摘要
目的获得能够用于制备旋毛虫病快速检测试纸条的ES Ag的单克隆抗体。方法应用旋毛虫肌幼虫排泄分泌抗原(ES Ag)免疫诱导Balb/c小鼠,使小鼠产生较强的免疫应答,将免疫小鼠的脾细胞与NS0瘤细胞融合,利用ES-45、ES-49抗原通过间接ELISA对大量杂交瘤细胞培养上清的筛选,筛选出分泌高亲和力单克隆抗体的4株杂交瘤细胞。结果ES-45和ES-49(分子质量分别为45ku,49ku)分别被Ts-2D4、Ts-4C5和Ts-4H6、Ts-2G8单克隆抗体识别,与猪肺丝虫(M etastrongylus pudendotectus.MP)、囊虫(Cysticercus cellulosae,CC)、细颈囊尾蚴(Cysticercus tenuicollis,CT)、蛔虫(Ascaris suum,AS)、弓形虫(Toxoplasma gondii,TG)、住肉孢子虫(Sarcocystism iescheriana,SM)抗原反应测试表明,4株单抗与参试抗原均无交叉反应,所有单抗上清ELISA平均效价为1∶1 120,腹水ELISA平均效价为1.1×106,亲和力常数的平均值为6.12×109L/mol。以金标免疫吸附试纸条原理为基础,利用制备的单抗成功研制了旋毛虫病快速检测试纸条,操作快速、无需设备及试剂,可以作为旋毛虫病的实时监测工具。结论ESAg单抗用于制备旋毛虫病快速诊断试纸条是理想的试剂。
Objective The aim of this study was to gain the excretory-secretory antigens （ES Ag） monoclonal antibodies that can be applied to produce immunochromatographic strip tbr detecting Trichinella spiralis. Methods Four lines of hybridoma cell wre produced which could secrete monoclonal antibodies against ES Ag of muscle larvae of Trichinella spiralis. The hybrid cells result from fusion of NS0 myeloma cells with splenocytes of Balb/c mice which had been immunized ES. Ag. Screening of supernatant fluid was performed by an indirect enzyme-linked immunosorbent assay （ELISA） using ES-45 and ES-49 antigens. Results ES-45 and ES-49 were recognized by Ts-2D4, Ts- 4C5 and Ts-4H6, Ts-2G8 respectively. The ELISA were used to characterize these McAbs by antigens of several parasite including Metastrongylus pudendotectus, Cysticercus cellulosae, Cysticercus tenuicollis, Ascaris suum, Toxoplasrna gondii, Sarcocystis miescheriana. None of the McAbs reacted with parasites tested. The average titers of all McAbs wre 1：1120 in supernatant, 1：1.1 × 10^6 in ascites and had a average affinity constant with 6.12 × 10^9 L/tool. Based on the principle of gold immunochromatog raphy assay, a strip for detecting trichinellosis in swine was developed with Ts-2D4, Ts-4C5, Ts-4H6 and Ts-2G8. The strip assay was rapid and easy to perform with no requirement of special skill, reagent or equipment, which can be used as a monifbring tool for the disease in real time. Conclusion The four McAbs were ideal reagents to produce strips used in diagnosis oftrichinellosis .
出处
《中国兽医寄生虫病》
2008年第1期7-11,共5页
Chinese Journal of Veterinary Parasitology
关键词
旋毛虫
单克降抗体
排泄分泌抗原
免疫吸附试纸条
Trichinella spiralis
monoclonat antibodies
excretory-secretory antigens
immunochromatographic strip
