检测棘球蚴病患者血清内循环抗原的免疫层析试条的制备和评价  

Development of an immunochromatographic test for diagnosis of echinococcosis through circulating antigen detection

作　　者：张璟 王颖 高春花 危芙蓉 贾孝凯 石锋 ZHANG Jing;WANG Ying;GAO Chun-hua;WEI Fu-rong;JIA Xiao-kai;SHI Feng(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Laboratory of Parasite and Vector Biology,Ministry of Public Health,WHO Collaborating Centre for Malaria,Schistosomiasis and Filariasis,Shanghai 200025,China)

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心),国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,国家级热带病国际联合研究中心,上海200025

出　　处：《中国人兽共患病学报》2024年第2期166-170,共5页Chinese Journal of Zoonoses

基　　金：国家重点研发计划项目(No.2021YFC2300800,2021YFC2300801)。

摘　　要：目的以棘球蚴囊液纯化抗原特异性单克隆抗体为基础建立一种快速、简便诊断棘球蚴病的胶体金免疫层析试条方法,并对其进行评价。方法纯化棘球蚴囊液抗原,并以此免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体,对所制备的单克隆抗体确定其亚类和效价。筛选基于棘球蚴囊液纯化抗原制备的单克隆抗体对,采用柠檬酸三钠还原法制备胶体金颗粒,标记筛选到的单克隆抗体,并将其吸附于交联垫;将另一筛选到的单克隆抗体划线包被于同一硝酸纤维素膜适当位置,制成免疫层析试条。用该试条检测手术确诊的细粒棘球蚴病(87例)、多房棘球蚴病(40例)、囊尾蚴病(25例)、日本血吸虫病(10例)、弓形虫病(5例)、并殖吸虫病(5例)、华支睾吸虫病(5例)患者血清,以及60例健康者血清,以评价其检测的敏感性和特异性。结果以棘球蚴囊液纯化抗原为免疫源制备单克隆抗体,共筛选了11株能高效分泌效价在1∶25600~1∶102400特异抗体的细胞株,抗体亚类为IgG_(1)或IgG_(2)a。筛选到的单克隆抗体F 3B 6作为标记抗体,单克隆抗体C 4H 6作为包被抗体制成免疫层析试条,用该试条检测127份棘球蚴病患者血清的敏感性为88.12%(112/127),其中试条检测囊型包虫病患者血清的敏感性为88.51%(77/87),检测泡型包虫病患者血清的敏感性为87.50%(35/40),试条法检测两型包虫病患者血清敏感性之间差异无统计学意义(χ^(2)=0.03,P=0.86)。与25份囊尾蚴病患者血清、10份日本血吸虫病患者血清、5份弓形虫病患者血清、5份并殖吸虫病患者血清和5份华支睾吸虫病患者血清均无交叉反应,60份健康者血清也均为阴性,总特异性为100.00%。结论成功制备了棘球蚴囊液纯化抗原的单克隆抗体,以此单抗为基础研制出的快速诊断棘球蚴病胶体金免疫层析试条敏感性、特异性均较高。This study was aimed at establishing and evaluating a colloid gold immunochromatographic strip test based on monoclonal antibodies(McAbs)in hydatid cyst fluid for echinococcosis detection.Antigens purified from hydatid cyst fluid in sheep were used to immunize BALB/c mice to prepare McAbs.The McAb pairs prepared against purified antigen from hydatid cyst fluid were screened.One screened McAb was conjugated with colloidal gold and served as a detection probe;another screened McAb was immobilized on nitrocellulose in the proper position to prepare an immunochromatographic strip test.Serum samples from 127 patients with echinococcosis were used for evaluating sensitivity.Serum samples from patients with cysticercosis(25),schistosomiasis japonica(10),toxoplasmosis(5),paragonimiasis(5),or clonorchiosis(5),and from 60 healthy people,were used to evaluate the specificity.Eleven cell lines with high titers of McAbs against hydatid cyst fluid protein were obtained with the purified hydatid cyst fluid antigen as immunogen.The McAb F3B6 was conjugated with colloidal gold and used as a detection probe;McAb C4H6 was immobilized on nitrocellulose in the proper position to prepare the immunochromatographic strip test.The sensitivity of the strip test for detection of serum from patients with echinococcosis was 88.12%(112/127),and the sensitivity of the test strip for detection of serum from patients with cyst echinococcosis and alveolar echinococcosis was 88.51%(77/87)and 87.5%(35/40),respectively.No statistically significant difference in strip test sensitivity was observed in the detection of serum from patients with cyst echinococcosis versus alveolar echinococcosis(χ^(2)=0.03,P=0.86).No cross reactivity with cysticercosis(25),schistosomiasis japonica(10),toxoplasmosis(5),paragonimiasis(5)or clonorchiosis(5)was observed,and sera from 60 healthy people were also negative.The total specificity of the strip test was 100.00%.The immunochromatographic strip test based on hydatid cyst fluid antigen is a sensitive,specific,simple

关 键 词：棘球蚴病 单克隆抗体 循环抗原 免疫层析试条 检测 评价 

分 类 号：R383.3[医药卫生—医学寄生虫学]