Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses

Abstract:

Abstract:［Objective］ To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses，we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli． ［Methods］We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector，designated as pET-1VP3- 3VP1．The fusion protein DHAV- 1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay．［Results］DHAV-1VP3-3VP1 fusion protein expressed in BL21 ( DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG)．The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay．The optimal working concentration for coating antigen was 1.0 μg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650≥0.38．［Conclusion］The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.