A Thymidine Kinase recombinant protein-based ELISA for detecting antibodies to Duck Plague Virus

Abstract

Background: Duck plague virus (DPV) is the causative agent of Duck Plague (DP) that causes significant morbidity and mortality throughout duck-producing areas of the world. The diagnosis of DP currently relies on the use of live or inactivated whole DPV virion as antigens in ELISA, but it is too laborious and expensive for routine application, and it is still difficult to get purified DPV virion with current technology. Results: In this study, we describe the expression and purification of a recombinant Thymidine Kinase (TK) protein which makes antigen in an in-house developed, optimized and standardized ELISA. The specificity of the optimized TKELISA was evaluated by antisera against Duck Plague Virus (DPV), Duck Hepatitis B Virus (DHBV), Duck Hepatitis Virus (DHV), Riemerella Anatipestifer(R. A), Escherichia coli (E. coli) and Salmonella anatum (S. anatum). Only antisera against DPV yielded a specific and strong signal. In order to determine the sensitivity of the TK-ELISA, a panel of diluted sera was tested, and the minimum detection limit of 1:2560 (OD450 nm = 0.401) was obtained according to the endpoint cut-off (0.2438). The repeatability and reproducibility under the experimental conditions demonstrates a low variability (P > 0.05). The suspected sera samples (n = 30) were determined by TK-ELISA and the positive rate is 90% (27/30), and the TK-ELISA showed 83.33% (22+3/30) coincidence rate with the Serum Neutralization Test (SNT) and 90% (24+3/30) coincidence rate with the whole DPV virion based-ELISA (DPV-ELISA). When defining the dynamics of antibody response to attenuated live DPV vaccine, the maximum antibodies is reached after 4 weeks. Conclusions: The results suggest that the TK-ELISA provides high specificity, sensitivity, repeatability and reproducibility for detection of anti-DPV antibodies in duck sera, and has the potential to be much simpler than DPVELISA and SNT for the sera epidemiological investigation.