Development of a Gag‐Based Indirect ELISA and Its Application for Serological Surveillance of Antibodies Against Small Ruminant Lentiviruses (SRLVs)

Small ruminant lentiviruses (SRLVs) cause lifelong persistent infections in sheep and goats, impairing productivity and restricting livestock trade. Serological enzyme‐linked immunosorbent assay (ELISA) testing is the primary method for SRLV epidemiological monitoring, and most commercial ELISAs use capsid (CA), envelope, or a combination of both proteins as antigens. We previously developed a CA‐based indirect ELISA (iELISA) to detect SRLV antibodies. Here, we expressed full‐length recombinant SRLV Gag protein and further developed an iELISA using Gag as the coating antigen. The cut‐off value of the established Gag‐based iELISA was determined to be 0.093 based on sample‐to‐positive (S/P) ratio analysis of 100 SRLV‐negative sera. Parallel testing of 228 sera using the Gag‐iELISA, CA‐iELISA, and a commercial ELISA showed that the positive detection rate of the Gag‐iELISA was 35.96 percentage points higher than that of the CA‐iELISA and 4.39 percentage points higher than that of the commercial ELISA. All samples that tested positive by either the CA‐iELISA or the commercial ELISA were also positive by the Gag‐iELISA. Samples testing positive by the Gag‐iELISA but negative by the CA‐iELISA or the commercial ELISA were confirmed as SRLV antibody‐positive by Western blot (WB). Concordance analysis of the Gag‐iELISA and commercial ELISA showed 95.61% overall agreement. Endpoint dilution assays demonstrated that the Gag‐iELISA showed twice the analytical sensitivity of the CA‐iELISA and sensitivity comparable to that of the commercial ELISA. Furthermore, testing of 761 ovine sera from Hulunbuir during 2024–2025 revealed an overall SRLV seropositivity rate of 54.80%, with regional positive rates ranging from 0.00% to 84.44%. Collectively, this Gag‐based iELISA is a sensitive and practical tool for large‐scale SRLV serological surveillance.