title: The use of African horse sickness virus VP7 antigen, synthesised in bacteria, and anti-VP7 monoclonal antibodies in a competitive ELISA.
abstract: A full-length cDNA clone of genome segment 7 of African Horse Sickness Virus, serotype 9 (AHSV9) was obtained using the PCR technique. The clone was sequenced and found to be 98.27% homologous to the previously published sequence of the equivalent cDNA clone from AHSV4 at the nucleotide level and to exhibit 99.7% identity at the amino acid level. The cDNA clone was transferred to pGEX-2T (Pharmacia), a bacterial expression vector, such that the reading frame of AHSV9 VP7 was continuous with that of the bacterial glutathione-S-transferase (GST) protein, under the control of the bacterial tac promoter. On induction with IPTG a fusion protein consisting of GST and VP7 was synthesised, which was readily purified on a GST-sepharose column (Pharmacia). The fusion protein reacted equally well in an indirect ELISA using monoclonal antibodies specific for AHSV9 VP7 or polyclonal guinea pig antisera raised against AHSV9 infectious sub-viral particles. This protein was also shown to be a suitable substitute for virus antigen, prepared from infected BHK cell extracts, in a competitive ELISA. Antibodies titres recorded for AHSV9 positive and negative horse sera were similar in the competitive ELISA using either bacterial AHSV VP7 or BHK extracted virus as the source of antigen, in combination with monoclonal or polyclonal antibodies, respectively, as the detectors.
authors.affiliation: AFRC, Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Wade-Evans
authors.forename: T
authors.initials: T
authors.lastname: Woolhouse
authors.forename: R
authors.initials: R
authors.lastname: O'Hara
authors.forename: C
authors.initials: C
authors.lastname: Hamblin
mesh_terms: D000355:African Horse Sickness / Q000175:diagnosis / Q000276:immunology; D000356:African Horse Sickness Virus / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D005814:Genes, Viral; D005822:Genetic Vectors; D006736:Horses; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014773:Virology / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D004279:DNA, Viral; C050779:VP7 protein, Rotavirus