More than 20,000 people suffer annually from ciguatera, a disease caused by consumption of reef fish. A problem in avoiding the disease is that there is currently no rapid and reliable method of detecting the causative toxins, namely the ciguatoxins, at fisheries. Furthermore, the content of ciguatoxin is extremely low, which has hampered the preparation of anti-ciguatoxin antibodies. Herein, we have demonstrated a rational approach for the preparation of specific antibodies to targeted sites of the ciguatoxins. Monoclonal antibodies (mAbs) elicited by using tricyclic ABC rings as haptens, however, were not bound to CTX3C itself. After considerable experimentations, specific mAbs with strong affinities to CTX3C itself were prepared by using pentacyclic haptens (ABCDE and IJKLM). The requisite surface area of the hydrophobic polycyclic ether parts was estimated to be more than 400A. We then developed a direct sandwich immunoassay employing the mAbs to both ends of CTX3C. This specific and sensitive sandwich protocol with detection limits of CTX3C [〜1ng/mL] will provide a reliable method for the screening of toxins for public health protection. This study therefore paves the way for the development of sandwich immunoassays using specific antibodies to targeted sites of non-biopolymer organic molecules.
