Development and Partial Characterization of High-affinity Monoclonal Antibodies for Botulinum Toxin Type A and their use in Analysis of Milk by Sandwich ELISA

Authors: Stanker, Larry; Merrill, Paul; Scotcher, Miles; Cheng, Luisa

Submitted to: Journal of Immunological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2008
Publication Date: 4/9/2008
Citation: Stanker, L.H., Merrill, P.A., Scotcher, M.C., Cheng, L.W. 2008. Development and Partial Characterization of High-affinity Monoclonal Antibodies for Botulinum Toxin Type A and their use in Analysis of Milk by Sandwich ELISA. Journal of Immunological Methods, 336:1-8.

Interpretive Summary: Botulism is a serious, often fatal neuroparalytic disease in humans and animals caused by a protein toxin (botulinum toxin, BoNT) produced by the bacterium Clostridium botulinum. BoNT is considered the most toxic biological toxin known. Three forms of botulism, food-borne, wound botulism, and infant botulism are known. Because of its high toxicity, the need for a long recovery period requiring extensive treatment, and the ease of producing BoNT, it is considered a class A bioterrorism agent. The ‘gold standard’ for detection of botulinum toxin is the mouse bioassay. Simple, non-rodent-based rapid detection methods are highly desirable to monitor food and environmental samples. We describe the development and characteristics of a set of high affinity monoclonal antibodies to BoNT, and the application of these antibodies to produce a simple but highly sensitive sandwich immunoassay for BoNT. Application of this assay to toxin spiked milk samples demonstrate the utility of this assay to measure toxin contamination in a complex food.

Technical Abstract: Botulinum neurotoxins (BoNT), produced by the anaerobic bacterium Clostridium botulinum, cause severe neuroparalytic disease and are considered the most toxic biological agents known. While botulism is rare in the US, it often is fatal if not treated quickly, and recovery is long, requiring intensive treatment. BoNT is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disulfide bond. The ‘gold standard’ for BoNT detection relies on the mouse bioassay. This is a time consuming (up to 4 days) assay and it lacks specificity, however, it gives a sensitivity (mouse LD50) of ~10 pg mL-1. Most BoNT immunoassays are much less sensitive. In this study we describe the development of four high affinity (Kd’s in the low pM range) monoclonal antibodies (mAbs) that specifically bind BoNT serotype A (BoNT/A). These antibodies, designated F1-2, F1-5, F1-40 and F2-43, are IgG1 subclass mAbs with kappa light chains and they specifically bind BoNT serotype A. Western blot analyses demonstrate that mAbs F1-2 and F1-5 bind the 100 kDa heavy-chain subunit and that mAb F1-40 binds the 50 kDa light-chain. The fourth antibody demonstrated strong binding in ELISA but no binding in western blots following SDS-PAGE was observed. A highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A was developed using mAbs F1-2 and F1-40. Such and assay represents a realistic, high sensitivity alternative to the mouse bioassay.