Dual-antigen immunization strategy for nanobody screening against canine parvovirus and canine distemper virus and development of a CPV-targeted competitive ELISA for Giant panda.

Canine parvovirus (CPV) and canine distemper virus (CDV) are major pathogens causing severe diseases in giant pandas and other carnivores. Serological monitoring of host antibody responses is critical for disease surveillance. Here, we constructed a high-quality nanobody library by immunizing Bactrian camels with recombinant CPV VP2 and CDV N proteins, generating a "dual-antigen immunized" library with a capacity of 7 × 109 pfu/mL and > 90 % positivity. Through phage display, we isolated five CPV-specific and three CDV-specific nanobodies. Five CPV nanobodies were expressed as nanobody-HRP fusion proteins, with Nb10-HRP showing superior performance in a competitive ELISA developed for anti-CPV antibody detection. This assay demonstrated high sensitivity (detecting antibodies at serum dilutions up to 1:1000), excellent specificity (no cross-reactivity), a cut-off value of 23 %, and good reproducibility. Testing 159 giant panda and 42 stray dog serum samples revealed no CPV infection in pandas, while a 12 % positivity in dogs indicated circulation of CPV, posing potential exposure risks. Although this study primarily developed a serological method for anti-CPV detection, the successful creation of a diverse nanobody library via simultaneous dual-antigen immunization highlights a cost-effective strategy that minimizes animal use and facilitates multi-pathogen antibody screening, laying a foundation for future CDV and other serological assays.