摘要
利用柞蚕微孢子虫孢子液直接免疫家兔获得柞蚕微孢子虫多克隆抗体后,分别采用双抗夹心法和竞争法制作胶体金免疫层析试纸条,建立能简便、快捷、准确诊断柞蚕微粒子病的柞蚕微孢子虫胶体金免疫层析检测法。双抗夹心法和竞争法分别是将柞蚕微孢子虫多克隆抗体与25 nm和17 nm的胶体金颗粒结合并固定在金标垫上,然后均将羊抗兔二抗包被在硝酸纤维素膜上作为质控点(C),但是2种方法制作的胶体金免疫层析试纸条的反应模式不同:前者是以柞蚕微孢子虫多克隆抗体作为检测点(T),而后者以柞蚕微孢子虫孢壁蛋白作为检测点(T)。2种方法制作的胶体金免疫层析试纸条的检测时间均为10 min,检测灵敏度为0.8×107个/mL,与柞蚕血淋巴无交叉反应,特异性强,操作简单,其中,以双抗夹心法制作的试纸条显色更清晰,结果更可靠,更具实用价值。
The polyclonal antibody against Nosema pernyi was prepared by immunizing rabbits using the suspension containing spores of N.pernyi and was used to prepare colloidal gold test strips for immunochromatographic assay through double antibody sandwich method and competition method.The established colloidal gold immunochromatographic assay could have a simple,rapid and accurate diagnosis to pebrine disease in Antheraea pernyi.The double antibody sandwich method and competition method conjugated the polyclonal antibody with 25 nm and 17 nm colloidal gold particles respectively and bound the conjugated products on a gold-labeled pad.Then,goat anti-rabbit secondary antibody was spotted on nitrocellulose membrane as control dot（C） for both methods.However,the reaction patterns for preparing colloidal gold test strips were different for the two methods： the former took the polyclonal antibody against N.pernyi as the test dot（T）,and the latter took spore wall proteins of N.pernyi as the test dot（T）.The test strips prepared from both methods had a working time of 10 min and a diagnostic sensitivity of 0.8×107 per mL,and had no cross reaction with the hemolymph of Antheraea pernyi,being highly specific and easy to operate.Besides,the test strip from double antibody sandwich method had clearer staining result and is thus more reliable and valuable for practical application.
出处
《蚕业科学》
CAS
CSCD
北大核心
2012年第1期97-101,共5页
ACTA SERICOLOGICA SINICA
基金
现代农业产业技术体系专项(No.CARS-22)
辽宁省教育厅科研项目(No.L2010512)
沈阳农业大学校青年基金(No.201010002)
关键词
柞蚕微孢子虫
多克隆抗体
胶体金
免疫层析
双抗夹心法
竞争法
Nosema pernyi
Polyclonal antibody
Colloidal gold
Immunochromatography
Double antibody sandwichmethod
Competition method
