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

Area of Science:

• Marine Biology
• Food Safety
• Analytical Chemistry

Background:

• Azaspiracids (AZAs) are marine biotoxins found in shellfish, posing a risk of human food poisoning.
• Current quantification methods like LC-MS are limited to regulated AZAs and struggle with novel variants.
• Previous ELISA methods for total AZAs were inefficient, requiring substantial amounts of AZA-1 conjugate.

Purpose of the Study:

• To develop a more sensitive and efficient ELISA for detecting total Azaspiracids (AZAs) in shellfish.
• To create an assay capable of detecting both regulated and novel AZAs, including precursors.
• To reduce the amount of AZA-1 conjugate required per assay compared to existing methods.

Main Methods:

• Development of a new plate-coater, OVA-cdiAZA1, for an improved Enzyme-Linked Immunosorbent Assay (ELISA).
• Characterization of the ELISA's working range (0.30-4.1 ng/mL) and limit of quantification (37 μg/kg) for AZA-1.
• Testing the ELISA's cross-reactivity with various AZAs and precursors, and comparing results with LC-MS analysis.

Main Results:

• The new ELISA demonstrated a working range of 0.30-4.1 ng/mL and a limit of quantification of 37 μg/kg for AZA-1.
• The assay was nearly twice as sensitive as previous ELISAs while utilizing five times less plate-coater.
• The ELISA showed broad cross-reactivity, detecting all tested AZA reference materials and precursors, with excellent correlation (R² = 0.99) to LC-MS for total AZA-1-10.

Conclusions:

• The developed ELISA is a suitable and efficient tool for screening shellfish samples for total Azaspiracids (AZAs).
• This assay can detect novel and unregulated AZAs, addressing limitations of current LC-MS methods.
• The improved sensitivity and reduced reagent consumption make this ELISA a valuable advancement in marine toxin analysis and food safety.