A recombinant full-length VP2-2b-based ELISA for evaluating immunoprotection against canine parvovirus-2: Expression,...

Canine parvovirus-2 remains a major threat to canine health, with maternal antibody interference and limitations of traditional diagnostic methods posing challenges to effective disease control. Maternal-derived antibodies can persist at levels that interfere with the development of active immunity following vaccination. Therefore, accurate detection of existing antibody levels prior to vaccination is essential to ensure induction of protective immunity. Hemagglutination inhibition tests are labor-intensive and highly susceptible to external variability, emphasizing the need for reliable and cost-effective alternatives. In this study, we developed and optimized an indirect ELISA using a full-length, recombinant VP2-2b protein expressed in E. coli to detect CPV-2 antibodies. To our knowledge, this is the first ELISA developed around a soluble full-length CPV-2b protein, with comprehensive optimization and clinical validation. VP2-2b was selected for its unique bidirectional neutralization kinetics, enhancing the diagnostic accuracy of CPV-2 serology. The ELISA demonstrated excellent sensitivity and specificity, effectively distinguishing between seropositive and seronegative samples. A gray zone (OD450 = 0.26-0.30) was defined to represent borderline antibody titers, where retesting is recommended to determine accurate vaccination timing. Optimal assay conditions were established as 7.5 μg antigen coating concentration and 1:300 serum dilution, yielding a high signal-to-noise ratio (SNR) of 5.6. Validation against HI tests showed strong correlation with minimal non-specific binding. This ELISA system provides a reliable and economical alternative to traditional methods, helping overcome the limitations of the HI test in field settings and supporting informed decisions in vaccination planning.