Comparative evaluation of lateral flow immunoassay and real time PCR for detection of canine morbillivirus antigen in live attenuated vaccines.

Canine morbillivirus (CDV) vaccine quality control requires rapid and reliable analytical methods to ensure vaccine consistency and compliance with regulatory standards. In this study, a gold nanoparticle-based lateral flow immunoassay (LFA-CDV) was developed and evaluated as a rapid antigen detection platform for modified live virus (MLV) vaccine assessment. A total of 100 CDV vaccine batches were analyzed and compared with multiplex real-time RT-PCR targeting the H gene, which served as an analytical reference method. Gold nanoparticles with a mean diameter of approximately 20 ± 5 nm were synthesized and conjugated with monoclonal antibodies specific to the CDV envelope protein to enable visual immunochromatographic detection. The assay demonstrated high analytical specificity, with no cross-reactivity against tested heterologous viral or bacterial agents and no interference from common substances. Analytical sensitivity testing established a limit of detection of 10 ³ TCID₅₀/0.1 mL, which is compatible with regulatory potency requirements for CDV vaccines. Comparative evaluation showed a sensitivity of 98.84% (95% CI: 93.7-99.9), specificity of 85.71% (95% CI: 57.2-98.2), and overall accuracy of 97.0% (95% CI: 91.5-99.4). Positive and negative predictive values were 97.70% and 92.31%, respectively. Because RT-qPCR served as an analytical comparator, positive percent agreement (PPA) and negative percent agreement (NPA) were also calculated and reached 98.84% and 85.71%, respectively. Overall agreement between methods was 97%, with a Cohen's kappa coefficient of 0.90 (95% CI: 0.78-1.00), indicating almost perfect agreement. These findings demonstrate that the developed LFA-CDV assay is a rapid, specific, and analytically robust tool for vaccine quality assessment. Integration of LFA with molecular methods may provide an efficient complementary approach for vaccine evaluation and batch-release testing.