This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level PI4KIIIbeta-IN-9 of 0.01 ppb. Keywords: ciguatera, ciguatoxin, monoclonal antibody, sandwich ELISA 1. Introduction Ciguatera fish poisoning (CFP) is one of the well-known food-borne diseases caused by the ingestion of various reef fish that have accumulated trace amounts of ciguatoxins (CTXs) [1,2]. Typical human symptoms of CFP are severe neurological, gastrointestinal, and cardiovascular symptoms, which may last week, months, or even years. It is estimated that over 50,000 people throughout the world suffer from CFP each year, making it the most common form of PI4KIIIbeta-IN-9 seafood poisoning caused by natural toxins [3,4,5]. The spread of CFP gives rise to a considerable burden on public health systems, and damage to fishery resources and the economies in tropical and subtropical regions [6]. In addition to climate change, globalization of trade might contribute to the expansion of CFP, even in temperate zones. It is difficult to prevent CFP due to the lack of reliable analytical methods to detect causative CTXs, along with the normal appearance, taste, and smell of CTX-contaminated fish. Yasumoto and his coworkers characterized CTXs as 3 nm-long ladder-shaped polycyclic ethers. CTXs were originally produced by species of marine dinoflagellates and accumulate in various types of reef fish through bioaccumulation [1,2,7]. CTX1B and its congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) (Figure 1) were initially isolated in the Pacific region [8,9,10,11,12], then detected in the Atlantic [13,14]. These CTXs are extremely toxic to mammals and the lethal potencies of CTXs by intraperitoneal injection into mice (median lethal dose (LD50) 0.25C4 g/kg) are much greater than those of the structurally related red-tide toxins, brevetoxins (LD50 > 100 g/kg) and the pufferfish toxin, tetrodotoxin (LD50~10 g/kg) [8,9,10,15,16,17]. The structural differences between the CTX congeners arise from the substituents on the A and M terminal rings as well as the size of PI4KIIIbeta-IN-9 the E-ring. The CTX1B series (CTX1B and 54-deoxyCTX1B) possesses a dihydroxybutenyl group on the A-ring and a seven-membered E-ring, while members the CTX3C series (CTX3C and 51-hydroxyCTX3C) lack the dihydroxybutenyl group and have an eight-membered E-ring. Open in a separate window Figure 1 Chemical structures of the four major ciguatoxin (CTX) congeners: CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C. Considerable attention has recently been directed to the development of analytical methods for detecting CTXs. To replace the traditional mouse bioassay (MBA), several analytical methods have been developed in recent years to detect CTXs in a contaminated fish. Examples include a neuroblastoma cell-based assay (CBA-N2a) [18], radiolabeled and fluorescent receptor binding assays (RBA) [16,19,20], PI4KIIIbeta-IN-9 HPLC [21], MS [12,22,23], and LC-MS/MS assays [13,14,24,25,26,27,28,29]. However, there is no quick and reliable Rabbit Polyclonal to Cyclin H (phospho-Thr315) method to detect CTXs in the fishery or even at inspection stations for seafood. MAb-based immunoassays such as ELISA was expected to provide suitable methods for the sensitive, accurate, routine, and portable detection of CTXs. For the generation of anti-CTX antibodies, Hokama et al. claimed that an anti-CTX1B mAb was prepared by immunization with natural CTX1B [30], but the mAb cross-reacted with okadaic acid (OA) [31,32], and the results of immunochemical kit Cigua-Check? using this mAb have been controversial [33,34,35]. The minimal amount of CTX isolated from contaminated fish has prevented further development of anti-CTX antibodies. Thus, rationally designed synthetic haptens instead of natural toxins were planned to produce anti-CTX mAbs. Successful syntheses of CTX congeners based on a convergent synthetic strategy.