Identification of bluetongue virus protein-specific antibody responses in sheep by immunoblotting.

A western immunoblotting procedure was developed for identifying bluetongue virus protein-specific antibody responses in sheep. Assay conditions were optimized and included electrophoretic transfer of viral proteins to a nitrocellulose membrane (NCM), blocking of unbound sites on the NCM, and detection of NCM-bound primary immune complexes. A biotin-avidin-enzyme system was determined to be superior in terms of sensitivity for identification of the NCM-bound virus protein-antibody complexes. The biotin-avidin-enzyme detection system made use of biotinylated rabbit anti-sheep IgG and horseradish peroxidase-conjugated egg white avidin. This system permitted identification of antibodies specific for up to 10 bluetongue virus-associated proteins, 4 of which have not been characterized.