Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA), and domoic acid (DA), for PSP, DSP, and ASP, respectively. In this study a multidetection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multidetection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA, or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in the buffer was similar in single- and multidetection: 5.6 +/- 1.1 ng/mL for STX, 1.1 +/- 0.03 ng/mL for OA, and 1.9 +/- 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA, and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90% for STX, 80% for OA, and 100% for DA). This microsphere-based multidetection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.