High-affinity chicken scFv antibodies recognize recombinant VP1-CDEF' protein and native virions of feline calicivirus genotype II strain LZ2023.

Feline calicivirus (FCV) is a significant pathogen responsible for upper respiratory tract disease and oral ulceration in felids. Genetic and antigenic variation in circulating FCV strains limits the utility of current diagnostic assays, and no validated genotype II-specific antibodies exist for rapid test development. In this study, we targeted the C, D, E, and partial F regions of the major capsid protein VP1 (termed VP1-CDEF'). The recombinant VP1-CDEF' protein was expressed in a prokaryotic system and used to immunize SPF chickens. A chicken-derived phage display single-chain variable fragment (scFv) library was successfully constructed, with a capacity of 5.25 × 107 transformants and an insert-positive rate of approximately 80%. After three rounds of solid-phase biopanning, the phage recovery ratio increased from 3.6 × 10-6 to 6.4 × 10-3, representing an enrichment factor of 1,777.78-fold. Phage-ELISA screening of 184 randomly selected clones yielded 25 positive clones, and sequence analysis identified five scFv antibodies with unique amino acid sequences, designated 1A2, 2B2, 2C1, 2E1, and 2G5. The scFv genes were constructed into scFv-Fc fusion proteins and expressed in Expi293F cells. Western blot analysis demonstrated that all five scFv-Fc fusion proteins specifically recognized the recombinant VP1-CDEF' protein and exhibited no cross-reactivity with feline parvovirus VP2 protein. In indirect immunofluorescence assay (IFA)-based binding assays, however, only 2B2-Fc, 2E1-Fc, and 2G5-Fc were capable of binding genotype II FCV strain LZ2023 in infected F81 cells. Virion capture ELISA using purified intact FCV particles confirmed that 2B2-Fc, 2E1-Fc, and 2G5-Fc bind to surface-accessible epitopes on native virions, whereas 1A2-Fc and 2C1-Fc showed no detectable binding. Biolayer interferometry (BLI) analysis showed that 2B2-Fc and 2E1-Fc exhibited the highest binding affinities, with equilibrium dissociation constant (KD) values of 0.046 nmol/L and 0.032 nmol/L, respectively, whereas 2G5-Fc displayed moderate affinity (KD = 65.44 nmol/L). 1A2-Fc and 2C1-Fc showed relatively weak binding (KD > 450 nmol/L). This study reports the generation of chicken-derived scFv antibodies targeting the FCV VP1-CDEF' region. The high-affinity clones 2B2 and 2E1 are promising candidates for rapid diagnostic test development and warrant further evaluation. However, further validation of their diagnostic utility will require clinical sample studies and the development of colloidal gold strips based on these scFv.