Immunoassays for the detection of the marine toxins azaspiracids

Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium and Amphidoma spp. and accumulate in shellfish. More than 30... [ view full abstract ]

Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium and Amphidoma spp. and accumulate in shellfish. More than 30 different analogues have been identified, of which AZA-1, AZA-2 and AZA-3 are the most important ones based on occurrence and toxicity. Regarding this, the European Union (EU) has set a maximum level of 160 μg of AZA equivalents/kg shellfish for AZA-1, AZA-2 and AZA-3. However, existing methods for detecting and quantifying AZAs are still restricted to liquid chromatography coupled to mass spectrometry (LC-MS/MS) and the mouse bioassay (MBA).

The development and validation of new methods to reliably detect AZAs at the level of interest in a rapid, simple, and cost-effective way is highly desirable. Therefore, ovine polyclonal antibodies were obtained with immunizing antigens prepared from synthetic fragments of the constant region of AZAs. These were used to develop immunoassays, including an enzyme-linked immunosorbent assay (ELISA) and a direct magneto-controlled electrochemical immunoassay, for quantitating AZAs in shellfish, algal cells, and culture supernatants.

Calibration curves have been obtained for both assays and cross-reactivity factors for AZA2 to AZA10 (relative to AZA1) have been established in order to better understand correlations with LC-MS/MS, which used as a reference analytical technique. Both immunoassays are rapid, reliable and sensitive enough to quantify the AZA-content of shellfish at levels well below the regulatory limit, and detect all known azaspiracid analogues including the carboxylated precursors to AZA3 and AZA6.