Multi-detection of three marine toxin groups by a solid-phase inhibition immunoassay using a microsphere-flow cytometry system

M Fraga1, N Vilariño1, MC Louzao1, K Campbell2, CT Elliott2, LM Botana1. 1Universidad de Santiago de Compostela, Facultad de Veterinaria, Farmacología y terapeútica, 27002, Spain, 2Queen\'s University, School of Biological Sciences, Institute of Agri-Food and Land Use, BT9 5AG, Ireland

Marine toxins are biochemical substances that threat human health and ecosystems and have an important economic impact to the seafood industry. Paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP) toxins are common marine toxins that can easily reach human consumers through the trophic chain. The severe human health threat associated to the presence of these groups of toxins in food has prompted the control and limitation of toxin levels in shellfish destined for human consumption. In the European Union the current limits, established by the European Food Safety Authority (EFSA), are 800 μg of STX equivalents/kg of shellfish meat, 160 μg of OA equivalents/kg shellfish meat and 20 mg of DA/kg shellfish meat. Methods capable of detecting these toxins in shellfish in the range of the regulatory limits are essential to enforce regulations. Most of the existing screening methods detect only a toxin group per sample. Multiplexing methods would allow screening the presence of several toxin groups simultaneously in a single sample. We have developed a multi-detection method using a solid-phase microsphere assay coupled to flow cytometry detection, based on a Luminex 200™ system. The quantification of the compound of interest is achieved by the fluorescence signal attached to the surface of Luminex microspheres. Multiplexing is provided by the incubation of a sample with multiple classes of microspheres simultaneously, each class being specific for a certain analyte, and classification of microspheres by a flow cytometry-based platform. Luminex microspheres are internally dyed in order to differentiate microsphere classes by their unique spectral signal after excitation with a classification laser and contain surface carboxyl groups for covalent attachment of ligands. Saxitoxin (STX), okadaic acid (OA) and domoic acid (DA) are the main representatives of the PSP, DSP and ASP toxins, respectively. The three toxins were attached to the surface of three different classes of microspheres. The multiplexed technique is based on three competitive immunoassays. For each immunoassay the toxins present in the sample/calibration solution compete with the toxin immobilized on the surface of the microsphere for binding to a monoclonal antibody specific for each toxin group. The amount of these three primary antibodies bound to the surface of the microsphere was quantified by the addition of a secondary antibody labeled with phycoerythrin, which is detected by a detector laser. The immunoassays provided adequate dynamic ranges for the detection of the three toxins within the regulatory limits. The half maximal inhibitory concentrations (IC50) achieved were: 15 ± 2 nM STX, 1.4 ± 0.04 nM OA and 7 ± 0.5 nM DA. The combination of the three immunoassays to achieve multi-detection in one sample did not change the sensibility versus the individual assays. This microsphere-based multi-detection immunoassay provides an easy, rapid, screening method capable of detecting simultaneously in the same sample three of the main groups of marine toxins, considering prevalence and toxicity, that appear in European coasts.