Biosensor‐Based Assay for Domoic Acid: Comparison of Performance Using Polyclonal, Monoclonal, and Recombinant Antibodies

Abstract

Domoic acid (DA) is a potent neuroexcitatory toxin produced by the diatom Pseudonitszchia sp, and causes amnesic shellfish poisoning in humans through the ingestion of contaminated shellfish. Shellfish products placed on the market are, therefore, highly monitored. A panel of anti‐DA antibodies, which included a mouse monoclonal, sheep polyclonal antiserum, and single chain variable region fragments from sheep and chicken, were evaluated for suitability for assay development using the surface plasmon resonance biosensors from BIAcore AB. Kinetic analysis of the binding of each antibody to immobilized DA gave dissociation rates varying from 2.07×10⁻⁵ for the sheep polyclonal to 6.52×10⁻⁴ for the mouse monoclonal. Inhibition assays were established with each antibody and the variation in sensitivities and lower limits of detection of the standard curves could be predicted from the dissociation rates. The standard curve given by the sheep polyclonal assay was the most sensitive with an ED‐50 (the concentration of DA inhibiting binding by 50%) of 26 ng/mL and the mouse monoclonal the least (ED‐50=1.445 µg/mL). All assays showed a high level of reproducibility. The sheep polyclonal assay was shown to be suitable for determination of domoic acid in scallop extracts.

Keywords:

• Domoic acid
• amnesic shellfish poisoning
• anti‐DA antibodies
• inhibition assay
• ED‐50

This work was supported by grants from the Irish Higher Education Authority's Programme for Research in Third Level Institutions (PRTLI), as part of the Marine Science Research Programme in the Martin Ryan Institute, NUI Galway, Ireland, and from the Advanced Technologies Research Programme of Enterprise Ireland. The authors would like to thank Catherine Donohue, Aoife O'Reilly, Joanna Reilly, and Iain Shaw for the antidomoic acid antibodies and David Swords, Jenny Ronan, and Dr. Philipp Hess from the Marine Institute, Galway, Ireland, for the extraction protocol and for scallop extracts. The authors are grateful to John Butler, from Biacore AB for his assistance with kinetic analysis.