Development Of A VP3-ELISA For The Detection Of Goose And Muscovy Duck Parvovirus Antibodies

The muscovy ducklings were usually infected by Goose Parvovirus and Muscovy duck parvovirus, which both belong to the member of Parvoviridae. Several diagnostic methods have been employed for the detection of both GPV or MDPV antigen and their antibodies, including agar gel precipitation, virus neutralization tests, Western blotting assays, virus antigen-based enzyme-linked immunosorbent assays (ELISA), a plaque neutralization assay, an indirect ?uorescent antibody test, and polymerase chain reaction (PCR) for the rapid detection of GPV DNA. Virus antigen-based serologic tests have been reported during screenings for GPV infection, but whole-virus purification requires the propagation of large quantities of virus in eukaryotic systems and depends on difficult and expensive processes. Currently, a rapid and simple diagnosis is required to detect highly contagious GPV or MDPV infection. Parent ?ocks could be monitored for antibody titers after vaccination against GPV or MDPV to determine whether titers are high enough for the protection of offspring from infection. The cost of E. coil-expressed proteins is relatively low and the recombinant protein is easy to purify by either chromatography or elution. Many reports have demonstrated that E. coli-expressed proteins are useful antigens for detecting antibodies against a variety of viral diseases.The VP3 protein is the most abundant of three core proteins and can induce neutralizing antibodies in GPV- or MDPV-infected waterfowl. Therefore, VP3 may be a suitable candidate for the detection of GPV-or MDPV-specific antibodies. In this study, a VP3-ELISA test was developed to diagnose GPV and MDPV infection and to monitor serum antibody titers against GPV or MDPV.Western blot analysis showed that the purified recombinant protein reacted specifically with GPV and MDPV positive serum. Indirect-enzyme-linked immunosorbent assay (Indirect-ELISA) was developed for the detection of antibodies against Parvovirus in the muscovy duck using recombinant GPV VP3 gene prokaryotic expression product as coating antigen. The suitable concentration of incubating antigen and detected serum were 5μg/ml and 1:160, respectively. And the working concentration of goat anti-duck IgG-HRP antibody was 1:400. The standard of estimating positive was OD450 nm≥0.34.There was no cross reaction with positive serum of DHV/DRV, and the variant coefficients of less than 10% in the intro-batch and inter-batch tests indicated that the assay had good inter-plate repeatability and intra-plate reproducibility. The results of 50 sera sample detected in VP3-ELISA assay were consistent with that in serum neutralization, which was 84% in concordance. The result of stability test revealed that they were stable after they were stored in the refrigerator of 4℃, -20℃and -70℃, for 9 months, respectively, and the way in 4℃refrigerator was better comparatively. Therefore, it provides a basis for the study of bird parvovirus diagnosis kit.