Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by

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Multi-detection of Paralytic, Diarrheic and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System María Fraga, Natalia Vilariño, M Carmen Louzao, Paula Rodriguez, Katrina Campbell, Christopher T. Elliott, and Luis M. Botana Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401146m • Publication Date (Web): 17 Jul 2013 Downloaded from http://pubs.acs.org on July 23, 2013

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Analytical Chemistry

Multi-detection of Paralytic, Diarrheic and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System María Fraga1, Natalia Vilariño1*, M Carmen Louzao1, Paula Rodríguez1, Katrina Campbell2, Christopher T.Elliott2 and Luis M. Botana1* Departamento de Farmacología1, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain Institute for Global Food Security (IGFS)2, School of Biological Sciences, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast, Northern Ireland, BT9 5AG *To whom correspondence should be addressed: Luis M. Botana, Natalia Vilariño Departamento de Farmacología Facultad de Veterinaria Campus Universitario 27002 Lugo Spain

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e-mail: [email protected], [email protected] Telephone and Fax: +34 982822233


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ABSTRACT 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 multi-detection 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 multi-detection 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 buffer was similar in single- and multi-detection: 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 multi-detection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.


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Keywords: marine toxins, saxitoxin, okadaic acid, domoic acid, multiplex, multi-detection, flow-fluorimetry detection, shellfish poisoning, seafood, bead-based array.


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INTRODUCTION Marine phycotoxins are biochemical substances usually produced by toxic planktonic algae species. Occasionally, the proliferation of these phytoplankton species and/or an increase in the production of phycotoxins can generate toxic harmful algal blooms (HABs). Toxic HABs have increased its frequency, intensity and geographical distribution in the last few decades becoming a serious threat to human health and ecosystems, and having important economic impacts to the seafood industry and tourism 1, 2. In order to protect the human population many countries have regulated the levels of several toxin groups in seafood destined for human consumption. Historically, many of these groups have been named according to the human poisoning symptoms. Paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP) toxins appear commonly worldwide and they can easily reach human consumers through the trophic chain due to their accumulation in filter-feeding shellfish species. The main representatives of these groups are saxitoxin (STX), okadaic acid (OA) and domoic acid (DA) for PSPs, DSPs and ASPs respectively. STX and its analogs are hydrophilic toxins with a common tetrahydropurine backbone. They block ion transport through voltage-dependent sodium-channels 3. OA and its derivatives are lipophilic compounds known to inhibit serine/threonine (ser/thr) protein phosphatases PP1 and PP2A 4-6. Besides causing the DSP syndrome, OA is also a tumor promoter 7-9. DA and its isomers are amino acids that bind to and activate kainate receptors, a subclass of glutamate receptors 10, 11. PSP and ASP can be lifethreatening 12, 13. In order to protect human health many countries have regulated the limits for the presence of these toxins in seafood destined to humans. These limits are 800 µg of STX equivalents/kg of meat, 160 µg of OA equivalents/kg of meat and 20 mg of DA/kg of meat 14, 15. The


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implementation of these legal regulations demands the development and validation of toxin detection methods. In European countries the official methods for PSP detection are the mouse bioassay (MBA) and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) 16, 17. For DSPs the commission decision EC 15/2011 18 established the progressive substitution of the MBA by liquid chromatography-mass spectrometry (LC-MS/MS) 19. The official methods for detecting ASPs are high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and Enzyme-Linked ImmunoSorbent Assay (ELISA) 16, 20, 21. Animal bioassays have important disadvantages including lack of accuracy and ethical concerns related to the use of laboratory animals 22. Although HPLC and LC-MS do not raise these ethical issues, they require highly trained personnel, expensive instrumentation and demand specific standards for each analog to avoid estimation errors 23. For several decades researchers in the marine toxin field have been working on the development of alternative detection methods. The drive to improve screening techniques would allow the reduction in the number of samples to be analyzed by more expensive and ethically questionable methods. The current trend is to develop multiplexed methods designed for the simultaneous detection of several groups of toxins in the same sample. These methods allow the screening of multiple samples in a single assay, saving precious resources in terms of time and reagents. The co-occurrence of more than one toxin group reinforces the need of multi-detection methods 24, 25. Currently Luminex technology is widely employed for detection purposes in the research and clinical diagnosis fields 26-29. Luminex technology allows the multi-detection of different analytes present in the same sample. Luminex microspheres are internally encoded with a specific mixture of two internal fluorophores that provides the differentiation of microsphere classes by their unique spectral signal. Additionally, these microspheres contain surface carboxyl


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groups for covalent attachment of ligands, which endows them with specificity for a certain analyte. A system based on flow cytometry principles separates individual microspheres. A red laser excites the internal dyes in order to classify the microsphere class and a green laser excites the surface-attached fluorophore, which is related to the amount of analyte in the sample, in order to quantify the compound of interest. Multiplexing is provided by the incubation of a sample with multiple classes of analyte-specific microspheres simultaneously. The aim of this study was the development of a multi-detection method using a solid-phase microsphere-based assay coupled to flow-fluorimetry detection, using Luminex technology. This work was based in a previously developed method for PSP toxins detection 26. Three toxin groups were selected for the initial application of this technology to marine toxin detection: DSTs, PSTs and ASTs. The risk these toxins pose to human health and the frequency of toxic episodes were the main criteria for their selection. METHODS Materials. Certified reference standard material of okadaic acid (OA) and domoic acid (DA) were obtained from CIFGA (Lugo, Spain). Certified reference standard material of saxitoxin dihydrochloride (STX di-HCl) was obtained from the Institute for Marine Biosciences, National Research Council (Halifax, Canada). Domoic acid for immobilization was purchased from Merck Millipore (Darmstadt, Germany) and CIFGA (Lugo, Spain). N-hydroxysuccinimide (NHS), sodium tetraborate decahydrate, jeffamine (2,2′-(ethylenedioxy)bis(ethylamine)), ethylenediamine, boric acid, sodium phosphate monobasic, ethanolamine and Tween-20 were purchased from Sigma-Aldrich (Madrid, Spain). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Pierce (Rockford, Illinois).The


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monoclonal GT-13A, okadaic acid (OA-Ab) and domoic acid (DA-Ab) antibodies were obtained as previously described 30-32. Phycoerythrin (PE) Goat Anti-Mouse Ig was purchased from Invitrogen (Eugene, Oregon). Sodium azide was purchased from Fluka (Steinheim, Germany). Mussels (Mytilus galloprovincialis) and scallops (Pecten maximus) were purchased from the market (Lugo, Spain). Carboxylated microspheres (LC10038-01, LC10004-01 and LC10054-01) were from Luminex Corporation (Austin, Texas). Luminex sheath fluid, multiscreen 96 well filter plates (Durapore® membrane), 33 mm Millex filter with 0.22 µm pore size and 0.45 µm pore size Ultrafree-MC centrifugal filters (Durapore® membrane) and 0.45 µm pore size Ultrafree-CL centrifugal filters (Low binding Durapore® PVDF membrane) were purchased from Millipore (Madrid, Spain). Hydrochloric acid, acetic acid, formaldehyde 37 %, dimethyl sulfoxide, sodium acetate anhydrous, di-sodium hydrogen phosphate anhydrous and sodium chloride were from reagent grade commercial sources. Phosphate-buffered saline solution (PBS) was 130 mM NaCl, 1.5 mM NaH2PO4, 8.5 mM Na2HPO4, pH 7.4. PBS-BT solution was PBS supplemented with 0.1% w/v BSA and 0.02% v/v Tween-20. Buffer solutions were filtered through a 0.22 µm pore size filter. Toxin immobilization on microspheres. STX, OA and DA were immobilized on the surface of three different classes of microspheres. STX, OA and DA were covalently attached to the surface of LC10038-01, LC10004-01 and LC10054-01 microspheres, respectively. The immobilizations were performed as described by Campbell et al. 30, Llamas et al.33 and Traynor et al.34 for immobilization on carboxylated planar surfaces. Briefly, for STX the carboxylated surface of 2·106 microspheres was activated by adding 150 µL of 150 mg/mL EDC and 23 mg/mL NHS dissolved in water. After 30 min of incubation, this mixture was removed and 20 % jeffamine in borate buffer (pH 8.5) was added


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for 1 h. After removal of jeffamine, 36.3 µg of free-STX di-HCl in 13 µL of 0.1 M acetic acid, 22 µL of H2O and 16 µL of 37 % formaldehyde were added to pre-activated microspheres for 24 h. Subsequently, free-STX was removed and the unreacted carboxyl groups were inactivated by 1 M ethanolamine-HCl during 30 min. The immobilization of OA started with the activation of the carboxylated microsphere surface as described above. Then 1 M ethylenediamine in borate buffer (pH 8.5) was added for 1 h. The free NHS-ester groups were inactivated by 1 M ethanolamine-HCl for 20 min. A mixture of 50 µg of free-OA in 10 µL of DMSO, 10 µL of 108 mg/mL EDC and 48 mg/mL NHS in 10 mM sodium acetate buffer (pH 4.5) and 30 µL of 10 mM sodium acetate buffer (pH 4.5) was added to the microspheres after ethanolamine removal and allowed to react for 4 h. The immobilization of DA is quite similar to the immobilization of OA, using a solution of 154 mg/mL EDC and 46 mg/mL NHS. Free-DA (91 µg) was dissolved in 30 µL of H2O and mixed with 25 µL of 22 mg/mL EDC and 9 mg/mL NHS dissolved in 10 mM sodium acetate buffer (pH 4.5). This mixture was added to pre-activated microspheres for 2 h. At the end of each immobilization, each class of microspheres was washed with PBS and stored in PBS with 0.01 % sodium azide at 4 ˚C in the dark. The immobilization protocols were performed in a well of opaque 96 well 1.2 µm filter plates. Removal of solutions and washing steps were performed with vacuum. In order to achieve maximum specific binding capability the immobilized microspheres were let to equilibrate in PBS with sodium azide for one week after immobilization, with a storage solution renewal. All incubations took place in the dark with constant shaking. Inhibition immunoassay for STX, OA and DA multi-detection. The detection method was designed as three competition assays in which the three toxins attached to the surface of three different classes of microspheres compete with free toxins for


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binding to toxin-specific monoclonal antibodies. The protocol consisted of an incubation of the sample or calibration solution (125 µL) with a mixture of the three antibodies (125 µL), GT-13A Ab (1 µg/mL), OA-Ab (1.3 µg/mL) and DA-Ab (1.9 µg/mL) in a microtiter plate. This first incubation is aimed at increasing the sensitivity of the assay. The calibration solution contained a mixture of the three toxins at an adequate concentration. After 1 h incubation, 220 µL of this mixture were transferred to another microtiter plate containing previously washed 3x(2x103) toxin-microspheres (2x103 STX-microspheres, 2x103 OA-microspheres, 2x103 DAmicrospheres). After 1 h the microspheres were washed and a volume of 100 µL of PE-labeled anti-mouse antibody (0.5 µg/mL), was added for 1 h. After a washing step, the microspheres were suspended in 100 µL of PBS-BT by shaking. The incubations were carried out at room temperature in the dark with constant shaking. All washing steps consisted of 3 washes performed by addition of 200 µL of PBS-BT and subsequent removal using vacuum. Quantification of binding signal. The fluorophore attached to the surface of the toxin-microspheres was quantified with a Luminex 200™ analyzer (LuminexCorp, Austin, Texas). Microspheres were classified with a 635 nm wavelength laser and PE fluorescence was quantified after excitation with a 532 nm wavelength laser. The acquisition volume was 75 µl and the number of minimum bead count was 100. Shellfish extraction method. Shellfish meat (100 g) was removed from the shell, drained, homogenized with a blender and frozen at -20 ˚C. One gram of homogenate was mixed with 5 mL of the extraction solution (30 % 0.2 M sodium acetate buffer (pH 5) and 70 % methanol), vortexed for 10 s, roller mixed for 30 min and centrifuged at 3000 g during 10 min. The supernatant was collected and the pellet was


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re-extracted with 3 mL of the extraction solution according to the same procedure. The supernatants were combined and made up to 10 mL. Later the extract was diluted in PBS-BT and filtered through a 0.45 µm filter. The selected shellfish matrixes were mussel and scallop. For mussel samples whole body was homogenized, for scallops whole body or muscle and gonad were used as indicated in the text. The samples used in this study did not contain detectable amounts of PSPs when tested by HPLC-FLD 17 or ASPs when tested by HPLC-UV 35. Trace concentrations of DSPs were detected in some mussel samples when analyzed by LC-MS/MS 36 however those concentrations were more than three times lower than the limit of detection of our immune-based competition assay for OA. Safety. Toxins should be handled with gloves and eye protection should be worn at all times. Appropriate disposal methods should also be utilized. Data analysis. Each experiment was performed in duplicate. The Student's t-test for unpaired data was used for statistical analysis, but multiple comparisons were performed using ANOVA (p

Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by

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