Comparative evaluation of recombinant A4L, A27L, and A36R proteins for ELISA-based serodiagnosis of lumpy skin disease virus.

Lumpy skin disease (LSD) is a transboundary poxviral disease of cattle and buffalo that has been economically significant in various parts of Africa, the Middle East, Asia and some parts of Europe and therefore requires the development of reliable, high throughput diagnostic tools to support effective surveillance and control. Conventional serological assays are labor intensive, time consuming and involve handling of live virus, which makes them unsuitable for large-scale surveillance. Recombinant antigen based ELISAs offer a safer, scalable and high-throughput alternative; however, identifying optimal diagnostic antigens remains a challenge. In this study, three representative immunogenic proteins of different structural components of the virion of the lumpy skin disease virus (LSDV) were selected for indirect ELISA to detect antibodies against LSDV: A4L (conserved core protein), A27L (intracellular mature virion) and A36R (extracellular enveloped virion). The corresponding genes were cloned and expressed in E. coli using the pET-33b(+) system. Recombinant proteins were purified using Ni-NTA affinity chromatography, and their expression and immunoreactivity were confirmed by SDS-PAGE and western blotting using anti-His antibodies and anti-LSDV sera. Purified proteins were used to optimize indirect ELISAs, and their diagnostic performances were comparatively evaluated. All three ELISAs demonstrated high analytical specificity, with no detectable cross-reactivity against sera positive for other viral infections. The A4L-based ELISA had the highest diagnostic sensitivity and specificity (93% and 94%, respectively) among the antigens evaluated. The diagnostic sensitivity and specificity of the A27L-based ELISA compared to the A36R-based ELISA were 89% and 88%, respectively, while the A36R-based ELISA had a sensitivity of 90% and specificity of 86%. These findings indicate that the highly conserved and abundantly expressed core protein A4L is a more suitable serodiagnostic antigen than the evaluated envelope-associated proteins. Overall, among the evaluated antigens, A4L-based indirect ELISA showed the better potential for serodiagnosis and large-scale serosurveillance of LSDV. Further large-scale field validation is required to confirm the diagnostic robustness and applicability of the A4L-based indirect ELISA for routine LSDV serosurveillance.