Preparation of Monoclonal Antibodies against the Viral p54 Protein and a Blocking ELISA for Detection of the Antibody against African Swine Fever Virus

Figure 1. Strategy of the truncated recombinant p54 (rp54) expression.

Figure 2. Analysis of rp54 protein. (a). SDS-PAGE analysis of His-tagged rp54 protein expression, followed by Coomassie brilliant blue stain. M is protein ladder, lane 1 was supernatants of bacterial cell lysates after induction with IPTG, lane 2 was precipitates of bacterial cell lysates after induction with IPTG, lane 3 was whole bacterial cell lysates after induction with IPTG, lane 4 was whole bacterial cell lysates with no induction with IPTG, lane 5 was negative control with pET-28a vector, lane 6 was the purified rp54 protein. (b). Western blot analysis of rp54 protein with anti-His tag antibody. (c). Western blot analysis of rp54 protein with ASFV-positive serum.

Figure 3. The reactivity of monoclonal antibodies. (a). Western blot analysis of anti-rp54 antibodies. The seven anti-rp54 monoclonal antibodies specifically reacted with recombinant protein at 20 kDa. (b). IFA performed on ASFV-infected PAMs. Cells were incubated with rp54-specific mAbs annotated on the top left of each panel and stained with Coralite488-conjugated Affinipure Goat Anti-Mouse IgG(H+L). Scale bars, 20 μm.

Figure 4. Antigen epitope analysis. (a). Strategy for the identification of the epitopes within rp54 protein and reactivity between the truncated rp54 protein fragments and the seven mAbs. +: interaction; −: no interaction. (b). Western blot analysis of the reactivity of the truncated rp54 protein fragments and the seven mAbs.

Figure 5. Sequence conservation analysis of the identified epitopes among different ASFV genotypes.

Figure 6. Assessment of rp54 mAbs for application in blocking ELISA against ASFV antibodies. The percent of inhibition of different mAbs against strong-, medium-, weak-positive (S1, S2 and S3, respectively) and negative serum (N1) samples were determined.

Figure 7. Serum sample analysis by 6A5, 6F9 and 6A5/6F9-based blocking ELISAs. 61 ASFV-positive serum samples and 144 ASFV-negative serum samples were analyzed by the three blocking ELISAs. (a). ROC analysis of the three blocking ELISAs. The AUC was 0.802, 0.968 and 0.986 for 6A5, 6F9 and 6A5/6F9-based blocking ELISAs, respectively. (b). PI values of each pig serum sample, labelled with indicated cut-off values, sensitivity and specificity.

Figure 8. Specificity and analytical sensitivity assay. (a). Percent of inhibition of the polyclonal anti-sera against various porcine viruses analyzed by 6A5/6F9-based blocking ELISA. Only ASFV-positive pig serum showed a higher PI value than the cut-off value. (b). Two-fold serially diluted ASFV-positive sera (Red) and ASFV-negative sera (Black) ranging from 1:2 to 1:128 were detected by the blocking ELISA. Cut-off value was marked with a dashed line.