A truncated N protein-based ELISA method for the detection of antibodies against porcine deltacoronavirus.

Dongsheng WANG; Ruiming YU; Liping ZHANG; Yingjie BAI; Xia LIU; Yonglu WANG; Xiaohua DU; Xinsheng LIU

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A truncated N protein-based ELISA method for the detection of antibodies against porcine deltacoronavirus.

Author: Dongsheng WANG ¹ ; Ruiming YU ¹ ; Liping ZHANG ² ; Yingjie BAI ² ; Xia LIU ¹ ; Yonglu WANG ² ; Xiaohua DU ¹ ; Xinsheng LIU ²
Author Information

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, Gansu, China.
2. 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: IgG antibodies; indirect ELISA; porcine deltacoronavirus; truncated N protein
MeSH: Animals; Enzyme-Linked Immunosorbent Assay/methods*; Swine; Antibodies, Viral/blood*; Recombinant Proteins/genetics*; Deltacoronavirus/isolation & purification*; Coronavirus Infections/virology*; Swine Diseases/diagnosis*; Coronavirus Nucleocapsid Proteins; Sensitivity and Specificity
From: Chinese Journal of Biotechnology 2025;41(7):2760-2773
CountryChina
Language:Chinese
Abstract: This study aims to establish an antibody detection method for porcine deltacoronavirus (PDCoV). The recombinant proteins PDCoV-N1 and PDCoV-N2 were expressed via the prokaryotic plasmid pColdII harboring the N gene sequence of the PDCoV strain CH/XJYN/2016. The reactivity and specificity of PDCoV-N1 and PDCoV-N2 with anti-PEDV sera were analyzed after the recombinant proteins were analyzed by SDS-PAGE and purified by the Ni-NTA Superflow Cartridge. Meanwhile, Western blotting and indirect immunofluorescence assay were carried out separately to validate the recombinant proteins PDCoV-N1 and PDCoV-N2. Finally, we established an indirect ELISA method based on the recombinant protein PDCoV-N2 after optimizing the conditions and tested the sensitivity, specificity, and reproducibility of the method. Then, the established method was employed to examine 102 clinical serum samples. The recombinant protein PDCoV-N2 showed low cross-reactivity with anti-PEDV sera. The optimal conditions of the indirect ELISA method based on PDCoV-N2 were as follows: the antigen coating concentration of 1.25 μg/mL and coating at 37 ℃ for 1 h; blocking by BSA overnight at 4 ℃; serum sample dilution at 1:50 and incubation at 37 ℃ for 1 h; secondary antibody dilution at 1:80 000 and incubation at 37 ℃ for 1 h; color development with TMB chromogenic solution at 37 ℃ for 10 min. The S/P value ≥ 0.45, ≤0.38, and between 0.45 and 0.38 indicated that the test sample was positive, negative, and suspicious, respectively. The testing results of the antisera against porcine epidemic diarrhea virus (PEDV), porcine circovirus 2 (PCV2), transmissible gastroenteritis virus (TGEV), foot-and-mouth disease virus (FMDV), and African swine fever virus (ASFV) showed that the S/P values were all less than 0.38. The testing results of the 800-fold diluted anti-PDCoV sera were still positive. The results of the inter- and intra-batch tests showed that the coefficients of variation of this method were less than 10%. Clinical serum sample test results showed the coincidence rate between this method and neutralization test was 94.12%. In this study, an ELISA method for the detection of anti-PDCoV antibodies was successfully established based on the truncated N protein of PDCoV. This method is sensitive, specific, stable, and reproducible, serving as a new method for the clinical diagnosis of PDCoV.