Gasser R B, Lightowlers M W, Rickard M D
University of Melbourne, Werribee, Victoria, Australia.
Int J Parasitol. 1990 Nov;20(7):943-50. doi: 10.1016/0020-7519(90)90033-j.
Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.
从含有假定血清学诊断抗原的转移印迹上，从犬血清中亲和纯化出针对细粒棘球绦虫的特异性抗体。这些抗体以及来自感染细粒棘球绦虫犬的血清池，被用于筛选使用细粒棘球绦虫原头蚴mRNA构建的λgt11 cDNA文库。分离并鉴定出9个明确的抗原性克隆，其中一个（c10P1）在噬菌斑免疫测定中与感染细粒棘球绦虫犬的血清产生强烈的特异性反应。这些克隆均被亚克隆到质粒载体pGEX-1中。在菌落免疫测定和/或免疫印迹中证实了克隆的抗原性。各个亚克隆的谷胱甘肽S-转移酶（GST）融合蛋白在大肠杆菌中产生，通过亲和层析纯化，并使用感染细粒棘球绦虫、带绦虫属或线虫的犬血清以及无蠕虫犬血清在ELISA中进行评估。尽管GST融合蛋白10P1的敏感性相对较低，但其在ELISA中对犬细粒棘球绦虫感染诊断的特异性为100%。进一步的研究旨在鉴定其他重组抗原，并测试在替代表达系统中表达的10P1，以提高ELISA的诊断敏感性。
