Fathi Saeid, Jalousian Fatemeh, Hosseini Seyed Hossein, Najafi Ali, Darabi Enayat, Koohsar Faramarz
Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Molecular Biology Research Center, Systems Biology and Poisoning Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
APMIS. 2018 May;126(5):428-439. doi: 10.1111/apm.12838.
The immunodiagnostic tests for cystic echinococcosis (CE) are mostly serological tests based on ELISA that use hydatid cyst antigens for primary screening because of its simple preparation and availability. The challenge to develop new serological methods (as compared to those based on the hydatid cyst fluid antigens) to meet the gold standard remains. Appropriate sources of antigenic material are necessary for application to improve the efficacy of immunodiagnostic tests at a population level. In the current study, a fusion protein containing the coding sequence of antigen B2t and two sequences of EPC1 antigen with some modifications was reconstructed. Using bioinformatics tools, these sequences were joined together by applying the sequence of a rigid α-helix-forming linker to obtain an appropriate structure of a fusion protein. Synthetic recombinant fusion protein was expressed using pET28a as a vector and evaluated by indirect ELISA test for sera from patients with hepatic CE and other parasitic infections. The sensitivity of the fusion protein was lower (88.46%) than the available ELISA kit (96.15%). However, the differences in sensitivity were not statistically significant as compared to the recombinant fusion peptide with the commercial kit (p = 0.269). The specificity of the recombinant fusion protein (95.45%) was not significantly lower than the commercial kit (96.59%; p = 1.000). Moreover, surprisingly there was no difference in the cross-reactivity values of performance between the recombinant-ELISA and commercial kit. The positive and negative predictive values of the recombinant antigen were achieved as 92% and 93.33%, respectively, while for the commercial kit, they were obtained as 94.33% and 97.70%, respectively. In conclusion, as an early evaluation of these antigens the performance of our recombinant fusion protein in ELISA is relatively promising. Although, it seemed that this peptide with specific antigenic epitopes might be more appropriate for the serological evaluation of CE by use of bioinformatics tools, our findings showed that cross-reactions and a negative reaction could occur in clinical performance. This fusion protein may have utility for diagnosis in humans, but further evaluation is needed using the WHO ultrasound classification for CE.
囊性棘球蚴病（CE）的免疫诊断测试大多是基于酶联免疫吸附测定（ELISA）的血清学检测，由于其制备简单且容易获得，因此使用棘球蚴囊肿抗原进行初步筛查。与基于棘球蚴囊肿液抗原的方法相比，开发新的血清学方法以达到金标准仍然是一项挑战。为了在人群层面提高免疫诊断测试的效力，需要合适的抗原物质来源。在本研究中，构建了一种融合蛋白，其包含抗原B2t的编码序列以及经过一些修饰的EPC1抗原的两个序列。利用生物信息学工具，通过应用形成刚性α螺旋的接头序列将这些序列连接在一起，以获得融合蛋白的合适结构。使用pET28a作为载体表达合成重组融合蛋白，并通过间接ELISA试验对肝CE患者和其他寄生虫感染患者的血清进行评估。融合蛋白的敏感性（88.46%）低于现有的ELISA试剂盒（96.15%）。然而，与商业试剂盒相比，重组融合肽的敏感性差异无统计学意义（p = 0.269）。重组融合蛋白的特异性（95.45%）并不显著低于商业试剂盒（96.59%；p = 1.000）。此外，令人惊讶的是，重组ELISA和商业试剂盒之间的交叉反应性能值没有差异。重组抗原的阳性和阴性预测值分别为92%和93.33%，而商业试剂盒的阳性和阴性预测值分别为94.33%和97.70%。总之，作为对这些抗原的早期评估，我们的重组融合蛋白在ELISA中的性能相对有前景。尽管通过生物信息学工具，这种具有特定抗原表位的肽似乎可能更适合用于CE的血清学评估，但我们的研究结果表明，在临床应用中可能会出现交叉反应和阴性反应。这种融合蛋白可能对人类诊断有用，但需要使用世界卫生组织（WHO）的CE超声分类进行进一步评估。
