10-2 |
Use of immuno-affinity columns to increase the specificity of polyclonal rabbit antisera to Clostridium botulinum neurotoxin type E (BoNT/E) and application in a rapid in vitro immunoassay to detect BoNT/E in mixed cultures |
B. ST-JACQUES1, J. P. Smith2, and J. W. Austin1. (1) Bureau of Microbial Hazards, Health Canada, Sir Frederick Banting Building, P.L. 2204A2, Tunney's Pasture, Ottawa, ON K1A 0L2, Canada, (2) Dept. of Food Science & Agricultural Chemistry, McGill Univ., Macdonald Campus, 21111 Lakeshore, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada Botulism is a foodborne intoxication resulting from ingestion of food containing preformed neurotoxin produced by Clostridium botulinum (BoNT). Immunological assays have been developed for the detection of BoNT in cultures, clinical samples and food extracts. Use of polyclonal antisera is simpler and offers greater sensitivity than monoclonal antibodies. However, polyclonal antisera are less specific than monoclonal antibodies. Until the specificity of antisera is improved, immunoassays will not replace the mouse bioassay for the detection of BoNT. The objective of the study was to increase the specificity of polyclonal antisera to C. botulinum neurotoxin type E (BoNT/E) and use it in a rapid in vitro immunoassay to detect BoNT/E in mixed cultures. Cross-reactive antibodies were removed from whole rabbit immune antisera to BoNT/E with affinity columns made with proteins from culture supernatants of closely related and/or highly cross-reactive strains of clostridia. The adsorbed antisera were used for detection of BoNT/E using both slot blot and colony blot immunoassays. The isolates were confirmed to be C. botulinum type E by mouse bioassay. Seven out of ten strains cross-reacted, to varying degrees, when using unadsorbed antisera. After cross-adsorption, the specificity was increased such that the adsorbed antisera slightly reacted with only one out of ten strains. The adsorbed antisera were specific for C. botulinum type E when used to screen colonies of mixed cultures in colony blots, and also detected BoNT/E in liquid cultures with a sensitivity corresponding to approximately 10-100 mouse lethal doses. These results suggest that the adsorption of cross-reactive antibodies from rabbit antisera can increase the specificity of these sera for BoNT/E. Cross-adsorbed antisera can be used in immunoassays without obtaining false-positive results. The slot blot, when used in combination with the colony blot assay, has the potential to replace the mouse bioassay when testing cultures.
Session 10, International: Division Lecture and Paper Competition
|