Preparation and characterization of an immunoaffinity column for the selective extraction of azaspiracids

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2022-09, Vol.1207, p.123360-123360, Article 123360
• Main Authors: Samdal, Ingunn A., Sandvik, Morten, Vu, Jennie, Sukenthirarasa, Merii S., Kanesamurthy, Sinthuja, Løvberg, Kjersti L.E., Kilcoyne, Jane, Forsyth, Craig J., Wright, Elliott J., Miles, Christopher O.
• Format: Article
• Language: English
• Subjects: Antibody; AZA; ELISA; Immunoaffinity chromatography; LC–MS; Mussel; Shellfish toxin
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2022.123360

•Polyclonal antibodies to azaspiracids were immobilized on sepharose to prepare immunoaffinity columns.•An optimized procedure was developed to retain and release azaspiracids from shellfish extracts containing complex mixtures of toxins and matrix components.•Other algal toxins and color from the sample matrix were removed by the immunoaffinity columns. The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC–MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26–C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC–MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90–92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.