Establishment of a novel double-antibody sandwich fluorescence microsphere immunochromatographic test strip for rapid detection of swine acute diarrhea syndrome coronavirus (SADS-CoV) infection

Cong Xiao ¹ Tong Fei ² Liu Huizhen ³ Zhu Yujun ⁴ Tan Ningxin ¹ Gu Feng ¹ Wang Huanan ⁵ Feng Cong ^4*

• ¹ The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
• ² Fu Shun Vocational Technology Institute, Fu Shun, China
• ³ College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, Guangdong Province, China
• ⁴ Guangdong Laboratory Animals Monitoring Institute, Guangzhou, Guangdong Province, China
• ⁵ Center for Veterinary Sciences, Zhejiang University, Zhe Jiang, China

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enveloped, positive-sense, single-stranded RNA virus that causes clinical symptoms such as vomiting and diarrhea in 10-day-old piglets. SADS-CoV has caused significant economic losses in the swine industry in southern China. Currently, no effective treatments or vaccines are available for this disease, making it crucial to establish a point-of-care testing (POCT) technology for early diagnosis and prevention. In this study, we first verified the specificity and immunogenicity of four monoclonal antibodies (mAbs) targeting the SADS-CoV N protein. Through paired screening, mAb 12E1 was identified as the coating antibody, and mAb 5G12 was selected as the labeled antibody, forming the optimal antibody combination for the preparation of fluorescent microsphere-based immunochromatography assay (FM-ICA). Subsequently, the conditions for the FM-ICA test strip were optimized. The results showed that the optimal labeling concentration of the antibodies was 200 µg/mg, the optimal coating concentration was 1 mg/mL, the optimal incubation time was 10 min, and the optimal dilution factor was 10. Furthermore, the functionality of the established FM-ICA was analyzed, revealing its excellent specificity, sensitivity, repeatability, and stability. The highest dilution factor detected was 1280, with the limit of detection (LOD) of 78 PFU mL-1. Finally, 72 clinical samples were tested using FM-ICA and qPCR, with a concordance rate between the two methods of 97.22%. These results indicate that FM-ICA is an excellent POCT technology that can be used for the early diagnosis of SADS-CoV, providing support for disease prevention and treatment.