Development and validation of enzyme-linked immunosorbent assays for detection of equine encephalosis virus antibody and antigen

by Crafford, Jan Ernst.

DEVELOPMENT AND VALIDATION OF ENZYME LINKED IMMUNOSORBENT ASSAYS FOR DETECTION OF EQUINE ENCEPHALOSIS VIRUS ANTIBODY AND ANTIGEN BY JAN ERNST CRAFFORD PROMOTER: CO-PROMOTERS: DEPARTMENT: DEGREE: Prof M van Vuuren Prof A J Guthrie, Mr C Hamblin Department of Veterinary Tropical Diseases Faculty of Veterinary Science, University of Pretoria Magister Scientiae (Veterinary Science) The main purpose of this work was to develop rapid and reliable techniques that will prove valuable in epidemiological studies of equine encephalosis virus (EEV), the detection and identification of the virus for the laboratory confirmation of the clinical diagnosis and for the differential diagnosis between EEV and African horsesickness virus (AHSV). Two enzyme linked immunosorbent assays (ELISA) were developed. A polyclonal antibody-based, group-specific, indirect sandwich ELISA for the detection of EEV antigen was developed. The design of the assay was based on the methods currently used for the detection of AHSV. The cut-off value (absorbance of 0.15) was determined using populations of known negative specimens. No cross-reactions were recorded with viruses from other orbivirus serogroups or from other arboviruses. The assay proved to be sensitive and specific for the rapid detection of EEV and viral antigens in cell culture and mouse brain preparations. iv A polyclonal antibody-based, group-specific, competitive ELISA for the detection of antibodies to EEV was developed. No cross-reactions were recorded with the reference sera prepared against other orbivirus serogroups or other arboviruses. The cut-off (29.5% inhibition) value was determined using populations of known positive and negative sera. Analysis of the data showed the assay to be highly repeatable, sensitive and specific. University of Pretoria etd University of Pretoria etd – Crafford, J E (2005) v University of Pretoria etd University of Pretoria etd – Crafford, J E (2005)