A Practical ELISA for Azaspiracids in Shellfish via Development of a New Plate-Coating Antigen

Abstract

Azaspiracids (AZAs) are a group of biotoxins that appear periodically in shellfish and can cause food poisoning in humans. Current methods for quantifying the regulated AZAs are restricted to LC-MS but are not well suited to detecting novel and unregulated AZAs. An ELISA method for total AZAs in shellfish was reported recently, but unfortunately, it used relatively large amounts of the AZA-1-containing plate-coating conjugate, consuming significant amounts of pure AZA-1 per assay. Therefore, a new plate-coater, OVA–cdiAZA1 was produced, resulting in an ELISA with a working range of 0.30–4.1 ng/mL and a limit of quantification of 37 μg/kg for AZA-1 in shellfish. This ELISA was nearly twice as sensitive as the previous ELISA while using 5-fold less plate-coater. The new ELISA displayed broad cross-reactivity toward AZAs, detecting all available quantitative AZA reference materials as well as the precursors to AZA-3 and AZA-6, and results from shellfish analyzed with the new ELISA showed excellent correlation (R 2 = 0.99) with total AZA-1–10 by LC-MS. The results suggest that the new ELISA is suitable for screening samples for total AZAs, even in cases where novel AZAs are present and regulated AZAs are absent, such as was reported recently from Puget Sound and the Bay of Naples.

In Vitro Metabolism of Azaspiracids 1–3 with a Hepatopancreatic Fraction from Blue Mussels (Mytilus edulis)

“…Previous work found that ELISA analysis of AZAs in shellfish, using an antibody that recognizes only the ring F−I substructure, gave concentrations significantly higher than targeted LC−MS/MS methods, even when AZA1−10 were included in the LC−MS/MS method. 61 However, the broader array of AZA metabolites being revealed in mussels by modern untargeted LC−MS techniques, such as those identified here, and in other studies 10,44,57,62 provides a plausible explanation for this difference. This is because all of the metabolites identified to date in shellfish contain the rings F−I substructure that is recognized by the antibodies, but not all are detected with routine LC−MS/MS methods.…”

Emerging Optical Materials in Sensing and Discovery of Bioactive Compounds

“…The main advantages of ELISA are the simple mechanism, accuracy, and easy equipment operation [ 1 ]. Examples of ELISA applications include the detection of yessotoxins in shellfish and algal samples [ 61 ], gonyautoxin (a PSP toxin) [ 62 , 63 ], and azaspiracid [ 64 ] from shellfish.…”

Current Research Status of Azaspiracids

“…In 2015, Samdal [72] demonstrated an ELISA with a working range of 0.45-8.6 ng/mL and a limit of quantitation for total AZAs in whole shellfish of 57 µg/kg. He also produced a new plate coater, OVA-cdiAZA-1, resulting in an ELISA with a working range of 0.30-4.1 ng/mL and a limit of quantification of 37 µg/kg for AZA-1 in shellfish, in 2019 [74]. ELISA can also be employed in conjunction with gel methods [123].…”