Epitope mapping and establishment of a blocking ELISA for mAb targeting the p72 protein of African swine fever virus

Abstract

The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to the viral epidemic, the pig industry worldwide has suffered significant financial setbacks. The absence of a proven vaccine for ASFV necessitates the development of a sensitive and reliable serological diagnostic method, enabling laboratories to effectively and expeditiously detect ASFV infection. In this study, four strains of monoclonal antibodies (mAbs) against p72, namely, 5A1, 4C4, 8A9, and 5E10, were generated through recombinant expression of p72, the main capsid protein of ASFV, and immunized mice with it. Epitope localization was performed by truncated overlapping polypeptides. The results indicate that 5A1 and 4C4 recognized the amino acid 20-39 aa, 8A9 and 5E10 are recognized at 263-282 aa, which is consistent with the reported 265-280 aa epitopes. Conserved analysis revealed 20-39 aa is a high conservation of the epitopes in the ASFV genotypes. Moreover, a blocking ELISA assay for detection ASFV antibody based on 4C4 monoclonal antibody was developed and assessed. The receiver-operating characteristic (ROC) was performed to identify the best threshold value using 87 negative and 67 positive samples. The established test exhibited an area under the curve (AUC) of 0.9997, with a 95% confidence interval ranging from 99.87 to 100%. Furthermore, the test achieved a diagnostic sensitivity of 100% (with a 95% confidence interval of 95.72 to 100%) and a specificity of 98.51% (with a 95% confidence interval of 92.02 to 99.92%) when the threshold was set at 41.97%. The inter- and intra-batch coefficient of variation were below 10%, demonstrating the exceptional repeatability of the method. This method can detect the positive standard serum at a dilution as high as 1:512. Subsequently, an exceptional blocking ELISA assay was established with high diagnostic sensitivity and specificity, providing a novel tool for detecting ASFV antibodies. KEY POINTS: • Four strains of ASFV monoclonal antibodies against p72 were prepared and their epitopes were identified. • Blocking ELISA method was established based on monoclonal antibody 4C4 with an identified conservative epitope. • The established blocking ELISA method has a good effect on the detection of ASFV antibody.

Keywords: African swine fever virus; ELISA; Epitope mapping; Monoclonal antibodies; P72.

Fig. 1

Characterization of rp72 protein and mAbs. A An analysis was conducted on the recombinant rp72 protein using SDS-PAGE. The 81 kD recombinant protein is visible. B Western blot analysis of recombinant rp72 protein with ASFV positive serum. The 81 kD recombinant protein is visible. C Indirect ELISA detection of hybridoma cell supernatant. Purified rp72 was applied to coat the plate, followed by incubation with the hybridoma supernatants as the primary antibody and HRP-conjugated goat anti-mouse IgG as the secondary antibody. The negative control consisted of SP2/0 cell supernatant. D The IFA method was used to analyze the reactivity of 4C4 mAbs. At 48 h post-infection, the cells were immobilized. Hybridoma supernatants were used as the primary antibody, while FITC-conjugated goat anti-mouse IgG served as the secondary antibody during cell incubation. The nucleus of PAM cells is stained blue by DAPI

Fig. 2

Schematic representation of p72 fragments used for epitope mapping. The truncated fragment diagram of the antibody recognition epitope is gradually located, and the lower peptide is truncated from the upper peptide, in which the truncated peptide recognized by the monoclonal antibody is marked red

Fig. 3

Preliminary mapping of mAb epitopes. A–D Preliminary mapping of the 4C4, 5A1, 5E10, and 8A9 epitope with western blot. In the initial mapping, the complete rp72 protein was shortened to peptides A1 (1–500 aa), A2 (20–303 aa), and A3 (430–647 aa). A1, A2, and A3 were produced as MBP-fused proteins in E. coli. E–H To further map the 4C4, 5A1, 5E10, and 8A9 epitopes, western blotting was conducted. A2 was truncated into peptides B1 (20–150 aa), B2 (100–220 aa), and B3 (170–303 aa) for the mapping process. MBP-fused proteins of B1, B2, and B3 were then expressed in E. coli. M: protein weight marker for molecular

Fig. 4

Precise mapping of the mAb epitope. A, B The initial stage of accurate epitope mapping for 4C4 and 5A1 was conducted using western blotting. We divided 20–99 aa into three overlapping peptides, C1 (20–59 aa), C2 (40–79 aa), and C3 (60–99 aa). C, D The western blot technique was used for the second round of accurate epitope mapping of 4C4 and 5A1. The unique region (20–39 aa) of C1 was divided into three overlapping peptides, C4 (20–29 aa), C5 (25–34 aa), and C6 (30–39 aa). E, F The precise epitope identification of 5E10 and 8A9. We divided 221–303 aa into three overlapping peptides, D1 (221–262 aa), D2 (242–282 aa), and D3 (263–303 aa). The peptides C1-C6 and D1-D3 were inserted into the pMAL-c2x plasmid and produced as MBP-fused proteins in E. coli. The shortened peptides were identified using the corresponding monoclonal antibody. M: protein weight marker for molecular

Fig. 5

Sequence alignment of p72 proteins of different strains. The p72 sequences of 42 ASFV strains were aligned with GeneDoc. Black dots represent matching residues, while dashed lines indicate gap regions. The alignment specifies the amino acid’s coordinate on the top and right terminus for each sequence. Epitope regionsof 20–39 aa recognized by 4C4 are circled with red

Fig. 6

ASFV p72-based blocking ELISA analysis of serum samples. The analysis was performed on known ASFV-negative samples (n = 86) and known ASFV-positive samples (n = 67). A The test’s area under the curve (AUC) was 0.9997, as determined by a ROC analysis of blocking ELISA results. B Display a dynamic dot plot diagram illustrating the serum sample’s blocking value with a cut-off value of 41.97

Fig. 7

Evaluation of blocking ELISA assay. A Specificity analysis of the blocking ELISA. B The analytical sensitivity analysis of the blocking ELISA. C The analytical sensitivity analysis of the commercial kit. D Serum antibody response kinetics in pigs infected with ASFV. Serum samples were collected from six pigs infected by ASFV at 0, 7, 14, 21, 28, and 35 days post-inoculation. The dashed line indicates the threshold for blocking ELISA