Analysis of ciguatoxins in fish with a single-step sandwich immunoassay

Ciguatoxins (CTXs) are the primary cause of ciguatera poisoning (CP), one of the most prevalent non-bacterial seafood-borne illnesses worldwide. With no cure available beyond palliative treatments to alleviate symptoms, effective CP management relies on prevention. However, the detection of CTXs in seafood poses significant analytical challenges due to their typically low concentrations in specimens and the high variability among CTX congeners, many of which remain poorly characterized. These challenges have led to a growing demand for the development of rapid, sensitive, and user-friendly bioanalytical tools for CP surveillance. In this study, several simplified sandwich immunoassay strategies were evaluated for the detection of Pacific CTXs in fish. Among them, the single-step strategy was identified as the most promising, as it enables the detection of Pacific CTXs in complex fish matrixes within only 40 min at levels as low as 0.01 µg CTX1B equivalents/kg of fish, aligned with the safety guidance level proposed by the United States Food and Drug Administration (FDA). Unlike traditional sandwich immunoassays, which require several sequential incubation steps, the single-step strategy involves a simultaneous incubation of all components with the sample, uniquely followed by a washing and substrate incubation step prior to signal measurement. This approach significantly reduces both the complexity and time required for analysis, positioning this immunoassay as a highly promising tool for CP risk assessment and management.