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

## TL;DR

This study develops a sensitive and specific ELISA test for African Swine Fever Virus using a recombinant p30 protein, which could improve disease detection and surveillance.

## Contribution

The novel contribution is the development of an indirect ELISA using recombinant p30 protein produced in a prokaryotic system for efficient and economical ASFV antibody detection.

## Key findings

- The recombinant p30 protein showed high immunogenicity in a murine model.
- The developed ELISA had 95.6% sensitivity and 92.3% specificity for detecting ASFV antibodies.
- The assay demonstrated low variability and high reproducibility for potential use in seroepidemiological studies.

## Abstract

African Swine Fever (ASF) is a viral disease in pigs with major impacts on production and the economy. The development of highly sensitive and specific detection tools would enable early identification. Since the p30 protein is highly conserved in the virus, the objective of this study was to produce the p30 protein in a prokaryotic system and develop a highly sensitive and specific indirect ELISA to identify antibodies against ASFV. Our results indicate a good production yield and a good immunogenic response in a murine model. The developed ELISA showed high sensitivity and specificity with a good kappa index using reduced amounts of antigen and low conjugate titers, indicating that the test could be efficient for ASFV detection and economical for future commercialization.

African Swine Fever (ASF), caused by the African Swine Fever Virus (ASFV), is a highly contagious hemorrhagic disease with high mortality (≈100%) in pigs and is considered the most devastating disease to date. Given the importance of this disease, we aimed to assess the use of the recombinant p30 protein as the sole antigen for the development of an accurate and precise ELISA test (iELISA) for the virus. The recombinant p30 protein (rp30) was produced in a bacterial expression system using a SUMO-tagged expression vector. Protein expression was confirmed by Western blot analysis and purified using affinity chromatography. Antigenicity was evaluated in CF-1 mice, which demonstrated the ability to generate high levels of specific antibodies. The rp30 showed a sensitivity of 95.6% when used in the development of iELISA, a specificity of 92.3%, and a kappa index (κ) of 0.836. Furthermore, reference sera (OIE-ASF) were used to validate the assays, and the results demonstrated an excellent capacity to detect ASF antibodies using only the rp30 antigen up to a serum dilution of 1:100. The inter- and intra-assay variability coefficients were 4.27% and 4.85%, respectively, demonstrating that the assay was accurate and reproducible, allowing its use in seroepidemiological analyses for ASF surveillance.