Expression of Recombinant Proteins for the Development of a c-ELISA Kit for the Serological Diagnosis of African Horse Sickness

African horse sickness (AHS) is a devastating arboviral disease of equids with mortality rates reaching 95% in susceptible horses. Reliable serological diagnostic tools are essential for surveillance, outbreak management, and facilitating international equine trade. This study aimed to develop a competitive enzyme-linked immunosorbent assay (c-ELISA) using a recombinant VP7 protein for the serological diagnosis of African horse sickness virus (AHSV). The VP7 core protein was selected as the diagnostic antigen due to its high conservation across all nine AHSV serotypes and group-specific antigenic properties. Two variants of recombinant VP7 were evaluated: wild-type VP7 (WT-VP7) and a solubilityenhanced variant with a leucine to arginine substitution at position 345 (VP7-L345R). Both were expressed using a baculovirus expression system in Sf9 insect cells. A comprehensive optimization study identified extremely low multiplicity of infection (MOI 0.001) combined with extended culture periods (120 hours post-infection) as optimal for VP7-L345R expression, yielding approximately 10 mg of purified protein from 0.75 L culture at 1.5 × 10^6 cells/mL. The recombinant VP7-L345R demonstrated enhanced solubility with substantial secretion into culture supernatant, facilitating simplified purification via immobilized metal affinity chromatography, while preserving essential antigenic epitopes. A panel of five monoclonal antibodies (Mabs) against VP7 was characterized through immunoblotting and indirect ELISA. All recognized the recombinant VP7-L345R, confirming that the solubility-enhancing mutation did not compromise critical antigenic determinants. Systematic evaluation in a competitive format identified Mab 5D4F7 as optimal for c-ELISA development due to its superior dynamic range, discriminatory capacity, and reproducibility. The finalized assay utilized microplates coated with 1 μg/mL of purified VP7-L345R and Mab 5D4F7 at a 1:60,000 dilution. This study has successfully produced and characterized the key reagents necessary for developing a VP7-based c-ELISA for AHSV diagnosis. The solubility-enhanced VP7-L345R antigen, combined with characterized monoclonal antibodies, provides a robust foundation for serological detection of AHSV antibodies. These optimized reagents address an important need for enhanced surveillance capabilities in the face of changing global patterns of vector distribution and equine movement. Further validation studies are required to establish the full diagnostic performance of this assay under field conditions.