Homologous Double-Antibody Sandwich ELISA Targeting a Linear Epitope of FPV VP2 and Demonstration of Microtubule-Dependent Intracellular Trafficking

Feline panleukopenia (FP) is a highly fatal disease in cats caused by feline panleukopenia virus (FPV). The viral capsid protein VP2 determines immunogenicity and host range. We isolated an FPV strain, ZZ202501, and generated four monoclonal antibodies (mAbs) against VP2 by immunizing mice with purified virus and screening the resulting hybridomas on recombinant VP2 expressed in Escherichia coli, a strategy that biases selection toward linear denaturation-resistant epitopes. One mAb, 4D10, bound a linear denaturation-resistant epitope and gave the highest signal-to-noise ratio among the 16 capture and detection combinations tested, which allowed it to serve as both capture and detection antibody in a double-antibody sandwich ELISA (DAS-ELISA). The assay detected 101.67 TCID50/0.1 mL of virus and did not cross-react with feline calicivirus or feline herpesvirus. Across 136 clinical samples, it showed complete agreement with a conventional PCR assay. Using mAb 4D10, we also followed the intracellular distribution of the total VP2 pool during infection. VP2 underwent a time-dependent cytoplasmic-to-nuclear redistribution that was blocked by nocodazole, an inhibitor of microtubule polymerization, and microtubule disruption reduced progeny virus titer by more than 100-fold without detectable cytotoxicity. These data indicate that microtubule-dependent trafficking contributes to efficient FPV replication. The study provides an analytically characterized DAS-ELISA for FPV antigen detection, together with evidence linking cytoskeletal transport to viral propagation, and illustrates why the choice between linear and conformational epitopes matters in assay design.