title: Development and evaluation of a DAS-ELISA for rapid detection of Tembusu virus using monoclonal antibodies against the envelope protein.
abstract: Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Quanbin
authors.initials: Q
authors.lastname: Ou
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Xuhui
authors.initials: X
authors.lastname: Gao
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Wu
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Xue
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Chunmei
authors.initials: C
authors.lastname: Yu
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Jingteng
authors.initials: J
authors.lastname: Cui
authors.affiliation: Poultry Diseases Lab, College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong, China.
authors.forename: Youxiang
authors.initials: Y
authors.lastname: Diao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D004372:Ducks / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005416:Flavivirus / Q000276:immunology*; D018177:Flavivirus Infections / Q000175:diagnosis / Q000662:veterinary*; D008807:Mice, Inbred BALB C; D011201:Poultry Diseases / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D014759:Viral Envelope Proteins; C065696:glycoprotein E, Flavivirus