Masoumi Koushk Mehdi Abolfazl, Motedayyen Hossein, Fasihi Harandi Majid, Akbari Hossein, Moradi Hasan-Abad Amin, Arbabi Mohsen
Department of Medical Parasitology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
Autoimmune Diseases Research Center, Kashan University of Medical Sciences, Kashan, Iran.
Parasit Vectors. 2025 Jul 8;18(1):267. doi: 10.1186/s13071-025-06900-1.
Cystic echinococcosis (CE), one of the most important parasitic diseases, threatens global health for animals and, occasionally, humans. Accurate and timely diagnosis is crucial for effective disease management and control. Therefore, this study focused on designing and investigating a novel direct hemagglutination test (HAT) using a recombinant protein to diagnose hydatid disease.
The fusion protein IH4_Antigen_HisTag was designed by incorporating EgFABP1, EgTeg, and IH4 nanobody sequences. The fragment was cloned for expression in Escherichia coli BL21 (DE3) using the vector pET-28a. It was optimized through isopropyl β-D-thiogalactopyranoside (IPTG) induction. The results of a transfection were assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. For consistency, a HAT was developed using O-negative red blood cells (RBCs) with standardized antigen concentrations. The sensitivity and specificity of the test were assessed by the receiver operating characteristic (ROC) curve, using serum samples from 43 patients with CE and 43 healthy controls.
The results of SDS-PAGE and western blot revealed that the size and concentration of the produced protein were 27 KDa and 126.1 μg/ml, respectively. The highest sensitivity (95%) and maximum specificity (95.3%) were observed at the cutoff point of the HAT equal to 0.09375 [area under the curve (AUC) = 0.972].
The HAT, as a test with low cost, ease of use, and no need for advanced equipment, has acceptable sensitivity and specificity in determining antibodies against the parasite. The IH4-Antigen-HisTag protein can be used in healthcare systems as a simple, cost-effective diagnostic tool for hydatid disease.
囊性棘球蚴病（CE）是最重要的寄生虫病之一，对动物乃至人类的全球健康构成威胁。准确及时的诊断对于有效的疾病管理和控制至关重要。因此，本研究着重于设计和研究一种使用重组蛋白诊断包虫病的新型直接血凝试验（HAT）。
通过整合EgFABP1、EgTeg和IH4纳米抗体序列设计融合蛋白IH4_Antigen_HisTag。使用载体pET - 28a将该片段克隆至大肠杆菌BL21（DE3）中进行表达。通过异丙基β - D -硫代半乳糖苷（IPTG）诱导进行优化。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）和蛋白质印迹法评估转染结果。为保持一致性，使用标准化抗原浓度的O型阴性红细胞（RBC）开展HAT。采用来自43例CE患者和43例健康对照的血清样本，通过受试者工作特征（ROC）曲线评估该试验的敏感性和特异性。
SDS - PAGE和蛋白质印迹法结果显示，所产生蛋白质的大小和浓度分别为27 kDa和126.1 μg/ml。在HAT的截断点等于0.09375时观察到最高敏感性（95%）和最大特异性（95.3%）[曲线下面积（AUC）= 0.972]。
HAT作为一种成本低、易于使用且无需先进设备的试验，在确定针对该寄生虫的抗体方面具有可接受的敏感性和特异性。IH4 - Antigen - HisTag蛋白可在医疗保健系统中用作包虫病的一种简单、经济高效的诊断工具。
