatographic test strip （ICT） for white spot syndrome vi-rus （WSSV） was optimized and applied to detect WSSV in crayfishes （Procambarus clarkii）. The colloi-dal gold labeled anti-WSSV monoclonal antibodies （MAbs） mixture of 2E6 and 2A3 in a volume ratio of 1 ： 1served as detection reagent, and anti-WSSV MAbs mixture of 2A3, 3B7, 4G9 and 5D2 in a volume ra-tio 2 ： 4 ： 2 ： 1 served as capture antibodies, which were immobilized on Whatman AE99 nitrocellulose membrane （NCM）. The dectection limit of the ICT was 117.2 ng/mL. Gill, haemolymph, gut, heart, gonad, muscle and hepatopancreas of WSSV-infected crayfishes were detected every 12 hours post infec- tion （P. I. ） with the optimized ICT. At 12 h P. I. , gut was found to be WSSV-positive. At 24 h P. I. , gill, haemolymph, gut, heart, gonad, hepatopancreas were found to be WSSV-positive; while the muscle was found to be WSSV-negative. At 36 h P. I. , all the tissues were found to be WSSV-positive. These re-sults revealed that the optimized ICT was applicable in rapid on-site detection of WSSV at early stage of in-fection in shrimp without requirement of specialized equipments and skills, which may aid to preventing and controlling the spread of WSSV in shrimp aquaculture.