Engineering a high-affinity multiple antigenic peptide dendrimer for group-specific detection of bluetongue virus antibodies targeting the VP7 epitope.

Bluetongue disease remains a global economic threat, necessitating the development of rapid, high-precision diagnostics. The genome of the Bluetongue virus (BTV) encodes seven structural proteins, VP1-VP7. Among these proteins, VP7 is the major immunodominant structural protein conserved across BTV serotypes and is therefore targeted for the development of serogroup-specific immunodiagnostic assays. This study aimed to identify an immunodominant VP7 epitope recognized by BTV-specific monoclonal antibodies (MAbs) and to evaluate its utility in a peptide-based ELISA. The gene encoding VP7 protein were expressed in three overlapping fragments in a prokaryotic system and analyzed for its reactivity with the two MAbs by Western blot and indirect ELISA. By dissecting the VP7 gene into overlapping recombinant fragments, we identified the binding domain is located in Fragment II (27 kDa) region. However, linear monomeric peptides designed from this region failed to replicate the protein's native antigenicity and did not show any reactivity with the MAbs. To overcome this, we designed a chimeric peptide bridging the junction of Fragments II and III and harnessed Multiple Antigenic Peptide (MAP) technology to present the epitope in a four-armed dendrimeric scaffold. An indirect ELISA based on the MAP antigen was subsequently evaluated using field serum which demonstrated diagnostic sensitivity of 92.3% and diagnostic specificity of 98.2%. The findings demonstrate the utility of combining epitope mapping, in silico analysis, and MAP technology for the development of safe, standardized, and cost-effective peptide-based immunodiagnostic assays for bluetongue surveillance.