title: [Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].
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 microg 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 > OR = 0.38.
  
  
  CONCLUSIONS
  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.
authors.forename: Yuyao
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiuli
authors.initials: X
authors.lastname: Ma
authors.forename: Bing
authors.initials: B
authors.lastname: Huang
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Li
authors.forename: Kexiang
authors.initials: K
authors.lastname: Yu
authors.forename: Jianliang
authors.initials: J
authors.lastname: Li
authors.forename: Cunxia
authors.initials: C
authors.lastname: Liu
authors.forename: Hongyu
authors.initials: H
authors.lastname: Han
authors.forename: Yanshun
authors.initials: Y
authors.lastname: Cui
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis* / Q000276:immunology; D004372:Ducks; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004371:Hepatitis Virus, Duck / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006524:Hepatitis, Viral, Animal / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D010850:Picornaviridae Infections / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D011201:Poultry Diseases / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D014764:Viral Proteins