Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

Abstract

“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. 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. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

Probing the Compatibility of an Enzyme‐Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains

“…Enzyme‐linked immunosorbent assay (ELISA) has been widely used to detect toxins, [14] ligand‐receptor interactions, [15] and protein‐protein interactions [16] because it is a simple, precise, and sensitive technique. We have developed an ELISA for the study of A‐domains in NRPS enzymes using a set of active site‐directed probes coupled to a biotinylated 5′‐ O ‐[ N ‐(aminoacyl)‐sulfamoyl]adenosine (aminoacyl‐AMS; Figure 1A and B) [17,18] .…”

Advances in Detecting Ciguatoxins in Fish

“…P-CTX-1 could be detected specifically as low as 0.28 ng mL −1 without cross-reactivity with other related marine toxins; this concentration was still higher than the regulatory limit of 0.01 ppb [ 117 ]. As a further development, Tsumuraya et al combined these MAbs into a single sandwich ELISA assay to detect any of these four CTX congeners [ 50 , 120 ]. Detection of the immunoreaction was changed to a fluorescent method using alkaline phosphatase(ALP)-linked MAb and a fluorescent substrate system, 2′-(2-benzothiazoyl)-6′-hyrodxybenzothiazole phosphate (BBTP).…”

Neurological Disturbances of Ciguatera Poisoning: Clinical Features and Pathophysiological Basis

“…Currently, there are no portable assays available to detect CTXs in fish in the field prior to consumption. To achieve this goal, the development of an enzyme-linked immunoassay (ELISA) is attractive for its accuracy, sensitivity, routine and portable use, and recent progress is promising [ 18 , 19 , 20 , 21 ]. Laboratory methods such as cell-based assays and HPLC-MS(/MS) are useful to detect, quantify and/or identify CTXs after suitable extraction from fish meal remnants [ 8 , 17 , 22 , 23 , 24 , 25 , 26 , 27 ] and mammalian fluids or tissues following intoxication [ 28 , 29 , 30 ].…”