Phage Displayed Antibodies to C. botulinum toxin A

The neurotoxins of Clostridium botulinum are the most lethal proteins known. The bacterium is ubiquitous, and thus rapid, reliable, sensitive, and cost-effective assays are needed to detect the toxin. Current immunoassays provide a rapid means to detect the neurotoxin. Their sensitivity, however, is limited by the affinity of the detecting antibody. The objective of this research, therefore, is to develop high-affinity anti-botulinal antibody through recombinant DNA technology. During Phase I, we will take a two pronged approach to generate recombinant botulinal antibodies. First, we will construct a phage-display heavy and light chain variable gene combinatorial library from a botulinal toxin A hyperimmunized animal. Bacteriophage displaying the neurotoxin reactive antibody fragments will be affinity selected and clonally isolated for subsequent analysis. As the combinatorial library may not represent in vivo occurring heavy-light chain combinations or all anti-toxin A immunoglobulin classes, we will in the second approach produce monoclonal antibodies (Mabs) by traditional hybridoma techniques, following which the encoding cDNAs will be cloned. In future work, the cDNAs from the isolated antibody fragment-displaying phage and from selected Mabs will be engineered for higher affinity recombinant antibodies, providing an invariant, stable, and inexpensive immunoassay reagent.