Development of a colloidal gold immunochromatographic strip for the rapid on-site detection of Ecytonucleospora hepatopenaei (EHP).

The Pacific white shrimp (Penaeus vannamei), one of the world's most economically important aquatic species, is highly susceptible to Ecytonucleospora hepatopenaei (EHP), a pathogen that infects the hepatopancreas and causes hepatopancreatic microsporidiosis (HPM), leading to stunted growth and substantial economic losses in shrimp farming. Currently, no effective treatments for EHP exist, making rapid on-site detection and preventive measures essential for disease control. While nucleic acid-based detection methods are commonly employed, they require specialized equipment, controlled environments, and trained personnel, which increase costs. To address this limitation, we developed a colloidal gold immunochromatographic assay (GICA) strip for rapid on-site detection of EHP in shrimp farms. Using LC-MS/MS, 15 high-abundance EHP proteins were identified, with EhSWP3 ranked highest and selected as the optimal antigen detection target. Recombinant EhSWP3 was used to immunize mice, resulting in the development of monoclonal antibodies. The optimal capture and labeled antibody combination (1B6, 3A6) was identified and incorporated into the GICA strip. Testing with common shrimp pathogens and various microsporidia samples demonstrated the high specificity of the EHP test strip. The strip exhibited a sensitivity of 1.81 × 10 copies of the EHP-SSU rRNA gene for detecting EHP-infected shrimp and 1 × 10 purified EHP spores, indicating its strong sensitivity in practical applications. To facilitate on-site use, a simple GICA workflow was established using disposable pestles, Buffer A, and Buffer B, enabling detection within 15 min. Testing of 110 shrimp samples revealed a 90.0 % concordance between the GICA strip and qPCR results. This study marks the first development and application of an EHP antigen detection strip, offering a practical tool for rapid, on-site disease monitoring in shrimp farming.