In this study, nine monoclonal antibodies (mAbs) against the FCV VP1 protein were successfully prepared using traditional hybridoma technology, and all of them could simultaneously recognize linear and conformational epitopes. Their subtypes mainly included IgG1/kappa, IgG2a/kappa, IgG1/lambda, IgG2b/kappa, IgG2b/lambda, and IgM/kappa. Based on the mAb 8F9 with the highest blocking activity, a blocking enzyme-linked immunosorbent assay (ELISA) method for detecting cat calicivirus antibodies was established. After systematic optimization, the antigen coating concentration was determined to be 1.0 μg/mL, the serum dilution was 1:8, and the dilution of horseradish peroxidase (HRP)-labeled mAb was 1:400. The critical value for detecting cat serum was 23.5%, the sensitivity was 94.9%, and the specificity was 93.02%. There was no cross-reaction with common pathogens such as feline parvovirus and feline mycoplasma. The repeatability was good, with intra-batch and inter-batch coefficients of variation (CV) <10%. Clinical validation showed that the total coincidence rate with the indirect ELISA method recommended in the local standard was 96.19% (kappa = 0.9). The sensitive and specific blocking ELISA method established in this study can provide reliable technical support for FCV antibody screening, vaccine immune evaluation, and specific pathogen-free (SPF) cat breeding.

Keywords: VP1 protein; antibody detection; blocking ELISA; feline calicivirus; monoclonal antibody; vaccine evaluation.