Development of a cost-effective serodiagnosis for African swine fever using solubility-enhanced recombinant p54, p30, and p72.

Background and aim: The rapid spread of African swine fever (ASF) in Indonesia and other Asian countries has devastated domestic and wild pig populations. In the absence of a viable vaccine, ASF control depends on strict biosecurity measures and the prompt culling of infected animals. Accurate and timely detection is therefore essential to limit disease transmission, highlighting the urgent need for reliable diagnostic tools. This study aimed to develop serological assays for ASF virus (ASFV) antibody detection using recombinant ASFV proteins.

Materials and methods: Three key ASFV structural proteins-p30, p54, and p72-were heterologously expressed in Escherichia coli. Protein solubility, particularly for p54, was enhanced by targeted deletion of hydrophobic domains. Recombinant proteins were purified using nickel-nitrilotriacetic acid affinity chromatography and assessed for diagnostic performance through enzyme-linked immunosorbent assay (ELISA) and immunoblotting using 114 field serum samples.

Results: The solubility-optimized p54 antigen was successfully used to develop an indirect ELISA, while the insoluble p30 retained sufficient antigenicity for immunoblot-based detection. The p54-based ELISA showed high diagnostic performance, achieving an area under the curve of 0.936, with 91% sensitivity and 85% specificity. Agreement with a commercial ELISA kit was substantial (Cohen's kappa = 0.635). Immunoblotting confirmed that all recombinant proteins maintained strong antigenicity and diagnostic specificity.

Conclusion: Recombinant ASFV proteins p54 and p30 demonstrated strong potential for serological diagnostics when expressed in E. coli. Notably, this is the first study to report a successful domain truncation strategy for enhancing p54 solubility in E. coli, enabling the development of affordable, locally produced ELISA kits. The p30-based immunoblot assay serves as a confirmatory tool to strengthen ASF detection and outbreak response in resource-limited settings.