基于Stefin抗原兔豆状囊尾蚴病间接ELISA检测方法的建立  

Establishment of an indirect ELISA method for detecting cysticercosis in rabbits based on Stefin antigen

作　　者：王由森 石正发 车亮 张月玥 羊倩倩 李甲 肖琴 孙晓林[1] 王泽祥 WANG Yousen;SHI Zhengfa;CHE Liang;ZHANG Yueyue;YANG Qianqian;LI Jia;XIAO Qin;SUN Xiaolin;WANG Zexiang(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070;Lanzhou Bio-Pharmaceutical Factory of China Animal Husbandry Industry Co,Ltd,Lanzhou 730100,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中牧实业有限公司兰州生物药厂,甘肃兰州730100

出　　处：《甘肃农业大学学报》2025年第1期1-9,共9页Journal of Gansu Agricultural University

基　　金：国家自然科学基金项目(32160843);甘肃省自然科学基金项目(21JR7RA823)。

摘　　要：【目的】研究半胱氨酸蛋白酶抑制剂Stefin的生物活性,建立豆状囊尾蚴病新的、高效便捷的血清学诊断方法。【方法】用Bradford法测定纯化后的重组蛋白浓度。以重组蛋白stefin作为包被抗原,建立间接ELISA诊断方法,通过从抗原包被浓度、包被条件、封闭条件、血清稀释度、酶标二抗稀释度、反应时间、温度等条件,以阳性血清与阴性血清的D_(450 nm)比值(P/N)作为判断标准优化反应条件,并对ELISA方法的敏感性、特异性、重复性进行检测。【结果】测定了重组蛋白浓度。间接ELISA检测方法临界值为0.352。阳性血清稀释倍数至1∶12800时,D_(450 nm)的值仍大于临界值。兔球虫、兔弓形虫等阳性血清检测显示D_(450 nm)均小于0.352。【结论】Stefin蛋白可以大批量获得,以stefin作为抗原建立的间接ELISA检测方法可以应用于豆状囊尾蚴病的诊断和流行病学调查研究,对豆状囊尾蚴病的有效防控具有重要的意义。【Objective】The study aimed to investigate the biological activity of Stefin,a cysteine prote⁃ase inhibitor,with the goal of establishing a novel,efficient,and convenient serological method for diagnos⁃ing Cysticercus pisiformis.【Method】The concentration of the purified recombinant protein was determined using the Bradford method.An indirect ELISA diagnostic approach was developed with the recombinant protein Stefin serving as the coating antigen.The optimization of reaction conditions was conducted based on the D_(450 nm)ratio(P/N)of positive serum to negative serum,considering factors such as antigen coating concentration,coating conditions,containment conditions,serum dilution,dilution of enzyme-labeled secondary antibody,reaction time and temperature.The sensitivity,specificity and reproducibility of the ELISA method were also assessed.【Result】The concentration of the recombinant protein was successfully determined.The critical value for the indirect ELISA assay was identified as 0.352.Even when the positive serum was diluted to 1∶12800,the D_(450 nm)value remained higher than the critical value.Tests conducted with positive serum for rabbit Coccidia and rabbit Toxoplasma revealed D_(450 nm)values below 0.352.【Conclusion】Stefin can be produced in large quantities.The utilization of Stefin as an antigen in an indirect ELISA assay presented a novel approach for the future diagnosis and epidemiological investigation of cysti⁃cercus pisiformis,which was of great significance in the prevention and management of the disease.

关 键 词：豆状囊尾蚴 BRADFORD 间接ELISA Stefin 

分 类 号：S859[农业科学—临床兽医学]