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

Abstract

BackgroundDuck 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.ResultsIn 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 TK-ELISA 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.ConclusionsThe 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 DPV-ELISA and SNT for the sera epidemiological investigation.

Duck virus enteritis (duck plague) – a comprehensive update

“…A TK(thymidine kinase)-ELISA using thymidine kinase recombinant protein as an antigen has been developed for the detection of serum antibodies specific to DPV which could be highly useful for the epidemiological survey of DP. TK-ELISA has been reported to be highly specific as well as sensitive and showed good repeatability and reproducibility when evaluated against DPV, duck Hepatitis B virus, duck Hepatitis virus, R. anatipestifer, E. coli and Salmonella Anatum antisera, as strong positive signals were evident only against DPV anti-sera (Wen et al 2010). The DPV glycoprotein C, encoded by the DPV UL44 gene, has been suggested to be a suitable candidate for developing new diagnostics and vaccine against DPV (Sun et al 2014).…”

Evaluation of recombinant outer membrane protein C based indirect enzyme-linked immunoassay for the detection of Salmonella antibodies in poultry

“…The recombinant protein antigen can be prepared without involving the risk of handling the zoonotic microbe; hence it is non-infectious, safe and can be used without containment facilities [ 20 ]. Moreover, recombinant protein improves the specificity of the ELISA [ 21 ]. This enhanced specificity is especially important for the control and eradication programs in developing countries.…”

Duck Enteritis Virus VP16 Antagonizes IFN-β-Mediated Antiviral Innate Immunity

“…Duck enteritis virus (DEV) is a kind of enveloped, large DNA virus who belongs to α-herpesvirus. Most of the research about DEV mainly focus on etiology [1][2][3][4][5][6][7][8], pathogenesis [9][10][11][12][13], epidemiology, and diagnosis [14][15][16][17][18][19][20][21][22][23]. All of the waterfowls are apt to suffer from this disease which could cause considerable mortality and morbidity [24][25][26].…”