Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 10, 2006
Publication Date: November 13, 2006
Citation: Stanker, L.H., Merrill, P.A. 2006. Development of High Affinity Monoclonal Antibodies Specific for Botulinum Neurotoxin Type A and a Sensitive Immunoassay with Detection Near that of the Mouse Bioassay [abstract]. ICCVAN, NICEAM/ECVAM Scientific Workshop on alternative methods to refine reduce and replace the mouse LD50 assay for botulinum testing Silver Springs, MD, 28, Nov 13-14, 2006. Technical Abstract: Clostridium botulinum neurotoxins(BoNT)cause severe and potentially fatal neuroparalytic disease and are considered the most toxic biological agents known. BoNT is synthesized as a single 150 kDa precursor protein, which is cleaved to form two subunit polypeptides, linked by a single disulfide bond. The ‘gold standard’ for BoNT detection is the mouse bioassay. While the mouse bioassay it is time consuming (up to 4 days) and lacks specificity, it has a limit of detection in the low picogram range. Most BoNT immunoassays reported appear to have much less sensitive than the mouse bioassay. In this study we describe the development of high affinity monoclonal antibodies (Mab). These are IgG1 and IgG2b subclass MAb’s with kappa light chains. They specifically bind BoNT serotype A and have measured Kd values in the low pM range. Western blot analysis demonstrated that four of the Mabs specifically bind the 100 kDa heavy-chain subunit, while one of the antibodies specifically binds the 50Kda light-chain. Using a simple sandwich immunoassay format with a heavy-chain specific Mab for capture, a directly labeled anti light-chain Mab for detection and a chemiluminescent substrate, detection of BoNT type A in the low picogram range was observed. Further characterization of these MAb and their application to rapid immunoassay formats will be discussed.