Development and evaluation of a competitive ELISA based on a porcine neutralizing Fab antibody against Senecavirus A.

Yubin LIANG; Xueqing MA; Yixuan HE; Caihe WANG; Kun LI; Pinghua LI; Yuanfang FU; Zengjun LU; Xiaohua DU; Xia LIU; Pu SUN

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Development and evaluation of a competitive ELISA based on a porcine neutralizing Fab antibody against Senecavirus A.

Author: Yubin LIANG ¹ ; Xueqing MA ² ; Yixuan HE ² ; Caihe WANG ¹ ; Kun LI ² ; Pinghua LI ² ; Yuanfang FU ² ; Zengjun LU ² ; Xiaohua DU ¹ ; Xia LIU ¹ ; Pu SUN ¹
Author Information

1. College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China.
2. Gansu Province Research Center for Basic Disciplines of Pathogen Biology, WOAH/National Foot-and-Mouth Disease Reference Laboratory, State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, Gansu, China.
Publication Type:Journal Article
Keywords: Senecavirus A; competitive ELISA; diagnostic assay; neutralizing antibody
MeSH: Animals; Swine; Antibodies, Neutralizing/immunology*; Enzyme-Linked Immunosorbent Assay/methods*; Immunoglobulin Fab Fragments/immunology*; Antibodies, Viral/immunology*; Picornaviridae/immunology*; Humans; HEK293 Cells; Swine Diseases/diagnosis*; Picornaviridae Infections/diagnosis*
From: Chinese Journal of Biotechnology 2025;41(7):2748-2759
CountryChina
Language:Chinese
Abstract: Senecavirus A (SVA) is a major viral pathogen causing disease in pigs, and effective monitoring of SVA infection is critical for disease control. In this study, we aimed to develop a reliable ELISA method for rapidly detecting neutralizing antibodies against SVA. We used HEK293F cells to express an SVA-specific porcine Fab antibody and verified the biological activity of the Fab antibody by indirect ELISA, immunofluorescence assay, virus neutralization test, and Western blotting. The Fab antibody was biotinylated and used as a competitive antibody to establish a competitive ELISA (C-ELISA) for detecting neutralizing antibodies against SVA. We then evaluated the C-ELISA in terms of sensitivity, specificity, repeatability, and result agreement rate with the VNT. The results showed that we successfully prepared an SVA-specific porcine Fab antibody, which showed high affinity for SVA. We named this antibody 1M33Fab and designated it as Bio-1M33Fab after biotin labeling. The assay conditions were optimized as follows: the coating concentration of SVA particles being 1 μg/mL, the working concentration of Bio-1M33Fab being 0.5 μg/mL, the optimal serum dilution of 1:10, and the optimal dilution of enzyme-labeled avidin being 1:30 000. At a percent inhibition (PI) of 47%, the assay demonstrated the highest sensitivity (96.88%) and specificity (100%), with no cross-reactivity observed with the positive sera of major porcine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, while the inter-assay coefficient of variation ranged from 1.10% to 8.97%, indicating good repeatability. In the detection of 224 clinical pig serum samples, C-ELISA and VNT showed a result agreement rate of 93.75%. In conclusion, we successfully develop a C-ELISA method for detecting neutralizing antibodies against SVA by using a porcine-derived Fab antibody, which lays a foundation for the development of detection kits.