ELISA to determine anti-K99 pilus antibody in the sera of normal and diarrhoeic calves

Abstract

An ELISA to detect circulating antibodies against K99 pili, a major attachment factor to intestinal epithelial cells of Escherichia coli in calves, was performed. Two methods of K99 pili purification were attempted. Best results in terms of purity of the K99 antigen were achieved following the method described by Karkhanis and Bhogal (1986). This procedure included a heat shock at 65°C during 25 min to release the pili and ultracentrifugation steps to purify the antigen. SDS-PAGE showed an 18 KDa major band, identified as the K99 pilus antigen after immunoblotting against reference antisera. The purified K99 antigen was then adsorbed to the ELISA microplates. High optical density was obtained in the ELISA using a pool of sera from immunized cows. No differences in antibody levels (P ≥ 0.05) could be detected between clinically healthy calves and those showing diarrhoea