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. 2023 Feb 10:10:1093440.
doi: 10.3389/fvets.2023.1093440. eCollection 2023.

Novel p22 and p30 dual-proteins combination based indirect ELISA for detecting antibodies against African swine fever virus

Jianda Li  1 , Jian Jiao  1   2 , Na Liu  1   3 , Sufang Ren  1 , Hao Zeng  1 , Jun Peng  4 , Yuyu Zhang  1   3 , Lihui Guo  1 , Fei Liu  1 , Tingting Lv  1   3 , Zhi Chen  1 , Wenbo Sun  1 , Nataliia Hrabchenko  1 , Jiang Yu  1   2 , Jiaqiang Wu  1   2   3
Affiliations

Novel p22 and p30 dual-proteins combination based indirect ELISA for detecting antibodies against African swine fever virus

Jianda Li et al. Front Vet Sci. .

Abstract

Introduction: African swine fever virus (ASFV) infection is one of the most complex and fatal hemorrhagic viral diseases, causing a devastating loss to the swine industry. Since no effective vaccine is available, prevention and control of ASFV heavily depends on early diagnostic detection.

Methods: In this study, a novel indirect ELISA was established for detecting antibodies against ASFV using dual-proteins, p22 and p30. Recombinants p22 and p30 were expressed and purified from E.coli vector system by recombined plasmids pET-KP177R and pET-CP204L. p22 and p30 were mixed as antigens for developing the indirect ELISA.

Results: Through optimizing coating concentrations of p30 and p22, coating ratio (p30: p22 = 1:3), and serum dilution (as 1:600), the established ELISA performed higher specificity, sensitivity, and repeatability against ASFV-positive serum. Furthermore, 184 clinical serum samples from suspected diseased pigs were verified the established ELISA in clinical diagnosis. The results showed that compared with two commercial ELISA kits, the established ELISA possessed higher sensitivity and almost uniform coincidence rate.

Conclusion: The novel indirect ELISA based on dual-proteins p30 and p22 performed a valuable role in diagnostic detection of ASFV, providing a broad insight into serological diagnostic methods of ASFV.

Keywords: African swine fever virus; indirect ELISA; p22; p30; serological diagnosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Exploration of p22 and p30 proteins induction conditions. (A, B) Determination of optimal IPTG concentration of p22 (A) and p30 (B). M, Marker; (1) pET-32a empty carrier; (2) Before induction; (3–6) IPTG concentration at 0.1, 0.4, 0.7, and 1.0 mM. (C, D) Determination of the best OD600 of p22 (C) and p30 (D). M, Marker; (1) pET-32a empty carrier; (2) Before induction; (3–6) OD600 at 0.4, 0.6, 0.8, 1.
Figure 2
Figure 2
Soluble analysis and purification of p22 and p30 proteins. (A, B) Soluble analysis of p22 protein (A) and p30 protein (B). M, Marker; (1) Supernatant after ultrasound; (2) Precipitation after ultrasound. (C, D) The purification results of p22 protein (C) and p30 protein (D). (C) M, Marker; (1–7) Flow through fluid, supernatant after ultrasound, 20, 40, 50, 100, and 200 mM imidazole. (D) M, Marker; (1–9) Precipitation after ultrasound, supernatant after ultrasound, flow through fluid, 20, 40, 50, 100, 200, and 200 mM imidazole.
Figure 3
Figure 3
Determination the expression of p22 and p30. (A, C) The expression of p22 protein (A) and p30 protein (C) was determined by anti-His antibody. (B, D) The expression of p22 protein (B) and p30 protein (D) was immunoreactive with ASFV-positive serum.
Figure 4
Figure 4
Optimal conditions of ELISA. (A) Optimal concentration of coating protein and serum dilution. (B) Optimal blocking solution and best blocking time. (C) Determination of the optimum dilution of enzyme-labeled secondary antibody. (D) Optimum substrate-enzyme interaction time.
Figure 5
Figure 5
Determination of sensitivity and specificity. (A) The cut-off value of the established ELISA. (B) The specificity was determined using positive serums against PCV2, PRV, CSFV, PRRSV and HPS.
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