The Indirect ELISA and Monoclonal Antibody against African Swine Fever Virus p17 Revealed Efficient Detection and Application Prospects

Abstract

Since 2018, the outbreak and prevalence of the African swine fever virus (ASFV) in China have caused huge economic losses. Less virulent ASFVs emerged in 2020, which led to difficulties and challenges for early diagnosis and control of African swine fever (ASF) in China. An effective method of monitoring ASFV antibodies and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, ASFV p17 was successfully expressed in CHO cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 was established and optimized. The monoclonal antibody (mAb) against p17 recognized a conservative linear epitope (³TETSPLLSH¹¹) and exhibited specific reactivity, which was conducive to the identification of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17. The ELISA method efficiently detected clinical ASFV infection and effectively monitored the antibody levels in vivo after recombinant PRRSV live vector virus expressing p17 vaccination. Overall, the determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of the ASFV antigen protein. The indirect ELISA method and mAb against ASFV p17 revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

Keywords: ASFV p17; CHO cells; epitope; indirect ELISA; recombinant PRRSV.

Figure 1

The recombinant p17 was successfully expressed and purified from CHO cells. (A) PCR amplification of D117L gene. (B) Identification of p17 expression and purification in CHO cells by SDS-PAGE. (C) Identification of purified p17 by WB using anti-strep tag antibody. (D) Identification of purified p17 by WB using an ASFV-positive serum as primary antibody.

Figure 2

The anti-p17 mAb (6E3) specifically recognized CHO suspension cells transiently transfected with p17-expressing plasmid. (A) Identification of the mAb subtypes. (B) Identification of antibody titer of the purified 6E3 by the ELISA method. (C) 293T cells were transfected with pcDNA3.1-D117L-strep or control plasmid. Cells were fixed at 24 h post-transfection and immunostained with 6E3 as primary antibody and FITC-conjugated goat anti-mouse IgG as second antibody. Cellular nuclei were counterstained with 1 μg/mL of 4′,6′-diamidino-2-phenylindole (DAPI). (D) WB was conducted as treated in (C) to show the reactivity of 6E3.

Figure 3

6E3 recognized specific linear B-cell epitope ³TETSPLLSH¹¹. (A) Schematic diagram of D117L-truncated fragments. (B,C) A series of D117L-truncated fragments were constructed to pCold-TF and successfully expressed in E. coli BL21 (DE3) cells. 6E3 was used to detect the truncated p17 by WB using anti-His tag antibody or 6E3 as primary antibody, respectively. (D) Twelve different truncated peptides were synthesized and tested by ELISA to show the minimum epitope recognized by 6E3.

Figure 4

The epitope recognized by 6E3 was conservative among different strains. (A) The percent identity and divergence of p17 among the 24 representative ASFV strains were analyzed using MEGA. (B) Alignment analysis of the epitope (³TETSPLLSH¹¹) in 24 representative ASFV strains. (C) Prediction of the p17 structure using PyMOL. The epitope recognized by 6E3 is displayed in pink color.

Figure 5

6E3 specifically recognized the recombinant PRRSV expressing p17. (A) The schematic representation of recombinant PRRSV virus expressing ASFV p17 construction. (B,C) CPE and plaque morphology investigation. MARC-145 cells were infected with vA-ASFV-p17 and vHuN4-F112 (MOI = 0.001). The mock control represented non-infected MARC-145 cells. MARC-145 cells were monitored or stained with crystal violet at 3 days post-infection. (D,E) Growth characteristics of vA-ASFV-p17 and vHuN4-F112 were evaluated in MARC-145 cells (D) and PAMs (E). (F) MARC-145 cells were infected by vA-ASFV-p17 and vHuN4-F112. WB analysis of cell lysates using 6E3 and an anti-Nsp10 antibody. (G,H) IFA against PRRSV N protein or ASFV p17 in MARC-145 cells (G) and PAMs (H) at 36 hpi with vA-ASFV-p17 and vHuN4-F112 (MOI = 0.1). Cellular nuclei were counterstained with DAPI. Scale bar = 100 µm. (I) Detection of ASFV p17 expression in the recombinant PRRSVs (P5, P10, P15, and P20 viral stocks) using 6E3 as primary antibody by IFA.

Figure 6

The indirect ELISA method against p17 efficiently detected clinical samples and recombinant PRRSV virus expressing ASFV antigen. (A) Sensitivity testing of the ELISA method using ASFV-positive serum. (B) Specificity testing of the ELISA method using CSFV, PRV, PRRSV, PEDV, FMDV, PCV2, and ASFV-positive sera samples. (C) The PRRSV-specific humoral immune response was assessed by the S/P value identified from serum samples collected at the indicated time points from piglets in vA-ASFV-p17, vHuN4-F112, and mock groups. (D) The ASFV-specific humoral immune response against p17 was tested using the indirect ELISA method from serum samples collected at the indicated time points from three groups.