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Background <p>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.</p> Methods <p>The fusion protein IH4_Antigen_HisTag was designed by incorporating EgFABP1, EgTeg, and IH4 nanobody sequences. The fragment was cloned for expression in <i>Escherichia coli </i> BL21 (DE3) using the vector pET-28a. It was optimized through isopropyl β-<span>d</span>-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.</p> Results <p>The results of SDS-PAGE and western blot revealed that the size and concentration of the produced protein were 27 KDa and 126.1&#xa0;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].</p> Conclusions <p>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.</p> Graphical Abstract <p></p>

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Design and evaluation of a novel direct hemagglutination test based on a recombinant protein for diagnosis of cystic echinococcosis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月8日

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  • Abolfazl Masoumi Koushk Mehdi,
  • Hossein Motedayyen,
  • Majid Fasihi Harandi,
  • Hossein Akbari,
  • Amin Moradi Hasan-Abad,
  • Mohsen Arbabi

摘要

Background

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.

Methods

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.

Results

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].

Conclusions

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.

Graphical Abstract