Detection of African swine fever virus antibodies in serum using a pB602L protein-based indirect ELISA

Figure 1. Expression and purification of the pB602L protein. (A) Prediction of the full-length transmembrane region of pB602L protein (B) Expression of pB602L protein in vector pCold I. (C) Protein marker, 1- transformed cells of BL21/pCold after IPTG induction for 16 h, 2- whole bacteria without induction, 3- BL21/pCold-B602L whole bacteria after IPTG induction for 16 h, 4-BL21/pCold-B602L supernatant after induction at 16°C, 5-BL21/pCold-B602L precipitation after IPTG induction for 16 h, 6-Purification of recombinant pB602L protein flow-through by Ni+ spin column affinity chromatography, 7-Ni+ spin column affinity chromatography purification of recombinant protein pB602L 40mM imidazole washing solution, 8, 9-purified recombinant pB602L protein by affinity chromatography of Ni+ spin column, 10-Western blot analysis of p602L protein using ASFV-positive pig serum, 11-Western blot analysis of p602L protein using anti-His. A prominent band with the expected size 65 kDa appeared after incubation.

Figure 2. Optimization of pB602l-ELISA method conditions. (A) Optimization of the type of pB602L-ELISA blocking solution and working conditions. The best blocking solution was 2% BSA in PBST, and optimal blocking conditions are 2 h at 37°C. (B) Determination of serum action conditions. The optimal reaction time for serum was 1 h at 37 °C. (C) Determination of TMB solution action conditions. The optimal reaction time for TMB solution was 15 min at 37°C.

Figure 3. Sensitivity and specificity of the pB602L-ELISA method. (A) Determination of the cut-off value of pB602L-ELISA (OD450 = 0.340). Distributions of OD values determined for ASFV-negative (n = 64) serum samples using pB602L-ELISA. The average value (AV) of the negative sera was calculated, and the cutoff value was set at 0.340 (AV+3×SD). (B) The specificity test of the pB602L-ELISA method. ASFV-positive serum, ASFV-negative serum and PRRSV, JEV, GETV, CSFV, PCV2, PPV, PRV antibody-positive swine serum were measured using the pB602L-ELISA, and the OD450 values with all antisera except the ASFV-positive serum were lower than the cutoff value (0.340). (C) Sensitivity of the pB602L-ELISA method. Two-fold serial dilutions from 1:100 to 1:204,800 of ASFV-positive serum were tested by pB620L-ELISA method, and the highest dilution that could be detected using the cutoff value (0.340) was found to be 1:6,400, ***p < 0.001. Each dot represents a serum sample, and the dashed line indicates the cutoff value of the pB602L-ELISA.