Development of novel immunochemical techniques for species-specific detection of Karnal bunt
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The purpose of this project was to develop species-specific, ultrasensitive immunochemical techniques for rapid detection of the wheat pathogen, Karnal bunt. Karnal bunt, caused by the fungal organism Tilletia indica, is of regulatory significance. Detection is the primary means for controlling the spread of the disease, however, current diagnostic tools lack accuracy and speed. Both polyclonal and monoclonal antibodies were developed to distinguish Karnal bunt from other closely related fungal species. A novel subtractive hybridization approach was taken to produce polyclonal antibodies that displayed absolute specificity for Karnal bunt. A sandwich ELISA was constructed using the Karnal bunt specific antibodies that illustrated the capability of these immunoassays to detect whole spores in wheat and provide the ultra-sensitive detection required to control the spread of this wheat pathogen. This sandwich ELISA was converted to both a dipstick and immunomagnetic bead format to display the potential of these methods as rapid, on-site diagnostic tools. The Karnal bunt specific antibodies were also used to determine the species-specific antigenic determinants of T. indica, the causal agent of Karnal bunt.