A dual gC-gD glycoprotein-based indirect ELISA for enhanced detection of anatid herpesvirus 1 antibodies

Anatid herpesvirus 1 (AnHV-1) is still a significant limiting factor in the waterfowl industry, and there is a clear demand for field-applicable serodiagnostic assays that are robust, sensitive and reproducible. In this report, we designed and conducted a comprehensive comparative evaluation of whole virus or recombinant glycoprotein C (gC), glycoprotein D (gD) alone or a mixture of gC-gD ELISA, to determine which one had the better diagnostic performance. The recombinant proteins were produced, purified, and optimized for use in indirect ELISA, and subsequent validation was carried out using a set of 120 field serum samples. Receiver operating characteristic (ROC) curve analysis demonstrated that the combined gC-gD ELISA exhibited an almost perfect diagnostic performance (AUC =   0.9925), and high sensitivity (93%) and specificity (95%), surpassing those of two antigen-based assays and the whole virus assay. Although both gC ELISA (high sensitivity 95% but moderate specificity 80%) and gD ELISA (outstanding sensitivity 99%, but lower overall accuracy: AUC  =  0.9150) performed well, the sensitivity of the whole virus ELISA was lower (87%) with marked variation. In particular, intra-assay precision analysis showed a significant decrease in the coefficient of variation for the gC-gD ELISA (∼4%) compared with the gD (∼15%), gC (∼20%) and whole virus (∼40%) formats, indicating the highest reproducibility of the gC-gD ELISA. Taken together, these results demonstrate that the gC-gD ELISA provides a novel and robust tool for high-throughput AnHV-1 serosurveillance, which represents a major breakthrough in terms of economy when compared to traditional whole virus-based ELISAs for large field applications.