title: Development of a monoclonal sandwich ELISA for direct detection of bluetongue virus 8 in infected animals.
abstract: Bluetongue is an infectious viral disease which can cause mortality in affected ruminants, and tremendous economic damage via impacts upon fertility, milk production and the quality of wool. The disease is caused by bluetongue virus (BTV) which is transmitted by species of Culicoides biting midge. Rapid detection of BTV is required to contain disease outbreaks and reduce economic losses. The purpose of this study was to develop a monoclonal sandwich ELISA for direct detection of BTV in infected animals. Phage display technology was used to isolate BTV specific antibody fragments by applying the human scFv Tomlinson antibody libraries directly on purified BTV-8 particles. Three unique BTV-8 specific human antibody fragments were isolated which were able to detect purified BTV particles and also BTV in serum of an infected sheep. A combination of a human/mouse scFv-Fc chimeric fusion protein and a human Fab fragment in a sandwich ELISA format was able to detect BTV specifically with a limit of detection (LOD) of 104infectious virus particles, as determined by tissue culture titration. This approach provided pilot data towards the development of a novel diagnostic test that might be used for direct detection of BTV-8 particles.
authors.affiliation: Institute of Pathology, University Hospital Giessen, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany. Electronic address: Andre.T.Haaf@patho.med.uni-giessen.de.
authors.forename: Andre
authors.initials: A
authors.lastname: Ten Haaf
authors.affiliation: Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University Giessen, Frankfurter-Str. 98, 35392 Giessen, Germany. Electronic address: Johannes.kohl@vetmed.uni-giessen.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Kohl
authors.affiliation: Institute of Pathology, University Hospital Giessen, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany. Electronic address: Sibylle.Pscherer@patho.med.uni-giessen.de.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Pscherer
authors.affiliation: Hessian State Laboratory (LHL), Schubertstraße 60, 35392 Gießen, Germany. Electronic address: Hans-Peter.Hamann@lhl.hessen.de.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Hamann
authors.affiliation: Hessian State Laboratory (LHL), Schubertstraße 60, 35392 Gießen, Germany. Electronic address: HUEskensGB@aol.com.
authors.forename: Hans Ulrich
authors.initials: HU
authors.lastname: Eskens
authors.affiliation: Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Südufer 10, 17493, Greifswald-Insel Riems, Germany. Electronic address: Max.Bastian@fli.bund.de.
authors.forename: Max
authors.initials: M
authors.lastname: Bastian
authors.affiliation: Institute of Pathology, University Hospital Giessen, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany. Electronic address: Stefan.Gattenloehner@patho.med.uni-giessen.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Gattenlöhner
authors.affiliation: Institute of Pathology, University Hospital Giessen, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany. Electronic address: Mehmet.K.Tur@patho.med.uni-giessen.de.
authors.forename: Mehmet Kemal
authors.initials: MK
authors.lastname: Tur
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D000956:Antigens, Viral / Q000032:analysis*; D001819:Bluetongue / Q000175:diagnosis* / Q000821:virology; D001820:Bluetongue virus / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D012698:Serologic Tests / Q000379:methods; D044967:Serum / Q000821:virology*; D012756:Sheep
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral
keywords: Bluetongue virus (BTV); Monoclonal sandwich ELISA; Phage display technology; Serological test; Virus-specific antibody fragment