title: An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein.
abstract: An enzyme linked immunosorbent assay (ELISA) method to study serum antibodies to viral haemorrhagic septicemia virus (VHSV) was designed by using recombinant fragments of their G protein. By using this fragment-ELISA, we describe the binding of antibodies against recombinant G fragments of 45-445 amino acids present in VHSV-hyperimmunized trout sera. Fragments were designed by taking into account their tridimensional pH-dependent structure and functional domains. Sera were obtained from hyperimmunized trout following 4-5 intraperitoneal injections of VHSV antigens by using Freund's or saponin adjuvants. Sera from different hyperimmunized trout differed quantitatively rather than qualitatively in their recognition of solid-phase frg11 (56-110), frg12 (65-109), frg13 (97-167), frg14 (141-214), frg15 (65-250), frg16 (252-450) and G (G21-465) by Western blot and ELISA. However, titres were higher when using frg11, frg15 or frg16, rather than G21-465, suggesting higher accessibility to G epitopes. Further knowledge of the antigenicity of the G protein of rhabdoviruses by using fragments might be used to improve current vaccines. On the other hand, they might be used to dissect the trout antibody response to VHSV infections, to complement in vitro neutralizing assays, and/or to quantitate anti-VHSV antibodies in VHSV-infected/vaccinated trout, other fish and/or other body fluids such as mucus.
authors.affiliation: INIA, SGIT, Dept Biotecnología Crt. Coruña Km 7, Madrid, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Encinas
authors.forename: E
authors.initials: E
authors.lastname: Gomez-Casado
authors.forename: A
authors.initials: A
authors.lastname: Estepa
authors.forename: J M
authors.initials: JM
authors.lastname: Coll
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005393:Fish Diseases / Q000276:immunology* / Q000821:virology; D007114:Immunization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D029141:Novirhabdovirus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000276:immunology* / Q000821:virology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; C096089:G protein, viral hemorrhagic septicemia virus; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins