Development and Standardization of Indirect ELISA for African Swine Fever Virus Using Recombinant p30 Protein Produced in Prokaryotic System

Figure 1. (A) Amplification of the CP204L gene, with a size of 528 bp (M—marker; 1—amplicon CP204L). (B) Expression vector pET-SUMO-rp30, with the insert corresponding to the CP204L gene (ASFV-p30) and primers at the corresponding positions for targeting analysis. (C) Targeting analysis of the CP204L gene in the expression vector (M—marker; 1/2—blank control; 3/5—samples of the expression vector with the insert).

Figure 2. Analysis of rp30 protein expression in the BL21/DE3 expression system by SDS-PAGE (A). Protein specificity was determined by Western blot (B) using 6X His-tag antibody (M—marker; 1—negative [Untransformed BL21/DE3]; 2—positive [BL21—CAT]; 3/6—BL21-ASFV-p30). SDS-PAGE and Western blot analysis of IMAC-purified rp30 protein (C) (1—CI; 2—unbound; 3—washed; 4/7—specific expression bands of the rp30 protein in IMAC elutions).

Figure 3. Humoral evaluation of rp30 in CF-1 strain mice. * Statistically significant differences were considered when p < 0.05 between group 2 (rp30) and group 3 (rp30 + Matrix-MTM).

Figure 4. Evaluation of the optimal concentration of antigen (A) and serum (B) for iELISA standardization. Optimal conditions were considered when 50 ng was used for each plate and 1:100 dilutions were used for the sera evaluated. Statistically significant differences were considered at * p ≤ 0.05.

Figure 5. Serological distribution of positive and negative reference sera evaluated in iELISA with rp30 (A), with a cut-off point of 0.432. The 2 × 2 contingency table (B) for the analysis of 49 sera (24 positive and 25 negative) to determine sensitivity, specificity and κ.

Figure 6. Indirect immunoperoxidase test (IPT) as a confirmatory test for ASF-negative (A) and -positive (B) serum samples.