Browsing School of Medicine by Author "Carney, James P."
Investigation of Botulinum Toxin AntibodiesBuckley, Patricia; Carney, James P. (2013)Antibodies are used in both detection and diagnostic assays because of their specificity and sensitivity in recognizing their corresponding antigens, and there have been many antibody assays designed to detect and identify Botulinum neurotoxins (BotNT). Initially, polyclonal antibody serum was screened and found to recognize Botulinum A/B toxin complex, which contains both the neurotoxins as well as the nontoxic associated proteins. These antibodies were produced in bacteria and tested for sensitivity in a variety of assays; however, antibodies expressed in bacteria lack molecular modifications made post-translationally by animal (eukaryotic) cells. Based on this information, this project was designed to determine if modifications to the expression of the Fab fragment antibody genes increase the binding capacity or the neutralization capacity of this antibody. By keeping the variable region genes of an antibody intact, and producing that antibody in a variety of organisms, the effect of the production method can be evaluated. The anti-botulinum toxin antibody genes were produced in three different types of organisms: bacteria cells (E. coli), insect larvae (T. ni), and mammalian cells (CHO) and the resultant antibodies were compared, producing little change in the overall efficacy of the antibody. Each antibody produced was found to be pure and active, recognizing the intended target; there were differences found among the antibodies, but the overall activity remained the same. Once the antibodies were physically characterized, the toxin inhibiting properties of an entire panel of botulinum antibodies were investigated. The M17 neuroblastoma cell line was used as a primary screen for blocking the intracellular catalytic toxic cascade produced by BotNT exposure by measuring the amount of cleaved SNAP-25 protein after exposure. One antibody, ABE 366, which recognizes the heavy chain of BotNT did inhibit the BotNT activity. These neutralizing results were then corroborated in vivo using the nematode C. elegans. The information obtained in this study provides a better understanding of the capabilities and drawbacks of reagents that may be used to detect, identify and neutralize bioweapons now and in the future.