Development of a dual-label time-resolved fluorescence immunoassay platform for simultaneous detection of canine distemper virus and parvovirus.

Abstract

OBJECTIVE: Canine distemper virus (CDV) and canine parvovirus type 2 (CPV-2) are two highly contagious and severely pathogenic viruses commonly found in dogs, particularly lethal to puppies. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CDV and CPV-2 infection. METHODS: A sandwich TRFIA method was established and optimized using europium(III) (Eu)/samarium(III) (Sm) chelates dual labeling. Subsequently, it was formulated into a kit and constructed the TRFIA platform, and both its laboratory performance (limit of detection, specificity, accuracy and stability) and its ability to detect target viruses in canine clinical samples were evaluated. RESULTS: A dual-label TRFIA platform for simultaneous CDV and CPV-2 detection was constructed and rigorously validated. The TRFIA platform exhibited limit of detection of 0.43&#x202f;ng/mL for CDV and 0.73&#x202f;ng/mL for CPV-2, with high specificity for both targets. Clinical cut-offs were established at 5.47&#x202f;ng/mL (CDV) and 6.96&#x202f;ng/mL (CPV-2). Intra-assay coefficients of variation ranged from 1.11&#x202f;% to 5.53&#x202f;%, with spike-recoveries between 104.74&#x202f;% and 108.50&#x202f;%. Concordance with PCR was excellent (Pearson's &#x3c7;&#xb2; test, P&#x202f;<&#x202f;0.001). Clinical validation yielded diagnostic sensitivities/specificities of 90.24&#x202f;%/97.37&#x202f;% for CDV and 88.37&#x202f;%/98.65&#x202f;% for CPV-2, respectively. CONCLUSION: A TRFIA platform for simultaneous detection of CDV and CPV-2 demonstrated robust limit of detection, specificity, accuracy, plus reliable clinical sensitivity and specificity. It offers an additional tool for distinguishing CDV from CPV-2 infections and may enhance future disease-prevention strategies.