Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Mahidol-Bangkok School of Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Parasit Vectors. 2018 May 30;11(1):322. doi: 10.1186/s13071-018-2878-5.
Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis.
To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 μg/ml, and anti-human IgG diluted at 1:4000.
The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11).
Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis.
异形并殖吸虫是导致泰国并殖吸虫病的主要病原体。在并殖吸虫病的 Western blot 诊断检测中，粗制异形并殖吸虫体提取物中存在的 35 kDa 带是已知的诊断带之一。本研究旨在使用异形并殖吸虫 cDNA 文库创建该抗原的重组版本，用于并殖吸虫病的免疫诊断。
为了实现这一目标，从成虫 mRNA 构建了 cDNA 表达文库，并使用用 35 kDa 抗原免疫的小鼠的抗体进行免疫筛选。筛选结果鉴定出一个由克隆 CE3 编码的免疫反应性蛋白，该克隆包含一个由 1292 个碱基对组成的插入序列。由于其与 proactivator 多肽相似，因此选择该克隆用于构建重组异形并殖吸虫蛋白。用于重组蛋白表达，将 CE3 基因序列插入质粒载体 pRset，得到的产物具有预期的 35 kDa 分子量。使用来自健康个体、并殖吸虫病和其他寄生虫感染患者的血清评估基于 CE3 重组蛋白的 IgG-ELISA。该 ELISA 通过使用稀释度为 1:2000 的人血清、优化的抗原浓度为 1 μg/ml 和稀释度为 1:4000 的抗人 IgG 进行。
将截止光密度值设定为平均值+2 个标准差（0.54），结果该检测的灵敏度为 88.89%，特异性为 95.51%。重组抗原可与异形并殖吸虫、拟异形并殖吸虫和后殖吸虫感染的抗体发生反应。与少数蓝氏贾第鞭毛虫感染（2/3）、班氏丝虫病（1/10）、肝片吸虫病（3/10）、钩虫病（4/10）和脑囊虫病（1/11）发生交叉反应。
鉴于 IgG-ELISA 中观察到的低水平异源感染交叉反应（215 份血清样本中有 11 份），该 35 kDa 抗原的检测敏感性和特异性均较高，因此可能对检测并殖吸虫病有用。
