Ralston M J, Heath D D
AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand.
J Parasitol. 1995 Jun;81(3):422-8.
The evaginated scolex of Taenia ovis secretes an antigen complex into defined culture medium that has been used to develop a cestode-specific enzyme-linked immunosorbent assay (ELISA). We now describe an immunoblot test for antibodies to T. ovis based on the recognition of a 94-kDa antigen band in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of T. ovis scolex secretions. The test was specific for cestode infections in dogs and was 82% sensitive for the recognition of T. ovis infections. Affinity-purified antibody uniquely recognizing this 94-kDa band was used to screen a cDNA expression library constructed utilizing mRNA from newly evaginated T. ovis scoleces. The cDNA from putative positive bacteriophage were subcloned into the plasmid pGEX and expressed as fusion proteins with schistosome glutathione-S-transferase (GST). The expressed fusion proteins were purified using glutathione-agarose beads. The recombinant parasite antigen was either eluted as a fusion protein with GST or cleaved from GST using a restriction protease. Some dog sera reacted with the GST molecule. However, the recombinant cleaved antigen from 1 clone, T. ovis 40, showed 42% sensitivity and 100% specificity for cestodes in an ELISA using test sera from dogs monospecifically infected with T. ovis and preabsorbed with bacteria. Some sera from dogs monospecifically infected with other cestode species (Taenia pisiformis, 30%; Taenia hydatigena, 30%; Echinococcus granulosus, 20%) reacted with the cloned antigen.
绵羊带绦虫外翻的头节可向特定培养基中分泌一种抗原复合物,该复合物已被用于开发一种针对绦虫的酶联免疫吸附测定(ELISA)。我们现在描述一种针对绵羊带绦虫抗体的免疫印迹试验,该试验基于在绵羊带绦虫头节分泌物的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳图谱中识别出一条94 kDa的抗原带。该试验对犬类绦虫感染具有特异性,对识别绵羊带绦虫感染的敏感性为82%。使用唯一识别这条94 kDa带的亲和纯化抗体筛选了一个cDNA表达文库,该文库利用新外翻的绵羊带绦虫头节的mRNA构建。来自推定阳性噬菌体的cDNA被亚克隆到质粒pGEX中,并表达为与血吸虫谷胱甘肽 - S - 转移酶(GST)的融合蛋白。使用谷胱甘肽 - 琼脂糖珠纯化表达的融合蛋白。重组寄生虫抗原要么作为与GST的融合蛋白洗脱,要么使用限制性蛋白酶从GST上切割下来。一些犬血清与GST分子发生反应。然而,来自1个克隆(绵羊带绦虫40)的重组切割抗原则在使用单特异性感染绵羊带绦虫并经细菌预吸收的犬的试验血清进行的ELISA中,对绦虫显示出42%的敏感性和100%的特异性。一些单特异性感染其他绦虫物种(豆状带绦虫,30%;多头带绦虫,30%;细粒棘球绦虫,20%)的犬的血清与克隆抗原发生反应。