Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen

Figure 1. The process of tag-free p15 protein preparation. ITC, inverse transition cycling; ELP, Elastin-like polypeptide; TEVp, tobacco etch virus protease. The figure was drawn using the online software Figdraw (https://www.figdraw.com/, accessed on 5 June 2023).

Figure 2. (A) The schematic structure of pET-ELP-p15 expression vector. ENLYFQ/G indicates the TEVp recognition site; (B) SDS-PAGE analysis of the expression of ELP-p15 fusion protein. Lane 1: uninduced pET-ELP-p15 recombinant E. coli cell lysate; Lane 2: IPTG-induced E. coli cell lysate; Lane 3: soluble fraction of IPTG-induced E. coli cell lysate; Lane 4: insoluble fraction of IPTG-induced E. coli cell lysate.

Figure 3. (A) Purification of ELP-p15 fusion protein under different ITC temperatures; (B) purification of ELP-p15 fusion protein using ITC with different NaCl concentrations. ITC, inverse transition cycling.

Figure 4. (A) SDS-PAGE analysis for the cleavage of ELP-p15 fusion protein. Lane 1: clarified E. coli cell lysate before ITC purification; Lane 2: ELP-p15 purified using ITC; Lane 3: p15 cleaved from ELP-p15 using ELK16-TEVp; (B) Western blot analysis of p15 protein using the ASFV-positive serum. ITC, inverse transition cycling.

Figure 5. (A) Optimization of coating buffer. The 0.05 M carbonate (pH 9.6) was selected as the best coating buffer; (B) optimization of blocking buffer. The 5% skim milk in PBS was selected as the optimal blocking buffer; (C) determination of secondary antibody concentration. A dilution of 1:10,000 was selected as the best secondary antibody concentration.

Figure 6. (A) Determination of the cut-off value using ASFV-negative serum samples; (B) specificity of p15-iELISA for ASFV antibody detection; (C) sensitivity test of p15-iELISA.