Anuracpreeda Panat, Watthanadirek Amaya, Chawengkirttikul Runglawan, Sobhon Prasert
Division of Agricultural Science, Mahidol University, Kanchanaburi Campus, Saiyok, Kanchanaburi, 71150, Thailand.
Faculty of Environment and Resource Studies, Mahidol University, Phuttamonthon District, Nakhon Pathom, 73170, Thailand.
Parasitol Res. 2017 Jan;116(1):167-175. doi: 10.1007/s00436-016-5273-1. Epub 2016 Oct 22.
A number of monoclonal antibodies (MoAbs) against the 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg) were produced in vitro by hybridoma technique. Reactivity and specificity of these MoAbs were evaluated by ELISA and immunoblotting assays. Seven MoAb clones were selected from the stable hybridoma clones, namely 1D10, 2D7, 3B10, 3D9, 4F1, 4G4, and 5G12. It was found to be IgM and kappa light chain isotypes. By immunoblotting and ELISA, all MoAbs reacted with purified 16 kDaAgPg at molecular weight (MW) of 16 kDa and with the native 16 kDa antigen at MW of 16 kDa in the whole body (WB) and excretory-secretory (ES) fractions, but not with tegumental antigens (TA) of adult fluke. All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants, including Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Moniezia benedeni, Avitellina centripunctata, Haemonchus placei, Trichuris sp., and Setaria labiato-papillosa. Localization and distribution of the native 16 kDaAg in adult P. gracile by immunohistochemistry, using MoAbs as probes, showed that the native 16 kDaAg was present in high concentration in the cytoplasm of vitelline cells, eggshell globules, and the shells of eggs, but not in the tegument, muscle, parenchymal cells, and cecum of adult fluke. This finding indicated that the 16 kDaAg is a copiously expressed parasite protein that is released into the ES; thus, 16 kDaAg and its MoAb could be a good candidate for immunodiagnosis of paramphistomosis in ruminants.
通过杂交瘤技术在体外制备了多种针对纤细双腔吸虫16 kDa抗原（16 kDaAgPg）的单克隆抗体（MoAbs）。通过酶联免疫吸附测定（ELISA）和免疫印迹分析评估了这些单克隆抗体的反应性和特异性。从稳定的杂交瘤克隆中筛选出7个单克隆抗体克隆，即1D10、2D7、3B10、3D9、4F1、4G4和5G12。发现其为IgM和κ轻链同种型。通过免疫印迹和ELISA检测，所有单克隆抗体均能与分子量（MW）为16 kDa的纯化16 kDaAgPg以及虫体整体（WB）和排泄分泌（ES）组分中MW为16 kDa的天然16 kDa抗原发生反应，但不与成虫的皮层抗原（TA）发生反应。所有这些单克隆抗体均未与反刍动物中常见的其他寄生虫抗原发生交叉反应，这些寄生虫包括胰阔盘吸虫、扩展巨盘吸虫、纺锤血吸虫、贝氏莫尼茨绦虫、点状无卵黄腺绦虫、绵羊血矛线虫、毛首线虫属以及唇乳突丝虫。以单克隆抗体为探针，通过免疫组织化学方法对纤细双腔吸虫成虫中天然16 kDaAg的定位和分布进行研究，结果显示天然16 kDaAg在卵黄细胞、卵壳小球和卵壳的细胞质中高浓度存在，但在成虫的皮层、肌肉、实质细胞和盲肠中不存在。这一发现表明16 kDaAg是一种大量表达并释放到ES中的寄生虫蛋白；因此，16 kDaAg及其单克隆抗体可能是反刍动物双腔吸虫病免疫诊断的良好候选物。
